U.S. patent application number 09/854816 was filed with the patent office on 2002-10-17 for constrained helical peptides and methods of making same.
Invention is credited to Braisted, Andrew C., Judice, J. Kevin, McDowell, Robert S., Phelan, J. Christopher, Starovasnik, Melissa A., Wells, James A..
Application Number | 20020151473 09/854816 |
Document ID | / |
Family ID | 27367607 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020151473 |
Kind Code |
A1 |
Braisted, Andrew C. ; et
al. |
October 17, 2002 |
Constrained helical peptides and methods of making same
Abstract
Provided are cyclized peptides with a constrained region(s)
having an .alpha.-helical conformation. Constrained helical
peptides having amino acid sequences from HIV gp41 are provided, as
is their use in preparing antibodies that prevent viral membrane
fusion. Also provided are methods for making such cyclized
peptides.
Inventors: |
Braisted, Andrew C.; (San
Francisco, CA) ; Judice, J. Kevin; (San Francisco,
CA) ; McDowell, Robert S.; (San Francisco, CA)
; Phelan, J. Christopher; (San Francisco, CA) ;
Starovasnik, Melissa A.; (Burlingame, CA) ; Wells,
James A.; (Burlingame, CA) |
Correspondence
Address: |
PIPER MARBURY RUDNICK & WOLFE LLP
Supervisor, Patent Prosecution Services
1200 Nineteenth Street, N.W.
Washington
DC
20036-2412
US
|
Family ID: |
27367607 |
Appl. No.: |
09/854816 |
Filed: |
May 15, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09854816 |
May 15, 2001 |
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08965056 |
Nov 5, 1997 |
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09854816 |
May 15, 2001 |
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08876698 |
Jun 16, 1997 |
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09854816 |
May 15, 2001 |
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08743698 |
Nov 6, 1996 |
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60049787 |
Jun 16, 1997 |
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Current U.S.
Class: |
514/3.8 ;
514/21.1; 530/317 |
Current CPC
Class: |
A61K 39/00 20130101;
A61K 38/00 20130101; C12N 2740/16122 20130101; C07K 14/005
20130101 |
Class at
Publication: |
514/9 ;
530/317 |
International
Class: |
A61K 038/12; C07K
007/64 |
Claims
What is claimed is:
1. A method of constructing a constrained helical peptide,
comprising the steps of: (a) synthesizing a peptide, wherein the
peptide comprises a sequence of eight amino acid residues, wherein
the sequence of eight amino acid residues has a first terminal
residue and a second terminal residue, wherein the first terminal
residue and the second terminal residue flank an internal sequence
of six amino acid residues, and wherein the first and second
terminal residues have a side chain containing an amide
bond-forming substituent; (b) providing a difunctional linker
having a first functional group capable of forming an amide linkage
with the side chain amide bond-forming substituent of the first
terminal residue and having a second functional group capable of
forming an amide linkage with the side chain amide bond-forming
substituent of the second terminal residue; and (c) cyclizing the
peptide by reacting the side chain amide bond-forming substituent
of the first terminal residue with the first functional group of
the difunctional linker to form an amide linkage and reacting the
side chain amide bond-forming substituent of the second terminal
residue with the second functional group of the difunctional linker
to form an amide linkage, yielding a constrained helical
peptide.
2. The method of claim 1 wherein in step (a) the side chain amide
bond-forming substituent of the first terminal residue is protected
with a first protecting group and the side chain amide bond-forming
substituent of the second terminal residue is protected with a
second protecting group, wherein the first protecting group and the
second protecting group are differentially removable, and wherein
in step (c) the first protecting group is removed such that the
side chain amide bond-forming substituent of the first terminal
residue is deprotected and the side chain amide bond-forming
substituent of the second terminal residue is not deprotected
before the peptide is reacted with the difunctional linker, and
thereafter the peptide is reacted with the difunctional linker to
form an amide linkage between the side chain amide bond-forming
substituent of the first terminal residue and the first functional
group of the difunctional linker, and thereafter the second
protecting group is removed from the side chain amide bond-forming
substituent of the second terminal residue and the peptide is
cyclized by intramolecularly reacting the side chain amide
bond-forming substituent of the second terminal residue with the
second functional group of the difunctional linker to form an amide
linkage.
3. A method of constructing a constrained helical peptide,
comprising the steps of: (a) synthesizing a peptide, wherein the
peptide comprises a sequence of eight amino acid residues, wherein
the sequence of eight amino acid residues has a first terminal
residue and a second terminal residue, wherein the first terminal
residue and the second terminal residue flank an internal sequence
of six amino acid residues, wherein the first and second terminal
residues have a side chain containing an amide bond-forming
substituent, wherein the first terminal residue is coupled to a
difunctional linker having a first functional group and a second
functional group, wherein the first functional group is in an amide
linkage with the side chain amide bond-forming substituent of the
first terminal residue, and wherein the second functional group of
the difunctional linker is capable of forming an amide linkage with
the side chain amide bond-forming substituent of the second
terminal residue; and (b) cyclizing the peptide by intramolecularly
reacting the side chain amide bond-forming substituent of the
second terminal residue with the second functional group of the
difunctional linker to form an amide linkage and yield a
constrained helical peptide.
4. A compound selected from the group consisting of: the compound
represented by Formula (1): 171 wherein S is absent or is a
macromolecule, X is hydrogen or is any amino acid or amino acid
sequence, Y is absent, or is hydroxyl if S is absent, or is any
amino acid or amino acid sequence, Z is any amino acid sequence
consisting of six amino acids; m and p are independently selected
from the integers 0 to 6 inclusive, provided that m+p is less than
or equal to 6, and n is any integer in the range defined by
(7-(m+p)) to (9-(m+p)) inclusive, provided that n is greater than
1; the compound represented by Formula (6): 172 wherein S is absent
or is a macromolecule, X is hydrogen or is any amino acid or amino
acid sequence, Y is absent, or is hydroxyl if S is absent, or is
any amino acid or amino acid sequence, Z is any amino acid sequence
consisting of six amino acids, q is selected from the integers 1 to
7 inclusive, s is selected from the integers 0 to 6 inclusive,
provided that q+s is less than or equal to 7, and r is any integer
in the range defined by (7-(q+s)) to (9-(q+s)) inclusive, provided
that r is greater than 0; the compound represented by Formula (11):
173 wherein S is absent or is a macromolecule, X is hydrogen or is
any amino acid or amino acid sequence, Y is absent, or is hydroxyl
if S is absent, or is any amino acid or amino acid sequence, Z is
any amino acid sequence consisting of six amino acids; t is
selected from the integers 0 to 6 inclusive, and v is selected from
the integers 1 to 7 inclusive, provided that t+v is less than or
equal to 7; and u is any integer in the range defined by (7-(t+v))
to (9-(t+v)) inclusive, provided that u is greater than 0; and the
compound represented by Formula (16): 174 wherein S is absent or is
a macromolecule, X is hydrogen or is any amino acid or amino acid
sequence, Y is absent, or is hydroxyl if S is absent, or is any
amino acid or amino acid sequence, Z is any amino acid sequence
consisting of six amino acids; w and y are independently selected
from the integers 1 to 7 inclusive, provided that w+y is less than
or equal to 8, and x is any integer in the range defined by
(7-(w+y)) to (9w+y)) inclusive, provided that x is greater than or
equal to 0.
5. The compound of claim 4 that is the compound of Formula (1),
wherein Z is Gln-Gln-Arg-Arg-Phe-Tyr.
6. A constrained helical peptide made according to the method of
claim 1.
7. A constrained helical peptide made according to the method of
claim 3.
8. A compound according to claim 4, wherein Z is an amino acid
sequence consisting of six amino acids, wherein the internal
sequence of six amino acids has the form gabcde, defgab, or cdefga
and is selected from the group of sequences consisting of a
sequence of six contiguous amino acids in HIV-1LAI strain gp41
amino acid sequence 633 to 678, in its homolog sequence from
another HIV strain, in a consensus sequence of its homolog
sequences from any one HIV clade, or amino acid substituted variant
thereof, in which amino acid 633 or its corresponding amino acid in
the homolog, consensus or variant sequence is assigned position a
of a repeating abcdefg assignment.
9. The compound of claim 8, further comprising S' when S is absent
and X is any amino acid or amino acid sequence, wherein S' is a
macromolecule attached to X.
10. A compound comprising a first constrained helical peptide
comprising a peptide comprising a sequence of eight amino acid
residues, wherein the sequence of eight amino acid residues has a
first terminal residue and a second terminal residue, wherein the
first terminal residue and the second terminal residue flank an
internal sequence of six amino acids, wherein the first and second
terminal residues have a side chain that are linked to each other
forming a locking moiety to form a constrained helical peptide,
wherein the internal sequence of six amino acids has the form
gabede, defgab, or cdefga and is selected from the group of
sequences consisting of a sequence of six contiguous amino acids in
HIV-1LAI strain gp41 amino acid sequence 633 to 678, in its homolog
sequence from another HIV strain, in a consensus sequence of its
homolog sequences from any one HIV lade, or in an amino acid
substituted variant thereof, in which amino acid 633 or its
corresponding amino acid in the homolog, consensus or variant
sequence is assigned position a of a repeating abcdefg
assignment.
11. The compound of claims 8 or 10, wherein the homolog or
consensus sequence is shown in FIGS. 16A-16G.
12. A compound of claims 8 or 10, further comprising a second
constrained helical peptide.
13. An antibody that binds to a compound of claim 8, wherein the
antibody specifically binds an epitope comprising an amino acid at
position a, d, e, or g in the helical peptide.
14. A method to prophylactically or therapeutically treat a mammal
at risk for or infected with HIV, comprising administering a
prophylactically or therapeutically effective amount of a compound
of claims 8 or 10.
15. The method of claim 14, wherein the composition comprises
internal six amino acid sequences from different HIV strains or HIV
clades.
16. A vaccine comprising at least one compound of claims 8 or 10.
Description
[0001] This is a non-provisional application filed under 37 CFR
1.53(b), claiming priority under USC Section 119(e) to provisional
Application Serial No. 60/049,787 filed on Jun. 16, 1997 and
provisional Application Serial No. 60/(unassigned) [Attorney Docket
No. PR1005] filed Nov. 6, 1996.
BACKGROUND OF THE INVENTION
[0002] Field of the Invention
[0003] The invention relates to the conformational constraint of
peptides. In particular, the invention relates to constraining
peptides to an .alpha.-helical conformation. This invention also
relates to the rational design and preparation of HIV vaccines
based on HIV gp41 polypeptide sequences. This invention further
relates to improved methods for HIV infection diagnosis and
immunogens which induce antibodies useful in the diagnostic
methods.
[0004] A variety of methods for stabilizing .alpha.-helical
peptides have been described previously. Addition of
trifluoroethanol or hexafluoroisopropanol has frequently been used
to stabilize .alpha.-helices in aqueous solution. Dimerization of
.alpha.-helices at hydrophobic interfaces has also provided
exogenous stabilization. Short .alpha.-helical peptides have been
stabilized by incorporating groups at the termini to stabilize the
intrinsic helix dipole. Naturally occurring capping motifs as well
as organic templates have been used to stabilize .alpha.-helices by
end-nucleation. Several non-covalent side chain constraints have
been investigated for .alpha.-helix stabilization, including
hydrophobic interactions, salt bridges, and metal ion chelation by
both natural and unnatural amino acids.
[0005] Finally, .alpha.-helices have been stabilized by covalent
side chain tethers. Chorev et al, Biochemistry, 30: 5968-5974
(1991), Osapay et al., J. Am. Chem. Soc. 112: 6046-6051 (1990),
Osapay et al., J. Am. Chem. Soc., 114: 6966-6973 (1990), Bracken et
al., J. Am. Chem. Soc., 116: 6431-6432 (1994), and Houston et al.,
J. Peptide Science, 1: 274-282 (1995) described the stabilization
of .alpha.-helices by side chain to side chain lactamization. Ravi
et al, J. Am. Chem. Soc. 105: 105-109 (1983) and Jackson et al., J.
Am. Chem. Soc., 113: 9391-9392 (1991) described the constraint of
peptides by disulfide bonds between residues. The naturally
occurring peptide apamin has been used as a scaffold for the
presentation of .alpha.-helical peptide sequences constrained in
helical conformation by disulfide bonds to scaffold cysteine
residues.
[0006] Acquired immunodeficiency syndrome (AIDS) is caused by a
retrovirus identified as the human immunodeficiency virus (HIV).
There have been intense efforts to develop a vaccine that induces a
protective immune response based on induction of antibodies or
cellular responses. Recent efforts have used subunit vaccines where
an HIV protein, rather than attenuated or killed virus, is used as
the immunogen in the vaccine for safety reasons. Subunit vaccines
generally include gp120, the portion of the HIV envelope protein
which is on the surface of the virus.
[0007] The HIV envelope protein has been extensively described, and
the amino acid and nucleic acid sequences encoding HIV envelope
from a number of HIV strains are known (Myers, G. et al., 1992.
Human Retroviruses and AIDS. A compilation and analysis of nucleic
acid and amino acid sequences. Los Alamos National Laboratory, Los
Alamos, N.M.). The HIV envelope protein is a glycoprotein of about
160 kd (gp160) which is anchored in the membrane bilayer at its
carboxyl terminal region. The N-terminal segment, gp120, protrudes
into the aqueous environment surrounding the virion and the
C-terminal segment, gp41, spans the membrane. Via a host-cell
mediated process, gp160 is cleaved to form gp120 and the integral
membrane protein gp41. As there is no covalent attachment between
gp120 and gp41, free gp120 is sometimes released from the surface
of virions and infected cells.
[0008] gp120 has been the object of intensive investigation as a
vaccine candidate for subunit vaccines, as the viral protein which
is most likely to be accessible to immune attack. At present,
clinical trials using gp120 MN strain are underway.
[0009] However, effective vaccines based on gp120 or another HIV
protein for protection against additional strains of HIV are still
being sought to prevent the spread of this disease.
SUMMARY OF THE INVENTION
[0010] The invention provides a method for constructing a
constrained helical peptide comprising the steps of: (1)
synthesizing a peptide, wherein the peptide comprises a sequence of
eight amino acid residues, wherein the sequence of eight amino acid
residues has a first terminal residue and a second terminal
residue, wherein the first terminal residue and the second terminal
residue flank an internal sequence of six amino acid residues, and
wherein the first terminal residue has a side chain containing an
amide bond-forming substituent and the second terminal residue has
a side chain containing an amide bond-forming substituent; (2)
providing a difunction al linker having a first functional group
capable of forming an amide linkage with the side chain amide
bond-forming substituent of the first terminal residue and having a
second functional group capable of forming an amide linkage with
the side chain amide bond-forming substituent of the second
terminal residue; and (3) cyclizing the peptide by reacting the
side chain amide bond-forming substituent of the first terminal
residue with the first functional group of the difunctional linker
to form an amide linkage and reacting the side chain amide
bond-forming substituent of the second terminal residue with the
second functional group of the difunctional linker to form an amide
linkage, yielding a constrained helical peptide.
[0011] The invention also provides a method for constructing a
constrained helical peptide comprising the steps of: (1)
synthesizing a peptide, wherein the peptide comprises a sequence of
eight amino acid residues, wherein the sequence of eight amino acid
residues has a first terminal residue and a second terminal
residue, wherein the first terminal residue and the second terminal
residue flank an internal sequence of six amino acid residues,
wherein the first terminal residue has a side chain containing an
amide bond-forming substituent and the second terminal residue has
a side chain containing an amide bond-forming substituent, and
wherein the side chain amide bond-forming substituent of the first
terminal residue is protected with a first protecting group and the
side chain amide bond-forming substituent of the second terminal
residue is protected with a second protecting group such that the
first protecting group and the second protecting group are
differentially removable; (2) removing the first protecting group
such that the side chain amide bond-forming substituent of the
first terminal residue is deprotected and the side chain amide
bond-forming substituent of the second terminal residue is not
deprotected; (3) providing a difunctional linker having a first
functional group capable of forming an amide linkage with the side
chain amide bond-forming substituent of the first terminal residue
and having a second functional group capable of forming an amide
linkage with the side chain amide bond-forming substituent of the
second terminal residue; (4) reacting the peptide with the
difunctional linker to form an amide linkage between the first
functional group of the difunctional linker and the side chain
amide bond-forming substituent of the first terminal residue; (5)
removing the second protecting group to deprotect the side chain
amide bond-forming substituent of the second terminal residue; and
(6) cyclizing the peptide by intramolecularly reacting the side
chain amide bond-forming substituent of the second terminal residue
with the second functional group of the difunctional linker to form
an amide linkage and yield a constrained helical peptide.
[0012] The invention further provides a method for constructing a
constrained helical peptide, comprising the steps of: (a)
synthesizing a peptide, wherein the peptide comprises a sequence of
eight amino acid residues, wherein the sequence of eight amino acid
residues has a first terminal residue and a second terminal
residue, wherein the first terminal residue and the second terminal
residue flank an internal sequence of six amino acid residues,
wherein the first terminal residue has a side chain containing an
amide bond-forming substituent and the second terminal residue has
side chain containing an amide bond-forming substituent, wherein
the first terminal residue is coupled to a difunctional linker
having a first functional group and a second functional group,
wherein the first functional group is in an amide linkage with the
side chain amide bond-forming substituent of the first terminal
residue, and wherein the second functional group of the
difunctional linker is capable of forming an amide linkage with the
side chain amide bond-forming substituent of the second terminal
residue; and (b) cyclizing the peptide by intramolecularly reacting
the side chain amide bond-forming substituent of the second
terminal residue with the second functional group of the
difunctional linker to form an amide linkage and yield a
constrained helical peptide.
[0013] The invention additionally provides a method for
constructing a constrained helical peptide comprising the steps of:
(1) synthesizing a peptide, wherein the peptide comprises a
sequence of eight amino acid residues, and wherein the sequence of
eight amino acid residues has a first terminal residue and a second
terminal residue, wherein the first terminal residue and the second
terminal residue are independently selected from Asp and Glu; (2)
providing a diamine linker having a first amino group capable of
forming an amide linkage with the carboxy side chain of the first
terminal residue and a second amino group capable of forming an
amide linkage with the carboxy side chain of the second terminal
residue; and (3) cyclizing the peptide by reacting the first amino
group of the diamine linker with the carboxy side chain of the
first terminal residue to form an amide linkage and reacting the
second amino group of the diamine linker with the carboxy side
chain of the second terminal residue to form an amide linkage,
yielding a constrained helical peptide.
[0014] The invention also encompasses a method for constructing a
constrained helical peptide comprising the steps of: (1)
synthesizing a peptide, wherein the peptide comprises a sequence of
eight amino acid residues, wherein the sequence of eight amino acid
residues has a first terminal residue and a second terminal
residue, wherein the first terminal residue and the second terminal
residue flank an internal sequence of six amino acid residues,
wherein the first terminal residue and the second terminal residue
are independently selected from Asp and Glu, and wherein the
carboxy side chain of the first terminal residue is protected with
a first protecting group and the carboxy side chain of the second
terminal residue is protected with a second protecting group such
that the first protecting group and the second protecting group are
differentially removable; (2) removing the first protecting group
such that the carboxy side chain of the first terminal residue is
deprotected and the carboxy side chain of the second terminal
residue is not deprotected; (3) reacting the peptide with a diamine
linker having a first amino group and a second amino group to form
an amide linkage between the deprotected carboxy side chain of the
first terminal residue and the first amino group of the diamine
linker; (4) removing the second protecting group to deprotect the
carboxy side chain of the second terminal residue; and (5)
cyclizing the peptide by intramolecularly reacting the deprotected
carboxy side chain of the second terminal residue with the second
amino group of the diamine linker to form an amide linkage and
yield a constrained helical peptide.
[0015] The invention further encompasses a method for constructing
a constrained helical peptide comprising the steps of: (1)
synthesizing a peptide, wherein the peptide comprises a sequence of
eight amino acid residues, wherein the sequence of eight amino acid
residues has a first terminal residue and a second terminal
residue, wherein the first terminal residue and the second terminal
residue flank an internal sequence of six amino acid residues,
wherein the first terminal residue and the second terminal residue
are independently selected from Asp and Glu, and wherein the
carboxy side chain of the first terminal residue is coupled to a
diamine linker having a first amino group and a second amino group,
such that the carboxy side chain of the first terminal residue is
in an amide linkage with the first amino group of the diamine
linker; and (2) cyclizing the peptide by intramolecularly reacting
the carboxy side chain of the second terminal residue with the
second amino group of the diamine linker to form an amide linkage
and yield a constrained helical peptide.
[0016] The invention also encompasses a compound selected from the
group consisting of:
[0017] the compound represented by Formula (1): 1
[0018] wherein S is absent or is a macromolecule, X is hydrogen or
is any amino acid or amino acid sequence, Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence, Z is any amino acid sequence consisting of six amino
acids; m and p are independently selected from the integers 0 to 6
inclusive, provided that m+p is less than or equal to 6, and n is
any integer in the range defined by (7-(m+p)) to (9-(m+p))
inclusive, provided that n is greater than 1;
[0019] the compound represented by Formula (6): 2
[0020] wherein S is absent or is a macromolecule, X is hydrogen or
is any amino acid or amino acid sequence, Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence, Z is any amino acid sequence consisting of six amino
acids, q is selected from the integers 1 to 7 inclusive, s is
selected from the integers 0 to 6 inclusive, provided that q+s is
less than or equal to 7, and r is any integer in the range defined
by (7-(q+s)) to (9-(q+s)) inclusive, provided that r is greater
than 0;
[0021] the compound represented by Formula (11): 3
[0022] wherein S is absent or is a macromolecule, X is hydrogen or
is any amino acid or amino acid sequence, Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence, Z is any amino acid sequence consisting of six amino
acids; t is selected from the integers 0 to 6 inclusive, and v is
selected from the integers 1 to 7 inclusive, provided that t+v is
less than or equal to 7; and u is any integer in the range defied
by (7-(t+v)) to (9-(t+v)) inclusive, provided that u is greater
than 0; and
[0023] the compound represented by Formula (16): 4
[0024] wherein S is absent or is a macromolecule, X is hydrogen or
is any amino acid or amino acid sequence, Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence, Z is any amino acid sequence consisting of six amino
acids; w and y are independently selected from the integers 1 to 7
inclusive, provided that w+y is less than or equal to 8, and x is
any integer in the range defined by (7-(w+y)) to (9-(w+y))
inclusive, provided that x is greater than or equal to 0.
[0025] In a preferred embodiment is provided a compound containing
a constrained helical peptide that in turn contains a peptide of a
sequence of eight amino acid residues, in which the sequence of
eight amino acid residues has a first terminal residue and a second
terminal residue that flank an internal sequence of six amino acids
and that have a side chain that are linked to each other forming a
locking moiety to form a constrained helical peptide. The internal
sequence of six amino acids has the form gabcde, defgab, or cdefga
and is selected from the group of sequences consisting of a
sequence of six contiguous amino acids in HIV-1LAI strain gp41
amino acid sequence 633 to 678, in its homolog sequence from
another HIV strain, in a consensus sequence of its homolog
sequences from any one HIV lade, or an amino acid substituted
variant thereof, in which amino acid 633 or its corresponding amino
acid in the homolog, consensus or variant sequence is assigned
position a of a repeating abcdefg assignment for the 633-678
sequence (as shown in FIG. 18). In these compounds the locking
moiety or tether is between adjacent f positions when the internal
sequence is of the form gabcde, adjacent c positions when the
internal sequence is of the form defgab, or adjacent b positions
when the internal sequence is of the form cdefga. Most preferably
the lock is between adjacent f positions. FIG. 18 provides the
alignment of the repeating abcdefg assignment to the amino acids in
the 633-678 region. In a preferred embodiment the internal sequence
of six amino acids has the form gabcde. The compounds preferably
have HIV anti-fusogenic or anti-infection activity.
[0026] Preferred compounds are those selected from the group
consisting constrained helical peptides of each possible sequence
having any one or any combination of amino acid substitutions
indicated in the constrained helical peptide series I to XII as
shown in FIGS. 23A and 23B in combination with any one or any
combination of amino acid truncations indicated in the constrained
helical peptide series I to XII as shown in FIGS. 23A and 23B.
Peptides HIV24 and HIV31 are particularly preferred compounds of
this type.
[0027] In another embodiment the compounds of the invention are
used as haptens, preferably attached to carriers, for use as an
immunogen to raise antibodies that have a diagnostic use or as a
vaccine for prophylactic or therapeutic treatment of patients at
risk for or infected with HIV. Examples of such prophylactic use of
the peptides may include, but are not limited to, prevention of
virus transmission from mother to infant and other settings where
the likelihood of HIV transmission exists, such as, for example,
accidents in health care settings wherein workers are exposed to
HIV-containing blood products. The constrained peptides of the
invention can serve the role of a prophylactic vaccine, wherein the
host raises antibodies against the peptides of the invention, which
then serve to neutralize HIV viruses by, for example, inhibiting
further HIV infection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 is a diagram depicting the synthesis of peptide 1b
and 1c. Reagent a represents 20% piperidine/DMA; reagent b
represents H.sub.2NCH.sub.2CH.sub.2CH.sub.2NHR (R=H or BOC), BOP,
DIPEA, CH.sub.2Cl.sub.2; reagent c represents Pd(PPh.sub.3).sub.4,
20% piperidine/DMA, (R=BOC)
TFA/CH.sub.2Cl.sub.2/anisole/1,2-ethanedithiol 45:45:5:5 v/v;
reagent d represents BOP, DIPEA, CH.sub.2Cl.sub.2; reagent e
represents HF/anisole/EtSMe20:2:1 v/v, 0.degree. C.; and reagent f
represents CH.sub.3NH.sub.2, BOP, CH.sub.2Cl.sub.2.
[0029] FIG. 2 is a diagram depicting the synthesis of
N-Fmoc-S-Acm-D-thiolysine (compound 7). Reagent a represents
.sup.nBuLi, THF, -78.degree. C.; Br(CH.sub.2).sub.4Br; reagent b
represents 4-MeOBnSH, KO.sup.tBu, THF; reagent c represents 0.25 M
HCl, THF/H.sub.2O; reagent d represents Hg(OAc).sub.2, TFA;
H.sub.2S; reagent e represents acetamidomethanol, TFA; reagent f
represents LiOH, THF/H.sub.2O; and reagent g represents Fmoc-OSu,
dioxane, NaHCO.sub.3.
[0030] FIGS. 3a and 3b are graphs depicting the
H.sup.N-H.sup..alpha. and H.sup.N-H.sup.N sections, respectively,
of the ROESY spectrum of peptide 1c. The spectrum was collected at
280 K, pH 5.0, 500 MHZ and a peptide concentration of 1.5 mM with a
4.5 kHz spin-lock mixing pulse of 200 ms duration. Lines connect
the ROEs by which sequential assignments were made. Rectangular,
oval and diamond shaped boxes denote intra residue, sequential and
(I, I+3) correlations, respectively.
[0031] FIG. 4 is a graph depicting ROE and .sup.3J.sub.HN-H.alpha.
data for peptides 1c and 1b. For the d.sub.NN and d.sub..alpha.N
rows, observation of the sequential ROE is indicated by a bar
connecting two residues, the thickness of the bar indicating the
relative intensity of the ROE. The downward pointing arrows
indicate 3J.sub.HN-H.alpha. less than 6.0 Hz. Observed medium range
ROEs (H.sup..alpha.-H.sup.N I, I+3 and H.sup..alpha.-H.sup..beta.I,
I+3) are indicated by the lines in the lower part of the figure;
dotted lines and stars indicate ROEs that could not be
unambiguously observed because of chemical shift degeneracy. The
coil motif above the primary sequence indicates the region deduced
to have helical structure from the NMR data; the dashed coil
indicates sections of peptide where only some of the NMR data
indicate helical character.
[0032] FIG. 5 is a molecular model depicting an ensemble of 20 rMD
structures calculated using NMR data for peptide 1c. The structures
were overlayed using the N, C.alpha. and C atoms of residue Thr1 to
Gln10. Backbone and side-chain heavy atoms are connected by solid
and dotted lines, respectively. The side-chains of Arg8 and Arg9
are truncated at C.sup..gamma., and all side-chain atoms of Gln11
and Gln12 are omitted for clarity.
[0033] FIG. 6 is a graph depicting the CD spectra of peptide 1c at
280, 310, 330, 350, and 370 K.
[0034] FIG. 7 is a graph depicting the CD spectra of peptides 1 and
3 (Apamin-based sequences) at 280 K.
[0035] FIG. 8 is a graph depicting the CD spectra of peptides 2 and
4 (C-peptide-based sequences) at 280 K.
[0036] FIG. 9 is a graph depicting the thermal denaturation profile
of peptide 1c as determined by CD spectra obtained before, during
and after heating for 1 day at 87.degree. C. Circles indicate the
initial spectrum obtained from a sample before heating; squares
indicate the spectrum obtained from a sample at 87.degree. C.
during incubation; triangles indicate the spectrum obtained from a
sample after recooling to 7.degree. C. at 0.2.degree. C./min.
[0037] FIG. 10 is a graph depicting a section of the TOCSY spectrum
of peptide 1c. The data were collected at 280 K, pH 5.0, 500 MHZ
and a peptide concentration of 1.5 mM with a mixing time of 90 ms.
The solid lines connect cross-peaks between backbone amide and side
chain protons; assignments are indicated at the top of each line.
Dashed lines connect cross-peaks between the side chain amide
protons of Gln3 and Gln10 and the methylene linker resonances.
[0038] FIG. 11 is a diagram depicting the synthesis of a locked
helix species of the peptide
Asn-Met-Glu-Gln-Gln-Arg-Arg-Phe-Tyr-Glu-Ala-Leu-Hi- s where the
carboxy side chains of the Glu residues are covalently linked with
a 1,5-pentanediamine linker.
[0039] FIG. 12 depicts sequences and schematic representations of
the locked-helix peptide embodiments of the invention. The
cylinders represent .alpha.-helices, with the stippled faces
corresponding to the 4, 3 hydrophobic repeat. Covalent restraints
linking side chains at I and I+7 are represented as dark lines.
[0040] FIG. 13 is a circular dichroism spectra of peptides HIV24
(open squares), HIV30 (open circles), HIV31 (closed circles), and
HIV35 (closed squares). Spectra were acquired at 7.degree. C. in 10
mM Tris-HCl, pH 7.5(21).
[0041] FIGS. 14A and 14B are graphs depicting the effect of
inhibitory peptides in primary infectivity assays using PBMCs with
virus JRCSF, an NSI strain (FIG. 14A), and BZ167, an SI strain
(FIG. 14B) (22). HIV24 (closed triangles); HIV30 (open circles);
HIV31 (closed circles); HIV35 (closed squares); DP178, (open
squares).
[0042] FIG. 15 is a schematic of a proposed mechanism for assembly
of the fusogenic state of gp41 (top) and inhibition by constrained
peptides (bottom).
[0043] FIGS. 16A to 16G present amino acid sequences of gp41 from
known HIV virus strains and their consensus sequences based on
statistical amino acid frequency. Amino acids are represented by
the standard single letter code. The strains within each HIV clade
are presented. A "-" in a sequence represents the amino acid
present in the consensus sequence for that clade. A "." represents
an amino acid gap. A "?" in a consensus sequence represents any
amino acid at that corresponding position found in a viral sequence
within that clade. A lower case amino acid represents the most
frequent amino acid from among all amino acids at that
corresponding position in viral sequences within that clade. An
upper case amino acid in a consensus sequence indicates that only
that amino acid is found at that corresponding position in viral
sequences within that clade. Strain designations with no sequence
information indicate that the complete gp41 sequence has not been
determined.
[0044] FIG. 17 is a summary of consensus sequences from known
strains. The peptide sequence of DP 178 is delineated. The
nomenclature is the same as in FIGS. 16A to 16G.
[0045] FIG. 18 is a schematic presenting an alignment of sequences
from clades A, B, C, D, and E consensus sequences, peptides DP178,
HIV35 and the Neurathpeptide, in which the repeating heptad abcdefg
assignment as taught herein is provided, and positions of some
constraining locks are indicated. For example, amino acids in the
sequence ESQNQQ of DP178 are assigned positions g, a, b, c, d, and
e, respectively, and thus has the form gabcde, for purposes o the
present invention. This sequence is the internal sequence of six
amino acids present in peptide HIV24, which is a single-lock form
of the HIV35 sequence. Locations of internal sequences of the
invention are those found between locking residues, whose positions
are indicated by the ".vertline." symbols and each of which, in
this example, correspond to assigned position f. Positions for
placing either one, two or three locks in the representative
presented sequences are shown. The figure delineates five gabede
form helical sections suitable for locking when locks occur at
adjacent f positions. Also shown are locations of gabcde form
helical sections when one, two or three i to i+7 locks are present
in a 633-678 sequence or variant thereof. The two-lock variants are
labeled (II), (III), HIV31, (VI) and (VII), and the one-lock
variants (VIII), (IX), HIV24, (XI) and (XII). Three-lock variant is
labeled (I).
[0046] FIG. 19 is a helical wheel representation of the
representative gp41 fusion peptide sequence from the HIV-1LAI
strain, showing the "abcdefg" heptad reading frame and the heptad
repeat pattern as assigned herein (see FIG. 18) for the purposes of
the present invention.
[0047] FIG. 20 is a schematic depicting the use of the compounds of
the invention as haptens for immunization and shows the gp41 core
trimer, its DP178 binding groove and the 633-678 region that binds
this grove.
[0048] FIG. 21 is a schematic depicting a proposed mechanism for
antibody intervention in HIV viral infectivity.
[0049] FIG. 22 is presents a consensus sequence of the HIV gp41
sequences from FIG. 17 with all allowed amino acid substitutions in
each position listed. For example, at the fifth amino acid position
(starting from the N-terminal amino acid (left end)), the amino
acids E (glutamic acid), D (aspartic acid) and K (lysine) are
allowed without disrupting H-bonding, thus without disrupting
helicity or significantly interfering with the peptide's
interaction with the core coiled-coil trimer of gp41. "X" indicates
positions that can be substituted with any non-helix breaking amino
acid. The repeating heptad abcdefg assignment for each amino acid
position in the 633 to 678 sequence, for purposes of the present
invention, is shown. The "*" indicate b, c, and f positions that,
when not used for locking the helix, can be replaced with a non
helix-breaking amino acid without significantly disturbing
H-bonding, helicity and trimer groove binding.
[0050] FIG. 23 presents a shorthand notation of specific peptides
in peptide series I through XII (as in FIG. 18), indicating locking
positions, amino acid substitution variant peptides, and truncation
variant peptides of each. The "X" indicates a position that can be
substituted with any non helix-breaking amino acid, but preferably
with an amino acid present in that position from any one of the
known HIV sequences shown in FIG. 16. "B" indicates a position used
for the bridging (or tethering or locking) residues. Preferred f
positions are presented for locking; however in less preferred
embodiments the c and some b positions can be used for locking. As
in FIG. 18, locations of internal sequences relevant to the
invention are those found between locking residues whose positions
are indicated by the ".vertline." symbols and correspond to
assigned position f, in this example. Positions for placing either
one, two or three locks in the representative presented sequences
are shown. The figure delineates five gabcde form helical sections
suitable for locking when locks occur at adjacent f positions. The
"." indicates positions that can be optionally absent from the
final constrained helical peptide compound without substantially
effecting the helical properties and groove binding properties of
the final constrained helical peptide. For example, a peptide based
on peptide I, having the three locks placed as indicated, can
optionally lack any one or all of the five N-terminal amino acids
WXXWE, which are marked by a ".". Further, another series of
truncated variants is indicated in the figure--C-terminal truncated
variants--since the five C-terminal residues (LWNWF) are marked
with a "." can be absent. When the lock is placed more centrally in
the 633-678 sequence, as shown in peptide series II, peptides in
this series can lack additional amino acids at the C-terminal end
as indicated by the "." marked positions.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0051] A. Definitions
[0052] Amino acids and amino acid residues described herein may be
referred to according to the accepted one or three letter code
provided in the table below. Unless otherwise specified, these
amino acids or residues are of the naturally occurring L
stereoisomer form.
1 Three-Letter Common Name One-Letter Symbol Symbol Alanine A Ala
Arginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys
Glutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H His
Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine M Met
Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T Thr
Tryptophan W Trp Tyrosine Y Tyr
[0053] In general, unless otherwise specified, the abbreviations
used for the designation of amino acids and the protective groups
used therefor are based on the recommendations of the IUPAC-IUB
Commission of Biochemical Nomenclature (Biochemistry, 11:1726-1732
(1972)).
[0054] As used herein, the term --(CH.sub.2).sub.n is used to
denote a straight chain alkyl substituent of n carbons in length,
wherein --(CH.sub.2).sub.0-- is defined as a chemical bond, i.e.
indicating that no alkyl substituent is present,
--(CH.sub.2).sub.1-- is defined as a methyl substituent,
--(CH.sub.2).sub.2-- is defined as an ethyl substituent, etc.
[0055] As used herein, the term "C .sub.1-C.sub.6alkyl" means a
saturated aliphatic hydrocarbon substituent having the number of
carbon atoms specified. C.sub.1-C.sub.6alkyl encompasses cyclic and
straight chain hydrocarbons, unbranched and branched hydrocarbons,
substituted and unsubstituted hydrocarbons, and primary, secondary
and tertiary hydrocarbon substituents. Representative examples of
these alkyl substituents include methyl, fluorenylmethyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,
n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl,
2-methylpentyl, 2,2-dimethylbutyl, cyclohexyl, and the like. The
terms "lower alkyl", "simple alkyl" and "C.sub.1-C.sub.6alkyl" are
synonymous and used interchangeably.
[0056] As used herein, the terms "peptide", "polypeptide", and
"protein" are used synonymously and refer to any proteinaceous
compound comprising an amino acid sequence of two or more amino
acid residues.
[0057] As used herein, an "amide bond-forming substituent contained
in an amino acid side chain", a "side chain amide bond-forming
subsituent", and their grammatical variants, are defined to include
(1) any carboxy substituent contained in the side chain ("R" group)
of an amino acid wherein the carboxy substituent is capable of
forming an amide linkage with an amino group contained in another
molecule, i.e. the carboxy substitutent reacts with an amino group
contained in another molecule to form an amide linkage; and (2) any
amino substituent contained in the side chain ("R" group) of an
amino acid wherein the amino substituent is capable of forming an
amide linkage with a carboxy group contained in another molecule,
i.e. the amino substitutent reacts with a carboxy group contained
in another molecule to form an amide linkage.
[0058] As used herein, "differentially removable" protecting or
protective groups are defined as any pair of protective groups
capable of protecting a first amide bond-forming substituent and a
second amide bond-forming substituent, wherein it is possible to
deprotect the first amide bond-forming substituent protected with
one member of the pair under conditions which do not deprotect the
second amide bond-forming substituent protected with the other
member of the pair. Differentially removable protecting groups are
also referred to herein as "orthogonal" protecting groups, and the
differentially removable protection conferred by such protective
groups is referred to herein as "orthogonal" protection.
[0059] The term "epitope" as used herein, designates the structural
component of a molecule that is responsible for specific
interactions with corresponding antibody (immunoglobulin) molecules
elicited by the same or related antigen. More generally, the term
refers to a peptide having the same or similar immunoreactive
properties, such as specific antibody binding affinity, as the
antigenic protein or peptide used to generate the antibody.
Therefore, an epitope that is formed by a specific peptide sequence
generally refers to any peptide which is reactive with antibodies
directed against the specific sequence.
[0060] The term "antigen" as used herein, means a molecule which is
used to induce production of antibodies. The term is alternatively
used to denote a molecule which is reactive with a specific
antibody.
[0061] The term "immunogen" as used herein, describes an entity
that induces antibody production in a host animal. In some
instances the antigen and the immunogen are the same entity, while
in other instances the two entities are different.
[0062] The term "subunit vaccine" is used herein, as in the art to
refer to a viral vaccine that does not contain virus, but rather
contains one or more viral proteins or fragments of viral proteins.
As used herein, the term "multivalent", means that the vaccine
contains a constrained helical peptide or peptides having a
gp41-based sequence from at least two HIV isolates having different
amino acid sequences.
[0063] The term "breakthrough isolate" or "breakthrough virus" is
used herein, as in the art, to refer to a virus isolated from a
vaccinee.
[0064] B. General Methods
[0065] In general, the invention provides a method for removing
elements of .alpha.-helical secondary structure from the context of
a protein without losing the well defined structure found within
the protein's .alpha.-helix. In one aspect, the method is useful
for artificially reconstructing and characterizing the binding
determinants that exist within an .alpha.-helical binding domain of
a protein of interest. The design of molecules which are capable of
binding competitively at a protein interface requires the ability
to mimic the higher level structure of the natural ligand. If the
ligand's structure at the site of protein interface can be mimicked
with a short peptide, then the peptide can be used to determine
whether it is feasible to design small molecules that competitively
bind at the protein interface. A short peptide's ability to compete
with the natural ligand for binding at the protein interface would
indicate that the ligand's structure at the contact point with the
protein interface is such that the short peptide could be used as a
model for designing small molecules that compete with the natural
ligand for binding at the protein interface.
[0066] In another aspect, the methods of the invention are used to
stabilize the conformational structure of a protein or peptide. The
present methods can be employed to lock in place one (or more)
.alpha.-helical determinant(s) of interest in a protein or peptide
such that the protein (or peptide) retains an .alpha.-helical
conformation in environments or conditions that would destabilize
or deteriorate the .alpha.-helical secondary structure of an
unconstrained protein or peptide species.
[0067] The methods of the invention are also useful for the
replication of protein function without an intact protein or intact
functional domain. For example, the replication of a protein's
binding activity by a constrained helical peptide of the invention
would allow the use of affinity purification procedures for the
protein's ligand without requiring a supply of intact protein or
large fragments thereof. Thus, a constrained helical peptide
possessing a particular protein's binding activity could overcome
supply or cost problems preventing the use of the protein in
affinity purification. In yet another example, a constrained
helical peptide possessing the conformational structure at the site
of interest in a particular protein could be used to isolate a
conformational epitope from the rest of the protein and raise
antibodies against the single epitope of interest without
interference from the other antigenic sites existing in the intact
protein.
[0068] Particularly preferred are the use of the compounds of the
invention having constrained helical peptides having internal amino
acid sequences from the HIV isolate LAI gp41 amino acid sequence
633-678 and homologs thereof, for use as haptens, vaccines, and in
diagnostics.
[0069] In another aspect, the methods and peptides of the invention
can be used to create combinatorial constrained helical peptide
libraries that are useful in chemical selection systems.
[0070] I. Locked Helix Peptides and Uses Therefor
[0071] The invention provides locked helix peptides of formula (1):
5
[0072] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; m and p are independently selected from the integers 0 to 6
inclusive, provided that m+p is less than or equal to 6; and n is
any integer in the range defined by (7-(m+p)) to (9-(m+p))
inclusive, provided that n is greater than 1.
[0073] In another embodiment, the invention provides locked helix
peptides of formula (2): 6
[0074] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0075] In yet another embodiment, the invention provides locked
helix peptides of formula (3): 7
[0076] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0077] Z is any amino acid sequence consisting of six amino acids;
and n is any integer from 4 to 6 inclusive.
[0078] In still another embodiment, the invention provides locked
helix peptides of formula (4): 8
[0079] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0080] Z is any amino acid sequence consisting of six amino acids;
and n is any integer from 3 to 5 inclusive.
[0081] In still another embodiment, the invention provides locked
helix peptides of formula (5): 9
[0082] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0083] Z is any amino acid sequence consisting of six amino acids;
and n is any integer from 5 to 7 inclusive.
[0084] In still another embodiment, the invention provides locked
helix peptides of formula (6): 10
[0085] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0086] Z is any amino acid sequence consisting of six amino acids;
q is selected from the integers 1 to 7 inclusive, and s is selected
from the integers 0 to 6 inclusive, provided that q+s is less than
or equal to 7; and r is any integer in the range defined by
(7-(q+s)) to (9-(q+s)) inclusive, provided that r is greater than
0.
[0087] In still another embodiment, the invention provides locked
helix peptides of formula (7): 11
[0088] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0089] Z is any amino acid sequence consisting of six amino acids;
and n is any integer from 4 to 6 inclusive.
[0090] In still another embodiment, the invention provides locked
helix peptides of formula (8): 12
[0091] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0092] Z is any amino acid sequence consisting of six amino acids;
and n is any integer from 4 to 6 inclusive.
[0093] In still another embodiment, the invention provides locked
helix peptides of formula (9): 13
[0094] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0095] Z is any amino acid sequence consisting of six amino acids;
and n is any integer from 3 to 5 inclusive.
[0096] In still another embodiment, the invention provides locked
helix peptides of formula (10): 14
[0097] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0098] Z is any amino acid sequence consisting of six amino acids;
and n is any integer from 5 to 7 inclusive.
[0099] In still another embodiment, the invention provides locked
helix peptides of formula (11): 15
[0100] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0101] Z is any amino acid sequence consisting of six amino acids;
t is selected from the integers O to 6 inclusive, and v is selected
from the integers 1 to 7 inclusive, provided that t+v is less than
or equal to 7; and u is any integer in the range defined by
(7-(t+v)) to (9-(t+v)) inclusive, provided that u is greater than
zero.
[0102] In still another embodiment, the invention provides locked
helix peptides of formula (12): 16
[0103] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0104] Z is any amino acid sequence consisting of six amino acids;
and n is any integer from 4 to 6 inclusive.
[0105] In still another embodiment, the invention provides locked
helix peptides of formula (13): 17
[0106] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0107] Z is any amino acid sequence consisting of six amino acids;
and n is any integer from 4 to 6 inclusive.
[0108] In still another embodiment, the invention provides locked
helix peptides of formula (14): 18
[0109] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0110] Z is any amino acid sequence consisting of six amino acids;
and n is any integer from 5 to 7 inclusive.
[0111] In still another embodiment, the invention provides locked
helix peptides of formula (15): 19
[0112] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0113] Z is any amino acid sequence consisting of six amino acids;
and n is any integer from 3 to 5 inclusive.
[0114] In still another embodiment, the invention provides locked
helix peptides of formula (16): 20
[0115] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0116] Z is any amino acid sequence consisting of six amino acids;
w and y are independently selected from the integers 1 to 7
inclusive, provided that w+y is less than or equal to 8; and x is
any integer in the range defined by (7w+y)) to (9-(w+y)) inclusive,
provided that x is greater than or equal to 0.
[0117] In still another embodiment, the invention provides locked
helix peptides of formula (17): 21
[0118] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0119] Z is any amino acid sequence consisting of six amino acids;
and n is any integer from 4 to 6 inclusive.
[0120] In still another embodiment, the invention provides locked
helix peptides of formula (18): 22
[0121] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0122] Z is any amino acid sequence consisting of six amino acids;
and n is any integer from 4 to 6 inclusive.
[0123] In still another embodiment, the invention provides locked
helix peptides of formula (19): 23
[0124] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0125] Z is any amino acid sequence consisting of six amino acids;
and n is any integer from 5 to 7 inclusive.
[0126] In still another embodiment, the invention provides locked
helix peptides of formula (20): 24
[0127] wherein S is absent or is a macromolecule; X is hydrogen or
is any amino acid or amino acid sequence; Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence;
[0128] Z is any amino acid sequence consisting of six amino acids;
and n is any integer from 3 to 5 inclusive.
[0129] In a further embodiment, the invention provides locked helix
peptides of formula (1), formula (2), formula (3), formula (4),
formula (5), formula (6), formula (7), formula (8), formula (9),
formula (10), formula (11), formula (12), formula (13), formula
(14), formula (15), formula (16), formula (17), formula (18),
formula (19) and formula (20) wherein X, Y, and Z collectively
contain up to or about 35 amino acids (i.e. locked helix peptides
of formulas (1), (2), (3), (4), (5), (6), (7), (8), (9), (10),
(11), (12), (13), (14), (15), (16), (17), (18), (19) and (20) each
of which contains a total of no more than or about 35 amino acid
residues).
[0130] Also provided herein are locked helix peptides of formula
(1), formula (2), formula (3), formula (4), formula (5), formula
(6), formula (7), formula (8), formula (9), formula (10), formula
(11), formula (12), formula (13), formula (14), formula (15),
formula (16), formula (17), formula (18), formula (19) and formula
(20) wherein X and/or Y contain(s) up to or about 30 amino acid
residues.
[0131] Further provided herein are locked helix peptides of formula
(1), formula (2), formula (3), formula (4), formula (5), formula
(6), formula (7), formula (8), formula (9), formula (10), formula
(11), formula (12), formula (13), formula (14), formula (15),
formula (16), formula (17), formula (18), formula (19) and formula
(20) wherein X and/or Y contain(s) up to or about 25 amino acid
residues.
[0132] Additionally provided herein are locked helix peptides of
formula (1), formula (2), formula (3), formula (4), formula (5),
formula (6), formula (7), formula (8), formula (9), formula (10),
formula (11), formula (12), formula (13), formula (14), formula
(15), formula (16), formula (17), formula (18), formula (19) and
formula (20) wherein X and/or Y contain(s) up to or about 20 amino
acid residues.
[0133] Also encompassed herein are locked helix peptides of formula
(l), formula (2), formula (3), formula (4), formula (5), formula
(6), formula (7), formula (8), formula (9), formula (10), formula
(11), formula (12), formula (13), formula (14), formula (15),
formula (16), formula (17), formula (18), formula (19) and formula
(20) wherein X and/or Y contain(s) up to or about 15 amino acid
residues.
[0134] Further encompassed herein are locked helix peptides of
formula (1), formula (2), formula (3), formula (4), formula (5),
formula (6), formula (7), formula (8), formula (9), formula (10),
formula (11), formula (12), formula (13), formula (14), formula
(15), formula (16), formula (17), formula (18), formula (19) and
formula (20) wherein X and/or Y contain(s) up to or about 10 amino
acid residues.
[0135] Additionally encompassed herein are locked helix peptides of
formula (1), formula (2), formula (3), formula (4), formula (5),
formula (6), formula (7), formula (8), formula (9), formula (10),
formula (11), formula (12), formula (13), formula (14), formula
(15), formula (16), formula (17), formula (18), formula (19) and
formula (20) wherein X and/or Y contain(s) up to or about 5 amino
acid residues.
[0136] Also within the scope of the invention are locked helix
peptides of formula (1), formula (2), formula (3), formula (4),
formula (5), formula (6), formula (7), formula (8), formula (9),
formula (10), formula (11), formula (12), formula (13), formula
(14), formula (15), formula (16), formula (17), formula (18),
formula (19) and formula (20) wherein X and/or Y contain(s) up to
or about 3 amino acid residues.
[0137] The invention also provides locked helix peptides of formula
(1a): 25
[0138] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; m and p are independently selected from the integers 0 to 6
inclusive, provided that m+p is less than or equal to 6; and n is
any integer in the range defined by (7(m+p)) to (9m+p)) inclusive,
provided that n is greater than 1.
[0139] In another embodiment, the invention provides locked helix
peptides of formula (2a): 26
[0140] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0141] In yet another embodiment, the invention provides locked
helix peptides of formula (3a): 27
[0142] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0143] In still another embodiment, the invention provides locked
helix peptides of formula (4a): 28
[0144] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0145] In still another embodiment, the invention provides locked
helix peptides of formula (5a): 29
[0146] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0147] In still another embodiment, the invention provides locked
helix peptides of formula (6a): 30
[0148] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; q is selected from the integers 1 to 7 inclusive, and s is
selected from the integers 0 to 6 inclusive, provided that q+s is
less than or equal to 7; and r is any integer in the range defined
by (7-(q+s)) to (9-(q+s)) inclusive, provided that r is greater
than 0.
[0149] In still another embodiment, the invention provides locked
helix peptides of formula (7a): 31
[0150] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0151] In still another embodiment, the invention provides locked
helix peptides of formula (8a): 32
[0152] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0153] In still another embodiment, the invention provides locked
helix peptides of formula (9a): 33
[0154] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0155] In still another embodiment, the invention provides locked
helix peptides of formula (10a): 34
[0156] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0157] In still another embodiment, the invention provides locked
helix peptides of formula (11a): 35
[0158] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; t is selected from the integers 0 to 6 inclusive, and v is
selected from the integers 1 to 7 inclusive, provided that t+v is
less than or equal to 7; and u is any integer in the range defined
by (7-(t+v)) to (9-(t+v)) inclusive, provided that u is greater
than 0.
[0159] In still another embodiment, the invention provides locked
helix peptides of formula (12a): 36
[0160] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0161] In still another embodiment, the invention provides locked
helix peptides of formula (13a): 37
[0162] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0163] In still another embodiment, the invention provides locked
helix peptides of formula (14a): 38
[0164] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0165] In still another embodiment, the invention provides locked
helix peptides of formula (15a): 39
[0166] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0167] In still another embodiment, the invention provides locked
helix peptides of formula (16a): 40
[0168] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; w and y are independently selected from the integers 1 to 7
inclusive, provided that w+y is less than or equal to 8; and x is
any integer in the range defined by (7-(w+y)) to (9-(w+y))
inclusive, provided that x is greater than or equal to 0.
[0169] In still another embodiment, the invention provides locked
helix peptides of formula (17a): 41
[0170] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0171] In still another embodiment, the invention provides locked
helix peptides of formula (18a): 42
[0172] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0173] In still another embodiment, the invention provides locked
helix peptides of formula (19a): 43
[0174] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0175] In still another embodiment, the invention provides locked
helix peptides of formula (20a): 44
[0176] wherein X is hydrogen or is any amino acid or amino acid
sequence; Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0177] In a further embodiment, the invention provides locked helix
peptides of formula (1a), formula (2a), formula (3a), formula (4a),
formula (5a), formula (6a), formula (7a), formula (8a), formula
(9a), formula (10a), formula (11a), formula (12a), formula (13a),
formula (14a), formula (15a), formula (16a), formula (17a), formula
(18a), formula (19a) and formula (20a) wherein X, Y, and Z
collectively contain up to or about 12 amino acids (i.e. locked
helix peptides of formulas (1a), (2a), (3a), (4a), (5a), (6a),
(7a), (8a), (9a), (10a), (11a), (12a), (13a), (14a), (15a), (16a),
(17a), (18a), (19a) and (20a) each of which contains a total of no
more than about 12 amino acid residues).
[0178] Also provided herein are locked helix peptides of formula
(1a), formula (2a), formula (3a), formula (4a), formula (5a),
formula (6a), formula (7a), formula (8a), formula (9a), formula
(10a), formula (11a), formula (12a), formula (13a), formula (14a),
formula (15a), formula (16a), formula (17a), formula (18a), formula
(19a) and formula (20a) wherein X and/or Y contain(s) up to or
about 30 amino acid residues.
[0179] Further provided herein are locked helix peptides of formula
(1a), formula (2a), formula (3a), formula (4a), formula (5a),
formula (6a), formula (7a), formula (8a), formula (9a), formula
(10a), formula (11a), formula (12a), formula (13a), formula (14a),
formula (85a), formula (16a), formula (17a), formula (18a), formula
(19a) and formula (20a) wherein X and/or Y contain(s) up to or
about 25 amino acid residues.
[0180] Additionally provided herein are locked helix peptides of
formula (1a), formula (2a), formula (3a), formula (4a), formula
(5a), formula (6a), formula (7a), formula (8a), formula (9a),
formula (10a), formula (11a), formula (12a), formula (13a), formula
(14a), formula (15a), formula (16a), formula (17a), formula (18a),
formula (19a) and formula (20a) wherein X and/or Y contain(s) up to
or about 20 amino acid residues.
[0181] Also encompassed herein are locked helix peptides of formula
(1a), formula (2a), formula (3a), formula (4a), formula (5a),
formula (6a), formula (7a), formula (8a), formula (9a), formula
(10a), formula (11a), formula (12a), formula (13a), formula (14a),
formula (15a), formula (16a), formula (17a), formula (18a), formula
(19a) and formula (20a) wherein X and/or Y contain(s) up to or
about 15 amino acid residues.
[0182] Further encompassed herein are locked helix peptides of
formula (1a), formula (2a), formula (3a), formula (4a), formula
(5a), formula (6a), formula (7a), formula (8a), formula (9a),
formula (10a), formula (11a), formula (12a), formula (13a), formula
(14a), formula (15a), formula (16a), formula (17a), formula (18a),
formula (19a) and formula (20a) wherein X and/or Y contain(s) up to
or about 10 amino acid residues.
[0183] Additionally encompassed herein are locked helix peptides of
formula (1a), formula (2a), formula (3a), formula (4a), formula
(5a), formula (6a), formula (7a), formula (8a), formula (9a),
formula (10a), formula (11a), formula (12a), formula (13a), formula
(14a), formula (15a), formula (16a), formula (17a), formula (18a),
formula (19a) and formula (20a) wherein X and/or Y contain(s) up to
or about 5 amino acid residues.
[0184] Also within the scope of the invention are locked helix
peptides of formula (1a), formula (2a), formula (3a), formula (4a),
formula (5a), formula (6a), formula (7a), formula (8a), formula
(9a), formula (10a), formula (11a), formula (12a), formula (13a),
formula (14a), formula (15a), formula (16a), formula (17a), formula
(18a), formula (19a) and formula (20a) wherein X and/or Y
contain(s) up to or about 3 amino acid residues.
[0185] The invention also provides locked helix peptides of formula
(1b): 45
[0186] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; m and p are independently selected from the integers 0 to 6
inclusive, provided that m+p is less than or equal to 6; and n is
any integer in the range defined by (7-(m+p)) to (9-(m+p))
inclusive, provided that n is greater than 1.
[0187] In another embodiment, the invention provides locked helix
peptides of formula (2b): 46
[0188] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0189] In yet another embodiment, the invention provides locked
helix peptides of formula (3b): 47
[0190] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0191] In still another embodiment, the invention provides locked
helix peptides of formula (4b): 48
[0192] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0193] In still another embodiment, the invention provides locked
helix peptides of formula (5b): 49
[0194] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0195] In still another embodiment, the invention provides locked
helix peptides of formula (6b): 50
[0196] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; q is selected from the integers 1 to 7 inclusive, and s is
selected from the integers 0 to 6 inclusive, provided that q+s is
less than or equal to 7; and r is any integer in the range defined
by (7-(q+s)) to (9-(q+s)) inclusive, provided that r is greater
than 0.
[0197] In still another embodiment, the invention provides locked
helix peptides of formula (7b): 51
[0198] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0199] In still another embodiment, the invention provides locked
helix peptides of formula (8b): 52
[0200] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0201] In still another embodiment, the invention provides locked
helix peptides of formula (9b): 53
[0202] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0203] In still another embodiment, the invention provides locked
helix peptides of formula (10b): 54
[0204] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0205] In still another embodiment, the invention provides locked
helix peptides of formula (11b): 55
[0206] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; t is selected from the integers 0 to 6 inclusive, and v is
selected from the integers 1 to 7 inclusive, provided that t+v is
less than or equal to 7; and u is any integer in the range defined
by (7-(t+v)) to (9-(t+v)) inclusive, provided that u is greater
than 0.
[0207] In still another embodiment, the invention provides locked
helix peptides of formula (12b): 56
[0208] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0209] In still another embodiment, the invention provides locked
helix peptides of formula (13b): 57
[0210] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0211] In still another embodiment, the invention provides locked
helix peptides of formula (14b): 58
[0212] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0213] In still another embodiment, the invention provides locked
helix peptides of formula (11b): 59
[0214] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0215] In still another embodiment, the invention provides locked
helix peptides of formula (16b): 60
[0216] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; w and y are independently selected from the integers 1 to 7
inclusive, provided that w+y is less than or equal to 8; and x is
any integer in the range defined by (7-(w+y)) to (9-(w+y))
inclusive, provided that x is greater than or equal to 0.
[0217] In still another embodiment, the invention provides locked
helix peptides of formula (17b): 61
[0218] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0219] In still another embodiment, the invention provides locked
helix peptides of formula (18b): 62
[0220] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0221] In still another embodiment, the invention provides locked
helix peptides of formula (19b): 63
[0222] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0223] In still another embodiment, the invention provides locked
helix peptides of formula (20b): 64
[0224] wherein Y is hydroxyl or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0225] Also provided herein are locked helix peptides of formula
(1b), formula (2b), formula (3b), formula (4b), formula (5b),
formula (6b), formula (7b), formula (8b), formula (9b), formula
(10b), formula (11b), formula (12b), formula (13b), formula (14b),
formula (15b), formula (16b), formula (17b), formula (18b), formula
(19b) and formula (20b) wherein Y contains up to or about 30 amino
acid residues.
[0226] Further provided herein are locked helix peptides of formula
(1b), formula (2b), formula (3b), formula (4b), formula (5b),
formula (6b), formula (7b), formula (8b), formula (9b), formula
(10b), formula (11b), formula (12b), formula (13b), formula (14b),
formula (15b), formula (16b), formula (17b), formula (18b), formula
(19b) and formula (20b) wherein Y contains up to or about 25 amino
acid residues.
[0227] Additionally provided herein are locked helix peptides of
formula (1b), formula (2b), formula (3b), formula (4b), formula
(5b), formula (6b), formula (7b), formula (8b), formula (9b),
formula (10b), formula (11b), formula (12b), formula (13b), formula
(14b), formula (15b), formula (16b), formula (17b), formula (18b),
formula (19b) and formula (20b) wherein Y contains up to or about
20 amino acid residues.
[0228] Also encompassed herein are locked helix peptides of formula
(1b), formula (2b), formula (3b), formula (4b), formula (5b),
formula (6b), formula (7b), formula (8b), formula (9b), formula
(10b), formula (11b), formula (12b), formula (13b), formula (14b),
formula (15b), formula (16b), formula (17b), formula (18b), formula
(19b) and formula (20b) wherein Y contains up to or about 15 amino
acid residues.
[0229] Further encompassed herein are locked helix peptides of
formula (1b), formula (2b), formula (3b), formula (4b), formula
(5b), formula (6b), formula (7b), formula (8b), formula (9b),
formula (10b), formula (11b), formula (12b), formula (13b), formula
(14b), formula (15b), formula (16b), formula (17b), formula (18b),
formula (19b) and formula (20b) wherein Y contains up to or about
10 amino acid residues.
[0230] Additionally encompassed herein are locked helix peptides of
formula (1b), formula (2b), formula (3b), formula (4b), formula
(5b), formula (6b), formula (7b), formula (8b), formula (9b),
formula (10b), formula (11b), formula (12b), formula (13b), formula
(14b), formula (15b), formula (16b), formula (17b), formula (18b),
formula (19b) and formula (20b) wherein Y contains up to or about 5
amino acid residues.
[0231] Also within the scope of the invention are locked helix
peptides of formula (1b), formula (2b), formula (3b), formula (4b),
formula (5b), formula (6b), formula (7b), formula (8b), formula
(9b), formula (10b), formula (11b), formula (12b), formula (13b),
formula (14b), formula (15b), formula (16b), formula (17b), formula
(18b), formula (19b) and formula (20b) wherein Y contains up to or
about 3 amino acid residues.
[0232] The invention also provides locked helix peptides of formula
(1c): 65
[0233] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; m and p are independently selected from the integers 0 to 6
inclusive, provided that m+p is less than or equal to 6; and n is
any integer in the range defined by (7-(m+p)) to (9-(m+p))
inclusive, provided that n is greater than 1.
[0234] In another embodiment, the invention provides locked helix
peptides of formula (2c): 66
[0235] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0236] In yet another embodiment, the invention provides locked
helix peptides of formula (3c): 67
[0237] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0238] In still another embodiment, the invention provides locked
helix peptides of formula (4c): 68
[0239] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0240] In still another embodiment, the invention provides locked
helix peptides of formula (5c): 69
[0241] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0242] In still another embodiment, the invention provides locked
helix peptides of formula (6c): 70
[0243] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; q is selected from the integers 1 to 7 inclusive, and s is
selected from the integers 0 to 6 inclusive, provided that q+s is
less than or equal to 7; and r is any integer in the range defined
by (7-(q+s)) to (9-(q+s)) inclusive, provided that r is greater
than 0.
[0244] In still another embodiment, the invention provides locked
helix peptides of formula (7c): 71
[0245] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0246] In still another embodiment, the invention provides locked
helix peptides of formula (8c): 72
[0247] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0248] In still another embodiment, the invention provides locked
helix peptides of formula (9c): 73
[0249] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0250] In still another embodiment, the invention provides locked
helix peptides of formula (10c): 74
[0251] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0252] In still another embodiment, the invention provides locked
helix peptides of formula (11c): 75
[0253] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; t is selected from the integers 0 to 6 inclusive, and v is
selected from the integers 1 to 7 inclusive, provided that t+v is
less than or equal to 7; and u is any integer in the range defined
by (7-(t+v)) to (9-(t+v)) inclusive, provided that u is greater
than 0.
[0254] In still another embodiment, the invention provides locked
helix peptides of formula (12c): 76
[0255] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0256] In still another embodiment, the invention provides locked
helix peptides of formula (13c): 77
[0257] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0258] In still another embodiment, the invention provides locked
helix peptides of formula (14c): 78
[0259] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0260] In still another embodiment, the invention provides locked
helix peptides of formula (15c): 79
[0261] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0262] In still another embodiment, the invention provides locked
helix peptides of formula (16c): 80
[0263] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; w and y are independently selected from the integers 1 to 7
inclusive, provided that w+y is less than or equal to 8; and x is
any integer in the range defined by (7-(w+y)) to (9-(w+y))
inclusive, provided that x is greater than or equal to 0.
[0264] In still another embodiment, the invention provides locked
helix peptides of formula (17c): 81
[0265] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0266] In still another embodiment, the invention provides locked
helix peptides of formula (18c): 82
[0267] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0268] In still another embodiment, the invention provides locked
helix peptides of formula (19c): 83
[0269] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0270] In still another embodiment, the invention provides locked
helix peptides of formula (20c): 84
[0271] wherein X is hydrogen or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0272] Also provided herein are locked helix peptides of formula
(1c), formula (2c), formula (3c), formula (4c), formula (5c),
formula (6c), formula (7c), formula (8c), formula (9c), formula
(10c), formula (11c), formula (12c), formula (13c), formula (14c),
formula (15c), formula (16c), formula (17c), formula (18c), formula
(19c) and formula (20c) wherein X contains up to or about 30 amino
acid residues.
[0273] Further provided herein are locked helix peptides of formula
(1c), formula (2c), formula (3c), formula (4c), formula (5c),
formula (6c), formula (7c), formula (8c), formula (9c), formula
(10c), formula (11c), formula (12c), formula (13c), formula (14c),
formula (15c), formula (16c), formula (17c), formula (18c), formula
(19c) and formula (20c) wherein X contains up to or about 25 amino
acid residues.
[0274] Additionally provided herein are locked helix peptides of
formula (1c), formula (2c), formula (3c), formula (4c), formula
(5c), formula (6c), formula (7c), formula (8c), formula (9c),
formula (10c), formula (11c), formula (12c), formula (13c), formula
(14c), formula (15c), formula (16c), formula (17c), formula (18c),
formula (19c) and formula (20c) wherein X contains up to or about
20 amino acid residues.
[0275] Also encompassed herein are locked helix peptides of formula
(1c), formula (2c), formula (3c), formula (4c), formula (5c),
formula (6c), formula (7c), formula (.c), formula (9c), formula
(10c), formula (11c), formula (12c), formula (13c), formula (14c),
formula (18c), formula (16c), formula (17c), formula (18c), formula
(19c) and formula (20c) wherein X contains up to or about 15 amino
acid residues.
[0276] Further encompassed herein are locked helix peptides of
formula (1c), formula (2c), formula (3c), formula (4c), formula
(5c), formula (6c), formula (7c), formula (8c), formula (9c),
formula (10c), formula (11c), formula (12c), formula (13c), formula
(14c), formula (15c), formula (16c), formula (17c), formula (18c),
formula (19c) and formula (20c) wherein X contains up to or about
10 amino acid residues.
[0277] Additionally encompassed herein are locked helix peptides of
formula (1c), formula (2c), formula (3c), formula (4c), formula
(5c), formula (6c), formula (7c), formula (8c), formula (9c),
formula (10c), formula (11c), formula (12c), formula (13c), formula
(14c), formula (15c), formula (16c), formula (17c), formula (15c),
formula (19c) and formula (20c) wherein X contains up to or about 5
amino acid residues.
[0278] Also within the scope of the invention are locked helix
peptides of formula (1c), formula (2c), formula (3c), formula (4c),
formula (5c), formula (6c), formula (7c), formula (8c), formula
(9c), formula (10c), formula (11c), formula (12c), formula (13c),
formula (14c), formula (15c), formula (16c), formula (17c), formula
(18c), formula (19c) and formula (20c) wherein X contains up to or
about 3 amino acid residues.
[0279] The invention also provides locked helix peptides of formula
(1d): 85
[0280] wherein Z is any amino acid sequence consisting of six amino
acids; m and p are independently selected from the integers 0 to 6
inclusive, provided that m+p is less than or equal to 6; and n is
any integer in the range defined by (7-(m+p)) to (9-(m+p))
inclusive, provided that n is greater than 1.
[0281] In another embodiment, the invention provides locked helix
peptides of formula (2d): 86
[0282] wherein Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0283] In yet another embodiment, the invention provides locked
helix peptides of formula (3d): 87
[0284] wherein Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0285] In still another embodiment, the invention provides locked
helix peptides of formula (4d): 88
[0286] wherein Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0287] In still another embodiment, the invention provides locked
helix peptides of formula (5d): 89
[0288] wherein Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0289] In still another embodiment, the invention provides locked
helix peptides of formula (6d): 90
[0290] wherein Z is any amino acid sequence consisting of six amino
acids; q is selected from the integers 1 to 7 inclusive, and s is
selected from the integers 0 to 6 inclusive, provided that q+s is
less than or equal to 7; and r is any integer in the range defined
by (7-(q+s)) to (9q+s)) inclusive, provided that r is greater than
0.
[0291] In still another embodiment, the invention provides locked
helix peptides of formula (7d): 91
[0292] wherein Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0293] In still another embodiment, the invention provides locked
helix peptides of formula (8d): 92
[0294] wherein Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0295] In still another embodiment, the invention provides locked
helix peptides of formula (9d): 93
[0296] wherein Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0297] In still another embodiment, the invention provides locked
helix peptides of formula (10d): 94
[0298] wherein Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0299] In still another embodiment, the invention provides locked
helix peptides of formula (11d): 95
[0300] wherein Z is any amino acid sequence consisting of six amino
acids; t is selected from the integers 0 to 6 inclusive, and v is
selected from the integers 1 to 7 inclusive, provided that t+v is
less than or equal to 7; and u is any integer in the range defined
by (7-(t+v)) to (9-(t+v)) inclusive, provided that u is greater
than 0.
[0301] In still another embodiment, the invention provides locked
helix peptides of formula (12d): 96
[0302] wherein Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0303] In still another embodiment, the invention provides locked
helix peptides of formula (13d): 97
[0304] wherein Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0305] In still another embodiment, the invention provides locked
helix peptides of formula (14d): 98
[0306] wherein Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0307] In still another embodiment, the invention provides locked
helix peptides of formula (15d): 99
[0308] wherein Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0309] In still another embodiment, the invention provides locked
helix peptides of formula (16d): 100
[0310] wherein Z is any amino acid sequence consisting of six amino
acids; w and y are independently selected from the integers 1 to 7
inclusive, provided that w+y is less than or equal to 8; and x is
any integer in the range defined by (7-(w+y)) to (9-(w+y))
inclusive, provided that x is greater than or equal to 0.
[0311] In still another embodiment, the invention provides locked
helix peptides of formula (17d): 101
[0312] wherein Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0313] In still another embodiment, the invention provides locked
helix peptides of formula (18d): 102
[0314] wherein Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0315] In still another embodiment, the invention provides locked
helix peptides of formula (19d): 103
[0316] wherein Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0317] In still another embodiment, the invention provides locked
helix peptides of formula (20d): 104
[0318] wherein Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0319] The invention also provides locked helix peptides of formula
(le): 105
[0320] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; m and p are independently selected from the integers 0 to 6
inclusive, provided that m+p is less than or equal to 6; and n is
any integer in the range defined by (7m+p)) to (9-(m+p)) inclusive,
provided that n is greater than 1.
[0321] In another embodiment, the invention provides locked helix
peptides of formula (2e): 106
[0322] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0323] In yet another embodiment, the invention provides locked
helix peptides of formula (3e): 107
[0324] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0325] In still another embodiment, the invention provides locked
helix peptides of formula (4e): 108
[0326] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0327] In still another embodiment, the invention provides locked
helix peptides of formula (5e): 109
[0328] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0329] In still another embodiment, the invention provides locked
helix peptides of formula (6e): 110
[0330] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; q is selected from the integers 1 to 7 inclusive, and s is
selected from the integers 0 to 6 inclusive, provided that q+s is
less than or equal to 7; and r is any integer in the range defined
by (7-(q+s)) to (9-(q+s)) inclusive, provided that r is greater
than 0.
[0331] In still another embodiment, the invention provides locked
helix peptides of formula (7e): 111
[0332] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0333] In still another embodiment, the invention provides locked
helix peptides of formula (8e): 112
[0334] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0335] In still another embodiment, the invention provides locked
helix peptides of formula (9e): 113
[0336] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0337] In still another embodiment, the invention provides locked
helix peptides of formula (10e): 114
[0338] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0339] In still another embodiment, the invention provides locked
helix peptides of formula (11e): 115
[0340] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; t is selected from the integers 0 to 6 inclusive, and v is
selected from the integers 1 to 7 inclusive, provided that t+v is
less than or equal to 7; and u is any integer in the range defined
by (7-(t+v)) to (9-(t+v)) inclusive, provided that u is greater
than 0.
[0341] In still another embodiment, the invention provides locked
helix peptides of formula (12e): 116
[0342] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0343] In still another embodiment, the invention provides locked
helix peptides of formula (13e): 117
[0344] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0345] In still another embodiment, the invention provides locked
helix peptides of formula (14e): 118
[0346] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0347] In still another embodiment, the invention provides locked
helix peptides of formula (15e): 119
[0348] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0349] In still another embodiment, the invention provides locked
helix peptides of formula (16e): 120
[0350] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; w and y are independently selected from the integers 1 to 7
inclusive, provided that w+y is less than or equal to 8; and x is
any integer in the range defined by (7w+y)) to (9-(w+y)) inclusive,
provided that x is greater than or equal to 0.
[0351] In still another embodiment, the invention provides locked
helix peptides of formula (17e): 121
[0352] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0353] In still another embodiment, the invention provides locked
helix peptides of formula (18e): 122
[0354] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 4 to 6 inclusive.
[0355] In still another embodiment, the invention provides locked
helix peptides of formula (19e): 123
[0356] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 5 to 7 inclusive.
[0357] In still another embodiment, the invention provides locked
helix peptides of formula (20e): 124
[0358] wherein S is absent or is a macromolecule; Y is absent, or
is hydroxyl if S is absent, or is any amino acid or amino acid
sequence; Z is any amino acid sequence consisting of six amino
acids; and n is any integer from 3 to 5 inclusive.
[0359] Also provided herein are locked helix peptides of formula
(1e), formula (2e), formula (3e), formula (4e), formula (5e),
formula (6e), formula (7e), formula (8e), formula (9e), formula
(10e), formula (11e), formula (12e), formula (13e), formula (14e),
formula (15e), formula (16e), formula (17e), formula (18e), formula
(19e) and formula (20e) wherein Y contains up to or about 30 amino
acid residues.
[0360] Further provided herein are locked helix peptides of formula
(le), formula (2e), formula (3e), formula (4e), formula (5e),
formula (6e), formula (7e), formula (8e), formula (9e), formula
(10e), formula (11e), formula (12e), formula (13e), formula (14e),
formula (15e), formula (16e), formula (17e), formula (18e), formula
(19e) and formula (20e) wherein Y contains up to or about 25 amino
acid residues.
[0361] Additionally provided herein are locked helix peptides of
formula (le), formula (2e), formula (3e), formula (4e), formula
(5e), formula (6e), formula (7e), formula (8e), formula (9e),
formula (10e), formula (11e), formula (12e), formula (13e), formula
(14e), formula (15e), formula (16e), formula (17e), formula (18e),
formula (19e) and formula (20e) wherein Y contains up to or about
20 amino acid residues.
[0362] Also encompassed herein are locked helix peptides of formula
(1e), formula (2e), formula (3e), formula (4e), formula (5e),
formula (6e), formula (7e), formula (5e), formula (9e), formula
(10e), formula (11e), formula (12e), formula (13e), formula (14e),
formula (15e), formula (16e), formula (17e), formula (18e), formula
(19e) and formula (20e) wherein Y contains up to or about 15 amino
acid residues.
[0363] Further encompassed herein are locked helix peptides of
formula (1e), formula (2e), formula (3e), formula (4e), formula
(5e), formula (6e), formula (7e), formula (5e), formula (9e),
formula (10e), formula (11e), formula (12e), formula (13e), formula
(14e), formula (15e), formula (16e), formula (17e), formula (18e),
formula (19e) and formula (20e) wherein Y contains up to or about
10 amino acid residues.
[0364] Additionally encompassed herein are locked helix peptides of
formula (1e), formula (2e), formula (3e), formula (4e), formula
(5e), formula (6e), formula (7e), formula (5e), formula (9e),
formula (10e), formula (11e), formula (12e), formula (13e), formula
(14e), formula (1Se), formula (16e), formula (17e), formula (18e),
formula (19e) and formula (20e) wherein Y contains up to or about 5
amino acid residues.
[0365] Also within the scope of the invention are locked helix
peptides of formula (1e), formula (2e), formula (3e), formula (4e),
formula (5e), formula (6e), formula (7e), formula (5e), formula
(9e), formula (10e), formula (11e), formula (12e), formula (13e),
formula (14e), formula (15e), formula (16e), formula (17e), formula
(18e), formula (19e) and formula (20e) wherein Y contains up to or
about 3 amino acid residues.
[0366] The invention also provides locked helix peptides of formula
(1f): 125
[0367] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; m and p are independently
selected from the integers 0 to 6 inclusive, provided that m+p is
less than or equal to 6; and n is any integer in the range defined
by (7-(m+p)) to (9-(m+p)) inclusive, provided that n is greater
than 1.
[0368] In another embodiment, the invention provides locked helix
peptides of formula (2f): 126
[0369] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; and n is any integer from 4
to 6 inclusive.
[0370] In yet another embodiment, the invention provides locked
helix peptides of formula (3f): 127
[0371] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; and n is any integer from 4
to 6 inclusive.
[0372] In still another embodiment, the invention provides locked
helix peptides of formula (4f): 128
[0373] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; and n is any integer from 3
to 5 inclusive.
[0374] In still another embodiment, the invention provides locked
helix peptides of formula (5f): 129
[0375] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; and n is any integer from 5
to 7 inclusive.
[0376] In still another embodiment, the invention provides locked
helix peptides of formula (6f): 130
[0377] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; q is selected from the
integers 1 to 7 inclusive, and s is selected from the integers 0 to
6 inclusive, provided that q+s is less than or equal to 7; and r is
any integer in the range defined by (7-(q+s)) to (9-(q+s))
inclusive, provided that r is greater than 0.
[0378] In still another embodiment, the invention provides locked
helix peptides of formula (7f): 131
[0379] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; and n is any integer from 4
to 6 inclusive.
[0380] In still another embodiment, the invention provides locked
helix peptides of formula (8f): 132
[0381] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; and n is any integer from 4
to 6 inclusive.
[0382] In still another embodiment, the invention provides locked
helix peptides of formula (9f): 133
[0383] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; and n is any integer from 3
to 5 inclusive.
[0384] In still another embodiment, the invention provides locked
helix peptides of formula (10f): 134
[0385] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; and n is any integer from 5
to 7 inclusive.
[0386] In still another embodiment, the invention provides locked
helix peptides of formula (11f): 135
[0387] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; t is selected from the
integers 0 to 6 inclusive, and v is selected from the integers 1 to
7 inclusive, provided that t+v is less than or equal to 7; and u is
any integer in the range defined by (7-(t+v)) to (9-(t+v))
inclusive, provided that u is greater than 0.
[0388] In still another embodiment, the invention provides locked
helix peptides of formula (12f): 136
[0389] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; and n is any integer from 4
to 6 inclusive.
[0390] In still another embodiment, the invention provides locked
helix peptides of formula (13f): 137
[0391] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; and n is any integer from 4
to 6 inclusive.
[0392] In still another embodiment, the invention provides locked
helix peptides of formula (14f): 138
[0393] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; and n is any integer from 5
to 7 inclusive.
[0394] In still another embodiment, the invention provides locked
helix peptides of formula (15f): 139
[0395] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; and n is any integer from 3
to 5 inclusive.
[0396] In still another embodiment, the invention provides locked
helix peptides of formula (16f): 140
[0397] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; w and y are independently
selected from the integers 1 to 7 inclusive, provided that w+y is
less than or equal to 8; and x is any integer in the range defined
by (7-(w+y)) to (9-(w+y)) inclusive, provided that x is greater
than or equal to 0.
[0398] In still another embodiment, the invention provides locked
helix peptides of formula (17f): 141
[0399] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; and n is any integer from 4
to 6 inclusive.
[0400] In still another embodiment, the invention provides locked
helix peptides of formula (18f): 142
[0401] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; and n is any integer from 4
to 6 inclusive.
[0402] In still another embodiment, the invention provides locked
helix peptides of formula (19f): 143
[0403] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; and n is any integer from 5
to 7 inclusive.
[0404] In still another embodiment, the invention provides locked
helix peptides of formula (20f): 144
[0405] wherein S is hydroxyl or is a macromolecule; X is hydrogen
or is any amino acid or amino acid sequence; Z is any amino acid
sequence consisting of six amino acids; and n is any integer from 3
to 5 inclusive.
[0406] Also provided herein are locked helix peptides of formula
(1f), formula (2f), formula (3f), formula (4f), formula (5f),
formula (6f), formula (7f), formula (8f), formula (9f), formula
(10f), formula (11f), formula (12f), formula (13f), formula (14f),
formula (15f), formula (16f), formula (17f), formula (18f), formula
(19f) and formula (20f) wherein X contains up to or about 30 amino
acid residues.
[0407] Further provided herein are locked helix peptides of formula
(1f), formula (2f), formula (3f), formula (4f), formula (5f),
formula (6f), formula (7f), formula (8f), formula (9f), formula
(10f), formula (11f), formula (12f), formula (13f), formula (14f),
formula (15f), formula (16f), formula (17f), formula (18f), formula
(19f) and formula (20f) wherein X contains up to or about 25 amino
acid residues.
[0408] Additionally provided herein are locked helix peptides of
formula (1f), formula (2f), formula (3f), formula (4f), formula
(5f), formula (6f), formula (7f), formula (8f), formula (9f),
formula (10f), formula (11f), formula (12f), formula (13f), formula
(14f), formula (15f), formula (16f), formula (17f), formula (18f),
formula (19f) and formula (20f) wherein X contains up to or about
20 amino acid residues.
[0409] Also encompassed herein are locked helix peptides of formula
(1f), formula (2f), formula (3f), formula (4f), formula (5f),
formula (6f), formula (7f), formula (8f), formula (9f), formula
(10f), formula (11f), formula (12f), formula (13f), formula (14f),
formula (15f), formula (16f), formula (17f), formula (18f), formula
(19f) and formula (20f) wherein X contains up to or about 15 amino
acid residues.
[0410] Further encompassed herein are locked helix peptides of
formula (1f), formula (2f), formula (3f), formula (4f), formula
(5f), formula (6f), formula (7f), formula (8f), formula (9f),
formula (10f), formula (11f), formula (12f), formula (13f), formula
(14f), formula (15f), formula (16f), formula (17f), formula (18f),
formula (19f) and formula (20f) wherein X contains up to or about
10 amino acid residues.
[0411] Additionally encompassed herein are locked helix peptides of
formula (1f), formula (2f), formula (3f), formula (4f), formula
(5f), formula (6f), formula (7f), formula (8f), formula (9f),
formula (10f), formula (11f), formula (12f), formula (13f), formula
(14f), formula (15f), formula (16f), formula (17f), formula (18f),
formula (19f) and formula (20f) wherein X contains up to or about 5
amino acid residues.
[0412] Also within the scope of the invention are locked helix
peptides of formula (1f), formula (2f), formula (3f), formula (4f),
formula (5f), formula (6f), formula (7f), formula (8f), formula
(9f), formula (10f), formula (11f), formula (12f), formula (13f),
formula (14f), formula (15f), formula (16f), formula (17f), formula
(18f), formula (19f) and formula (20f) wherein X contains up to or
about 3 amino acid residues.
[0413] The invention also provides locked helix peptides of formula
(1g): 145
[0414] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; m and p are
independently selected from the integers 0 to 6 inclusive, provided
that m+p is less than or equal to 6; and n is any integer in the
range defined by (7-(m+p))to (9-(m+p)) inclusive, provided that n
is greater than 1.
[0415] In another embodiment, the invention provides locked helix
peptides of formula (2g): 146
[0416] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; and n is any integer
from 4 to 6 inclusive.
[0417] In yet another embodiment, the invention provides locked
helix peptides of formula (3g): 147
[0418] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; and n is any integer
from 4 to 6 inclusive.
[0419] In still another embodiment, the invention provides locked
helix peptides of formula (4g): 148
[0420] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; and n is any integer
from 3 to 5 inclusive.
[0421] In still another embodiment, the invention provides locked
helix peptides of formula (5g): 149
[0422] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; and n is any integer
from 5 to 7 inclusive.
[0423] In still another embodiment, the invention provides locked
helix peptides of formula (6g): 150
[0424] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; q is selected from the
integers 1 to 7 inclusive, and s is selected from the integers 0 to
6 inclusive, provided that q+s is less than or equal to 7; and r is
any integer in the range defined by (7-(q+s)) to (9-(q+s))
inclusive, provided that r is greater than 0.
[0425] In still another embodiment, the invention provides locked
helix peptides of formula (7g): 151
[0426] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; and n is any integer
from 4 to 6 inclusive.
[0427] In still another embodiment, the invention provides locked
helix peptides of formula (8g): 152
[0428] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; and n is any integer
from 4 to 6 inclusive.
[0429] In still another embodiment, the invention provides locked
helix peptides of formula (9g): 153
[0430] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; and n is any integer
from 3 to 5 inclusive.
[0431] In still another embodiment, the invention provides locked
helix peptides of formula (10g): 154
[0432] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; and n is any integer
from 5 to 7 inclusive.
[0433] In still another embodiment, the invention provides locked
helix peptides of formula (11g): 155
[0434] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; t is selected from the
integers 0 to 6 inclusive, and v is selected from the integers 1 to
7 inclusive, provided that t+v is less than or equal to 7; and u is
any integer in the range defined by (7-(t+v)) to (9t+v)) inclusive,
provided that u is greater than 0.
[0435] In still another embodiment, the invention provides locked
helix peptides of formula (12g): 156
[0436] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; and n is any integer
from 4 to 6 inclusive.
[0437] In still another embodiment, the invention provides locked
helix peptides of formula (13g): 157
[0438] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; and n is any integer
from 4 to 6 inclusive.
[0439] In still another embodiment, the invention provides locked
helix peptides of formula (14g): 158
[0440] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; and n is any integer
from 5 to 7 inclusive.
[0441] In still another embodiment, the invention provides locked
helix peptides of formula (15g): 159
[0442] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; and n is any integer
from 3 to 5 inclusive.
[0443] In still another embodiment, the invention provides locked
helix peptides of formula (16g): 160
[0444] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; w and y are
independently selected from the integers 1 to 7 inclusive, provided
that w+y is less than or equal to 8; and x is any integer in the
range defined by (7-(w+y)) to (9-(w+y)) inclusive, provided that x
is greater than or equal to 0.
[0445] In still another embodiment, the invention provides locked
helix peptides of formula (17g): 161
[0446] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; and n is any integer
from 4 to 6 inclusive.
[0447] In still another embodiment, the invention provides locked
helix peptides of formula (18g): 162
[0448] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; and n is any integer
from 4 to 6 inclusive.
[0449] In still another embodiment, the invention provides locked
helix peptides of formula (19g): 163
[0450] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; and n is any integer
from 5 to 7 inclusive.
[0451] In still another embodiment, the invention provides locked
helix peptides of formula (20g): 164
[0452] wherein S is hydroxyl or is a macromolecule; Z is any amino
acid sequence consisting of six amino acids; and n is any integer
from 3 to 5 inclusive.
[0453] For locked helix peptides of formulas (1), (2), (3), (4),
(5), (6), (7), (8), (9), (10), (11), (12), (13), (14),
(15),(16),(17), (18),(19), (20), (1e), (2e), (3e), (4e), (5e),
(6e), (7e), (8e), (9e), (10e), (11e), (12e), (13e), (14e), (15e),
(16e), (17e), (18e), (19e), (20e), (1f), (2f), (3f), (4f), (5f),
(6f), (7f), (8f), (9f), (10f), (11f), (12f), (13f), (14f), (15f),
(16f), (17f), (18f), (19f), (20f), (1g), (2g), (3g), (4g), (5g),
(6g), (7g), (8g), (9g), (10g), (11g), (12g), (13g), (14g), (15g),
(16g), (17g), (18g), (19g), or (20g) bound to a macromolecule, the
invention encompasses any macromolecule capable of serving as an
anchor for the C-terminus of the locked helix peptide. Typically,
the macromolecule functions as a solid support. In general, the
solid support is an inert matrix, such as a polymeric gel,
comprising a three dimensional structure, lattice or network of a
material. Almost any macromolecule, synthetic or natural, can form
a gel in a suitable liquid when suitably cross-linked with a
difunctional reagent. In one embodiment, the macromolecule selected
is convenient for use in affinity chromatography. Most
chromatographic matrices used for affinity chromatography are
xerogels. Such gels shrink on drying to a compact solid comprising
only the gel matrix. When the dried xerogel is resuspended in the
liquid, the gel matrix imbibes liquid, swells and returns to the
gel state. Xerogels suitable for use herein include polymeric gels,
such as cellulose, cross-linked dextrans (e.g. Sepharose), agarose,
cross-linked agarose, polyacrylamide gels, and
polyacrylamide-agarose gels.
[0454] The locked helix peptides provided herein can be constructed
according to the methods of the invention described in Sections II
and III below.
[0455] In one embodiment, the peptides of the invention are
designed to isolate the binding determinants from .alpha.-helical
binding domains of known proteins. Such peptides have a number of
uses, including the determination of whether a binding determinant
in an .alpha.-helical binding domain of a known protein can serve
as a structural model for the design of peptidomimetics or small
molecules capable of mimicking or antagonizing the binding activity
of the intact protein. In using the peptides of the invention for
this purpose, the practitioner selects a binding protein with a
helical domain that interacts with ligand, and then identifies a
candidate binding determinant situated within a sequence of six (or
more) contiguous amino acids in the helical binding domain. The
candidate binding determinant can be identified by using alanine
scanning mutagenesis to determine whether the candidate sequence
contains one or more amino acid residues that are critical for
ligand binding. Next, a constrained peptide containing the
candidate sequence is designed by selecting two residues in the
candidate sequence (designated I and I+7) which are separated by an
intervening sequence of six amino acids and which do not interact
with ligand (as determined by alanine scanning mutagenesis in the
previous step) for substitution with amino acid residues having a
side chain containing an amide bond-forming substitutent. The
peptide is synthesized and the side chain amide bond-forming
substitutent of the foreign I and I+7 residues are used to tether
the peptide in an .alpha.-helical conformation according to the
methods of the invention described in Section II below. Finally,
the locked helix peptide's binding activity with the ligand is
assayed, e.g., in a binding competition assay with the intact
binding protein, and the results of the assay can be used to
determine whether a peptidomimetic or small molecule antagonist
could be developed using the binding determinant as a structural
model.
[0456] In another embodiment, the locked helix peptides of the
invention are used to replace intact binding proteins or protein
binding domains in the affinity purification of ligand. For
example, Protein A is commonly used for affinity chromatographic
purification of IgG molecules. The Z-domain of Protein A is a three
helix bundle, 59 residues in length, which binds to the Fc portion
of IgG. As described in Example 2 below, a locked helix species of
the peptide Phe-Asn-Met-(1)-Gln-Gln-Arg-Arg-Phe-T-
yr-(2)Ala-Leu-His (wherein the amino acid residues at the (1) and
(2) positions in the corresponding z-domain sequence are both
replaced with glutamic acid residues), corresponding to a binding
determinant in helix 1 of the Z-domain can be used to bind IgG.
Accordingly, the invention provides constrained helix species
containing binding determinants in helix I of the Z-domain in
Protein A, including molecules of formula (4) above wherein Z is
Gln-Gln-Arg-Arg-Phe-Tyr. In one embodiment, the constrained helix
species is a molecule of formula (4) wherein Z is
Gln-Gln-Arg-Arg-Phe-Tyr, X is Phe-Asn-Met, and Y is Ala-Leu-His.
The IgG binding molecules of the invention are conveniently
synthesized using the solid phase peptide synthesis methods
described in Section II below, such that the molecules are anchored
to resin beads suitable for column or batch affinity
chromatography.
[0457] In still another embodiment, the locked helix peptides of
the invention are designed to mimic epitopes in proteins and are
used to selectively raise polyclonal or monoclonal antibodies
against such individual epitopes. Since the peptides will
frequently be too small to generate an immune response, the
peptides can be conjugated to carriers known to be immunogenic in
the species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
difunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups.
[0458] The locked helix peptides of the invention are particularly
useful in isolating synthetic antibody clones with a selected
binding activity from phage display combinatorial libraries. The
locked helix peptide provides a significant advantage over the
intact protein or protein domain in that using the locked helix
peptide allows the isolation of binding activities for the
particular conformational epitope of interest. Without the locked
helix peptides of the invention, the conformation epitope of
interest would likely require structural support from other regions
of the protein or protein domain whose presence in the ligand would
result in the concomitant isolation of undesired clones. In
addition, the synthesis of locked helix peptides anchored to
polymeric resins as described in Sections II and III below would
provide material that can be conveniently packed into columns for
panning phage display libraries.
[0459] In another aspect, the locked helix peptides of the
invention are used to provide conformationally stable variants of
peptides or proteins which exhibit "floppy" or unstable
.alpha.-helical secondary structure at one or more site(s) in
unrestrained form under conditions of interest. In particular, the
methods of the invention can solve problems presented by some
antigens which relate to the instability of conformational
epitopes. A conformational epitope can fail to present the desired
antigenic determinant because of "floppy" or unstable
.alpha.-helical secondary structure element(s) in the epitope. The
restraint of such "floppy" .alpha.-helical structure(s) according
to the methods of the invention would stabilize the conformational
epitope and allow presentation of the desired antigenic
determinant. This application of the present methods and peptides
is particularly useful, for example, in vaccine design and in
generating polyclonal or monoclonal antibodies from host animals or
isolating antibody clones from phage display libraries.
[0460] In one embodiment the invention, where the locked helix
peptides of the invention are used to provide conformationally
stable variants of peptides or proteins which exhibit "floppy" or
unstable .alpha.-helical secondary structure at one or more sites
in unrestrained form under conditions of interest, a compound
containing a constrained helical peptide that is useful as an
immunogen, vaccine and diagnostic for human immunodeficiency virus
(HIV) is provided. Acquired immunodeficiency syndrome (AIDS) is
caused by a retrovirus identified as the human immunodeficiency
virus (HIV). There have been intense efforts to develop a vaccine
that induces a protective immune response based on induction of
antibodies or cellular responses. Recent efforts have used subunit
vaccines where an HIV protein, rather than attenuated or killed
virus, is used as the immunogen in the vaccine for safety
reasons.
[0461] The human immunodeficiency virus 1 (HIV-1) envelope
glycoproteins gp120 and gp41 mediate viral tropism to and
subsequententry into target cells (Freed et al., The Journal of
Biological Chemistry 270, 23883-23886 (1995)). The role of gp120 is
to bind to target cells by interactions with CD4 and one of several
co-receptors (D'Souza et al., Nature Medicine 2,
1293-1300(1996)),after which gp41 promotes the fusion of viral and
cellular membranes. The mechanism by which gp41 mediates membrane
fusion has recently been the subject of intensive study. Evidence
suggests that the process may involve the formation of a
coiled-coil trimer, which is thought to drive the transition from
resting to fusogenic states, as modeled for influenza hemagglutinin
(Wilson et al., Nature 289, 366-373 (1981); Carr, et al., Cell 73,
823-832 (1993); Bullough et al., Nature 371, 3743 (1994)).
[0462] Two linear peptides derived from HIV-1 gp41 have been found
to inhibit viral fusion. The first of these, DP107, represents a
portion of gp41 near the N-terminal fusion peptide and has been
shown to be helical in solution and oligomerizein a manner
consistent with coiled-coil formation (Gallaheret al., AIDS Res.
Hum. Retroviruses 5, 431-440 (1989); Weissenhorn et al., Nature
387,426-430 (1997)). A more potent peptide, DP178, was derived from
the C-terminal region of the gp41 ectodomain (Wild, et al., PNAS
91: 9770-9774 (1994); Jiang et al., Nature, 365:113 (1993)).
Although this region of gp41 was predicted to be .alpha.-helical
(Gallaheret al., AIDS Res. Hum. Retroviruses 5, 431-440 (1989)),
DP178 itself lacks discernable structure in solution (Wild, et al.,
PNAS 91: 9770-9774 (1994). Attempts to explore the mechanism of
action of DP178 have been complicated by a lack of understanding of
its bioactive conformation. Recently, crystallographic (Chan et
al., Cell 89, 263-273 (1997); Weissenhorn et al., Nature 387,
426-430 (1997)) and solution (Lawless, et al., Biochemistry 35,
13697-13708 (1996); Lu et al., Nature Structural Biology 2,
1075-1082 (1995); Rabenstein et al., Biochemistry 35, 13922-13928
(1996)) studies have shown that disconnected segments of HIV-1 gp41
that overlap DP107 and DP178 associate in a tightly-packed helical
bundle. The C-terminal segment, corresponding to DP178, forms an
extended helix which packs in an antiparallel fashion against a
groove created by an N-terminal (DP107) coiled-coil trimer. While
these data suggest one possible conformation for DP178, they do not
provide conclusive information about the mechanism of peptide
inhibition during viral fusion events.
[0463] The present invention provides helical constrained forms of
DP178 and homologous sequences and variants, overcoming the
limitations in the art concerning DP178 and providing more
effective use of DP-178 like sequences. Accordingly, in one
embodiment of the invention is provided a constrained helical
peptide having at least its internal amino acid sequence (and
preferably adjacent amino acid sequences) selected from the
C-terminal region of the HIV-1LAI isolate transmembrane protein
gp41 ectodomain amino acids 633 to 678, which overlap with the
sequence corresponding to peptide DP-178 (amino acid residues 643
to 673). This region is a 46-amino acid sequence (reading from the
amino to carboxy terminus):
NH2-WMEWEREIDNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-COOH.
[0464] Peptides in an alpha-helical coiled-coil conformation
interact with one another in a characteristic manner that is
determined by the primary sequence of each peptide. The tertiary
structure of an alpha-helix is such that 7 amino acid residues in
the primary sequence correspond to approximately 2 turns of the
alpha-helix. Accordingly, a primary amino acid sequence giving rise
to an alpha-helical conformation may be broken down into units of 7
residues each, termed heptads (having the form abcdefg). The core
polypeptides are comprised of a series of heptads in tandem. When
the sequence of a heptad is repeated in a particular core
polypeptide, the heptad may be referred to as a "heptad repeat", or
simply "repeat".
[0465] According to the invention, embodiments are provided as
compounds containing a constrained helical peptide that is composed
of a peptide which contains a sequence of eight amino acid
residues, where the sequence of eight amino acid residues has a
first terminal residue and a second terminal residue, where the
first terminal residue and the second terminal residue flank an
internal sequence of six amino acids, wherein the first and second
terminal residues have a side chain that are linked to each other
forming a locking moiety to constrain the peptide to a helical
form. The internal sequence of six amino acids has the form gabode,
defgab, or cdefga and has a sequences of six contiguous amino acids
found in HIV-1LAI strain transmembrane protein gp41 amino acid
sequence 633 to 678, in its homolog sequence from another HIV
strain, in a consensus sequence of its homolog sequences from any
one HIV lade, or amino acid substituted variant thereof. According
to the invention, each of the amino acids in the aforementioned
sequences is assigned a position of a, b, c, d, e, f, or g. The
assignment is based on assigning the amino acid 633 of the HIV LAI
gp41 633-678 sequence to position a of a repeating abcdefg heptad
assignment. Subsequent amino acids in the sequence are assigned
positions accordingly. FIG. 18 indicates the heptad positional
assignment of each amino acid in the sequence. The assignment can
be readily applied to homologs and consensus sequences by aligning
their amino acids to the corresponding amino acid in the
representative HIV LAI sequence. The 633 amino acid or its
corresponding amino acid in a homolog or consensus sequence is
assigned position a, which begins the repeating abcdefg assignment
pattern.
[0466] In these representative compounds and sequences shown in
FIGS. 16-18, the locking moiety or tether is between adjacent f
positions when the internal sequence is of the form gabede,
adjacent c positions when the internal sequence is of the form
defgab, or adjacent b positions when the internal sequence is of
the form cdefga. In the most preferred embodiments the locking
occurs between adjacent f positions, in which case the f position
amino acids are replaced by amino acids suitable for providing a
helix locking moiety. FIG. 18 provides the alignment of the
repeating abcdefg assignment with the sequences relevant to the
invention. In a preferred embodiment the internal sequence of six
amino acids has the form gabcde. These "internal sequence" of six
amino acids from g$ l can substitute for moiety "Z" in any of the
compounds, formulas, and synthetic methods taught herein.
[0467] While the internal amino acid sequence is preferably from a
sequence of six contiguous amino acids in HIV-1LAI strain gp41
amino acid sequence 633 to 678, in its homolog sequence from
another HIV strain, or in a consensus sequence of its homolog
sequences from any one HIV lade, it may be an amino acid
substituted variant thereof. The sequences of the invention also
include analogs of HIV gp41 sequence 33-678, truncations which may
include, but are not limited to, peptides comprising the 633-678
sequence, containing one or more amino acid substitutions,
insertions and/or deletions. The analogs of the sequence will
exhibit antiviral activity when in constrained peptides of the
invention, and may, further, possess additional advantageous
features, such as, for example, increased bioavailability, and/or
stability, or generate antibodies with increased HIV strain
recognition.
[0468] HIV-1 and HIV-2 envelope proteins are structurally distinct,
but there exists a striking amino acid conservation within the gp41
633-678 corresponding regions of HIV-1 and HIV-2. Amino acid
substitutions may be of a conserved or non-conserved nature.
Conserved amino acid substitutions consist of replacing one or more
amino acids of the 633-678 peptide sequence with amino acids of
similar charge, size, and/or hydrophobicity characteristics, such
as, for example, a glutamic acid (E) to aspartic acid (D) amino
acid substitution. Non-conserved substitutions consist of replacing
one or more amino acids of the 633-678 peptide sequence with amino
acids possessing dissimilar charge, size, and/or hydrophobicity
characteristics, such as, for example, a glutamic acid (E) to
valine (V) substitution.
[0469] Deletions of the 633-678 region or its homologs are also
within the scope of the invention. Such deletions consist of the
removal of one or more amino acids with the lower limit length of
the resulting peptide sequence being 6 amino acids for use as an
internal sequence of a constrained helical peptide. Preferably the
deletions retain sufficient amino acids such that at least two
locks may be incorporated as taught herein. Examples of such
deletions are the HIV35 peptide and its constrained helix compounds
of the invention that have one lock (e.g. HIV 24) and two locks
(e.g. HIV 31). Most preferably, the deletions are terminal
truncations, but in any event result in peptides which, when
constrained along the f-b-c helical face, are still recognized by
the coiled coil search algorithms used herein, or alternatively,
retain the a-d helical face orientation and spatial arrangement of
the parent molecule, or alternatively, can exhibit antifusogenic or
antiviral activity.
[0470] Most preferred compounds are those that, when used as
immunogens, generate antibodies that neutralize HIV viral fusogenic
activity or infectivity.
[0471] The peptides of the invention may further include homolog
sequences of the HIV LAI strain 633-678 sequence which exhibit
antiviral activity when in constrained helical form. Most
preferably, the constrained peptides, when used as happens, will
generate antibodies that block viral fusion events, leading to an
inhibition of viral infectivity. Such homologs are peptides whose
amino acid sequences are comprised of the amino acid sequences of
peptide regions of other (i.e., other than HIV-1LAI) viruses that
correspond to the gp41 peptide region of 633-678. Such viruses may
include, but are not limited to, other HIV-1 isolates and HIV-2
isolates. Homologs derived from the corresponding gp41 peptide
region of other (i.e., non HIV-1LAI) HIV-1 isolates may include
those provide in FIGS. 16A to 16G, or other known corresponding
sequences. Particularly preferred are those derived from HIV-1SF2,
HIV-1RF, and HIV-1MN, GNE6, GNE8, and Thai strain isolate A244.
[0472] In a particularly preferred embodiment, amino acids at
positions a and d of the internal sequence of six amino acids are
not amino acid substituted in the helical peptide, but are the
amino acids in the known isolates or consensus sequences (see FIGS.
16A-16G and 17).
[0473] Also most preferred are embodiments where the amino acids at
positions g and e of the internal sequence of six amino acids are
not amino acid substituted in the helical peptide. An amino acid at
any one of positions a, d, g, or e of the internal sequence of six
amino acids can be conservatively substituted in the helical
peptide in preferred embodiments. The a and d positions, and less
directly the g and e positions, are believed to be those that are
on the face of the constrained helix that interacts with the gp41
core trimer (see FIG. 19). Since positions f, b and c are believed
to not directly participate in binding, but rather serve to allow
helix structure, preferred variations at positions b and c of the
internal sequence of six amino acids are not amino acid substituted
in the helical peptide, when not the locking (tethering) residues.
Less preferred are compounds wherein an amino acid at any one of
positions b, c, or f of the internal sequence of six amino acids is
conservatively substituted or is any non-helix-breaking amino acid
in the helical peptide, that does not interfere with the locking
moiety. Preferred internal sequence chimeras are those in which an
amino acid at any one of positions a, d, g, or e of the internal
sequence of six amino acids is substituted in the helical peptide
with an amino acid from the corresponding position of a different
HIV virus strain.
[0474] Preferred compounds of the invention can include sequences
from HIV-1 clade consensus sequences:
2 (clade B consensus) W m e W e r E I d n Y T ? l I y t L I e e s Q
n Q Q e k N e q e L L e L d k W a s L w n (SEQ ID NO: 109); W f
(clade A consensus) W L q W d K E I s n Y T ? I I Y n L I E e S q n
Q Q E k N E q d L L A L D K W a n L (SEQ ID NO: 110); w n W F
(clade C consensus) W M q W D R E I S N Y T d t I Y r L L E D S Q N
Q Q E r N E K D L L A L D S W k (SEQ ID NO: 111); N L W N W F
(clade D consensus) W m e W E r E I d N Y T G l I Y s L I E e S Q I
Q Q E K N E k e L L e L D K W A S (SEQ ID NO: 112); L W N W F and
(clade E consensus) W I E W e R E I S N Y T N q I Y e I L T e S Q n
Q Q D R N E K D L L e L D K W A (SEQ ID NO: 113). S L W n W f
[0475] The amino acids in these sequences are represented by a
single letter code, wherein a lower case letter is the represented
amino acid or is substituted with an amino acid from that
corresponding position in a sequence within the same clade, and
wherein a ? is any amino acid from that corresponding position in a
sequence from within the same clade. Most preferred are homologs or
consensus sequences from FIGS. 16A-16G. The internal sequences are
preferably found virus sequences in the group of HIV-1 clades
consisting of clades A, B, C, D, E, F, G and F/B.
[0476] While the locking moiety can be any structure that
constrains the internal sequence to a helical peptide form and does
not interfere with the a-d face (active face) of the constrained
peptide, the preferred compounds use the locking chemistry taught
herein. Compounds of the invention can have the first and second
terminal residues with a side chain containing an amide
bond-forming substituent that are linked to each other forming an
amide bond to form a constrained helical peptide. The side chain
amide bond-forming substituent of the first terminal residue and
the side chain amide bond-forming substituent of the second
terminal residue are independently selected from the group
consisting of an amino substituent and a carboxy substituent The
side chain amide bond-forming substituent of the first terminal
residue is a carboxy substituent, the side chain amide bond-forming
substituent of the second terminal residue is a carboxy
substituent, and the difunctional linker is a diamine wherein the
first and second functional groups are amino groups. In preferred
form the first terminal residue and the second terminal residue are
independently selected from the group consisting of Asp and Glu,
more preferred the first terminal residue and the second terminal
residue are both Glu. The first terminal residues can have a
D-thio-lysine side chain and the second terminal residue a
L-thio-lysine that are linked to each other forming a disulfide
bonded locking moiety to form a constrained helical peptide.
[0477] In another embodiment the constrained peptide is a hapten
that is attached to a carrier macromolecule, preferably covalently
linked to the constrained helical peptide, as discussed herein. The
macromolecule can be linked to the helical peptide at the locking
moiety or at amino acids at positions f, b, or c of the constrained
helical peptide, and can be any carrier that does not interfere
with the a-d face of the constrained helical peptide. A preferred
carrier for immunogenic purposes is keyhole limpet hemocyanin, or
other carriers discussed herein.
[0478] In other embodiments the compounds contain more than one
constrained helical peptide. The internal sequences of a first and
a second constrained helical peptide in these embodiments are
preferably different. The internal sequences of the first and
second constrained helical peptides are from the same HIV gp41
sequence or the same HIV clade consensus sequence, or amino acid
substituted variant thereof. The internal sequences of the first
and second constrained helical peptides are chosen from those that
were separated by at least two helical turns (or six residues) in
the HIV gp41 sequence or the same HIV clade consensus sequence, or
amino acid substituted variant thereof. The compounds can further
comprise a third constrained helical peptide. Again, the internal
sequences of the first, second, and third constrained helical
peptides are preferably different. In one example, the three
sequences are present as separate constrained helical segments in a
super helix of the polypeptide backbone of a 633-678 sequence as
depicted in FIG. 18.
[0479] In other embodiments the compounds of the invention contain
1 to 38, 1 to 35, or more preferably, 1 to 19 amino acids flanking
either or both terminal residues of the helical peptide. The
flanking amino acids preferably are the flanking amino acids for
the internal sequence as found in a sequence from an HIV gp41
sequence.
[0480] In yet other embodiments. the compounds further comprising a
blocking group attached at either or both of the terminal residues
of the helical peptide to prevent proteolytic degradation. The
blocking group can contain a D-amino acid or a non-amide bond
between adjacent flanking amino acids.
[0481] Particularly preferred compounds include those in which a
single lock is placed within sequence YTSLIHSLIEESQNQQEKNEQELLELD
(SEQ ID NO: 2) sequence or a homolog sequence thereof, within
EWDREINNYTSLIHSLIEESQNQQEK- NEQE (SEQ ID NO: 107) sequence or a
homolog sequence thereof, within
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNF (SEQ ID NO: 108) sequence or a
homolog sequence thereof, to yield a constrained helical peptide.
More than one constraint, preferably two, can be placed in these
sequences, with examples shown in FIG. 18. Shown in FIG. 18 are
locations of gabcde form helical sections when one, two or three i
to i+7 locks are present in a 633-678 sequence or variant
(truncated or sequence variant) thereof. The two-lock variants
(II), (III), and HIV31, and the one-lock variants HIV24, (IX) and
(XI) (FIG. 18) are preferred compounds demonstrating preferred
locking positions. Less preferred are the three-lock variant, the
two-lock (VI) and (VII) variants, and the one lock VIII and XII
variants. Particularly preferred are the truncated variants HIV24
and HIV31 and their homologs from other HIV strains or consensus
sequences or substitution variants thereof . Much less preferred
are i to i+4 lock to constrain a "floppy" helical segment.
[0482] In a preferred embodiment there are at least two constrained
helical peptides in the compound, for example attached as different
and independent haptens to KHL or a synthetic TASP or lysine
network, or as two or more locked helical segments within a longer
polypeptide sequence, preferably one that has a tendency to form an
extended or super helical structure. The internal sequences of the
first and second constrained helical peptides are preferably
different, for example as multiple haptens on a single hapten
carrier or two or more locked helical segments within a longer
super helix polypeptide sequence. In the latter case, the internal
sequences of the first and second constrained helical peptides are
preferably from the same HIV gp41 sequence, the same HIV lade
consensus sequence, or the same amino acid substituted variant
thereof. The two helical peptides are attached to each other by a
separating amino acid sequence, which can comprises from 5 to 7, 12
to 14, or 19 to 21 non helix-breaking natural or unnatural amino
acids, and where preferably, the internal sequences of the first
and second constrained helical peptides are from the same HIV gp41
sequence, the same HIV clade consensus sequence, or the same amino
acid substituted variant thereof. The separating sequence can be a
contiguous amino acid sequence selected from an intervening
sequence that is located between the two internal sequences present
in the same HIV gp41 sequence, the same HIV clade consensus
sequence, or the same amino acid substituted variant thereof, and
that excludes the two amino acids that correspond to the helical
peptide locking positions immediately flanking the intervening
sequence. An example is HIV31, in which the two constrained
segments (internal amino acid sequences) are separated in the
parent sequence (HIV35) by an eight amino acid sequence of which
the amino acids at adjacent f positions used in locking are not
considered part of the intervening sequence, such that the
intervening sequence is a six amino acid sequence which is
synthesized into the final peptide as a six amino acid separating
sequence. The separating sequence is most preferably 6, 13, or 20
amino acids, in order to maintain alignment of a-d faces in between
to constrained helical peptides.
[0483] The amino acids in the separating sequence retain abcdefg
assignment positions of the intervening sequence, wherein
preferably the amino acids in positions a and d in the separating
sequence are identical to their corresponding intervening sequence
amino acids. In addition, in preferred embodiments the amino acids
in the separating sequence positions g and e also are identical to
their corresponding intervening sequence amino acids. Less
preferably, an amino acid at any one of positions a, d, g, ore is
conservatively substituted in the separating sequence (with a
sequence other than that represented in the clade at that
position). Most preferably, the amino acids in the separating
sequence retain abcdefg assignment positions of the intervening
sequence and an amino acid at any one of positions a, d, g, or e is
substituted in the separating sequence with a corresponding amino
acid from its homolog sequence from another HIV strain, from a
consensus sequence of its homolog sequences from any one HIV clade,
or from an amino acid substituted variant thereof. The amino acids
in the separating sequence positions b, c, or f can be any
non-helix-breaking amino acid, with the preferences given in FIGS.
22 and 23A and B. Chimeras can be formed where an amino acid at any
one of positions a, d, g, or e of the internal sequence of six
amino acids is substituted in the helical peptide with an amino
acid from the corresponding position of a different HIV virus
strain. Likewise substitutions of the same nature can be made in
flanking or in separating sequences. Preferred are compounds
wherein the internal amino acid sequence is from any one of the
peptide sequences from FIG. 23A and 23B. More preferably, the
compound of the invention is selected from the group consisting of
constrained helical peptides of each possible sequence having any
one or any combination of amino acid substitutions indicated in the
constrained helical peptide series I to XII as shown in FIGS. 23A
and 23B. In other embodiments, the compound is selected from the
group consisting of constrained helical peptides of each possible
sequence having any one or any combination of amino acid
truncations indicated in the constrained helical peptide series I
to XII as shown in FIGS. 23A and 23B. In yet other embodiments, the
compound is selected from the group consisting of constrained
helical peptides of each possible sequence having any one or any
combination of amino acid substitutions indicated in the
constrained helical peptide series I to XII as shown in FIGS. 23A
and 23B in combination with any one or any combination of amino
acid truncations indicated in the constrained helical peptide
series I to XII as shown in FIGS. 23A and 23B. X in these sequences
can be any non helix-breaking amino acid.
[0484] In yet another embodiment of the invention, peptides
comprising the sequences described herein can be synthesized with
additional chemical groups present at their amino and/or carboxy
termini, such that, for example, the stability, bioavailability,
and/or inhibitory activity of the peptides is enhanced. For
example, hydrophobic groups such as carbobenzoxyl, dansyl, or
t-butyloxycarbonyl groups, may be added to the amino termini. An
acetyl group or a 9-fluorenylmethoxy-carbonyl group may be placed
at the amino termini. A hydrophobic group, t-butyloxycarbonyl, or
an amido group may be added to carboxy termini. Furthermore, the
peptides of the invention can be synthesized such that their steric
configuration is altered. For example, the D-isomer of one or more
of the amino acid residues of the peptide can be used, rather than
the usual L-isomer. The compounds can contain at least one bond
linking adjacent amino acids that is a non-peptide bond, and is
preferably not helix breaking. Non-peptide bonds for use in
flanking sequences include an imino, ester, hydrazine,
semicarbazide, oxime, or azo bond. Still further, at least one of
the amino acid residues of the peptides of the invention can be
substituted by one of the well known non-naturally occurring amino
acid residues, that is preferably not helix breaking. Most
preferably the non-natural amino acid or non-amide bond linking
adjacent amino acids, when present, is present outside of the
internal sequence, and is, more preferably, not helix breaking.
Still further, at least one of the amino acid residues of the
peptides of the invention can be substituted by one of the well
known non-naturally occurring amino acid residues. Alterations such
as these can serve to increase the stability, bioavailability,
immunogenicity, and/or inhibitory action of the peptides of the
invention.
[0485] While not wishing to be limited by any one theory, the
constrained helical peptides are believed to derive their activity
by interaction of the a-d face of the helix. The potent anti-HIV
activity of the compounds of the invention derive from the gp41
633-678 region which corresponds to a putative alpha-helix region
located in the C-terminal end of the gp4 ectodomain, and which
appears to associate with a distal site on gp4 whose interactive
structure is influenced by the leucine zipper motif, a coiled-coil
structure, referred to as DP-107. The association of these two
domains may reflect a molecular linkage or "molecular clasp"
intimately involved in the fusion process (see FIGS. 18 and 19).
The DP 107 region forms a core truner complex with a groove that
recognizes and binds the a-d face of the helical peptides of the
invention.
[0486] When synthesized as peptides both DP-107 and DP-178 are
potent inhibitors of HIV infection and fusion, probably by virtue
of their ability to form complexes with viral gp41 and interfere
with its fusogenic process; e.g., during the structural transition
of the viral protein from the native structure to the fusogenic
state, the DP-107 and DP-178 peptides may gain access to their
respective binding sites on the viral gp41, and exert a disruptive
influence. Consequently, when more than one constrained helical
peptide is present, as part of a super helix or extended helix
polypeptide backbone, the positions a and d of a first constrained
helical peptide are in the same plane as positions a and d of the
second constrained helical peptide. In other words, the a-d face of
the two helices are aligned in the same plane. To achieve this
orientation when the helices are in a polypeptide super helix, the
first and second constrained helical peptides are separated by
either 5 to 7, 12 to 14 or 19 to 21 natural or unnatural
helix-forming amino acids. Preferably, the first and second
constrained helical peptides are separated by either 6, 13, or 20
natural or unnatural helix-forming amino acids. A most preferred
spatial alignment of the first, second, and any additional
constrained helical peptides is that found in DP107, wherein the
a-d faces are aligned in the same plane to allow interaction with
the grove in the core trimer.
[0487] When the particularly preferred tethering chemistry as
taught herein is used, the compounds of the invention are selected
from the group consisting of:
[0488] the compound represented by Formula (1): 165
[0489] wherein S is absent or is a macromolecule,
[0490] X is hydrogen or is any amino acid or amino acid
sequence,
[0491] Y is absent, or is hydroxyl if S is absent, or is any amino
acid or amino acid sequence,
[0492] Z is an amino acid sequence consisting of six amino acids,
wherein the internal sequence of six amino acids has the form
gabede, defgab, or cdefga and is selected from the group of
sequences consisting of a sequence of six contiguous amino acids in
HIV-1LAI strain gp41 amino acid sequence 633 to 678, in its homolog
sequence from another HIV strain, in a consensus sequence of its
homolog sequences from any one HIV clade, or amino acid substituted
variant thereof, in which amino acid 633 or its corresponding amino
acid in the homolog, consensus or variant sequence is assigned
position a of a repeating abcdefg assignment;
[0493] m and p are independently selected from the integers 0 to 6
inclusive, provided that m+p is less than or equal to 6, and
[0494] n is any integer in the range defined by (7-(m+p)) to
(9-(m+p)) inclusive, provided that n is greater than 1;
[0495] the compound represented by Formula (6): 166
[0496] wherein S is absent or is a macromolecule, X is hydrogen or
is any amino acid or amino acid sequence, Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence, Z is an amino acid sequence consisting of six amino
acids, wherein the internal sequence of six amino acids has the
form gabcde, defgab, or cdefga and is selected from the group of
sequences consisting of a sequence of six contiguous amino acids in
HIV-1LAI strain gp41 amino acid sequence 633 to 678, in its homolog
sequence from another HIV strain, in a consensus sequence of its
homolog sequences from any one HIV clade, or amino acid substituted
variant thereof, in which amino acid 633 or its corresponding amino
acid in the homolog, consensus or variant sequence is assigned
position a of a repeating abcdefg; q is selected from the integers
1 to 7 inclusive, s is selected from
[0497] the integers 0 to 6 inclusive, provided that q+s is less
than or equal to 7, and r is any integer in the range defined by
(7-(q+s)) to (9-(q+s)) inclusive, provided that r is greater than
0;
[0498] the compound represented by Formula (11): 167
[0499] wherein S is absent or is a macromolecule, X is hydrogen or
is any amino acid or amino acid sequence, Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence, Z is an amino acid sequence consisting of six amino
acids, wherein the internal sequence of six amino acids has the
form gabcde, defgab, or cdefga and is selected from the group of
sequences consisting of a sequence of six contiguous amino acids in
HIV-1LAI strain gp41 amino acid sequence 633 to 678, in its homolog
sequence from another HIV strain, in a consensus sequence of its
homolog sequences from any one HIV clade, or amino acid substituted
variant thereof, in which amino acid 633 or its corresponding amino
acid in the homolog, consensus or variant sequence is assigned
position a of a repeating abcdefg assignment; t is selected from
the integers 0 to 6 inclusive, and v is selected from the integers
1 to 7 inclusive, provided that t+v is less than or equal to 7; and
u is any integer in the range defined by (7-(t+v))to (9-(t+v))
inclusive, provided that u is greater than 0; and
[0500] the compound represented by Formula (16): 168
[0501] wherein S is absent or is a macromolecule, X is hydrogen or
is any amino acid or amino acid sequence, Y is absent, or is
hydroxyl if S is absent, or is any amino acid or amino acid
sequence, Z is an amino acid sequence consisting of six amino
acids, wherein the internal sequence of six amino acids has the
form gabcde, defgab, or cdefga and is selected from the group of
sequences consisting of a sequence of six contiguous amino acids in
HIV-1LAI strain gp41 amino acid sequence 633 to 678, in its homolog
sequence from another HIV strain, in a consensus sequence of its
homolog sequences from any one HIV lade, or amino acid substituted
variant thereof, in which amino acid 633 or its corresponding amino
acid in the homolog, consensus or variant sequence is assigned
position a of a repeating abcdefg assignment; w and y are
independently selected from the integers 1 to 7 inclusive, provided
that w+y is less than or equal to 8, and x is any integer in the
range defined by (7-(w+y)) to (9w+y)) inclusive, provided that x is
greater than or equal to 0.
[0502] These compounds can further contain S' when S is absent and
X is any amino acid or amino acid sequence, wherein S' is a
macromolecule attached to X. The X or Y can contain a blocking
group that prevents enzymatic degradation. Standard terminal
blacking groups as known in the art are suitable. X or Y can also
contain a D-amino acid or a non-amide bond between adjacent amino
acids to prevent enzymatic degradation.
[0503] The compounds can be formulated with a carrier as taught
herein. When the helical peptide is to be used as a hapten the
carrier can be an adjuvant. Typically, compositions of the
invention are sterile. Compositions can contain at least two
compounds of the invention, ether free or covalently or ionically
attached to one another. The peptides of the invention that have a
virus fusion inhibitor activity, can be used in combination with
other therapeutic agents, preferably in combination with another
antiviral agent, to enhance its antiviral effect. Such antiviral
agents include but are not limited to those which function on a
different target molecule involved in viral replication, e.g.,
reverse transcriptase inhibitors, viral protease inhibitors,
glycosylation inhibitors; those which act on a different target
molecule involved in viral transmission; those which act on a
different loci of the same molecule; and those which prevent or
reduce the occurrence of viral resistance.
[0504] In treating mammals, including humans, having a viral
infection, a therapeutically effective amount of the compounds of
the invention, or a pharmaceutically acceptable derivative, is
administered is a dose sufficient to inhibit viral replication,
either alone or in combination with other virus inhibiting drugs.
For example HIV31 or HIV 24 can be administered as an infusion at
about 0.1 mg/kg to 1.0 mg/kg per day for about 12 weeks. A
preferable dose is from 20 mg to 35 mg. Doses can be administered
in intervals of from about once per day to 4 times per day and
preferably from about once every two days to once per day. A
preferred dose is administered to achieve peak plasma
concentrations of compound of from about 1 mg/ml to 10 mg/ml. This
may be achieved by the sterile injection of about a 2.0% solution
of the administered ingredients in buffered saline (any suitable
saline solutions known to those skilled in the art of chemistry may
be used). Desirable blood levels may be maintained by a continuous
infusion as ascertained by plasma levels measured by HPLC.
Pharmaceutical compositions containing the compounds of the
invention can be administered to a human patient, by itself, or in
pharmaceutical compositions where it is mixed with suitable
carriers or excipient(s), as taught herein, at doses to treat a
viral infection, in particular HIV infection. Suitable routes of
administration include oral, rectal, transmucosal, or intestinal
administration; parenteral delivery, including intramuscular,
subcutaneous, intramedullary injections, as well as intrathecal,
direct intraventricular, intravenous, intraperitoneal, intranasal,
or intraocular injections; transdermal, topical, vaginal and the
like. Dosage forms include but are not limited to tablets, troches,
dispersions, suspensions, suppositories, solutions, capsules,
creams, patches, minipumps and the like.
[0505] As discussed herein the compounds of the invention are
particularly suited as haptens to raise an antibody that binds to
the compound, preferably the antibody specifically binds an epitope
comprising an amino acid at position a, d, e, or g in the helical
peptide. Preferred antibodies are monoclonal. Antibodies of the
invention, not only recognize the peptides of the invention, but
preferably recognize the corresponding sequence when present in the
virus. They may also bind unconstrained DP178. More preferably, the
antibody neutralizes HIV viral infectivity and/or neutralizes HIV
virus membrane fusion. Thus the antibodies can recognize and bind
gp41 sequence.
[0506] In another embodiment is provided a method to immunize an
animal, comprising administering to the animal an immunogenic
amount of a compound of the invention. In yet another embodiment is
provided a method to prophylactically or therapeutically treat a
mammal at risk for or infected with HIV, comprising administering a
composition comprising a prophylactically or therapeutically
effective amount of a compound of the invention and a carrier.
While antibodies of the invention are expected to have broad viral
activity, preferably, the composition comprises internal six amino
acid sequences from different HIV strains or HIV clades. The
compositions include a vaccine formulation. The formulations can
contain one or more (multivalent) constrained helical peptides form
different HIV strains, for use as a vaccine or immunogen. The
composition can be administered, prophylactically or
therapeutically, to a patient at risk of infection or in need of
such treatment using the dosages and routes and means of
administration that are readily determined. However, chronic
administration may be preferred and dosages can be adjusted
accordingly. Administration of the compounds containing the
constrained helical peptides of the invention as a prophylactic
vaccine (or therapeutic vaccine), can comprise administering to a
host a concentration of peptides effective in raising an immune
response which is sufficient to neutralize HIV, by, for example,
inhibiting HIV ability to infect cells. The exact concentration
will depend upon the specific peptide to be administered, but may
be determined by using standard techniques for assaying the
development of an immune response which are well known to those of
ordinary skill in the art. The peptides to be used as vaccines are
usually administered intramuscularly. The peptides may be
formulated with a suitable adjuvant in order to enhance the
immunological response. Such adjuvants may include, but are not
limited to, mineral gels such as aluminum hydroxide; surface active
substances such as lysolecithin, pluronic polyols, polyanions;
other peptides; oil emulsions; and potentially useful human
adjuvants such as BCG and Corynebacterium parvum. Many methods may
be used to introduce the vaccine formulations described here. These
methods include but are not limited to oral, intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous, and
intranasal routes.
[0507] A compound of this invention in a suitable carrier or
excipient is used to make a vaccine. The polypeptide can be used
alone, but is preferably administered in a multivalent subunit
vaccine that includes internal sequences from MN strain. The
vaccine usually includes constrained helices representing 3 to
about 5 different strains, but 30 or more different gp41-based
constrained helical polypeptides can be used to provide a more
effective vaccine. Of particular interest are gp41 sequences from
breakthrough isolates of HIV vaccine trials. Use of a homolog gp41
sequence from one or more of breakthrough isolates in a subunit
vaccine, usually together a sequence from a commonly present
isolate like the MN sequence, can provide protection against HIV
strains that are sufficiently different from the common strain
(e.g., MN) that the typical single subunit vaccine does not confer
protection against those strains.
[0508] Preparation of polypeptides for use in a vaccine is well
known. The compound with the desired degree of purity and at a
sufficient concentration to induce antibody formation is mixed with
a physiologically acceptable carrier. A physiologically acceptable
carrier is nontoxic to a recipient at the dosage and concentration
employed in the vaccine. Generally, the vaccine is formulated for
injection, usually intramuscular or subcutaneous injection.
Suitable carriers for injection include sterile water, but
preferably are physiologic salt solutions, such as normal saline or
buffered salt solutions such as phosphate-buffered saline or
ringer's lactate. The vaccine generally contains an adjuvant.
Useful adjuvants include QS21 (Quillaja saponaria, commercially
available from Cambridge Biotech, Worcester, Mass.), which
stimulates cytotoxic T-cells, and alum (aluminum hydroxide
adjuvant). Formulations with different adjuvants which enhance
cellular or local immunity can also be used.
[0509] Additional excipients that can be present in the vaccine
include low molecular weight polypeptides (less than about 10
residues), proteins, amino acids, carbohydrates including glucose
or dextrans, chelating agents such as EDTA, and other excipients
that stabilize the protein or inhibit growth of microorganisms.
[0510] The vaccine can also contain other HIV proteins. In
particular, gp120, or the extracellular portion of gp41 or HIV-1
core proteins such as P24, P17, and P55 can be present in the
vaccine. Preferably, any gp120 present in the vaccine is from an
HIV isolate sequence represented in a constrained helical peptide
present in the vaccine.
[0511] Vaccine formulations generally include a total of about 10
to 5,000 .mu.g of compound, more preferably about 100 to 1000
.mu.g, even more preferably about 300 to 600 jig , conveniently in
about 1.0 ml to 1.5 ml of carrier. The amount of compound
representing any one isolate or clade present in the vaccine will
vary depending on the immunogenicity of the compound. For example,
a constrained helical peptide with sequences from some strains of
HIV may be less immunogenic than those from the MN strain. If
peptides representing two strains having different immunogenicity
are used in combination, empirical titration of the amount of each
virus would be performed to determine the percent of the peptide of
each strain in the vaccine. For isolates having similar
immunogenicity, approximately equal amounts of each isolate's
peptide would be present in the vaccine. Methods of determining the
relative amount of an immunogenic protein in multivalent vaccines
are well known and have been used, for example, to determine
relative proportions of various isolates in multivalent polio
vaccines.
[0512] The vaccines are generally administered at 0, 1, and at 6, 8
or 12 months, depending on the protocol. A preferred protocol
includes administration at 0, 1, 6, and 12 months. Following the
immunization procedure, annual or bi-annual boosts can be
administered. However, during the immunization process and
thereafter, neutralizing antibody levels can be assayed and the
protocol adjusted accordingly.
[0513] The vaccine is administered to uninfected individuals. In
addition, the vaccine can be administered to seropositive
individuals to augment immune response to the virus.
[0514] Although the compounds described herein can be used as a
vaccine as described above, the compounds can also be used alone or
in combinations in the same type of formulation, for use as an
immunogen, to induce antibodies that recognize the isolate(s)
present in the immunogen. Immunogens are formulated in the same
manner as vaccines and can include the same excipients, etc.
Antibodies induced by the immunogens can be used in a diagnostic to
detect the HIV strain in patient sera or body fluid samples, or to
affinity purify the particular gp41 molecule or virus. The
compounds also find use in diagnostic assays to detect the presence
of antibodies in HIV in sera from individuals suspected of being
infected.
[0515] In a further embodiment, the locked helix peptides of the
invention are used to create constrained combinatorial peptide
libraries. Combinatorial peptide libraries are uniquely suited to
incorporate constrained peptides. The libraries are constructed
with a "split synthesis" method in which a solid support (e.g.
beads) is aliquoted equally and a different amino acid is coupled
separately to each portion. The portions are pooled, resplit and
the process is repeated. In the "peptides-on-beads" technique, this
process yields a mixture of beads, each of which is coupled to a
peptide of unique sequence. The bead mixture can be used directly
in a binding selection, with binding detected calorimetrically and
positive beads physically removed from the mixture for
microsequencing (Clackson and Wells, Tibtech, 12: 173-184 (1994)).
To produce a library of peptides containing a random sequence of
six (or more) amino acids locked into a helical conformation by I
and I+7 residues according to the invention, the split synthesis
technique is modified to place I and I+7 residues in set positions
separated by six residues in each random amino acid sequence, and
the peptides are cyclized by linking the side chain amide
bond-forming substituents of the I and I+7 residues in each peptide
using any of the methods described in Section II below.
[0516] Combinatorial libraries containing the constrained peptides
of the invention are a particularly powerful tool for
identification of high affinity ligands in drug design. Given the
prevalence of the .alpha.-helical motif in active sites of binding
proteins, including DNA binding proteins, and the absence of amino
acid sequence constraints in the invention's tethering system, the
locked helix peptides of the invention greatly increase the utility
of combinatorial peptide libraries in screening methods for
specific binding activities, such as the methods of U.S. Pat. No.
5,306,619 used to screen for DNA sequence-specific binding
molecules.
[0517] II. Methods for Constructing Synthetic Locked Helix
Peptides
[0518] According to the present method, an element of
.alpha.-helical structure is removed from its context in a native
protein by constructing a peptide with an amino acid sequence
spanning the .alpha.-helical secondary structure of interest in the
native protein, and constraining the short peptide into an
.alpha.-helical conformation that mimics the .alpha.-helical
secondary structure of interest. The present methods enable the
practitioner to lock into a helical conformation any peptide that
is six amino acids in length by placing an amino acid with a side
chain amide bond-forming substitutent at the N-terminus of the
peptide and placing another amino acid with a side chain amide
bond-forming substitutent at the C-terminus of the peptide, and
then joining the side chain amide bond-forming substituents of the
N-terminal and C-terminal residues to form a cyclized structure
which mimics the conformation of an .alpha.-helix. The present
methods also enable the practitioner to lock into a helical
conformation any sequence of six amino acid residues in a larger
peptide by importing two residues with side chain amide
bond-forming substituents into the N-terminal amino acid position
and the C-terminal position amino acid position flanking the
sequence (of six amino acid residues) of interest within a larger
peptide, and then joining the side chain amide bond-forming
substituents of the N-terminal and C-terminal flanking residues to
form a cyclized structure which mimics the conformation of an
.alpha.-helix.
[0519] There are at least two general methods for constructing the
constrained helix peptides of the invention: (1) synthesis of a
linear peptide comprising a pair of residues that flank an amino
acid sequence that is six residues in length, wherein the two
flanking residues are independently selected from the group
consisting of amino acid residues with side chain amide
bond-forming substituents, followed by bridging the side chain
amide bond-forming substituents of the flanking residues with a
difunctional linker to cyclize the peptide; and (2) synthesis of a
linear peptide comprising a pair of residues that flank an amino
acid sequence that is six residues in length, wherein the two
flanking residues are independently selected from the group
consisting of amino acid residues with side chain amide
bond-forming substituents, and wherein one of the flanking residues
is added to the peptide chain carrying a difunctional linker such
that one functional group of the linker is coupled to the residue's
side chain amide bond-forming substitutent, followed by coupling of
the linker's free functional group to the side chain amide
bond-forming substitutent on the other flanking residue to cyclize
the peptide.
[0520] Any amino acid that has a side chain containing a
substitutent capable of forming an amide bond can be used as a
flanking residue herein. Suitable flanking amino acid residues
include amino acids with side chains carrying a free carboxy group,
such as aminopropanedioic acid, Asp, Glu, 2-aminohexanedioic acid,
and 2-aminoheptanedioic acid, and amino acids with side chains
carrying a free amino group, such as 2,3-diaminopropanoic acid
(2,3-diaminopropionic acid), 2,4-diaminobutanoic acid
(2,4-diaminobutyric acid), 2,5-diaminopentanoic acid, and Lys.
[0521] (1) Synthesis of Linear Peptide without Difunctional
Linker-coupled Flanking Amino Acid
[0522] a. Peptide Synthesis
[0523] The desired peptide sequence is designed such that the
sequence of six amino acid residues to be helicized extends between
two flanking residues independently selected from the group
consisting of amino acid residues with side chain amide
bond-forming substituents. In one embodiment, the side chain amide
bond-forming substituents of the N-terminal and C-terminal flanking
residues are independently selected from the group consisting of a
carboxy substitutent and an amino substitutent. In another
embodiment, the side chain amide bond-forming substituents of the
N-terminal and C-terminal flanking residues are both carboxy
substituents. In yet another embodiment, the side chain amide
bond-forming substitutent of one of the flanking residues is a
carboxy substitutent and the side chain amide bond-forming
substitutent of the other flanking residue is an amino
substitutent. In still another embodiment, the side chain amide
bond-forming substituents of the flanking residues are both amino
substituents. In yet another embodiment, the flanking residues are
independently selected from the group consisting of
aminopropanedioic acid, Asp, Glu, 2-aminohexanedioic acid,
2-aminoheptanedioic acid, 2-aminooctanedioic acid,
2-aminononanedioic acid, 2,3-diaminopropanoic acid,
2,4-diaminobutanoic acid, 2,5-diaminopentanoic acid, Lys,
2,7-diaminoheptanoic acid, 2,8-diaminooctanoic acid, and
2,9-diaminononanoic acid.
[0524] In some embodiments, the desired peptide contains an
additional amino acid or amino acids extending from the C-terminal
flanking residue and/or N-terminal flanking residue.
[0525] Once the desired peptide sequence is selected, chemical
synthesis can be employed to construct the constrained helix
peptide of the invention. This can be accomplished by modifying any
one of a number of methodologies well known in the art (see Kelley,
R. F. & Winkler, M. E. in Genetic Engineering Principles and
Methods, Setlow, J. K, ed., Plenum Press, N.Y., vol. 12, pp 1-19
(1990), Stewart, J. M. Young, J. D., Solid Phase Peptide Synthesis,
Pierce Chemical Co., Rockford, Ill. (1984); see also U.S. Pat. Nos.
4,105,603; 3,972,859; 3,842,067; and 3,862,925) to produce a the
desired peptide.
[0526] Peptides of the invention can be conveniently prepared using
solid phase peptide synthesis (Merrifield, J. Am. Chem. Soc. 85:
2149 (1964); Houghten, Proc. Natl. Acad. Sci. USA, 82: 5132 (1985).
Solid phase synthesis begins at the carboxy terminus of the
putative peptide by coupling a protected amino acid to an inert
solid support. The inert solid support can be any macromolecule
capable of serving as an anchor for the C-terminus of the initial
amino acid. Typically, the macromolecular support is a cross-linked
polymeric resin (e.g. a polyamide or polystyrene resin) as shown in
FIGS. 1-1 and 1-2, on pages 2 and 4 of Stewart and Young, supra. In
one embodiment, the C-terminal amino acid is coupled to a
polystyreneres in to form a benzyl ester. A macromolecular support
is selected such that the peptide anchor link is stable under the
conditions used to deprotect the .alpha.-amino group of the blocked
amino acids in peptide synthesis. If an base-labile a-protecting
group is used, then it is desirable to use an acid-labile link
between the peptide and the solid support. For example, an
acid-labile ether resin is effective for base-labile Fmoc-amino
acid peptide synthesis as described on page 16 of Stewart and
Young, supra. Alternatively, a peptide anchor link and
.alpha.-protecting group that are differentially labile to
acidolysis can be used. For example, an aminomethyl resin such as
the phenylacetamidomethyl (Pam) resin works well in conjunction
with Boc-amino acid peptide synthesis as described on pages 11-12
of Stewart and Young, supra.
[0527] After the initial amino acid is coupled to an inert solid
support, the .alpha.-amino protecting group of the initial amino
acid is removed with, for example, trifluoroacetic acid (TFA) in
methylene chloride and neutralizing in, for example, triethylamine
(TEA). Following deprotection of the initial amino acid's
.alpha.-amino group, the next .alpha.-amino and side chain
protected amino acid in the synthesis is added. The remaining
.alpha.-amino protected and, if necessary, side chain protected
amino acids are then coupled sequentially in the desired order by
condensation to obtain an intermediate compound connected to the
solid support. Alternatively, some amino acids may be coupled to
one another to form a fragment of the desired peptide followed by
addition of the peptide fragment to the growing solid phase peptide
chain.
[0528] The condensation reaction between two amino acids, or an
amino acid and a peptide, or a peptide and a peptidecan be carried
out according to the usual condensation methods such as the axide
method, mixed acid anhydride method, DCC
(N,N'-dicyclohexylcarbodiimide) or DIC
(N,N'-diisopropylcarbodiimide) methods, active ester method,
p-nitrophenyl ester method, BOP (benzotriazole-1-yl-oxy-tris
[dimethylamino] phosphoniumhexafluorophosphate)method,
N-hydroxysuccinicacid imido ester method, etc, and Woodward reagent
K method.
[0529] It is common in the chemical syntheses of peptides to
protect any reactive side-chain groups of the amino acids with
suitable protecting groups. Ultimately, these protecting groups are
removed after the desired polypeptide chain has been sequentially
assembled. Also common is the protection of the .alpha.-amino group
on an amino acid or a fragment while that entity reacts at the
carboxy group followed by the selective removal of the
.alpha.-amino protecting group to allow subsequent reaction to take
place at that location. Accordingly, it is common in polypeptide
synthesis that an intermediate compound is produced which contains
each of the amino acid residues located in the desired sequence in
the peptide chain with various of these residues having side chain
protecting groups attached. These protecting groups are then
commonly removed at substantially the same time so as to produce
the desired product following cleavage from the resin. Protecting
groups and procedures for their use in peptide synthesis are
reviewed in Protective Groups in Organic Synthesis, 2d ed., Greene,
T. W. and Wuts, P. G. M., Wiley & Sons (New York: 1991).
[0530] Suitable protecting groups for .alpha.-amino and side chain
amino groups are exemplified by benzyloxycarbonyl (abbreviated Z),
isonicotinyloxycarbonyl (iNOC), o-chlorobenzyloxycarbonyl [Z(2Cl)],
p-nitrobenzyloxycarbonyl [Z(NO.sub.2)], p-methoxybenzyloxycarbonyl
[Z(OMe)], t-butoxycarbonyl (Boc), t-amyloxycarbonyl (Aoc),
isobornyloxycarbonyl, adamantyloxycarbonyl,
2-(4-biphenyl)-2-propyloxycar- bonyl (Bpoc),
9-fluorenylmethoxycarbonyl (Fmoc), methylsulfonyethoxycarbon- yl
(Msc), trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulfenyl
(NPS), diphenylphosphinothioyl (Ppt), and dimethylphosphinothioyl
(Mpt) groups, and the like.
[0531] Protective groups for the carboxy functional group are
exemplified by benzyl ester, (Obz), cyclohexyl ester (Chx),
4-nitrobenzyl ester (Onb), t-butyl ester (Obut), 4-pyridylmethyl
ester (Opic), and the like. It is often desirable that amino acids
such as arginine, cysteine, and serine possessing a functional
group other than amino and carboxy groups be protected by a
suitable protecting group. For example, the guanidino group of
arginine may be protected with nitro, p-toluenesulfonyl,
benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzenesulfonyl,
4-methoxy-2,6-dimethylbenzenesulfonyl (Nds),
1,3,5-trimethylphenysulfonyl (Mts), and the like. The thiol group
of cysteine can be protected with p-methoxybenzyl, trityl, and the
like.
[0532] In one embodiment, the peptides of the invention are
synthesized with the help of blocking groups that protect the side
chain amide bond-forming substituents of the N-terminal and
C-terminal flanking residues. The protecting group or groups used
for the side chain amide bond-forming substituents of the
N-terminal and C-terminal flanking residues can be the same or
different than the protecting group or groups used to block the
side chain functional groups of other residues in the peptide. In a
preferred embodiment, the protecting group or groups used to block
the side chain amide bond-forming substituents is (are)
differentially removable with respect to the protecting groups used
for other side chain functional groups in the peptide, i.e. the
side chain amide bond-forming substituents can be deprotected
without deprotecting the other side chain functional groups in the
peptide, in addition to being differentially removable with respect
to the .alpha.-amino protecting group used in peptide synthesis. In
another preferred embodiment, the side chain amide bond-forming
substituents of the flanking residues are orthogonally protected
with respect to each other such that the side chain amide
bond-forming substituent of one flanking residue can be deprotected
without deprotecting the side chain amide bond-forming substituent
of the other flanking residue.
[0533] Suitable protecting groups for use in orthogonally
protecting the side chain amide bond-forming substituents of the
flanking residues with respect to other functional groups and/or
with respect to each other include pairs of differentially
removable carboxy protective groups, such as a reduction-labile
carboxy protective group, e.g. allyl or benzyl esters, paired with
a base-labile carboxy protective group, e.g. fluorenylmethylesters,
methyl or other primary alkyl esters. Fluorenylmethyl, methyl or
other primary alkyl groups or other base-labile carboxy protective
groups can be removed from their corresponding esters to yield a
free carboxy group (without deprotecting allyl or benzyl esters or
other reduction-labile esters) by saponification of the esters with
a suitable base such as piperidine and sodium hydroxide in a
suitable solvent such as dimethylacetamide, or methanol and water,
for a period of 10 to 120 minutes, and preferably 20 minutes, at 0
to 50.degree. C. The allyl or benzyl or other reduction-labile
esters can be removed when desired by reduction in the presence of
a suitable transition metal catalyst, such as Pd(PPh.sub.3).sub.4,
Pd(PPh.sub.3).sub.2Cl.sub.2, Pd(OAc).sub.2 or Pd on carbon with a
source of hydrogen, e.g. H.sub.2 gas, in a suitable solvent such as
dimethylacetamide, dimethylformamide, N-methylpyrrolidinoneor
methanol for a period of 10 to 500 minutes, and preferably 100
minutes, at 0 to 50.degree. C. For the sake of simplicity and
convenience, all carboxy protective groups that are removable by
Pd-catalyzed methods which result in the reduction of the protected
carboxy substitutent are included in the term "reduction-labile
protective groups" as used herein, even though such Pd-catalyzed
deprotection methods may not result in the reduction of the
protective group in question.
[0534] In embodiments wherein pd catalysis involves the formation
of intermediates of Pd derivatized with reduction-labile protecting
groups, e.g. Pd-allyl derivatives, the Pd catalyst can be restored
by reaction with a suitable nucleophile, such as piperidine or
tributyltin hydride. When such reduction-labile groups are used to
provide orthogonal protection in combination with base-labile
protecting groups, it is preferable to either (1) utilize a
synthetic scheme that calls for the removal of the base-labile
protecting groups before the removal of the reduction-labile
protecting groups or (2) restore the Pd catalyst with a nucleophile
that does not deprotect the base-labile protecting groups.
[0535] Alternatively, the carboxy substituents of the flanking
residues can be orthogonally protected with respect to other
functional groups and/or with respect to each other by using an
acid-labile protecting group, such as a tertiary alkyl ester, e.g.
t-butyl ester, in combination with a reduction-labile protecting
group, such as the allyl or benzyl esters described above. The
tertiary alkyl or other acid-labile ester group can be removed by
acidolysis, e.g. with trifluoroacetic acid in methylene chloride,
and the allyl or benzyl or other reduction-labile esters can be
removed by reduction in the presence of a transition metal catalyst
as described above.
[0536] In another embodiment, the carboxy substituents of the
flanking residues can be orthogonally protected with respect to
other functional groups and/or with respect to each other by using
a fluoride ion-labile protecting group, such as
2-(trimethylsilyl)ethyl and silyl esters, in combination with a
reduction-labile protecting group, such as the allyl or benzyl
esters described above, or in combination with a base-labile
protecting group, such as the fluorenylmethyl, methyl or other
primary allyl esters described above, without deprotecting the
reduction-labile or base-labile esters. The 2(trimethylsilyl)ethyl,
silyl or other fluoride-labile ester group can be removed by
reaction with a suitable fluoride ion source, such as
tetrabutylammonium fluoride in the presence of a suitable solvent,
such as dimethylacetamide (DMA), dimethylformamide (DMF),
tetrahydrofuran (THF), or acetonitrile.
[0537] Suitable protecting groups for use in orthogonally
protecting the side chain amide bond-forming substituents of the
flanking residues with respect to other functional groups and/or
with respect to each other also include pairs of differentially
removable amino protective groups, such as an allyloxycarbonyl or
other reduction-labile amino protective group paired with a
t-butoxycarbonyl (Boc) or other acid-labile amino protective group,
and a reduction-labile amino protective group paired with a
fluorenylmethoxycarbonyl (Fmoc) or other base-labile amino
protective group. An allyloxycarbonyl (or other reduction-labile
blocking group) protected amino group can be deprotected by
reduction using a transition metal catalyst as in the procedures
for removing reduction-labile carboxy protective groups described
above, without deprotecting a Boc or Fmoc protected amino group.
Likewise, an acid-labile amino protective group and a base-labile
amino protective group can be removed by acidolysis and base
saponification, respectively, without removing a reduction-labile
amino protective group. For the sake of simplicity and convenience,
all amino protective groups that are removable by Pd-catalyzed
methods which result in the reduction of the protected amino
substitutent are included in the term "reduction-labile protective
groups" as used herein, even though such Pd-catalyzed deprotection
methods may not result in the reduction of the protective group in
question.
[0538] In another embodiment, the amino substituents of the
flanking residues can be orthogonally protected with respect to
other functional groups and/or with respect to each other by using
a fluoride-labile protecting group, such as
2-trimethylsilylethylcarbamate (Teoc), in combination with a
reduction-labile protecting group, such as allyloxylcarbonyl, or in
combination with a base-labile protecting group, such as
fluorenylmethoxycarbonyl, as described above. The Teoc or other
fluoride-labile group can be removed by reaction with a suitable
fluoride ion source, such as tetrabutylammonium fluoride, as in the
procedures for removal of fluoride-labile carboxy protective groups
described above, without deprotecting an allyloxycarbonyl or
fluorenylmethoxycarbonyl protected amino group. Likewise, a
reduction-labile amino protective group and a base-labile amino
protective group can be removed by reduction and base
saponification, respectively, without removing a fluoride-labile
amino protective group.
[0539] In embodiments that use a carboxy substituent as the side
chain amide bond-forming substituent of one flanking residue and
that use an amino substituent as the side chain amide bond-forming
substituent of the other flanking residue, the carboxy substituent
and the amino substituent can be orthogonally protected with
respect to each other by using a reduction-labile protecting group
to block one substituent, e.g. allyl ester or allyloxycarbonyl, and
a fluoride-labile, acid-labile or base-labile protecting group to
block other substituent, e.g. silyl ester, t-butyl ester,
fluorenylmethyl ester, Teoc, Boc, or Fmoc.
[0540] In a preferred embodiment, a reduction-labile protecting
group is used to block the side chain amide bond-forming
substituent of one flanking residue and the protecting group for
the side chain amide bond-forming substituent of the other flanking
residue is selected such that it provides orthogonal protection
with respect to both the reduction-labile protecting group and the
.alpha.-amino protecting group used in peptide synthesis. For
example, in an embodiment using Fmoc chemistry for peptide
synthesis, orthogonal protection of the side chain amide
bond-forming substituents would be provided by a reduction-labile
protecting group and an acid-labile protecting group. Likewise, in
an embodiment using Boc chemistry for peptide synthesis, orthogonal
protection of the side chain amide bond-forming substituents would
be provided by a reduction-labile protecting group and a
base-labile protecting group.
[0541] In yet another preferred embodiment, the side chain amide
bond-forming substituents of the flanking residues are orthogonally
protected with respect to each other, with respect to .alpha.-amino
protecting group used in peptide synthesis, and with respect to the
protecting groups used to block other side chain functional groups
in the peptide chain.
[0542] In still another preferred embodiment, the side chain amide
bond-forming substituents of the flanking residues are orthogonally
protected with respect to each other, and with respect to
.alpha.-amino protecting group used in peptide synthesis, but only
one of the side chain amide bond-forming substituents is
orthogonally protected with respect to the protecting groups used
to block other side chain functional groups in the peptide chain.
In this embodiment, it is preferable to use the side chain amide
bond-forming substituent with fully orthogonal protection as the
target for initial coupling of the peptide to the difunctional
linker. Since the side chain amide bond-forming substituent with
fully orthogonal protection can be deprotected without deprotecting
other functional groups, the coupling reaction will be specific to
the desired side chain amide bond-forming substituent, and will
reduce the production of unwanted peptide/difunctional linker
derivatives. Although cyclization will require the deprotection of
the side chain amide bond-forming substituent of the other flanking
residue, and may cause concomitant deprotection of other side chain
functional groups, unwanted derivatives are less likely to form
given that the peptide chains are anchored to a solid support and
that the linker length will regioselectively favor a coupling
reaction between the unbound functional group of the linker and the
side chain amide bond-forming substituent of the other flanking
residue. If further peptide chain synthesis is desired after
cyclization, any side chain functional groups on other amino acid
residues left unprotected by the cyclization reactions can be
reprotected before chain synthesis is resumed.
[0543] Many of the blocked amino acids described above can be
obtained from commercial sources such as Novabiochem (San Diego,
Calif.), Bachem Calif. (Torrence, Calif.) or Peninsula Labs
(Belmont, Calif.).
[0544] In addition, the methods of the invention can be practiced
in conjunction with solution phase peptide synthesis, for example,
the solution phase peptide synthesis methods described in
Principles of Peptide Synthesis, 2d ed, M. Bodanszky,
Springer-Verlag (1993) or in The Practice of Peptide Synthesis 2d
ed, M. Bodanszky and A. Bodanszky, Springer-Verlag (1994). It will
be appreciated that solution phase peptide synthesis methods can be
easily modified to incorporate the desired flanking residues, with
or without orthogonally-protected side chain amide bond-forming
substituents, into the peptide chain of interest, using procedures
similar to those used in the solid phase peptide synthesis methods
described herein. It will be further appreciated that all
references to peptide synthesis herein encompass both solid phase
and solution (or liquid) phase peptide synthesis methods, unless
otherwise indicated.
[0545] b. Peptide Cyclization
[0546] After the desired amino acid sequence has been completed,
the linear peptide is cyclized in order to constrain the peptide in
a helical conformation. Any method of bridging the side chain amide
bond-forming substituents of the flanking residues with a
difunctional linker is suitable for producing the constrained
helical peptides of the invention.
[0547] (i) Selection of Difunctional Linker
[0548] Typically, the difunctional linker suitable for use herein
is capable of presenting two functional groups separated by a
distance of or about 5 .ANG. to or about 30 .ANG., and preferably
of or about 8 .ANG. to or about 14 .ANG., and more preferably of or
about 10 .ANG., such that the side chain amide bond-forming
substituent of one of the flanking residues can form an amide
linkage with one or either of the functional groups of the linker
and the side chain amide bond-forming substituent of the other
flanking residue can form an amide linkage with the remaining
functional group of the linker. It will be appreciated that the
nature of the molecular scaffold used to present the desired
functional groups in the proper spatial relationship is not
critical to the practice of the invention. Although straight chain
and branched alkyl scaffolds are suitable for use herein, the
invention is not so limited. For example, alkenyl, alkynyl,
cycloalkyl, or other aliphatic hydrocarbon species, with or without
heteroatoms, and monophenyl, biphenyl, naphthyl, and other aromatic
hydrocarbon species, with or without heteroatoms, that are
substituted with the desired functional groups in the proper
spatial relationship (e.g. para- or meta-substitutions in ring
structures such as monophenyl, biphenyl, naphthyl and the like) can
be used to link the side chain amide bond-forming substituents of
the flanking residues. The functional groups used in the
difunctional linker are selected such that they are capable of
forming amide linkages with the side chain amide bond-forming
substituents of the flanking residues used in the peptide to be
cyclized. In embodiments wherein the side chain amide bond-forming
substituent of each flanking residue is a carboxy substituent, the
peptide can be conveniently cyclized with a diamine linker. In one
example, the flanking residues and the diamine linker are selected
according to Table 1 below. It will be appreciated that each of the
flanking residues and linker molecules listed in Table 1 below is
considered to represent not only the particular molecule
corresponding to the given chemical name under IUPAC rules, but
also any variant of the molecule containing additional substituents
or modified substituents which do not prevent or substantially
alter the functioning of the amino and/or carboxy groups contained
in the molecule, which functioning is necessary for use of the
molecule in the methods of the invention. Accordingly, each
molecule listed will be understood to encompasses variant molecules
containing alkenyl, alkynyl and other unsaturated bonds,
heteroatoms, cycloalkyl substituents, aromatic substituents, or
other substituents in the carbon backbone of the molecule, and/or
variants containing the foregoing or other substituents or groups
in place of hydrogen atoms on the carbon backbone of the
molecule.
3TABLE 1 Item Flanking Flanking Diamine No. Residue #1 Residue #2
Linker 1 aminopropanedioic aminopropanedioic 1,7-diaminoheptane;
acid acid 1,8-diaminooctane; 1,9-diaminononane 2 aminopropanedioic
aspartic acid 1,6-diaminohexane; acid 1,7-diaminoheptane;
1,8-diaminooctane 3 aminopropanedioic glutamic acid
1,5-diaminopentane; acid 1,6-diaminohexane; 1,7-diaminoheptane 4
aminopropanedioic 2-aminohexanedioic 1,4-diaminobutane; acid acid
1,5-diaminopentane; 1,6-diaminohexane 5 aminopropanedioic
2-aminoheptanedioic 1,3-diaminopropane; acid acid
1,4-diaminobutane; 1,5-diaminopentane 6 aminopropanedioic
2-aminooctanedloic 1,2-diaminoethane; acid acid 1,3-diaminopropane;
1,4-diaminobutane 7 aminopropanedioic 2-aminononanedioic
1,2-diaminoethane; acid acid 1,3-diaminopropane 8 aspartic acid
aspartic acid 1,5-diaminopentane; 1,6-diaminohexane;
1,7-diaminoheptane 9 aspartic acid glutamic acid 1,4-diaminobutane;
1,5-diaminopentane; 1,6-diaminohexane 10 aspartic acid
2-aminohexanedioic 1,3-diaminopropane; acid 1,4-diaminobutane;
1,5-diaminopentane 11 aspartic acid 2-aminoheptanedioic
1,2-diaminoethane; acid 1,3-diaminopropane; 1,4-diaminobutane 12
aspartic acid 2-aminooctanedioic 1,2-diaminoethane; acid
1,3-diaminopropane; 13 glutamic acid glutamic acid
1,3-diaminopropane; 1,4-diaminobutane; 1,5-diaminopentane 14
glutamic acid 2-aminohexanedioic 1,2-diaminoethane; acid
1,3-diaminopropane; 1,4-diaminobutane 15 glutamic acid
2-aminoheptanedioic 1,2-diaminoethane; acid 1,3-diaminopropane 16
2-aminohexanedioic 2-aminohexanedioic 1,2-diaminoethane; acid acid
1,3-diaminopropane
[0549] In embodiments wherein the side chain amide bond-forming
substituent of each flanking residue is an amino substituent, the
peptide can be conveniently cyclized with a dicarboxylic acid
linker. In one example, the flanking residues and the dicarboxylic
acid linker are selected according to Table 2 below. It will be
appreciated that each of the flanking residues and linker molecules
listed in Table 2 below is considered to represent not only the
particular molecule corresponding to the given chemical name under
IUPAC rules, but also any variant of the molecule containing
additional substituents or modified substituents which do not
prevent or substantially alter the functioning of the amino and/or
carboxy groups contained in the molecule, which functioning is
necessary for use of the molecule in the methods of the invention.
Accordingly, each molecule listed will be understood to encompasses
variant molecules containing alkenyl, alkynyl and other unsaturated
bonds, heteroatoms, cycloalkyl substituents, aromatic substituents,
or other substituents in the carbon backbone of the molecule,
and/or variants containing the foregoing or other substituents or
groups in place of hydrogen atoms on the carbon backbone of the
molecule.
4TABLE 2 Item Flanking Flanking Dicarboxylic No. Residue #1 Residue
#2 acid Linker 1 2,3- 2,3-diaminopropanoic heptanedioic acid;
diaminopropanoic acid octanedioic acid; acid nonanedioic acid 2
2,3- 2,4-diaminobutanoic hexanedioic acid; diaminopropanoic acid
heptanedioic acid; acid octanedioic acid 3 2,3-
2,5-diaminopentanoic pentanedioic acid; diaminopropanoic acid
hexanedioic acid; acid heptanedioic acid 4 2,3- lysine butanedioic
acid; diaminopropanoic pentanedioic acid; acid hexanedioic acid 5
2,3- 2,7-diaminoheptanoic propanedioic acid; diaminopropanoic acid
butanedioic acid; acid pentanedioic acid 6 2,3- 2,8-diaminooctanoic
ethanedioic acid; diaminopropanoic acid propanedioic acid; acid
butanedioic acid 7 2,3- 2,9-diaminononanoic ethanedioic acid;
diaminopropanoic acid propanediolic acid acid 8 2,4-diaminobutanoic
2,4-diaminobutanoic pentanedioic acid; acid acid hexanedioic acid;
heptanedioic acid 9 2,4-diaminobutanoic 2,5-diaminopentanoic
butanedioic acid; acid acid pentanedioic acid; hexanedioic acid 10
2,4-diaminobutanoic lysine propanedioic acid; acid butanedioic
acid; pentanedioic acid 11 2,4- 2,7-diaminoheptanoic ethanedioic
acid; diaminobutanoic acid propanedioic acid; acid butanedioic acid
12 2,4- 2,8-diaminooctanoic ethanedioic acid; diaminobutanoic acid
propanedioic acid acid 13 2,5- 2,5-diaminopentanoic propanedioic
acid; diaminopentanoic acid butanedioic acid; acid pentanedioic
acid 14 2,5- lysine ethanedioic acid; diaminopentanoic propanedioic
acid; acid butanedioic acid 15 2,5- 2,7-diaminoheptanoic
ethanedioic acid; diaminopentanoic acid propanedioic acid acid 16
lysine lysine ethanedioic acid; propanedioic acid
[0550] In embodiments using an amino substituent as the side chain
amide bond-forming substituent of one flanking residue and a
carboxy substituent as the side chain amide bond-forming
substituent of the other flanking residue, the peptide can be
conveniently cyclized with an amino-substituted carboxylic acid
(aminocarboxylic acid) linker. In one example, the flanking
residues and the aminocarboxylic acid linker are selected according
to Table 3 below. It will be appreciated that each of the flanking
residues and linker molecules listed in Table 3 below is considered
to represent not only the particular molecule corresponding to the
given chemical name under IUPAC rules, but also any variant of the
molecule containing additional substituents or modified
substituents which do not prevent or substantially alter the
functioning of the amino and/or carboxy groups contained in the
molecule, which functioning is necessary for use of the molecule in
the methods of the invention. Accordingly, each molecule listed
will be understood to encompasses variant molecules containing
alkenyl, alkynyl and other unsaturated bonds, heteroatoms,
cycloalkyl substituents, aromatic substituents, or other
substituents in the carbon backbone of the molecule, and/or
variants containing the foregoing or other substituents or groups
in place of hydrogen atoms on the carbon backbone of the
molecule.
5TABLE 3 Item Flanking Flanking Aminocarboxylic No. Residue #1
Residue #2 acid Linker 1 aminopropanedioic 2,3-diaminopropanoic
7-aminoheptanoic acid acid acid; 8-aminooctanoic acid;
9-aminononanoic acid 2 aminopropanedioic 2,4-diaminobutanoic
6-aminohexanoic acid acid acid; 7-aminoheptanoic acid;
8-aminooctanoic acid 3 aminopropanedioic 2,5-diaminopentanoic
5-aminopentanoic acid acid acid; 6-aminohexanoic acid;
7-aminoheptanoic acid 4 aminopropanedioic 2,6-diaminohexanoic
4-aminobutanoic acid acid acid; 5-aminopentanoic acid;
6-aminohexanoic acid 5 aminopropanedioic 2,7-diaminoheptanoic
3-aminopropanoic acid acid acid; 4-aminobutanoic acid;
5-aminopentanoic acid 6 aminopropanedioic 2,8-diaminooctanoic
aminoethanoic acid acid acid; 3-aminopropanoic acid;
4-aminobutanoic acid 7 aminopropanedioic 2,9-diaminononanoic
aminoethanoic acid acid acid; 3-aminopropanoic acid 8 aspartic acid
2,3-diaminopropanoic 6-aminohexanoic acid acid; 7-aminoheptanoic
acid; 8-aminooctanoic acid 9 aspartic acid 2,4-diaminobutanoic
5-aminopentanoic acid acid; 6-aminohexanoic acid; 7-aminoheptanoic
acid 10 aspartic acid 2,5-diaminopeinanoic 4-aminobutanoic acid
acid; 5-aminopentanoic acid; 6-aminohexanoic acid 11 aspartic acid
2,6-diaminohexanoic 3-aminopropanoic acid acid; 4-aminobutanoic
acid; 5-aminopentanoic acid 12 aspartic acid 2,7-diaminoheptanoic
aminoethanoic acid acid; 3-aminopropanoic acid; 4-aminobutanoic
acid 13 aspartic acid 2,8-diaminooctanoic aminoethanoic acid acid;
3-aminopropanoic acid 14 glutamic acid 2,3-diaminopropanoic
5-aminoheptanoic acid acid; 6-aminohexanoic acid; 7-aminoheptanoic
acid 15 glutamic acid 2,4-diaminobutanoic 4-aminobutanoic acid
acid; 5-aminoheptanoic acid; 6-aminohexanoic acid 16 glutamic acid
2,5-diaminopentanoic 3-aminopropanoic acid acid; 4-aminobutanoic
acid; 5-aminoheptanoic acid 17 glutamic acid 2,6-diaminohexanoic
aminoethanoic acid acid; 3-aminopropanoic acid; 4-aminobutanoic
acid 18 glutamic acid 2,7-diaminoheptanoic aminoethanoic acid acid;
3-aminopropanoic acid 19 2-aminohexanedioic 2,3-diaminopropanoic
4-aminobutanoic acid acid acid; 5-aminoheptanoic acid;
6-aminohexanoic acid 20 2-aminohexanedioic 2,4-diaminobutanoic
3-aminopropanoic acid acid acid; 4-aminobutanoic acids
5-aminoheptanoic acid 21 2-aminohexanedioic 2,5-diaminopentanoic
aminoethanoic acid acid acid; 3-aminopropanoic acid;
4-aminobutanoic acid 22 2-aminohexanedioic lysine aminoethanoic
acid acid; 3-aminopropanoic acid 23 2- 2,3-diaminopropanoic
3-aminopropanoic aminoheptanedioic acid acid; acid 4-aminobutanoic
acid; 5-aminoheptanoic acid 24 2- 2,4-diaminobutanoic aminoethanoic
aminoheptanedioic acid acid; acid 3-aminopropanoic acid;
4-aminobutanoic acid 25 2- 2,5-diaminopentanoic aminoethanoic
aminoheptanedioic acid acid; acid 3-aminopropanoic acid; 26
2-aminooctanedioic 2,3-diaminopropanoic aminoethanoic acid acid
acid; 3-aminopropanoic acid; 4-aminobutanoic acid 27
2-aminooctanedioic 2,4-diaminobutanoic aminoethanoic acid acid
acid; 3-aminopropanoic acid 28 2-aminononanedioic
2,3-diaminopropanoic aminoethanoic acid acid acid; 3-aminopropanoic
acid
[0551] (ii) Cyclization Methods
[0552] Once the flanking residues and difunctional linker have been
selected and the peptide chain spanning the flanking residues has
been synthesized on solid phase, the difunctional linker can be
used to cyclize the solid phase-bound peptide by any convenient
method. It will be appreciated that the invention encompasses
methods of cyclizing a peptide after the finished peptide chain is
fully synthesized, and methods of cyclizing the peptide at any
point during peptide synthesis in which the peptide chain contains
the flanking residues that are to be cross linked by the
difunctional linker. Methods for cyclizing the peptide include (1)
deprotecting the side chain amide bond-forming substituents of the
flanking residues and reacting the solid phase peptide with the
difunctional linker to simultaneously form amide linkages between
the two functional groups of the linker and the side chain amide
bond-forming substituents of both flanking residues; (2)
deprotecting the side chain amide bond-forming substituent of only
one of the flanking residues (without deprotecting the side chain
amide bond-forming substituent of the other flanking residue),
reacting the difunctional linker with the solid phase peptide to
form an amide linkage between one functional group on the linker
and the side chain amide bond-forming substituent of the
deprotected flanking residue, deprotecting the side chain amide
bond-forming substituent of the other flanking residue, and then
intramolecularly reacting the free functional group on the linker
and the side chain amide bond-forming substituent of the other
flanking residue, thereby cyclizing the peptide; and (3)
deprotecting the side chain amide bond-forming substituents of both
of the flanking residues, obtaining a monoprotected difunctional
linker wherein only one of the linker's two amide bond-forming
functional groups is capable of reacting with a counterpart side
chain amide bond-forming substituent in a flanking residue,
reacting the monoprotected, difunctional linker with the solid
phase peptide to form an amide linkage between the free functional
group on the linker and the side chain amide bond-forming
substituent of one of the deprotected flanking residues,
deprotecting the blocked functional group on the linker, and then
intramolecularly reacting the free functional group on the linker
and the side chain amide bond-forming substituent of the other
flanking residue, thereby cyclizing the peptide. The orthogonal
deprotection reactions, non-orthogonal deprotection reactions, and
amide bond formation reactions can be performed as described in
Section (B)(II)(1)(a) above.
[0553] In implementing the methods of the invention generally
described as methods (2) and (3) above, it is desirable to use
synthesis schemes that exploit the advantages of orthogonal
protection and deprotection of functional groups to avoid formation
of unwanted derivatives. It will be evident to the practitioner
from the following representative synthetic schemes that the
protecting groups for the side chain amide bond-forming
substituents of the flanking residues, the method of peptide
synthesis used, and the sequence of peptide cyclization reactions
can be selected such that each of these components of the synthetic
scheme increases the specificity of the reactions and improves
yield of the desired product
[0554] (iii) Cyclization Using Diamine Linkers
[0555] In an example using carboxy substituents for the side chain
amide bond-forming substituents of both flanking residues, a
diamine linker for cyclization, and Fmoc chemistry for peptide
synthesis, the carboxy substituents are orthogonally protected with
respect to each other and with respect to the Fmoc-protected
.alpha.-amino group of the N-terminal residue in the peptide chain
by using an allyl group to protect the carboxy substituent of one
flanking residue and a t-butyl ester to protect the carboxy
substituent of the other flanking residue. In this example, the
peptide can be cyclized by (1) using reduction to deprotect the
allyl-protected carboxy substituent of one flanking residue
(without deprotecting the t-butyl ester-protected carboxy
substituent of the other flanking residue); (2) reacting an
unprotected or monoprotected (e.g. allyloxycarbonyl- or
Boc-monoprotected)diamine linker with the solid phase peptide to
form an amide linkage between one of the linker's amino groups and
the deprotected carboxy substituent; (3) using acidolysis to
deprotect the t-butyl ester-protected carboxy substituent of the
other flanking residue and deprotect the Boc-protected amino group
of the linker if a Boc-monoprotected diamine linker is used as the
linker; (4) using reduction to deprotect the
allyloxycarbonyl-protected amino group of the linker if an
allyloxycarbonyl-monoprotected diamine linker is used as the
linker; and (5) intramolecularly reacting the free carboxy
substituent of the other flanking residue with the free amino group
of the linker to form an amide linkage that cyclizes the
peptide.
[0556] Alternatively, the peptide can be cyclized by (1) using
acidolysis to deprotect the t-butyl ester-protected carboxy
substituent of one flanking residue (without deprotecting the
allyl-protected carboxy substituent of the other flanking residue);
(2) reacting an unprotected or monoprotected (e.g.
allyloxycarbonyl- or Boc-monoprotected)diamine linker with the
solid phase peptide to form an amide linkage between one of the
linker's amino groups and the deprotected carboxy substituent; (3)
using reduction to deprotect the allyl-protected carboxy
substituent of the other flanking residue and deprotect the
allyloxycarbonyl-protected amino group of the linker if an
allyloxycarbonyl-monoprotected diamine linker is used as the
linker; (4) using acidolysis to deprotect the Boc-protected amino
group of the linker if a Boc-monoprotected diamine linker is used
as the linker; and (5) intramolecularly reacting the free carboxy
substituent of the other flanking residue with the free amino group
of the linker to form an amide linkage that cyclizes the
peptide.
[0557] In an example using carboxy substituents for the side chain
amide bond-forming substituents of both flanking residues, a
diamine linker for cyclization, and Boc chemistry for peptide
synthesis, the carboxy substituents are orthogonally protected with
respect to each other and with respect to the Boc-protected
.alpha.-amino group of the N-terminal residue in the peptide chain
by using an allyl group to protect the carboxy substituent of one
flanking residue and a fluorenylmethyl (Fm) ester to protect the
carboxy substituent of the other flanking residue. In this example,
the peptide can be cyclized by (1) using reduction to deprotect the
allyl-protected carboxy substituent of one flanking residue
(without deprotecting the Fm ester-protected carboxy substituent of
the other flanking residue); (2) reacting an unprotected or
monoprotected (e.g. allyloxycarbonyl- or Fmoc-monoprotected)
diamine linker with the solid phase peptide to form an amide
linkage between one of the linker's amino groups and the
deprotected carboxy substituent; (3) using base saponification to
deprotect the Fm ester-protected carboxy substituent of the other
flanking residue and deprotect the Fmoc-protected amino group of
the linker if a Fmoc-monoprotected diamine linker is used as the
linker; (4) using reduction to deprotect the
allyloxycarbonyl-protected amino group of the linker if an
allyloxycarbonyl-monoprotected diamine linker is used as the
linker; and (5) intramolecularly reacting the free carboxy
substituent of the other flanking residue with the free amino group
of the linker to form an amide linkage that cyclizes the
peptide.
[0558] Alternatively, the peptide can be cyclized by (1) using base
saponification to deprotect the Fm ester-protected carboxy
substituent of one flanking residue (without deprotecting the
allyl-protected carboxy substituent of the other flanking residue);
(2) reacting an unprotected or monoprotected (e.g.
allyloxycarbonyl- or Fmoc-monoprotected) diamine linker with the
solid phase peptide to form an amide linkage between one of the
linker's amino groups and the deprotected carboxy substituent; (3)
using reduction to deprotect the allyl-protected carboxy
substituent of the other flanking residue and deprotect the
allyloxycarbonyl-protected amino group of the linker if an
allyloxycarbonyl-monoprotected diamine linker is used as the
linker; (4) using base saponification to deprotect the
Fmoc-protected amino group of the linker if a Fmoc-monoprotected
diamine linker is used as the linker; and (S) intramolecularly
reacting the free carboxy substituent of the other flanking residue
with the free amino group of the linker to form an amide linkage
that cyclizes the peptide.
[0559] (iv) Cyclization Using Dicarboxylic Acid Linkers
[0560] In an example using amino substituents for the side chain
amide bond-forming substituents of both flanking residues, a
dicarboxylic acid linker for cyclization, and Fmoc chemistry for
peptide synthesis, the amino substituents are orthogonally
protected with respect to each other and with respect to the
Fmoc-protected .alpha.-amino group of the N-terminal residue in the
peptide chain by using an allyloxycarbonyl group to protect the
amino substituent of one flanking residue and a Boc group to
protect the amino substituent of the other flanking residue. In
this example, the peptide can be cyclized by (I) using reduction to
deprotect the allyloxycarbonyl-protected amino substituent of one
flanking residue (without deprotecting the Boc-protected amino
substituent of the other flanking residue); (2) reacting an
unprotected or monoprotected (e.g. allyl- or t-butyl
ester-monoprotected) dicarboxylic acid linker with the solid phase
peptide to form an amide linkage between one of the linker's
carboxy groups and the deprotected amino substituent; (3) using
acidolysis to deprotect the Boc-protected amino substituent of the
other flanking residue, and to deprotect the t-butyl
ester-protected carboxy group of the linker if a t-butyl
ester-monoprotected dicarboxylic acid linker is used as the linker;
(4) using reduction to deprotect the allyl-protected carboxy group
of the linker if an allyl-monoprotected dicarboxylic acid linker is
used as the linker; and (5) intramolecularly reacting the free
amino substituent of the other flanking residue with the free
carboxy group of the linker to form an amide linkage that cyclizes
the peptide.
[0561] Alternatively, the peptide can be cyclized by (I) using
acidolysis to deprotect the Boc-protected amino substituent of one
flanking residue (without deprotecting the
allyloxycarbonyl-protected amino substituent of the other flanking
residue); (2) reacting an unprotected or monoprotected (e.g. allyl-
or t-butyl ester-monoprotected)dicarboxylic acid linker with the
solid phase peptide to form an amide linkage between one of the
linker's carboxy groups and the deprotected amino substituent; (3)
using reduction to deprotect the allyloxycarbonyl-protected amino
substituent of the other flanking residue, and to deprotect the
allyl-protected carboxy group of the linker if an
allyl-monoprotected dicarboxylic acid linker is used as the linker;
(4) using acidolysis to deprotect the t-butyl ester-protected
carboxy group of the linker if a t-butyl ester-monoprotected
dicarboxylic acid linker is used as the linker; and (5)
intramolecularly reacting the free amino substituent of the other
flanking residue with the free carboxy group of the linker to form
an amide linkage that cyclizes the peptide.
[0562] In an example using amino substituents for the side chain
amide bond-forming substituents of both flanking residues, a
dicarboxylic acid linker for cyclization, and Boc chemistry for
peptide synthesis, the amino substituents are orthogonally
protected with respect to each other and with respect to the
Boc-protected .alpha.-amino group of the N-terminal residue in the
peptide chain by using an allyloxycarbonyl group to protect the
amino substituent of one flanking residue and a Fmoc group to
protect the amino substituent of the other flanking residue. In
this example, the peptide can be cyclized by (1) using reduction to
deprotect the allyloxycarbonyl-protected amino substituent of one
flanking residue (without deprotecting the Fmoc-protected amino
substituent of the other flanking residue); (2) reacting an
unprotected or monoprotected (e.g. allyl- or Fm
ester-monoprotected) dicarboxylic acid linker with the solid phase
peptide to form an amide linkage between one of the linker's
carboxy groups and the deprotected amino substituent; (3) using
base saponification to deprotect the Fmoc-protected amino
substituent of the other flanking residue, and to deprotect the Fm
ester-protected carboxy group of the linker if a Fm
ester-monoprotected dicarboxylic acid linker is used as the linker;
(4) using reduction to deprotect the allyl-protected carboxy group
of the linker if an allyl-monoprotected dicarboxylic acid linker is
used as the linker; and (5) intramolecularly reacting the free
amino substituent of the other flanking residue with the free
carboxy group of the linker to form an amide linkage that cyclizes
the peptide.
[0563] Alternatively, the peptide can be cyclized by (1) using base
saponification to deprotect the Fmoc-protected amino substituent of
one flanking residue (without deprotecting the
allyloxycarbonyl-protected amino substituent of the other flanking
residue); (2) reacting an unprotected or monoprotected (e.g. allyl-
or Fm ester-monoprotected) dicarboxylic acid linker with the solid
phase peptide to form an amide linkage between one of the linker's
carboxy groups and the deprotected amino substituent; (3) using
reduction to deprotect the allyloxycarbonyl-protected amino
substituent of the other flanking residue, and to deprotect the
allyl-protected carboxy group of the linker if an
allyl-monoprotected dicarboxylic acid linker is used as the linker,
(4) using base saponification to deprotect the Fm ester-protected
carboxy group of the linker if a Fmoc-monoprotected dicarboxylic
acid linker is used as the linker; and (5) intramolecularly
reacting the free amino substituent of the other flanking residue
with the free carboxy group of the linker to form an amide linkage
that cyclizes the peptide.
[0564] (v) Cyclization Using Aminocarboxylic Acid Linkers
[0565] In an example using an amino substituent for the side chain
amide bond-forming substituent of one flanking residue, a carboxy
substituent for the side chain amide bond-forming substituent of
the other flanking residue, an aminocarboxylic acid linker for
cyclization, and Fmoc chemistry for peptide synthesis, the side
chain amide bond-forming substituents of the flanking residues are
orthogonally protected with respect to each other and with respect
to the Fmoc-protected .alpha.-amino group of the N-terminal residue
in the peptide chain by using an allyloxycarbonyl group to protect
the amino substituent of one flanking residue and a t-butyl ester
to protect the carboxy substituent of the other flanking residue.
In this example, the peptide can be cyclized by (I) using reduction
to deprotect the allyloxycarbonyl-protect- ed amino substituent of
one flanking residue (without deprotecting the t-butyl
ester-protected carboxy substituent of the other flanking residue);
(2) reacting an unprotected or amino-protected (e.g.
allyloxycarbonyl-protected amino or Boc-protected amino)
aminocarboxylic acid linker with the solid phase peptide to form an
amide linkage between the linker's carboxy group and the
deprotected amino substituent; (3) using acidolysis to deprotect
the t-butyl ester-protected carboxy substituent of the other
flanking residue, and to deprotect the Boc-protected amino group f
the linker if an aminocarboxylic acid with a Boc-protected amino
group is used as the linker; (4) using reduction to deprotect the
allyloxycarbonyl-protected amino group of the linker if an
aminocarboxylic acid with an allyloxycarbonyl-protected amino group
is used as the linker; and (5) intramolecularly reacting the free
carboxy substituent of the other flanking residue and the free
amino group of the aminocarboxylic acid linker to cyclize the
peptide.
[0566] Alternatively, the peptide can be cyclized by (1) using
acidolysis to deprotect the t-butyl ester-protected carboxy
substituent of one flanking residue (without deprotecting the
allyloxycarbonyl-protected amino substituent of the other flanking
residue); (2) reacting an unprotected or carboxy-protected (e.g.
allyl- or t-butyl ester-protected carboxy) aminocarboxylic acid
linker with the solid phase peptide to form an amide linkage
between the linker's amino group and the deprotected carboxy
substituent; (3) using reduction to deprotect the
allyloxycarbonyl-protected amino substituent of the other flanking
residue, and to deprotect the allyl-protected carboxy group of the
linker if an aminocarboxylic acid with an allyl-protected carboxy
group is used as the linker; (4) using acidolysis to deprotect the
t-butyl ester-protected carboxy group of the linker if an
aminocarboxylic acid with a t-butyl ester-protected carboxy group
is used as the linker; and (5) intramolecularly reacting the free
amino substituent of the other flanking residue and the free
carboxy group of the aminocarboxylic acid linker to cyclize the
peptide.
[0567] In another example using an amino substituent for the side
chain amide bond-forming substituent of one flanking residue, a
carboxy substituent for the side chain amide bond-forming
substituent of the other flanking residue, an aminocarboxylic acid
linker for cyclization, and Fmoc chemistry for peptide synthesis,
the side chain amide bond-forming substituents of the flanking
residues are orthogonally protected with respect to each other and
with respect to the Fmoc-protected .alpha.-amino group of the
N-terminal residue in the peptide chain by using a Boc group to
protect the amino substituent of one flanking residue and an allyl
group to protect the carboxy substituent of the other flanking
residue. In this example, the peptide can be cyclized by (1) using
acidolysis to deprotect the Boc-protected amino substituent of one
flanking residue (without deprotecting the allyl-protected carboxy
substituent of the other flanking residue); (2) reacting an
unprotected or amino-protected (e.g. allyloxycarbonyl-protect- ed
amino or Boc-protected amino) aminocarboxylic acid linker with the
solid phase peptide to form an amide linkage between the linker's
carboxy group and the deprotected amino substituent; (3) using
reduction to deprotect the allyl-protected carboxy substituent of
the other flanking residue, and to deprotect the
allyloxycarbonyl-protected amino group of the linker if an
aminocarboxylic acid with a allyloxycarbonyl-protected amino group
is used; (4) using acidolysis to deprotect the Boc-protected amino
group of the linker if an aminocarboxylic acid with an
Boc-protected amino group is used as the linker, and (5)
intramolecularly reacting the free carboxy substituent of the other
flanking residue and the free amino group of the aminocarboxylic
acid linker to cyclize the peptide.
[0568] Alternatively, the peptide can be cyclized by (1) using
acidolysis to deprotect the Boc-protected amino substituent of one
flanking residue (without deprotecting the allyl-protected carboxy
substituent of the other flanking residue); (2) reacting an
unprotected or amino-protected (e.g. allyloxycarbonyl-protected or
Boc-protected amino) aminocarboxylic acid linker with the solid
phase peptide to form an amide linkage between the linker's carboxy
group and the deprotected amino substituent; (3) using reduction to
deprotect the allyl-protected carboxy substituent of the other
flanking residue, and to deprotect the allyloxycarbonyl-protect- ed
amino group of the linker if an aminocarboxylic acid with an
allyloxycarbonyl-protected amino group is used as the linker; (4)
using acidolysis to deprotect the Boc-protected amino group of the
linker if an aminocarboxylic acid with a Boc-protected amino group
is used as the linker; and (5) intramolecularly reacting the free
carboxy substituent of the other flanking residue and the free
amino group of the aminocarboxylic acid linker to cyclize the
peptide.
[0569] In an example using an amino substituent for the side chain
amide bond-forming substituent of one flanking residue, a carboxy
substituent for the side chain amide bond-forming substituent of
the other flanking residue, an aminocarboxylic acid linker for
cyclization, and Boc chemistry for peptide synthesis, the side
chain amide bond-forming substituents of the flanking residues are
orthogonally protected with respect to each other and with respect
to the Boc-protected .alpha.-amino group of the N-terminal residue
in the peptide chain by using an allyloxycarbonyl group to protect
the amino substituent of one flanking residue and a Fm ester to
protect the carboxy substituent of the other flanking residue. In
this example, the peptide can be cyclized by (1) using reduction to
deprotect the allyloxycarbonyl-protected amino substituent of one
flanking residue (without deprotecting the Fm ester-protected
carboxy substituent of the other flanking residue); (2) reacting an
unprotected or amino-protected (e.g. allyloxycarbonyl-protect- ed
amino or Fmoc-protected amino) aminocarboxylic acid linker with the
solid phase peptide to form an amide linkage between the linker's
carboxy group and the deprotected amino substituent; (3) using base
saponification to deprotect the Fm ester-protected carboxy
substituent of the other flanking residue, and to deprotect the
Fmoc-protected amino group of the linker if an aminocarboxylic acid
with a Fmoc-protected amino group is used as the linker; (4) using
reduction to deprotect the allyloxycarbonyl-protected amino group
of the linker if an aminocarboxylic acid with an
allyloxycarbonyl-protected amino group is used as the linker; and
(5) intramolecularly reacting the free carboxy substituent of the
other flanking residue and the free amino group of the
aminocarboxylic acid linker to cyclize the peptide.
[0570] Alternatively, the peptide can be cyclized by (1) using base
saponification to deprotect the Fm ester-protected carboxy
substituent of one flanking residue (without deprotecting the
allyloxycarbonyl-protected amino substituent of the other flanking
residue); (2) reacting an unprotected or carboxy-protected (e.g.
allyl- or Fm ester-protected carboxy) aminocarboxylic acid linker
with the solid phase peptide to form an amide linkage between the
linker's amino group and the deprotected carboxy substituent; (3)
using reduction to deprotect the allyloxycarbonyl-protected amino
substituent of the other flanking residue, and to deprotect the
allyl-protected carboxy group of the linker if an aminocarboxylic
acid with an allyl-protected carboxy group is used as the linker;
(4) using base saponification to deprotect the Fm ester-protected
carboxy group of the linker if an aminocarboxylic acid with a Fm
ester-protected carboxy group is used as the linker; and (5)
intramolecularly reacting the free amino substituent of the other
flanking residue and the free carboxy group of the aminocarboxylic
acid linker to cyclize the peptide.
[0571] In another example using an amino substituent for the side
chain amide bond-forming substituent of one flanking residue, a
carboxy substituent for the side chain amide bond-forming
substituent of the other flanking residue, an aminocarboxylic acid
linker for cyclization, and Boc chemistry for peptide synthesis,
the side chain amide bond-forming substituents of the flanking
residues are orthogonally protected with respect to each other and
with respect to the Boc-protected .alpha.-amino group of the
N-terminal residue in the peptide chain by using a Fmoc group to
protect the amino substituent of one flanking residue and an allyl
group to protect the carboxy substituent of the other flanking
residue. In this example, the peptide can be cyclized by (1) using
base saponification to deprotect the Fmoc-protected amino
substituent of one flanking residue (without deprotecting the
allyl-protected carboxy substituent of the other flanking residue);
(2) reacting an unprotected or amino-protected (e.g.
allyloxycarbonyl-protected amino or Fmoc-protected amino)
aminocarboxylic acid linker with the solid phase peptide to form an
amide linkage between the linker's carboxy group and the
deprotected amino substituent; (3) using reduction to deprotect the
allyl-protected carboxy substituent of the other flanking residue,
and to deprotect the allyloxycarbonyl-protect- ed amino group of
the linker if an aminocarboxylic acid with a
allyloxycarbonyl-protected amino group is used; (4) using base
saponification to deprotect the Fmoc-protected amino group of the
linker if an aminocarboxylic acid with an Fmoc-protected amino
group is used as the linker; and (5) intramolecularly reacting the
free carboxy substituent of the other flanking residue and the free
amino group of the aminocarboxylic acid linker to cyclize the
peptide.
[0572] Alternatively, the peptide can be cyclized by (I) using
reduction to deprotect the allyl-protected carboxy substituent of
one flanking residue (without deprotecting the Fmoc-protected amino
substituent of the other flanking residue); (2) reacting an
unprotected or carboxy-protected (e.g. allyl-protected or Fm
ester-protected carboxy) aminocarboxylic acid linker with the solid
phase peptide to form an amide linkage between the linker's amino
group and the deprotected carboxy substituent; (3) using base
saponification to deprotect the Fmoc-protected amino substituent of
the other flanking residue, and to deprotect the Fm ester-protected
carboxy group of the linker if an aminocarboxylic acid with a Fm
ester-protected carboxy group is used as the linker; (4) using
reduction to deprotect the allyl-protected carboxy group of the
linker if an aminocarboxylic acid with an allyl-protected carboxy
group is used as the linker; and (5) intramolecularly reacting the
free amino substituent of the other flanking residue and the free
carboxy group of the aminocarboxylic acid linker to cyclize the
peptide.
[0573] In yet another embodiment using an amino substituent for the
side chain amide bond-forming substituent of one flanking residue,
a carboxy substituent for the side chain amide bond-forming
substituent of the other flanking residue, an aminocarboxylic acid
linker for cyclization, and Fmoc chemistry for peptide synthesis,
the regioselectivity of the cyclization procedure is provided by
orthogonally protecting the side chain amide bond-forming
substituents of the flanking residues with respect to the
Fmoc-protected .alpha.-amino group of the N-terminal residue in the
peptide chain but not with respect to each other, and orthogonally
protecting one of the aminocarboxylic acid linker's functional
groups with respect to the Fmoc-protected .alpha.-amino group of
the N-terminal residue in the peptide chain.
[0574] In an example of the foregoing embodiment using an
allyloxycarbonyl-protected amino substituent as the side chain
amide bond-forming substituent of one flanking residue, an
allyl-protected carboxy substituent as the side chain amide-bond
forming substituent of the other flanking residue, a monoprotected
aminocarboxylic acid linker, and Fmoc chemistry for peptide
synthesis, the peptide can be cyclized by (1) using reduction to
orthogonally deprotect the side chain amide bond-forming
substituents of the flanking residues (without deprotecting the
Fmoc-protected .alpha.-amino group of the N-terminal residue in the
peptide chain); (2) reacting a carboxy-protected (e.g. allyl- or
t-butyl ester protected carboxy) or amino-protected (e.g.
allyloxycarbonyl- or Boc-protected amino) aminocarboxylic acid
linker with the solid phase peptide to form an amide linkage
between the unprotected functional group of the linker and the
corresponding side chain amide bond-forming substituent on one of
the flanking residues; (3) using reduction or acidolysis, as
appropriate, to deprotect the protected functional group of the
linker; and (4) intramolecularly reacting the free side chain amide
bond-forming substituent of the other flanking residue and the free
functional group of the linker to cyclize the peptide.
[0575] In an example of the foregoing embodiment using a
Boc-protected amino substituent as the side chain amide
bond-forming substituent of one flanking residue, a t-butyl
ester-protected carboxy substituent as the side chain amide-bond
forming substituent of the other flanking residue, a monoprotected
aminocarboxylic acid linker, and Fmoc chemistry for peptide
synthesis, the peptide can be cyclized by (1) using acidolysis to
orthogonally deprotect the side chain amide bond-forming
substituents of the flanking residues (without deprotecting the
Fmoc-protected .alpha.-amino group of the N-terminal residue in the
peptide chain); (2) reacting a carboxy-protected (e.g. allyl or
t-butyl ester-protected carboxy) or amino-protected (e.g.
allyloxycarbonyl- or Boc-protected amino) aminocarboxylic acid
linker with the solid phase peptide to form an amide linkage
between the unprotected functional group of the linker and the
corresponding side chain amide bond-forming substituent on one of
the flanking residues; (3) using reduction or acidolysis, as
appropriate, to deprotect the protected functional group of the
linker; and (4) intramolecularly reacting the free side chain amide
bond-forming substituent of the other flanking residue and the free
functional group of the linker to cyclize the peptide.
[0576] In still another embodiment using an amino substituent for
the side chain amide bond-forming substituent of one flanking
residue, a carboxy substituent for the side chain amide
bond-forming substituent of the other flanking residue, an
aminocarboxylic acid linker for cyclization, and Boc chemistry for
peptide synthesis, the regioselectivity of the cyclization
procedure is provided by orthogonally protecting the side chain
amide bond-forming substituents of the flanking residues with
respect to the Boc-protected .alpha.-amino group of the N-terminal
residue in the peptide chain but not with respect to each other,
and orthogonally protecting one of the aminocarboxylic acid
linker's functional groups with respect to the Boc-protected
.alpha.-amino group of the N-terminal residue in the peptide
chain.
[0577] In an example of the foregoing embodiment using an
allyloxycarbonyl-protected amino substituent as the side chain
amide bond-forming substituent of one flanking residue, an
allyl-protected carboxy substituent as the side chain amide-bond
forming substituent of the other flanking residue, an
aminocarboxylic acid linker, and Boc chemistry for peptide
synthesis, the peptide can be cyclized by (I) using reduction to
orthogonally deprotect the side chain amide bond-forming
substituents of the flanking residues (without deprotecting the
Boc-protected .alpha.-amino group of the N-terminal residue in the
peptide chain); (2) reacting a carboxy-protected (e.g. allyl- or Fm
ester-protected carboxy) or amino-protected (e.g. allyloxycarbonyl-
or Fmoc-protected amino) aminocarboxylic acid linker with the solid
phase peptide to form an amide linkage between the unprotected
functional group of the linker and the corresponding side chain
amide bond-forming substituent on one of the flanking residues; (3)
using reduction or base saponification, as appropriate, to
deprotect the protected functional group of the linker; and (4)
intramolecularly reacting the free side chain amide bond-forming
substituent of the other flanking residue and the free functional
group of the linker to cyclize the peptide.
[0578] In an example of the foregoing embodiment using a
Fmoc-protected amino substituent as the side chain amide
bond-forming substituent of one flanking residue, a Fm
ester-protected carboxy substituent as the side chain amide-bond
forming substituent of the other flanking residue, an
aminocarboxylic acid linker, and Boc chemistry for peptide
synthesis, the peptide can be cyclized by (1) using base
saponification to orthogonally deprotect the side chain amide
bond-forming substituents of the flanking residues (without
deprotecting the Boc-protected .alpha.-amino group of the
N-terminal residue in the peptide chain); (2) reacting a
carboxy-protected (e.g. allyl- or Fm ester-protected carboxy) or
amino-protected (e.g. allyloxycarbonyl- or Fmoc-protected amino)
aminocarboxylic acid linker with the solid phase peptide to form an
amide linkage between the unprotected functional group of the
linker and the corresponding side chain amide bond-forming
substituent on one of the flanking residues; (3) using reduction or
base saponification, as appropriate, to deprotect the protected
functional group of the linker; and (4) intramolecularly reacting
the free side chain amide bond-forming substituent of the other
flanking residue and the free functional group of the linker to
cyclize the peptide.
[0579] Following cyclization, the helix-constrained peptide is
optionally cleaved away from the solid support, recovered and
purified. The peptide can be removed from the solid support by a
reagent capable of disrupting the peptide-solid phase link, and
optionally deprotecting blocked side chain functional groups on the
peptide. In one embodiment, the peptide is cleaved away from the
solid phase by acidolysis with liquid hydrofluoric acid (HF), which
also removes any remaining side chain protective groups.
Preferably, in order to avoid alkylation of residues in the peptide
(for example, alkylation of methionine, cysteine, and tyrosine
residues), the acidolysis reaction mixture contains thio-cresol and
cresol scavengers. Following HF cleavage, the resin is washed with
ether, and the free peptide is extracted from the resin with
sequential washes of acetic acid solutions. The combined washes are
lyophilized, and the residue is purified.
[0580] c. Liquid Phase Cyclization
[0581] Alternatively, the peptide can be cleaved away from the
solid support prior to the cyclization step. In one embodiment,
after the difunctional linker is coupled to the side chain amide
bond-forming substituent of the fist flanking residue in the
peptide, the peptide is cleaved away from the solid support. The
peptide is recovered, deblocked at the side chain amide
bond-forming substituent of the second flanking residue (if
necessary), and then cyclized at low concentration in a reaction
mixture to maximize intramolecular amide bond formation. Typically,
a maximum level of intramolecular amide bond formation can be
achieved under conditions in which the concentration of the peptide
provides an intramolecular concentration of free carboxy and amino
groups that exceeds the intermolecular concentration of free
carboxy and amino groups in the reaction mixture. In one
embodiment, a peptide concentration of 1 nM to 1 M, and preferably
1 .mu.M to 1 mM, and more preferably 1 .mu.M to 100 .mu.M, is used
to maximize cyclization. The cyclization of free peptide can be
conducted with any of the amino acid coupling reactions used to
helicize peptide bound to a solid support described above.
[0582] d. Synthetic Schemes
[0583] In one embodiment, any helix constrained compound of
formulas (1), (1a), (1b), (1c), (1d), (1e), (1f), and (1g) is made
by utilizing (in peptide synthesis as described in Section
(B)(II)(1)(a) above) the particular combination of flanking
residues and diamine linker shown in Table 1 above that provides
the values of n, m and p characterizing the compound of interest,
and cyclizing the resulting peptide according to the methods
described in Section (B)(II)(1)(b)(ii) or (iii) above. For example,
any compound of formulas (1), (1a), (1b), (1c), (1d), (1e), (1f),
and (1g) characterized by m=0, p=0, and n=7, 8, or 9 can be made by
utilizing (in peptide synthesis as described in Section
(B)(II)(1)(a) above) the flanking residues and any diamine linker
listed in Item No. 1 in Table 1 above, and cyclizing the resulting
peptide according to the methods described in Section
(B)(II)(1)(b)(ii) or (iii) above. In another example, any compound
of formulas (1), (1a), (1b), (1c), (1d), (1e), (1f), and (1g)
characterized by m=0, p=6, and n=2 or 3, or characterized by m=6,
p=0, and n=2 or 3, can be made by utilizing (in peptide synthesis
as described in Section (B)(II)(1)(a) above) the flanking residues
and any diamine linker listed in Item No. 7 in Table 1 above, and
cyclizing the resulting peptide according to the methods described
in Section (B)(II)(1)(b)(ii) or (iii) above. In yet another
example, any compound of formulas (1), (1a), (1b), (1c), (1d),
(1e), (1f), and (1g) characterized by m=3, p=3, and n=2 or 3, can
be made by utilizing (in peptide synthesis as described in Section
(B)(II)(1)(a) above) the flanking residues and any diamine linker
listed in Item No. 16 in Table 1 above, and cyclizing the resulting
peptide according to the methods described in Section
(B)(II)(1)(b)(ii) or (iii) above.
[0584] In another embodiment, any helix constrained compound of
formulas (2), (2a), (2b), (2c), (2d), (2e), (2f), (2g), (3), (3a),
(3b), (3c), (3d), (3e), (3f), and (3g) is made by utilizing (in
peptide synthesis as described in Section (B)(II)(1)(a) above) the
flanking residues and any diamine linker listed in Item No. 9 in
Table 1 above, and cyclizing the resulting peptide according to the
methods described in Section (B)(II)(1)(b)(ii) or (iii) above.
[0585] In another embodiment, any helix constrained compound of
formulas (4), (4a), (4b), (4c), (4d), (4e), (4f), and (4g) is made
by utilizing (in peptide synthesis as described in Section
(B)(II)(1)(a) above) the flanking residues and any diamine linker
listed in Item No. 13 in Table 1 above, and cyclizing the resulting
peptide according to the methods described in Section
(B)(II)(1)(b)(ii) or (iii) above.
[0586] In another embodiment, any helix constrained compound of
formulas (5), (5a), (5b), (5c), (5d), (5e), (5f), and (5g) is made
by utilizing (in peptide synthesis as described in Section
(B)(II)(1)(a) above) the flanking residues and any diamine linker
listed in Item No. 8 in Table 1 above, and cyclizing the resulting
peptide according to the methods described in Section
(B)(II)(1)(b)(ii) or (iii) above.
[0587] In another embodiment, any helix constrained compound of
formulas (6), (6a), (6b), (6c), (6d), (6e), (6f), (6g), (11),
(11a), (11b), (11c), (11d), (11e), (11f), and (11g) is made by
utilizing (in peptide synthesis as described in Section
(B)(II)(1)(a) above) the particular combination of flanking
residues and aminocarboxylic acid linker shown in Table 3 above
that provides the values of q, r and s characterizing the compound
of interest, or the values of t, u and v characterizing the
compound of interest, as appropriate, and cyclizing the resulting
peptide according to the methods described in Section
(B)(II)(1)(b)(ii) or (v) above. For example, any compound of
formulas (6), (6a), (6b), (6c), (6d), (6e), (6f), (6g), (11),
(11a), (11b), (11c), (11d), (11e), (11f), and (11g) characterized
by q=1, s=0, and r=6, 7, or 8, or characterized by t=0, v=1, and
u=6, 7, or 8, as appropriate, can be made by utilizing (in peptide
synthesis as described in Section (B)(II)(1)(a) above) the flanking
residues and any aminocarboxylic acid linker listed in Item No. 1
in Table 3 above, and cyclizing the resulting peptide according to
the methods described in Section (B)(II)(1)(b)(ii) or (v)
above.
[0588] In another example, any compound of formulas (6), (6a),
(6b), (6c), (6d), (6e), (6f), (6g), (11), (11a), (11b), (11c),
(11d), (11e), (11f), and (11g) characterized by q=1, s=6, and r=1
or 2, or characterized by t=6, v=1, and u--1 or 2, as appropriate,
can be made by utilizing (in peptide synthesis as described in
Section (B)(II)(1)(a)(above) the flanking residues and any
aminocarboxylic acid linker listed in Item No. 28 in Table 3 above,
and cyclizing the resulting peptide according to the methods
described in Section (B)(II)(1)(b)(ii) or (v) above.
[0589] In another example, any compound of formulas (6), (6a),
(6b), (6c), (6d), (6e), (6f), (6g), (11), (11a), (11b), (11c),
(11d), (11e), (11f), and (11g) characterized by q=7, s=0, and r=1
or 2, or characterized by t=0, v=7, and u=1 or 2, as appropriate,
can be made by utilizing (in peptide synthesis as described in
Section (B)(II)(1)(a) above) the flanking residues and any
aminocarboxylic acid linker listed in Item No. 7 in Table 3 above,
and cyclizing the resulting peptide according to the methods
described in Section (B)(II)(1)(b)(ii) or (v) above.
[0590] In another example, any compound of formulas (6), (6a),
(6b), (6c), (6d), (6e), (6f), (6g), (11), (11a), (11b), (11c),
(11d), (11e), (11f), and (11g) characterized by q=3, s=4, and r=1
or 2, or characterized by t=, v=3, and u=1 or 2, as appropriate,
can be made by utilizing (in peptide synthesis as described in
Section (B)(II)(1)(a)above) the flanking residues and any
aminocarboxylic acid linker listed in Item No. 25 in Table 3 above,
and cyclizing the resulting peptide according to the methods
described in Section (B)(II)(1)(b)(ii) or (v) above.
[0591] In another embodiment, any helix constrained compound of
formulas (7), (7a), (7b), (7c), (7d), (7e), (7f), (7g), (13),
(13a), (13b), (13c), (13d), (13e), (13f), and (13g) is made by
utilizing (in peptide synthesis as described in Section
(B)(II)(1)(a) above) the flanking residues and any aminocarboxylic
acid linker listed in Item No. 14 in Table 3 above, and cyclizing
the resulting peptide according to the methods described in Section
(B)(II)(1)(b)(ii) or (v) above.
[0592] In another embodiment, any helix constrained compound of
formulas (8), (8a), (8b), (8c), (8d), (8e), (8f), (8g), (12),
(12a), (12b), (12c), (12d), (12e), (12f), and (12g) is made by
utilizing (in peptide synthesis as described in Section
(B)(II)(1)(a) above) the flanking residues and any aminocarboxylic
acid linker listed in Item No. 9 in Table 3 above, and cyclizing
the resulting peptide according to the methods described in Section
(B)(II)(1)(b)(ii) or (v) above.
[0593] In another embodiment, any helix constrained compound of
formulas (9), (9a), (9b), (9c), (9d), (9e), (9f), (9g), (15),
(15a), (15b), (15c), (15d), (15e), (15f), and (15g) is made by
utilizing (in peptide synthesis as described in Section
(B)(II)(1)(a) above) the flanking residues and any aminocarboxylic
acid linker listed in Item No. 15 in Table 3 above, and cyclizing
the resulting peptide according to the methods described in Section
(B)(II)(1)(b)(ii) or (v) above.
[0594] In another embodiment, any helix constrained compound of
formulas (10), (10a), (10b), (10c), (10d), (10e), (10f), (10g),
(14), (14a), (14b), (14c), (14d), (14e), (14f), and (14g) is made
by utilizing (in peptide synthesis as described in Section
(B)(II)(1)(a) above) the flanking residues and any aminocarboxylic
acid linker listed in Item No. 8 in Table 3 above, and cyclizing
the resulting peptide according to the methods described in Section
(B)(II)(1)(b)(ii) or (v) above.
[0595] In one embodiment, any helix constrained compound of
formulas (16), (16a), (16b), (16c), (16d), (16e), (16f), and (16g)
is made by utilizing (in peptide synthesis as described in Section
(B)(II)(1)(a) above) the particular combination of flanking
residues and dicarboxylic acid linker shown in Table 2 above that
provides the values of w, x and y characterizing the compound of
interest, and cyclizing the resulting peptide according to the
methods described in Section (B)(I)(1)(b)(ii)or (iv) above. For
example, any compound of formulas (16), (16a), (16b), (16c), (16d),
(16e), (16f), and (16g) characterized by w=1, y=1, and x=5, 6, or 7
can be made by utilizing (in peptide synthesis as described in
Section (B)(II)(1)(a) above) the flanking residues and any
dicarboxylic acid linker listed in Item No. 1 in Table 2 above, and
cyclizing the resulting peptide according to the methods described
in Section (B)(II)(1)(b)(ii)or (iv) above. In another example, any
compound of formulas (16), (16a), (16b), (16c), (16d), (16e),
(16f), and (16g) characterized by w=1, y=7, and x=0 or 1, or
characterized by w=7, y=1, and x=0 or 1, can be made by utilizing
(in peptide synthesis as described in Section (B)(II)(1)(a)above)
the flanking residues and any dicarboxylic acid linker listed in
Item No. 7 in Table 2 above, and cyclizing the resulting peptide
according to the methods described in Section (B)(II)(1)(b)(ii) or
(iv) above. In yet another example, any compound of formulas (16),
(16a), (16b), (16c), (16d), (16e), (16f), and (16g) characterized
by w=4, y=4, and x=0 or 1, can be made by utilizing (in peptide
synthesis as described in Section (B)(II)(1)(a) above) the flanking
residues and any dicarboxylic acid linker listed in Item No. 16 in
Table 2 above, and cyclizing the resulting peptide according to the
methods described in Section (B)(II)(1)(b)(ii) or (iv) above.
[0596] In another embodiment, any helix constrained compound of
formulas (17), (17a), (17b), (17c), (17d), (17e), (17f), (17g),
(18), (18a), (18b), (18c), (18d), (18e), (18f), and (18g) is made
by utilizing (in peptide synthesis as described in Section
(B)(II)(1)(a) above) the flanking residues and any dicarboxylic
acid linker listed in Item No. 2 in Table 2 above, and cyclizing
the resulting peptide according to the methods described in Section
(B)(II)(1)(b)(ii) or (iv) above.
[0597] In another embodiment, any helix constrained compound of
formulas (19), (19a), (19b), (19c), (19d), (19e), (19f), and (19g)
is made by utilizing (in peptide synthesis as described in Section
(B)(II)(1)(a) above) the flanking residues and any dicarboxylic
acid linker listed in Item No. 1 in Table 2 above, and cyclizing
the resulting peptide according to the methods described in Section
(B)(II)(1)(b)(ii) or (iv) above.
[0598] In another embodiment, any helix constrained compound of
formulas (20), (20a), (20b), (20c), (20d), (20e), (20f), and (20g)
is made by utilizing (in peptide synthesis as described in Section
(B)(II)(1)(a) above) the flanking residues and any dicarboxylic
acid linker listed in Item No. 8 in Table 2 above, and cyclizing
the resulting peptide according to the methods described in Section
(B)(II)(1)(b)(ii) or (iv) above.
[0599] (2) Synthesis of Linear Peptide with Difunctional
Linker-Coupled Flanking Amino Acid
[0600] The peptide is designed such that the sequence to be
helicized comprises an amino acid sequence that is six residues in
length that extends between flanking residues as described in
Section (B)(II)(1)(a) above. The peptide can be constructed using a
modification of the solid phase synthesis methods described in
Section (B)(II)(1)(a)above wherein one of the flanking residues is
coupled to a difunctional linker before addition to the peptide
chain. This allows the linker to be incorporated into the peptide
as part of a standard amino acid.
[0601] The flanking residue can be coupled to the difunctional
linker by any convenient means. Typically, the side chain amide
bond-forming substituent of the flanking residue is used to form an
amide linkage with one of the functional groups on the linker. In
one embodiment designed for use in conjunction with Fmoc chemistry,
the linker-derivatized flanking residue is created by obtaining
from a commercial source an amino acid residue with an
Fmoc-protected .alpha.-amino substituent, a t-butyl ester-protected
.alpha.-carboxy substituent, and an unprotected side chain amino
substituent, and then reacting the a-substituent protected amino
acid with a difunctional linker having a free carboxy group to form
an amide linkage between the linker's free carboxy group and the
unprotected side chain amino substituent of the amino acid using
any of the condensation methods described in Section (B)(II)(1)(a)
above. The t-butyl ester-protected .alpha.-carboxy substituent of
the derivatized amino acid residue is then removed by acidolysis to
permit incorporation of the derivatized amino acid into the peptide
chain.
[0602] In another embodiment designed for use in conjunction with
Fmoc chemistry, the linker-derivatized flanking residue is created
by obtaining from a commercial source an amino acid residue with an
Fmoc-protected .alpha.-amino substituent, an allyl-protected
.alpha.-carboxy substituent, and an unprotected side chain amino
substituent, and then reacting the .alpha.-substituent protected
amino acid with a difunctional linker having a free carboxy group
to form an amide linkage between the linker's free carboxy group
and the unprotected side chain amino substituent of the amino acid
using any of the condensation methods described in Section
(B)(II)(1)(a) above. The allyl-protected .alpha.-carboxy
substituent of the derivatized amino acid residue is then removed
by reduction to permit incorporation of the derivatized amino acid
into the peptide chain.
[0603] In one embodiment designed for use in conjunction with Boc
chemistry, the linker-derivatized flanking residue is created by
obtaining from a commercial source an amino acid residue with an
Boc-protected .alpha.-amino substituent, a Fm ester-protected
.alpha.-carboxy substituent, and an unprotected side chain amino
substituent, and then reacting the .alpha.-substituent protected
amino acid with a difunctional linker having a free carboxy group
to form an amide linkage between the linker's free carboxy group
and the unprotected side chain amino substituent of the amino acid
using any of the condensation methods described in Section
(B)(II)(1)(a) above. The Fm ester-protected .alpha.-carboxy
substituent of the derivatized amino acid residue is then removed
by base saponification to permit incorporation of the derivatized
amino acid into the peptide chain.
[0604] In another embodiment designed for use in conjunction with
Boc chemistry, the linker-derivatized flanking residue is created
by obtaining from a commercial source an amino acid residue with an
Boc-protected .alpha.-amino substituent, an allyl-protected
.alpha.-carboxy substituent, and an unprotected side chain amino
substituent, and then reacting the a-substituent protected amino
acid with a difunctional linker having a free carboxy group to form
an amide linkage between the linker's free carboxy group and the
unprotected side chain amino substituent of the amino acid using
any of the condensation methods described in Section (B)(II)(1)(a)
above. The allyl-protected .alpha.-carboxy substituent of the
derivatized amino acid residue is then removed by reduction to
permit incorporation of the derivatized amino acid into the peptide
chain.
[0605] In one embodiment designed for use in conjunction with Fmoc
chemistry, the linker-derivatized flanking residue is created by
obtaining from a commercial source an amino acid residue with an
Fmoc-protected .alpha.-amino substituent, a t-butyl ester-protected
.alpha.-carboxy substituent, and an unprotected side chain carboxy
substituent, and then reacting the .alpha.-substituent protected
amino acid with a difunctional linker having a free amino group to
form an amide linkage between the linker's free amino group and the
unprotected side chain carboxy substituent of the amino acid using
any of the condensation methods described in Section (B)(II)(1)(a)
above. The t-butyl ester-protected .alpha.-carboxy substituent of
the derivatized amino acid residue is then removed by acidolysis to
permit incorporation of the derivatized amino acid into the peptide
chain.
[0606] In another embodiment designed for use in conjunction with
Fmoc chemistry, the linker-derivatized flanking residue is created
by obtaining from a commercial source an amino acid residue with an
Fmoc-protected .alpha.-amino substituent, an allyl-protected
.alpha.-carboxy substituent, and an unprotected side chain carboxy
substituent, and then reacting the .alpha.-substituent protected
amino acid with a difunctional linker having a free amino group to
form an amide linkage between the linker's free amino group and the
unprotected side chain carboxy substituent of the amino acid using
any of the condensation methods described in Section (B)(II)(1)(a)
above. The allyl-protected .alpha.-carboxy substituent of the
derivatized amino acid residue is then removed by reduction to
permit incorporation of the derivatized amino acid into the peptide
chain.
[0607] In one embodiment designed for use in conjunction with Boc
chemistry, the linker-derivatized flanking residue is created by
obtaining from a commercial source an amino acid residue with an
Boc-protected .alpha.-amino substituent, a Fm ester-protected
.alpha.-carboxy substituent, and an unprotected side chain carboxy
substituent, and then reacting the .alpha.-substituent protected
amino acid with a difunctional linker having a free amino group to
form an amide linkage between the linker's free amino group and the
unprotected side chain carboxy substituent of the amino acid using
any of the condensation methods described in Section (B)(II)(1)(a)
above. The Fm ester-protected .alpha.-carboxy substituent of the
derivatized amino acid residue is then removed by base
saponification to permit incorporation of the derivatized amino
acid into the peptide chain.
[0608] In another embodiment designed for use in conjunction with
Boc chemistry, the linker-derivatized flanking residue is created
by obtaining from a commercial source an amino acid residue with an
Boc-protected .alpha.-amino substituent, an allyl-protected
.alpha.-carboxy substituent, and an unprotected side chain carboxy
substituent, and then reacting the .alpha.-substituent protected
amino acid with a difunctional linker having a free amino group to
form an amide linkage between the linker's free amino group and the
unprotected side chain carboxy substituent of the amino acid using
any of the condensation methods described in Section (B)(II)(1)(a)
above. The allyl-protected .alpha.-carboxy substituent of the
derivatized amino acid residue is then removed by reduction to
permit incorporation of the derivatized amino acid into the peptide
chain.
[0609] It is desirable to protect one of the functional groups on
the difunctional linker either before the linker is coupled to the
flanking residue that is selected to carry the linker or after the
coupling but before the addition of the linker-coupled flanking
residue to the peptide chain. This improves yield by avoiding
unwanted reaction of the free functional group on the flanking
residue-coupled linker during peptide synthesis. The free
functional group on the linker can be blocked with any of the amino
or carboxy protective groups described in Section (B)(II)(1)(a)
above. In one embodiment, the free functional group on the linker
and the .alpha.-amino groups are orthogonally protected such that
the .alpha.-amino groups can be deprotected in peptide synthesis
without deprotecting the free functional group on the linker. It
will be appreciated that any of the foregoing procedures for
coupling difunctional linkers to flanking residues can be easily
modified to derivative a particular flanking residue with a
selected orthogonally monoprotected difunctional linker.
[0610] In one embodiment designed for use in conjunction with Fmoc
chemistry, an orthogonally monoprotected difunctional
linker-derivatized flanking residue is created by obtaining from a
commercial source an amino acid residue with an Fmoc-protected
.alpha.-amino substituent, a t-butyl ester-protected
.alpha.-carboxy substituent, and an unprotected side chain amino
substituent, and then reacting the a-substituent protected amino
acid with a difunctional linker carrying a free carboxy group and
either an allyl-protected carboxy group or an
allyloxycarbonyl-protected amino group to form an amide linkage
between the linker's free carboxy group and the unprotected side
chain amino substituent of the amino acid using any of the
condensation methods described in Section (B)(II)(1)(a) above. The
t-butyl ester-protected .alpha.-carboxy substituent of the
derivatized amino acid residue is then removed by acidolysis to
permit incorporation of the derivatized amino acid into the peptide
chain.
[0611] In another embodiment designed for use in conjunction with
Fmoc chemistry, an orthogonally monoprotected difunctional
linker-derivatized flanking residue is created by obtaining from a
commercial source an amino acid residue with an Fmoc-protected
.alpha.-amino substituent, an allyl-protected .alpha.-carboxy
substituent, and an unprotected side chain amino substituent, and
then reacting the a-substituent protected amino acid with a
difunctional linker carrying a free carboxy group and either a
Boc-protected amino group or a t-butyl ester-protected carboxy
group to form an amide linkage between the linker's free carboxy
group and the unprotected side chain amino substituent of the amino
acid using any of the condensation methods described in Section
(B)(II)(1)(a) above. The allyl-protected .alpha.-carboxy
substituent of the derivatized amino acid residue is then removed
by reduction to permit incorporation of the derivatized amino acid
into the peptide chain.
[0612] In one embodiment designed for use in conjunction with Boc
chemistry, an orthogonally -monoprotected difunctional
linker-derivatized flanking residue is created by obtaining from a
commercial source an amino acid residue with an Boc-protected
.alpha.-amino substituent, a Fm ester-protected .alpha.-carboxy
substituent, and an unprotected side chain amino substituent, and
then reacting the a-substituent protected amino acid with a
difunctional linker carrying a free carboxy group and either an
allyloxycarbonyl-protected amino group or an allyl-protected
carboxy group to form an amide linkage between the linker's free
carboxy group and the unprotected side chain amino substituent of
the amino acid using any of the condensation methods described in
Section (B)(II)(1)(a) above. The Fm ester-protected .alpha.-carboxy
substituent of the derivatized amino acid residue is then removed
by base saponification to permit incorporation of the derivatized
amino acid into the peptide chain.
[0613] In another embodiment designed for use in conjunction with
Boc chemistry, an orthogonally monoprotected difunctional
linker-derivatized flanking residue is created by obtaining from a
commercial source an amino acid residue with an Boc-protected
.alpha.-amino substituent, an allyl-protected .alpha.-carboxy
substituent, and an unprotected side chain amino substituent, and
then reacting the a-substituent protected amino acid with a
difunctional linker carrying a free carboxy group and either a
Fmoc-protected amino group or a Fm ester-protected carboxy group to
form an amide linkage between the linker's free carboxy group and
the unprotected side chain amino substituent of the amino acid
using any of the condensation methods described in Section
(B)(II)(1)(a)above. The allyl-protected .alpha.-carboxy substituent
of the derivatized amino acid residue is then removed by reduction
to permit incorporation of the derivatized amino acid into the
peptide chain.
[0614] In one embodiment designed for use in conjunction with Fmoc
chemistry, an orthogonally monoprotected difunctional
linker-derivatized flanking residue is created by obtaining from a
commercial source an amino acid residue with an Fmoc-protected
.alpha.-amino substituent, a t-butyl ester-protected
.alpha.-carboxy substituent, and an unprotected side chain carboxy
substituent, and then reacting the a-substituent protected amino
acid with a difunctional linker carrying a free amino group and
either an allyloxycarbonyl-protected amino group or an
allyl-protected carboxy group to form an amide linkage between the
linker's free amino group and the unprotected side chain carboxy
substituent of the amino acid using any of the condensation methods
described in Section (B)(II)(1)(a) above. The t-butyl
ester-protected .alpha.-carboxy substituent of the derivatized
amino acid residue is then removed by acidolysis to permit
incorporation of the derivatized amino acid into the peptide
chain.
[0615] In another embodiment designed for use in conjunction with
Fmoc chemistry, an orthogonally monoprotected difunctional
linker-derivatized flanking residue is created by obtaining from a
commercial source an amino acid residue with an Fmoc-protected
.alpha.-amino substituent, an allyl-protected .alpha.-carboxy
substituent, and an unprotected side chain carboxy substituent, and
then reacting the a-substituent protected amino acid with a
difunctional linker carrying a free amino group and either a
Boc-protected amino group or a t-butyl ester-protected carboxy
group to form an amide linkage between the linker's free amino
group and the unprotected side chain carboxy substituent of the
amino acid using any of the condensation methods described in
Section (B)(II)(1)(a)above. The allyl-protected .alpha.-carboxy
substituent of the derivatized amino acid residue is then removed
by reduction to permit incorporation of the derivatized amino acid
into the peptide chain.
[0616] In one embodiment designed for use in conjunction with Boc
chemistry, an orthogonally monoprotected difunctional
linker-derivatized flanking residue is created by obtaining from a
commercial source an amino acid residue with an Boc-protected
.alpha.-amino substituent, a Fm ester-protected .alpha.-carboxy
substituent, and an unprotected side chain carboxy substituent, and
then reacting the a-substituent protected amino acid with a
difunctional linker carrying a free amino group and either an
allyloxycarbonyl-protected amino group or an allyl-protected
carboxy group to form an amide linkage between the linker's free
amino group and the unprotected side chain carboxy substituent of
the amino acid using any of the condensation methods described in
Section (B)(II)(1)(a) above. The Fm ester-protected .alpha.-carboxy
substituent of the derivatized amino acid residue is then removed
by base saponification to permit incorporation of the derivatized
amino acid into the peptide chain.
[0617] In another embodiment designed for use in conjunction with
Boc chemistry, an orthogonally monoprotected difunctional
linker-derivatized flanking residue is created by obtaining from a
commercial source an amino acid residue with an Boc-protected
.alpha.-amino substituent, an allyl-protected .alpha.-carboxy
substituent, and an unprotected side chain carboxy substituent, and
then reacting the a-substituent protected amino acid with a
difunctional linker carrying a free amino group and either a
Fmoc-protected amino group or a Fm ester-protected carboxy group to
form an amide linkage between the linker's free amino group and the
unprotected side chain carboxy substituent of the amino acid using
any of the condensation methods described in Section (B)(II)(1)(a)
above. The allyl-protected .alpha.-carboxy substituent of the
derivatized amino acid residue is then removed by reduction to
permit incorporation of the derivatized amino acid into the peptide
chain.
[0618] In another aspect, the foregoing embodiments utilizing a
difunctional linker-derivatized flanking residue can be modified by
orthogonally protecting the side chain amide bond-forming
substituent of the underivatized (not pre-coupled to difunctional
linker) flanking residue with respect to the .alpha.-amino
protection chemistry used in peptide synthesis and with respect to
any or all of the amide bond-forming substituents found in the side
chains of other amino acid residues in the peptide. In this aspect,
the side chain amide bond-forming substituent of the underivatized,
flanking residue can be selectively deblocked, yielding a peptide
that can be cyclized by a condensation reaction that is
specifically targeted to be between the deprotected side chain
amide bond-forming substituent of the underivatized, flanking
residue and the free functional group of the difunctional linker.
Suitable methods for orthogonal protection of side chain amide
bond-forming substituents are described in Section (B)(II)(1)(a)
above.
[0619] Following completion of solid phase peptide synthesis, the
peptide can be cyclized by a coupling reaction between the free
functional group of the difunctional linker and the side chain
amide bond-forming substituent of the underivatized, flanking
residue as described in Section (B)(II)(1)(b) above. Any blocking
group(s) protecting the underivatized, flanking residue's side
chain amide bond-forming substituent and/or the free functional
group of the difunctional linker is (are) removed, and the
deprotected groups are coupled to form an amide linkage using any
of the condensation methods described in Section (B)(II)(1)(a)
above. Optionally, the resulting cyclized (constrained helix)
peptide is cleaved away from the solid support, recovered and
purified.
[0620] Alternatively, the peptide can be cleaved away from the
solid support prior to the cyclization step. In one embodiment,
after synthesis of the linear peptide chain is complete, the
peptide is cleaved away from the solid support. The peptide is
recovered, deblocked at the side chain amide bond-forming
substituent of the underivatized, flanking residue and/or the free
functional group of the difunctional linker, and then cyclized at
low concentration in a reaction mixture in order to maximize
intramolecular amide bond formation. Typically, a maximum level of
intramolecular amide bond formation can be achieved under
conditions in which the concentration of the peptide provides an
intramolecular concentration of free amide bond-forming
substituents or groups that exceeds the intermolecular
concentration of free amide bond-forming substituents or groups in
the reaction mixture. In one embodiment, a peptide concentration of
1 nM to 1 M, and preferably 1 .mu.M to 1 mM, and more preferably 1
.mu.M to 100 .mu.M, is used to maximize cyclization. The
cyclization of free peptide can be conducted with any of the
condensation reactions used to helicize solid phase peptide
described above.
[0621] III. Methods for Constructing Semisynthetic Locked Helix
Proteins
[0622] Also provided herein are semisynthetic proteins comprising
locked helix peptides attached onto or incorporated in between one
or more larger, recombinantly synthesized protein molecules. The
semisynthetic, locked helix peptides of the invention can be made
by any convenient method, including ligation of the locked helix
peptides synthesized as described in Section (B)(II) above to one
or more recombinantly synthesized protein sequences. For example,
protein ligases such as the "subtiligases" can be used to
concatenate the locked helix peptides made as described herein to
larger, recombinantly synthesized protein fragments.
[0623] In one embodiment, the methods of the invention are modified
in order to produce a locked helix peptide that functions as "first
ligation substrate" in the subtiligase catalyzed peptide ligation
methods described in International Patent Application No. PCT/US
91/05480 (WO 92/02615 published Feb. 20, 1992) or as "donor ester",
"donor peptide", and "P.sub.n . . .
P.sub.4-P.sub.3-P.sub.2P.sub.1-glc-F-amide ester", respectively, in
the subtiligase catalyzed peptide ligation methods described in
Abrahmsen et al., Biochem. 30:4151-4159(1991), Jackson et al.,
Science, 266: 243-247 (1994), and Chang et al., Proc. Natl. Acad.
Sci. USA, 91: 12544-12548 (1994). The locked helix peptide can be
synthesized such that the C-terminal amino acid residue of the
cyclized peptide is in an ester linkage with the 2-hydroxyl group
of a 2-hydroxycarboxylic acid, such as glycolic acid or lactic
acid, to form a leaving group favored by the particular subtiligase
of interest, i.e. such that the 2-hydroxycarboxylic acid ester,
shown as the X residue of the "first ligation substrate" in FIG. 2B
of WO 92/02615, resembles the first residue positioned on the
N-terminal side of the hydrolyzable amide bond in the normal
peptide substrate of subtilisin, shown as residue P.sub.1' of the
"hydrolysis substrate" in FIG. 2B.
[0624] In another embodiment, the leaving group comprises a
2-hydroxycarboxylic acid and another amino acid residue, shown as
the R.sub.2" residue of the first ligation substrate in FIG. 2B of
WO 92/02615, wherein the carboxy group of the 2-hydroxycarboxylic
acid residue is in an amide linkage with the .alpha.-amino group of
the additional amino acid residue. In such embodiments, the amino
acid residue in the leaving group can be selected to resemble the
second residue positioned on the N-terminal side of the
hydrolyzable amide bond in the normal peptide substrate of
subtilisin, shown as residue Pi of the "hydrolysis substrate" in
FIG. 2B of WO 92/02615. In a preferred embodiment, the leaving
group is a glycolate-phenylalanyl (glc-F) moiety such as the
glycolate-phenylalanyl-amide (glc-F-NH.sub.2) moiety described in
Example 2 of WO 92/02615.
[0625] In one aspect, the glc-F leaving group is placed in its
proper position at the C-terminus of the locked helix peptide by
obtaining a Boc- or Fmoc-.alpha.-amino protected phenylalanine,
linking the .alpha.-amino protected phenylalanine to solid phase
resin with an .alpha.-carboxy ester or amide linkage, deprotecting
the protected .alpha.-amino group, adding a glycolic acid residue
in the form of a t-butyl ether to form an amide linkage between the
carboxy group of the glycolic acid and the free .alpha.-amino group
of the solid phase phenylalanine, removing the t-butyl ether group
from the glycolic acid residue with acid and forming an ester
linkage between the free hydroxyl of the glycolic acid residue and
the .alpha.-carboxy of the next amino acid residue in the
C-terminal sequence desired for the locked helix peptide.
Subsequentamino acids can be added and the resulting peptide can be
helicized according to any of the above described methods which
utilize standard Boc or Fmoc chemistry for peptide synthesis. In
one embodiment, a glc-F-NH.sub.2 leaving group is incorporated into
the desired peptide chain essentially as described in Example 2 of
WO 92/02615 or as described in Jackson et al., Science, 266:
243-247 (1994).
[0626] In yet another embodiment, the "donor peptide" includes a
flexible linker sequence between the C-terminal residue of the
locked helix peptide sequence and the leaving group sequence, such
as a di- or tri-glycine linker, to promote flexibility and
accessibility of the donor peptide's leaving group to
subtiligase.
[0627] After the donor peptide (with the helix locking tether in
place) is obtained, a subtiligase can be used to ligate a peptide
or protein fragment (produced by recombinant or other synthetic
methods), designated the "second ligation substrate" in FIG. 2C of
WO 92/02615, the "acceptor peptide" in FIG. 1 on page 244 of
Jackson et al., Science, 266: 243-247 (1994), and the "Nucleophile"
peptide in the synthetic scheme on page 12545 of Chang et al.,
Proc. Natl. Acad. Sci. USA, 21: 12544-12548 (1994), to the
C-terminus of the donor peptide by displacement of the leaving
group according to any of the subtiligase-catalyzed peptide
ligation methods described above. In embodiments using acceptor
peptides or proteins having a relatively inaccessible N-terminus
due to higher order protein structure, ligation efficiency can be
improved by altering the design of the acceptor peptide to
incorporate a flexible linker sequence, such as a di- or
tri-glycine sequence, at the N-terminus to promote flexibility and
accessibility of the acceptor peptide N-terminus in the peptide
ligation reaction. Alternatively, the accessibility of the accept
or peptide N-terminus and/or donor peptide C-terminus to
subtiligase can be improved by conducting the ligation reaction
under denaturing conditions which eliminates unfavorable structural
conformations that may be assumed by the peptide substrates. In
such embodiments, it is preferable to use a denaturation-stable
subtiligase, such as the "stabiligase" described in Chang et al.,
supra (capable of retaining nearly 50% of catalytic activity in 4 M
guanidine hydrochloride).
[0628] It will be appreciated that additional peptides can be
synthesized with a suitable leaving group at the C-terminus and
successively ligated to the N-terminus of the semisynthetic peptide
containing the locked helix moiety by repeating the foregoing
procedures until a completed peptide with the desired N-terminus is
obtained.
[0629] In the event that the completed, semisynthetic, locked helix
protein is obtained in a denatured, incorrectly folded, or
otherwise in active form as a result of the synthetic procedures
used, the inactive species can be refolded into the native or
active conformation by renaturation techniques that are well known
in the art. Typical renaturation procedures use a chaotrope, such
as urea at high pH or guanidine hydrochloride, to unfold inactive
material followed by dilution of the denaturant to permit refolding
to occur, while preventing the formation of random disulfide bonds
prior to the assumption of the biologically active conformation
through non-covalent, intramolecular interactions (see, U.S. Pat.
Nos. 4,512,922; 4,518,256; 4,511,502; and 4,511,503). Reversed
micelles or ion exchange chromatography are used to assist
refolding of denatured proteins by enclosing a single protein
molecule within micelles or isolating proteins on a resin and then
removing the denaturant (Hagen et al., Biotechnol Bioeng.
35:966-975(1990); Creightonin Protein Structure Folding and Design,
Oxender, D. L., ed., Alan R. Liss, Inc. (New York: 1985), pp.
249-251. In addition, conformation-specific refolding can be
performed with ligands and antibodies to the native structure of
the protein (Cleland and Wang in Biotechnology, Rehm, H. -J., and
Reed, G., eds, VCH (New York), pp.528-555. Since they are more
likely to interact with the protein in its native conformation,
these binding molecules can be used to guide the folding reactions
towards native state protein. The foregoing recovery methods are
regarded as being universally applicable, with minor modifications,
to the recovery of biologically active recombinant proteins from
inclusion bodies, and are equally applicable to the recovery of
biologically active proteins from the semisynthetic methods of the
invention.
[0630] IV. Methods for Constructing Macromolecule-bound Locked
Helix Peptides
[0631] In one embodiment, the constrained, helical peptides of the
invention bound to a macromolecular solid support can be obtained
by constructing the locked helix peptides with the solid phase
synthesis techniques described in Section (II) above and recovering
the intact, solid support-peptide conjugate. Alternatively, the
cyclized peptide can be cleaved away from solid phase following
synthesis and then attached to the macromolecule of choice by any
convenient method known in the art. For example, a commonly
employed technique for attaching peptide ligands to polysaccharide
matrices, e.g. agarose, dextran or cellulose, involves activation
of the carrier with cyanogen halides and subsequent coupling of the
peptide's primary aliphatic or aromatic amines to the activated
matrix. The activation of polysaccharides with cyanogen bromide
(CNBr) at alkaline pH was introduced to affinity chromatography by
Axen et al, Nature, 214:1302 (1967). In one aspect of the
invention, the activation of polysaccharide matrices, particularly
agarose matrices, is performed according to the
titration-activation method. In this procedure, for example, 20 g
of exhaustively washed moist agarose cake is added to 20 ml of
water in a 100 ml beaker equipped with a 0-100.degree. C.
thermometer, a pH meter and a 25 mm magnetic stirring bar. The
suspension is stirred slowly, the temperature lowered to about
10-15.degree. C. by the addition of crushed ice and the pH adjusted
to 10.8.+-.0.1 by the addition of 1-2 drops of 4 N NaOH. The
activation procedure is initiated by the addition of the CNBr and
the pH of the reaction maintained at 10.8.+-.0.1 by manual
titration with the 4 N NaOH. The CNBr (100 g/mg moist weight gel)
can be added as a crystalline solid, a crushed solid, an aqueous
solution or by adding an aliquot of a stock solution. The latter
can be prepared by dissolving CNBr in acetonitrile (1 g/ml) and
storing in a tightly stoppered vial at -20.degree. C. The
temperature is subsequently allowed to rise to 18-20.degree. C.
[0632] Despite the relative simplicity of the titration method, it
may be preferable to use the faster and technically simplified
method of March et al., Anal. Biochem., 60: 149 (1974). The
activation procedure is performed in concentrated carbonate buffer.
The required amount of washed gel is suspended in an equal volume
of 2 M NaHCO.sub.3--NaCO.sub.3 buffer (pH 10.9) in a beaker
equipped with a thermometer and magnetic stirring bar. The slurry
is cooled to approximately 4-5.degree. C., the activated gel is
transferred to a sintered funnel and washed.
[0633] The concentration of CNBr recommended in the procedures
described above is satisfactory for moderate levels of peptide
substitution. When lower or higher levels of activation are
required, 50 mg and 200-300 mg CNBr/g moist weight gel respectively
can be employed together with 2 M and 8 M NaOH for the
titration.
[0634] It is generally recognized that the CNBr-activated
intermediate functional groups of polysaccharide gels display
limited stability and therefore it is preferable that the gel be
washed as rapidly as possible prior to transferring the gel to the
coupling-reaction medium. At the end of the activation step, the
gel is rapidly cooled by the addition of crushed ice and poured
into a large sintered glass funnel which has been pre-cooled with
crushed ice. The suspension is rapidly filtered into a Buchner
flask (2 liter) containing solid ferrous sulfate to remove
unreacted CNBr and cyanides as harmless ferrocyanide. The gel is
subsequently washed under suction with 1 liter ice-cold distilled
water and 1 liter of the buffer to be used in the coupling stage,
typically ice-cold 0.1 M NaHCO.sub.3--NaCO.sub.3 buffer (pH
8.5-9.5).
[0635] CNBr-activated Sepharose 4B is available commercially from
Pharmacia and obviates the hazardous manipulation of CNBr. The
activated gel is freeze dried in the presence of dextran and
lactose to preserve the beaded form and supplied in 15 g air-tight
packs. The required amount of freeze-dried powder is swollen in 1
mM HCl on a glass filter and washed with at least 200 ml of the
same solution per gram of powder. 1 g of freeze-dried material is
roughly equivalent to 3.5 ml final gel volume. The peptide
ligand-binding capacity of the gel is conserved more effectively by
washing with solutions of low pH than with solutions of pH greater
than 7. The gel is then ready to couple peptide ligand as soon as
the washing is completed.
[0636] Pharmacia also markets CNBr-activated Sepharose 6 MB for use
in cell biology and immunology for the separation of "functionally
homogeneous cell populations". It is produced by activation of
Sepharose 6MB macrobeads (diameter 200-300 .mu.m) with cyanogen
bromide and is handled in a manner analogous to CNBr-activated
Sepharose 4B.
[0637] The peptide to be coupled is suspended in a volume of the
cold buffer equal to the volume of the packed gel, added to the
moist, washed gel, and then the suspension is immediately mixed (in
a Buchner funnel) with a glass stirring rod. The entire procedure
of washing, adding the peptide solution, and mixing preferably
consumes less than 90 seconds. The suspension is transferred from
the Buchner funnel to a beaker containing a magnetic mixing bar and
is gently stirred at 4.degree. C. Although the reaction is
essentially complete in 2 to 3 hours, the mixture is allowed to
stand at 4.degree. C. for 16 to 20 hours to insure complete loss of
reactive polysaccharide groups. The peptide-linked gel is then
washed with large volumes of water until it is established that
peptide is no longer being removed.
[0638] The quantity of peptide coupled to the polysaccharide gel
can in part be controlled by the amount of peptide added to the
activated matrix. When highly substituted polysaccharide gel
derivatives are desired, the amount of peptide added should be 20
to 30 times greater than that which is desired in the final
product. For ordinary procedures, 100 to 150 mg of cyanogen bromide
are used per ml of packed polysaccharide gel, but much higher
coupling yields can be obtained if this amount is increased to 250
to 300 mg. The pH at which the coupling reaction is performed also
affects the degree of coupling, since it is only the unprotonated
form of a peptide's amino groups that reacts with CNBr-activated
polysaccharides. Preferably, the N-terminal .alpha.-amino group of
the peptide ligand is used for coupling with the activated
polysaccharide matrix. .alpha.-amino groups will couple optimally
at a pH of about 9.5 to 10.0. If coupling at the e-amino group(s)
of the selected peptide ligand (such as the E-amino groups of the
lysinyl residues) is desired, the coupling reaction should be
conducted at a pH value of about 10.0, and a large excess of
peptide should be added. If coupling at the aromatic amino groups
in the histidyl or tryptophanyl residues of the selected peptide is
desired, very high coupling efficiency can be obtained at pH values
between 8 and 9.
[0639] Further details of the invention can be found in the
following examples, which further define the scope of the
invention. All references cited throughout the specification, and
the references cited therein, are hereby expressly incorporated
herein by reference in their entirety.
EXAMPLES
Example 1
[0640] Experimental Section
[0641] Computational Methods
[0642] All calculations were performed with the DISCOVER program
(Biosym Technologies, San Diego) using the all-atom AMBER force
field (Weiner, S. J.; Kollman, P. A.; Case, D. A.; Singh, U. C.;
Ghio, C.; Alagona, G.; Profeta, S., Jr.; Weiner, P. J. Am. Chem.
Soc. 1984, 106, 765-784; Weiner, S. J.; Kollman, P. A.; Nguyen, D.
T.; Case, D. A. J. Comp. Chem. 1986, 7, 230-252) with a distance
dependent dielectric constant
[0643] Synthesis
[0644] Materials and Methods
[0645] Peptides were synthesized using standard solid phase
synthesis techniques (Merrifield, R. B. J. Am. Chem. Soc. 1963, 85,
2149-2154; Kaiser, E.; Colescot, R. L.; Bossinger, C. D.; Cook, P.
I. Anal. Biochem. 1970, 34, 595-598). Organic chemicals were
purchased from Aldrich (Milwaukee Wis.) or Fluka (Ronkonkoma,
N.Y.). Protected amino acids were purchased from Bachem Calif.
(Torrance Calif.) or Peninsula Labs (Belmont Calif.). BOP
(benzotriazole-1-yl-oxy-tris [dimethylamino] phosphonium
hexafluorophosphate) was purchased from Richelieu Biotechnologies
(Montreal). Solvents were purchased from Baxter (McGaw Park Ill.),
Baker (Phillipsburg N.J.), or Mallinckrodt (Paris Ky.). Polystyrene
supports were purchased from Advanced ChemTech (Louisville
Ky.).
[0646] Mono-t-butyloxycarbonyl (BOC) 1,3-propanediamine was
prepared as follows.
2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (18 g, 73
mmol) was added portion wise over 10 minutes to a solution of
1,3-diaminopropane (12.5 g, 184 mmol) in 100 mL of tetrahydrofuran
cooled to 0.degree. C. After four hours at 0.degree. C. the
reaction was allowed to warm to 25.degree. C. for two hours. The
reaction was diluted with 150 mL of ethyl acetate and washed twice
with 100 mL of saturated aqueous sodium chloride. The organic phase
was extracted with three 100 mL volumes of 10% aqueous citric acid,
the combined aqueous portions were then washed twice with 100 mL of
ethyl acetate. The aqueous phase was cooled in an ice bath and the
pH was adjusted to approximately 13 with 50% sodium hydroxide. The
basic aqueous phase was then extracted with three 100 mL volumes of
dichloromethane. The organic portion was then dried with potassium
carbonate and filtered. Solvent was removed by rotary evaporation
to yield mono-tert-butyloxycarbonyl-1,3-diaminopropane- .
[0647] Peptides were purified by reverse-phase HPLC on a Vydac C-18
column, eluted with acetonitrile-water gradients containing 0.1%
v/v trifluoroacetic acid (TFA). Peptides were characterized by
electrospray MS on a PE SCIEX API III+triple quadrupole mass
spectrometer and by quantitative amino acid analysis on a Beckmann
6300 automated amino acid analyzer. Organic intermediates were
analyzed by .sup.1H and .sup.13C nuclear magnetic resonance (NMR)
on a Varian VXR-300S and by high-resolution mass spectrometry (MS)
on a JEOL JMS-HX110HF/HX110HF tandem mass spectrometer.
[0648] AcTNE(OFm)DLAARRE(OAllyl)QQnh-MBHA-polystyrene (1a):
[0649] Linear peptide 1a with the sequence shown was synthesized on
p-MBHA resin (4.25 grams (g), 0.57 milliequivalents/gram (meq/g),
2.42 millimoles (mmol)) using standard coupling cycles with three
molar equivalents of BOC-amino acid, 3.3 molar equivalents of BOP
and 3.3 molar equivalents of N-methylmorpholine in dichloromethane
(CH.sub.2Cl.sub.2) and dimethyl acetamide (DMA) if needed for
solubility, for one hour at room temperature. The N-acetyl cap was
attached by treatment with 5 milliliters (mL) of acetic anhydride
in 3% triethylamine (TEA) in CH.sub.2Cl.sub.2 for 20 minutes at
room temperature. The resin was dried and weighed (4.41g, estimated
at 0.22 meq/g).
[0650] AcTNE(OFm)DLAARRE(OFm)QQnh-MBHA-polystyrene (1f):
[0651] AcAEE(OFm)AAAKFLE(OAllyl)AHAnh-MBHA-polystyrene (2a):
[0652] Linear peptides 1f and 2a as shown were synthesized as
described above for 1a.
[0653]
AcTNQ(.gamma.-NHCH.sub.3)DLAARRQ(.gamma.-NHCH.sub.3)QQnh.sub.2
(1b):
[0654] Linear resin-bound peptide 1f (0.60 g, 0.17 mmol) was doubly
deprotected with 20% piperidine/DMA for 20 minutes. The free
carboxylic acids were coupled to methylamine (CH.sub.3NH.sub.3Cl,
0.26 g, 3.85 mmol) with BOP (1.57 g, 3.55 mmol) and
N-methylmorpholine (0.90 mL, 8.2 mmol) in CH.sub.2Cl.sub.2/DMA for
1.5 hours. The resin was washed and dried, and the peptide-resin
bond was cleaved with anhydroushydrofluoric acid (HF) (10 mL) at
0.degree. C. for one hour with anisole (1 mL) and
ethylmethylsulfide (EtSMe) (0.5 mL) as scavengers. The resin was
washed twice with ether, once with ethyl acetate, and again with
ether. The free peptide was then extracted from the resin with
sequential washes of 10% acetic acid, glacial acetic acid,
acetonitrile, 10% acetic acid, and water. The combined solutions
were lyophilized and the residue was purified.
_CH.sub.2CH.sub.2CH.sub.2.sub.--
[0655] cyclo-AcTNQ(.gamma.-NH)DLAARRQ(.gamma.-NH)QQnh.sub.2
(1c):
[0656] 1. Using Unprotected Propanediamine:
[0657] Linear peptide 1a on the resin (0.51 g, 0.11 mmol)was
deprotected at the fluorenylmethyl ester with 20% piperidine/DMA
for 20 minutes and the resulting piperidine salt was neutralized by
washing twice with 1% TFA in CH.sub.2Cl.sub.2. The free carboxylic
acid was coupled to 1,3-propanediamine (0.12 mL, 1.44 mmol) with
BOP (0.40 g, 0.90 mmol) and diisopropylethylamine (DIPEA) (0.17 mL,
0.98 mmol) in CH.sub.2Cl.sub.2 for one hour, followed by addition
of DMA and continued coupling for an additional 45 minutes. The
glutamic acid allyl ester was deprotected with tetrakis
(triphenylphosphine)palladium (0)(Pd(PPh.sub.3).sub.4) (0.21 g,
0.18 mmol) in 20% piperidine/DMA for 1.5 hours and the piperidine
was removed by washing twice with 1% TFA in CH.sub.2Cl.sub.2. The
resulting amino acid was cyclized with BOP (0.32 g, 0.72 mmol) and
DIPEA (0.13 mL, 0.75 mmol) in CH.sub.2Cl.sub.2 for 3.5 hours. A
Kaiser test gave a noticeable purple color, so the cyclization was
repeated with BOP (0.44 g, 0.99 mmol) and DIPEA (0.19 mL, 1.09
mmol) in CH.sub.2Cl.sub.2 for three days. The peptide was cleaved
from the resin as described above for 1b.
[0658] 2. Using Mono-BOC Propanediamine:
[0659] Linear peptide 1a on the resin (0.57 g, 0.13 mmol) was
deprotected at the fluorenylmethyl ester with 20% piperidine/DMA
for 0.5 hour and the resulting piperidine salt was neutralized by
washing twice with 1% TFA in CH.sub.2Cl.sub.2. The free carboxylic
acid was coupled to mono-tert-butyloxycarbonyl-1,3-propanediamine
(0.23 g, 1.32 mmol) with BOP (0.52 g, 1.18 mmol) and DIPEA (0.25
mL, 1.44 mmol) in CH.sub.2Cl.sub.2/DMA for one hour. The glutamic
acid allyl ester was deprotected with Pd(PPh.sub.3).sub.4 (0.21 g,
0.18 mmol) in 20% piperidine/DMA for 1.5 hours and the piperidine
was removed by washing twice with 1% TFA in CH.sub.2Cl.sub.2; the
Kaiser test was negative at this point. The BOC group was removed
with TFA/CH.sub.2Cl.sub.2/anisole/1- ,2-ethanedithiol (45:45:5:5
vol/vol); the free amine then gave a positive Kaiser test. The
resulting amino acid was cyclized with BOP (0.58 g, 1.31 mmol) and
DIPEA (0.30 mL, 1.72 mmol) in CH.sub.2Cl.sub.2 for two hours,
whereupon the Kaiser test gave only a faint blue-green color. The
peptide was cleaved from the resin as described above for 1b.
[0660]
AcAEQ(.gamma.-NHCH.sub.3)AAAKFLQ(.gamma.-NHCH.sub.3)AHAnh.sub.2
(2b):
[0661] Linear peptide 2a on the resin (0.60 g, 0.19 mmol) was
deprotected at both the allyl and the fluorenylmethyl esters with
Pd(PPh.sub.3).sub.4 (0.1 g, 0.09 mmol) in 20% piperidine/DMA for 30
minutes and the resulting piperidine salt was neutralized by
washing with 50% TFA in CH.sub.2Cl.sub.2 containing anisole and
1,2-ethanedithiol. The free carboxylic acids were coupled to
methylamine (40% aqueous, 0.16 mL, 1.86 mmol) with BOP (0.32 g,
0.72 mmol) and DIPEA (0.35 mL, 2.01 mmol) in CH.sub.2Cl.sub.2/DMA
for 1 hour. The peptide was cleaved from the resin as described
above for 1b. 1d, 1e, 2c, 2d, 2e:
[0662] 1d and 1e were prepared from 1a and 2c-2e were prepared from
2a by the same procedures as described above for 1c using
unprotected 1,3-propanediamine, coupling with 1,4-butanediamine for
1d and 2d and with 1,5-pentanediamine for 1e and 2e.
[0663] AcTNk(S-Acm)DLAARRK(S-Acm)QQnh.sub.2 (3a):
[0664] AcAEk(S-Acm)AAAKFLK(S-Acm)AHAnh.sub.2 (4a):
[0665] Linear peptides 3a and 4a were synthesized by standard
Merrifield techniques using FMOC chemistry (Atherton, E.; Sheppard,
R. C. J. Chem. Soc., Chem. Commum 1985, 165-166).
Fmoc-D-Thiolys(Acm)-OH (7, k(S-Acm)) and Fmoc-L-Thiolys(Acm)-OH
(10, k(S-Acm)) were prepared as described below.
[0666] AcTNk(S)DLAARRK(S)QQnh.sub.2 (3b):
[0667] AcAEk(S)AAAKFLK(S)AlAnh.sub.2 (4b):
[0668] Cyclic peptides 3b and 4b were prepared from 3a and 4a
respectively by simultaneous deprotection and oxidation.
Approximately 16 mg of Acm-protected peptide was dissolved in 1.5
mL of water containing 10% acetic acid and then diluted to 50 mL
total volume with trifluoroethanol to give a final concentration of
approximately 200 micromoles/liter (.mu.M). A total of 12 mL of a 6
millimoles/liter (mM) solution of iodine (80 milligrams (mg)
dissolved in 3 mL of acetic acid and diluted to 50 mL with
trifluoroethanol) was added in 1 mL portions over the course of 10
hours while the reaction progress was monitored by HPLC. When the
starting material had been consumed, the reaction was diluted with
water and lyophilized and the crude oxidized material was
purified.
[0669]
(2S,5R)-2,5-dihydro-3,6-diethoxy-2-isopropyl-5-(4bromobutyl)pyrazin-
e (5):
[0670] n-butyllithium (13.3 mL of a 1.6 M solution in hexanes, 21.3
mmol) was added to a solution of
(2S)-2,5-dihydro-3,6-diethoxy-2-isopropyl pyrazine (Schollkopf
reagent)(4.30g, 20.3 mmol)in tetaahydrofuran (THF) over the course
of five minutes. The solution was maintained at -78.degree. C. for
15 minutes after which 1,4-dibromobutane (9.75 mL, 81.2 mmol) was
added in a single portion. After 2.5 hours at -78.degree. C. the
reaction was allowed to warm to room temperature and diluted with
diethyl ether (100 mL). The organic phase was washed with water
(100 mL), brine (100 mL) and then dried with magnesium sulfate
(MgSO.sub.4). Following filtration and concentration most of the
residual 1,4-dibromobutane was removed under high vacuum. The
remaining oil was purified by silica gel chromatography (2% ethyl
acetate in hexanes) to provide 5 (3.7 g, 57%) as a colorless
liquid; [.alpha.].sup.25.sub.D -1.13.degree. (c=4.5, CHCl); IR
(thin film) 2957, 1689, 1456, 1364, 1304, 1230, 1144, 1038
cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 4.04-4.18 (m,
4H) 3.94-4.02 (m, 1H)3.90 (t, J=3.9, 1H) 3.39 (t, J=6.9, 2H)
2.21-2.32 (m, 1H) 1.68-1.92 (m, 4H) 1.33-1.47 (m, 2H) 1.274 (t,
J=7.2, 3H) 1.268 (t, J=7.2, 3H) 1.03 (d, J=6.9, 3H) 0.70 (d, J=6.9,
3H); .sup.--C NMR (100.6 MHz, CDCl.sub.3) d 163.12, 163.05, 60.72,
60.48, 55.09, 33.60, 33.11, 32.66, 31.80, 23.22, 19.03, 16.6,
14.34, 14.31; Mass Spectrum (FAB+) 347.1 (MH+).
[0671]
(2S,5R)-2,5-dihydro-3,6-diethoxy-2-isopropyl-5(44-metboxybenzyl)-th-
iobutyl)pyrazine (6):
[0672] Potassium tert-butoxide (11.8 mL of a 1 M solution in THF)
was added over five minutes to a solution of
4-methoxy-.alpha.-toluenethiol (1.85 mL of 90%, 11.8 mmol) in THF
(20 mL) at 25.degree. C., generating a thick white precipitate
which was stirred for 25 minutes. A solution of 5 (3.7 g, 10.7
mmol) in THF (20 mL) was added and stirring continued for three
hours. The reaction was concentrated by rotary evaporation and then
partitioned between water (50 mL) and diethyl ether (100 mL). The
organic portion was washed with brine, dried (MgSO.sub.4), and
concentrated. The residue was purified by silica gel chromatography
(1% increasing to 2.5% ethyl acetate in hexanes) to provide 6 (2.90
g, 91%) as a colorless oil; [.alpha.].sup.25.sub.D -3.77.degree.
(c=3.5, CHCl.sub.3); IR (thin film) 2957, 1689, 1510, 1238, 1038
cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.21 (d,
J=8.7, 2H) 6.84 (d, J=8.4, 2H) 4.02-4.20 (m, 4H) 3.93-3.99 (m, 1H)
3.88 (t, J=3.3, 1H) 3.80 (s, 3H) 3.65 (s, 2H) 2.39 (t, J=7.5, 2H)
2.20-2.32 (m, 1H) 1.64-1.82 (m, 2H) 1.50-1.62 (m, 2H) 1.22-1.38 (m,
2H) 1.27 (t, J=7.2, 6H) 1.03 (d, J=6.9, 3H) 0.70 (d, J=6.9, 3H);
.sup.13C NMR (100.6 MHz CDCl.sub.3) .delta. 163.20, 163.00, 158.50,
130.57, 129.80, 113.80, 60.67, 60.45, 60.40, 55.24, 55.20, 35.57,
33.68, 31.74, 31.14, 29.19, 23.92, 19.05, 16.60, 14.36, 14.32; Mass
Spectrum (FAB+) 421.2 (MH+).
[0673] (2R)
2-(9-Fluorenylmethoxycarbonyl)amino-6acetamidomethylthiohexano- ic
acid (D-Thio(Acm)lysine) (7):
[0674] Water (30 mL) and 3N HCl (6.5 mL) was added to a solution of
6 (2.90 g, 9.32 mmol) in THF (50 mL). The mixture was stirred at
25.degree. C. for 12 hours and then the THF was removed by rotary
evaporation. The solution was adjusted to pH 10 with aqueous
potassium carbonate (K.sub.2CO.sub.3) and then extracted twice with
ethyl acetate (75 mL). The organic extracts were dried
(MgSO.sub.4), concentrated and the residue was purified by silica
gel chromatography (1:1 increasing to 2:1 ethyl acetateihexanes
with 0.5% triethylamine) to yield crude
S-methoxybenzylthiolysineethyl ester (2.65 g, 91%) as a colorless
oil which was carried on directly to the next step. This ester
(3.02 g, 9.71 mmol) was dissolved in a mixture of trifluoroacetic
acid (50 mL) and anisole (1 mL) at 0.degree. C., and
mercuricacetate (3.09 g, 9.71 mmol)was added to give a clear
solution. After 15 minutes the trifluoroacetic acid was removed by
rotary evaporation and the residue was first diluted with water (75
mL), then washed with diethyl ether (75 mL). The aqueous portion
was treated with hydrogen sulfide (H.sub.2S) (bubbled through the
solution) for 30 minutes and the resulting black precipitate was
removed by filtration through a bed of celite. The filtrate was
concentrated by rotary evaporation, redissolved in water (20 mL)
and filtered through a 0.45 micrometer (.mu.m) nylon filter. The
solution was again concentrated and the residual foam was dried
under high vacuum overnight. The residue was dissolved in
trifluoroacetic acid (15 mL) and acetamidomethanol (Fluka, 0.95 g,
10.7 mmol) was added. After 45 minutes the reaction was
concentrated by rotary evaporation and then dried under high vacuum
overnight. The residue was dissolved in THF (20 mL) and cooled to
0.degree. C. A solution of lithium hydroxide (1.22 g, 29.1 mmol) in
water (20 mL) was added in two portions at 15 minute intervals.
After two hours the reaction was allowed to warm to room
temperature and the pH was adjusted to 7.0 with 1 mole/liter (M)
aqueous citric acid. The solvent was removed by rotary evaporation,
the residue was dissolved in dioxane (80 mL), and Fmoc N-hydroxy
succinimide (3.3 g, 9.71 mmol) was added followed by saturated
aqueous sodium bicarbonate (NaHCO.sub.3) (15 mL). After one hour,
the solvent was removed by rotary evaporation and the residue was
partitioned between water (50 mL) and ethyl acetate (50 mL). The
aqueous portion was adjusted to pH 2.5 with 1 M aqueous citric acid
and then extracted three times with ethyl acetate (75 mL each). The
combined organic phases were dried (MgSO.sub.4) and concentrated.
The residue was purified by silica gel chromatography (first with
2:1 ethyl acetate/hexanes with 0.5% acetic acid, then ethyl acetate
with 0.5% acetic acid, then 5% methanol in ethyl acetate with 0.5%
acetic acid), product containing fractions were concentrated from
toluene (150 mL, three times) prior to dissolution in water with
acetonitrile and lyophilization to provide 7 (3.16 g, 71% over four
steps) as a white powder, [.alpha.].sup.25.sub.D -1.8.degree. (c=2,
EtOH); IR (thinfilm) 2800-3400, 1709, 1536, 1260, 759, 740
cm.sup.-1; .sup.1H NMR (300 MHZ, DMSO-d6) .delta. 8.42 (t, J=6.0,
1H) 7.88 (d, J=7.3, 2H) 7.72 (d, J=7.5, 2H) 7.58 (d, J=8.1, 1H)
7.407 (t, J=7.5, 2H) 7.32 (t, J=7.5, 2H) 4.16-4.30 (m, 5H) 3.91 (m,
1H) 2.53 (t, J=7.2, 2H) 1.82 (s, 3H) 1.33-1.76 (m, 6H); .sup.13C
NMR (100.6 MHZ, DMSO-d6) .delta. 174.00, 169.16, 156.08, 143.86,
143.80, 140.71, 127.61, 127.05, 125.28, 120.08, 65.57, 53.90,
46.68, 30.54, 30.01, 28.79, 24.91, 22.54; High Resolution Mass
Spectrum (FAB+) 457.1785, Err[ppm/mmu] -2.7/-1.2.
[0675]
(2R,5S)-2,5-dihydro-3,6-dimethoxy-2-isopropyl-5(4bromobutane)pyrazi-
ne (8):
[0676] [.alpha.].sup.25.sub.D +1.73.degree. (c=4.4, CHCl.sub.3); IR
(thin film) 2959, 1696, 1458, 1434, 1237, 1196, 1007 cm.sup.-1;
.sup.1-H NMR (300 MHZ, CDCl.sub.3) .delta. 3.98-4.07 (m, 1H) 3.95
(t, J=3.6, 1H) 3.70 (s, 3H) 3.68 (s, 3H)3.40 (t, J=6.9, 2H)
2.19-2.32(m, 1H) 1.66-1.94 (m, 4H)1.34-1.48(m, 2H) 1.05 (d, J=6.9,
3H)0.69 (d, J=6.9, 3H); .sup.13C NMR (100.6 MHZ, CDCl.sub.3)
.delta. 163.62, 163.60, 60.77, 55.11, 52.31, 33.54, 33.10, 32.61,
31.73, 23.26, 19.02, 16.56; Mass Spectrum (FAB+) 319.1 (MH+).
[0677]
(2R,5S)2,5-dizydro-3,6-dimethoxy-2-isopropyl-5-(4-(4-methoxybenzyl)-
-thiobutyl)pyrazine (9):
[0678] [.alpha.].sup.25.sub.D +3.96.degree. (c=3.63, CHCl.sub.3);
IR (thin film) 2944, 1696, 1510, 1238 cm.sup.-1; .sup.1H NMR (300
MHZ, CDCl.sub.3) .delta. 7.21 (d, J=8.7, 2H) 6.83 (d, J=8.4, 2H)
3.97-4.25 (m, 1H) 3.93 (t, J=3.3, 1H) 3.79 (s, 3H) 3.68 (s, 3H)
3.67 (s, 3H) 3.65 (s, 2H) 2.39 (t, J=7.5, 2H) 2.18-2.30 (m, 1H)
1.60-1.84(m, 2H) 1.49-1.62 (m, 2H) 1.23-1.38 (m, 2H) 1.27 (t,
J=7.2, 6H) 1.04 (d, J=6.9,33H) 0.68 (d, J=6.9, 3H); .sup.13C NMR
(100.6 MHZ, CDCl.sub.3) .delta. 163.72, 163.47, 158.48, 130.55,
129.78, 113.78, 60.68, 55.22, 55.18, 52.27, 35.58, 33.63, 31.64,
31.12, 29.13, 23.88, 19.02, 16.51; Mass Spectrum (FAB+) 393.2
(MH+).
[0679] (2S)
2-(9-Fluorenylmetboxycarbonyl)amino-6-acetamidomethylthiohexan-
oicacid: (L-Thio(Acm)lysine) (10):
[0680] [.alpha.].sup.25.sub.D +1.3.degree. (c=2, EtOH); IR (thin
film) 2800-3400, 1709, 1536, 1260, 759, 740 cm.sup.-1; .sup.1H NMR
(300 MHZ, DMSO-d6) .delta. 8.42 (t, J=6.0, 1H) 7.88 (d, J=7.3, 2H)
7.72 (d, J=7.5, 2H) 7.58 (d, J=8.1, 1H) 7.407 (t, J=7.5, 2H) 7.32
(t, J=7.5, 2H) 4.16-4.30 (m, 5H) 3.91 (m, 1H) 2.53 (t, J=7.2, 2H)
1.82 (s, 3H) 1.33-1.76 (m, 6H); .sup.13C NMR (100.6MHZ, DMSO-d6)
.delta.174.00, 169.16, 156.08, 143.86, 143.80, 140.71, 127.61,
127.05, 125.28, 120.08, 65.57, 53.90, 46.68, 30.54, 30.01, 28.79,
24.91, 22.54; High Resolution Mass Spectrum (FAB+) 457.1776,
Err[ppm/mmu] -4.6/-2.1.
[0681] These materials were prepared in the same manner as 5,6, and
7 starting from (2R)-2,5-dihydro-3,6-dimethoxy-2-isopropyl pyrazine
(Merck).
[0682] NMR Spectroscopy
[0683] For each peptide, 2-4 mg of purified material was dissolved
in 440 microliters (.mu.l) of 25 mM d.sub.3-sodium acetate
containing 5% deuterium oxide (D.sub.2O) and 0.1 millimoles/liter
(mM) sodium azide yielding a total peptide concentration of 1-6 mM;
the pH was adjusted to 4.5 by microliter additions of 1M sodium
hydroxide (NaOH). All spectra were acquired at 5.degree. C. or
10.degree. C. on a Bruker AMX-500 spectrometer. Two dimensionalCOSY
(Aue, W. P., Bartholdi, E. & Ernst, R. R. J. Chem. Phys. 1976,
64,2229-2246), ROESY (Bothner-By, A. A., Stephens, R L., Lee, J.
-m., Warren, C. D. & Jeanloz, R. W. J. Am. Chem. Soc. 1984,
106, 811-813; Rance, M. J. Magn Reson 1987, 74,557-564) and TOCSY
(Braunschweiler, L. & Ernst, R. R. J. Magn. Reson. 1983, 53,
521-528; Bax, A. & Davis, D. G. J. Magn. Reson. 1985, 65,
355-360) spectra were acquired with phase discrimination in
.omega..sub.1 achieved with TPPI (Marion, D. & Wuthrich, K.
Biochem. Biophys. Res. Commun. 1983, 113, 967-974). Total
acquisition times were approximately 2, 4, and 12 hours for COSY,
TOCSY and ROESY spectra, respectively. Water suppression was
achieved by coherent low power irradiation of the water resonance
for the 1.5 second (s) recycle delay. ROESY and TOCSY spectra were
acquired as described by Akke, M., Skelton, N. J., Kordel, J. &
Chazin, W. J. In Techniques in Protein Chemistry II; Villafranca,
J. J., Ed.; Academic Press, Inc.: Boca Raton, Fla., 1991; pp.
401-408; in addition, first-order phase corrections were avoided by
acquisition in a sine-modulated fashion in .omega..sub.1. TOCSY
mixing was achieved with a clean DIPSI-2rc sequence applied for 90
milliseconds (ms) (Cavanagh, J. & Rance, M. J. Magn. Reson
1992, 96, 670-678). The ROESY spectra were collected with a 4.0
kilohertz (kHz) spin-lock pulse of 200 ms duration. The spectra
were processed and analyzed using the Felix software package
(Biosym Technologies, San Diego, Calif.). .sup.3J.sub.HN-H.alpha.
were obtained by fitting an antiphase pair of Lorentzian lines to
.omega..sub.2 slices of high digital resolution COSY spectra.
[0684] Amide proton exchange rates with solvent were measured for
1b and 1c by lyophilizing the peptide from H.sub.2O and acquiring a
series of one dimensional (1D) NMR spectra immediately after
dissolving the peptide in D.sub.2O. Exchange rate constants were
determined by performing a three parameter exponential fit to the
decaying amide signals. Protection factors were calculated as the
ratio of exchange rate in the cross-linked and uncross-linked
peptide.
[0685] Structure Calculation
[0686] NOESY (Kumar, A., Ernst, R. R & Wuthrich, K. Biochem.
Biophys. Res. Commun. 1980, 95, 1-6; Bodenhausen, G., Kogler, H.
& Ernst, R. R. J. Magn. Reson. 1984, 58, 370-388) and ROESY
data were collected with mixing times of 300 ms and 200 ms,
respectively, from water (H.sub.2O) and D.sub.2O using a sample of
1c (approximately 8 mM). Total acquisition times were 24 hours per
experiment. Distance restraints were generated from these data by
categorizing cross-peaks as strong, medium, weak or very weak
according to the integrated peak volume, and assigning an upper
bound of 2.9, 3.5, 4.6, or 5.6 angstroms (.ANG.), respectively, to
the corresponding interproton distance. The dihedral angle .phi.
was restrained between -90.degree. and -40.degree. for residues in
which .sup.3J.sub.HN-H.alpha. less than 6.0 hertz (Hz). Values of
.sup.3J.sub.H.alpha.-H.beta. were determined from a COSY spectrum
acquired in D.sub.2O solution with a 35.degree. mixing pulse.
.chi..sub.1 restraints and H.sub..beta. stereospecific assignments
were obtained for four side-chains on the basis of these coupling
constants and the results of initial structure calculations
(Skelton, N. J., Garcia, K. C., Goeddel, D. V., Quan, C. &
Burnier, J. P. Biochemistry 1994, 33, 13581-13592).
[0687] Structures were calculated using the program DGII using the
CVFF force field parameters (Biosym Technologies, San Diego,
Calif.). Input restraints consisted of 141 interproton distances, 9
.phi. dihedral angle restraints and 5.chi..sub.1 dihedral angle
restraints. Explicit hydrogen bonds were not included. Structures
were generated with triangle and tetrangle smoothing prior to
perspective embedding of all atoms. The embedded structures were
annealed for 10,000 steps in four-dimensional space while cooling
from 200 degrees kelvin (K) with all atom masses set to 1000. The
DG structures were refined by rMD using the AMBER force field
within the DISCOVER program (Biosym Technologies). Structures were
annealed at 600 K for 3 picoseconds (ps), cooled to 0 K over 1.8 ps
and finally subjected to 200 cycles of rEM. Charges on Glu, Asp,
Arg, C-terminal and N-terminal residues were reduced to 0.2 e and a
distance dependent dielectric pf 1/4r was employed. Restraints were
employed as square well potentials with force constraints of 25
kilocalories/mole/angstrom.sup.2 (kcal.multidot.mol.sup.-1
.ANG..sup.-2) and 100 kilocalories/mole/radian.sup.2
(kcal.multidot.mol.sup.-1rad.sup.-- 2) for distances and dihedral
angles, respectively. In the final round of calculation, 60
structures were embedded in DGII and refined by rMD.
[0688] Circular Dichroism
[0689] CD spectra were acquired on an Aviv 62 spectrometer with a
0.1 centimeter (cm) path length temperature-controlled cell.
Solutions for analytical spectra were prepared by dilution of NMR
samples to approximately 100 micromoles/liter (AM) with additional
NMR buffer. Points were taken every 0.2 nanometers (nm) with 0.2 nm
bandwidth and 2 seconds(s) averaging time. The shortest wavelength
attainable was limited by absorption of the acetate buffer. Curves
shown are smoothed with standard parameters (10-point
smoothing).
RESULTS AND DISCUSSION
[0690] Design Considerations
[0691] Given synthetic and geometric considerations, it was
determined that amide chemistry should be used to link the I and
I+7 side-chains. Disulfide bonds, while synthetically feasible,
introduced an unwanted 90.degree. twist into the linkage. In order
to exploit the ability of simple alkyl chain linkers to avoid
steric crowding in the region near the I+3 and I+4 residues,
linkage methods for bridging either Gln or Asn at land I+7 with an
alkanediyl chain were considered. Gln was chosen because its
greater length allows use of the minimum size tether to link these
side-chains. A representative set of protein crystal structures
from the Brookhaven Database (Bernstein, F. C.; Koetzle, T. F.;
Williams, G. J. B.; Meyer, E. F.; Brice, M. D.; Rodgers, J. R.;
Kennard, O.; Shimanouchi, T.; Tasumi, M. J. Mol. Biol. 1977, 112,
535-542) was searched for all occurrences of glutamine in an a
helical context (with .phi.=-60.degree..+-.30.degree. and
45.degree..+-.30.degree.). The resulting data set was used to
determine the side-chain rotamer distributions of naturally
occurring helical glutamine residues. In general, amino acid
residues in an a helical context have
.chi..sub.1.apprxeq.-60.degree., a conformation suitable for the
I+7 position of a side chain linker. Glutamine has a relatively
high population (14.6%) of the .chi..sub.1=180.degree. rotamer,
representing a significant natural conformation that points the
side chain towards the C terminal end of the helix. Rotamer
combinations were identified that minimized the
N.epsilon.2-N.epsilon.2 distance between the I and I+7 side-chains
in a model helical peptide. Depending on .chi..sub.3 values,
distances ranging from 5.3 .ANG. to 7 .ANG. were found if the I
glutamine assumes .chi..sub.1 and .chi..sub.2 angles of 180.degree.
and 60.degree. and the I+7 glutamine assumes X and .chi..sub.2
values of -60.degree. and 180.degree., respectively.
[0692] Model building indicated that a 4-methylene "bridge" could
efficiently link these two glutamine side-chains without incurring
unfavorable torsional interactions. Models of 3-, 4-, and
5-methylene-bridged helical peptides were constructed using
distance geometry methods (Quantum Chemistry Program Exchange,
Program #590, entitled DGEOM by Blaney et al) followed by energy
minimization. All residues except the linked glutamines were
alanine. The conformational stabilities of helical peptides were
assessed using 1 nanosecond (ns) of unconstrained molecular
dynamics at 298 K following an initial 100 picoseconds (ps)
equilibration period during which harmonic restraints (25
kilocalories/mole/angstrom (kcal-mol.sup.-1 .ANG..sup.-1)) were
applied to maintain helicity. As a control, a polyalanine helix was
calculated for 1 ns in the presence of identical restraints.
[0693] Peptides containing a 3-methylene bridge maintained a
consistent helical conformation but showed significant "bending" of
the helix axis. Peptides containing a 4-methylene bridge maintained
helicity with little distortion, having comparable backbone
dihedral angles to the control peptide; .chi..sub.1 and .chi..sub.2
angles of the tethered glutamines did not change during the
simulation. Peptides based on a 5-methylene bridge transiently
escaped out of a helical conformation into nested turns centered
around the I+5 residue. Multiple side-chain rotamers were also
observed in the I+7 residue. Based on these observations, it was
determined that the 4-methylene bridge would provide the preferred
tether length.
[0694] Synthesis and Characterization
[0695] Amino acid sequences for trial peptides were based on the
C-terminal helix of apamin (Habermann, E. and Reiz, K. G., Biochem.
Z 1%5, 343, 192-203; Callewaert, G. L., Shipolini, R., and Vernon,
C.A., FEBS Lett. 1968, 1, 111-113; Shipolini, R., Bradbury, A. F.,
Callewaert, G. L., and Vernon, C. A., Chem. Commun. 1967, 679-680)
and on S peptide derived from the C-peptide from RNAse A (Brown, J.
E.; Klee, W. A. Biochemistry 1971, 10, 470-476). The sequences of
these peptides are shown in Table 1 below.
6TABLE 1 Structures of peptides 1-4 Peptide Sequence Side Chain
Protection 1 Ac T N X D L A A R R Z Q Q NH.sub.2 a: protected, on
resin, X=Glu(OAll), Z=Glu(OFm) b: X=Z=Gln(NMe) 2 Ac A E X A A A K F
L Z A H A NH.sub.2 c: X-Z=Gln(N(CH.sub.2).sub.3N)Gln d:
X-Z=Gln(N(CH.sub.2).sub.4N)Gln e: X-Z=Gln(N(CH.sub.2).sub.5N)Gln f:
protected, on resin, X=Z=Glu(OFm) 3 Ac T N X D L A A R R Z Q Q
NH.sub.2 a: X=D-Thiolys(Acm), Z=L-Thiolys(Acm) b:
X-Z=D-Thiolys-S-S-L-Thioly- s 4 Ac A E X A A A K F L Z A H A
NH.sub.2
[0696] Linear protected peptides 1a, 1f, and 2a were synthesized by
standard Merrifield methods using t-butyloxycarbonyl (BOC)
chemistry. Control peptides 1b and 2b were elaborated from if and
2a by simultaneous deprotection of both glutamate residues followed
by coupling with methylamine (FIG. 1). Synthesis of Id from if by
double deprotection and coupling with 1,4-butanediamine was
achieved in low yield/purity. Constrained peptides 1c-e and 2c-e
were elaborated from 1a and 2a by removal of the
fluorenylmethylester from Glu3, coupling with the appropriate
alkanediamine, removal of the allyl ester from Glu10, and
cyclization (FIG. 1). Yields were improved by the use of mono-BOC
protected alkanediamine in the first coupling step and by the use
of a polystyrene resin with 2% divinylbenzene (DVB) crosslinker.
The completed peptides were cleaved from the resin with
hydrofluoric acid (HF) and purified by preparative high performance
liquid chromatography (HPLC). Installation of the tether on the
solid phase allowed the completion of the synthesis with only a
single purification.
[0697] Thiolysine based peptides 3a and 4a were synthesized in the
linear acetamidomethyl-protected form using standard Merrifield
methods and FMOC chemistry, followed by cleavage from the resin
with trifluoroaceticacid/triethylsilane (9:1 v/v) and purification
by preparative HPLC. These were converted into the disulfide forms
3b and 4b in solution by simultaneous deprotection and oxidation
with acetic acid and molecular iodine in trifluoroethanol.
[0698] Peptides 14 were characterized by mass spectrometry and by
quantitative amino acid analysis. All peptides gave results
consistent with the intended structures.
[0699] Protected D- (7) and L-Thiolysine (10) were prepared as
shown in FIG. 2. The Schollkopf reagent (Schollkpf, U.; Groth, U.;
Deng, C. Angew. Chem. Int. Ed. Engl. 1981, 20, 798-799) was treated
with n-butyllithiumfollowedby 1,4-dibromobutaneto give the known
bromobutyl pyrazine 5. The bromide was displaced with the potassium
salt of methoxytoluenethiol to give 6. The pyrazine was hydrolyzed
with aqueous hydrochloric acid (HCl) and the thiol was deprotected
with mercuric acetate (Hg(OAc).sub.2) in TFA followed by H.sub.2S.
The crude thiolysine ethyl ester was then reprotected with
acetamidomethanolin TFA. The ester was hydrolyzed with lithium
hydroxide (LiOH) and the free S-protected amino acid was
N-protected with Fmoc N-hydroxysuccinimide in dioxane to give 7.
The same procedures were used for the synthesis of 10.
[0700] Proton NMR
[0701] Peptides 1-4 were studied by 2D .sup.1H NMR. Resonance
positions were obtained by standard sequential assignment methods
(Wuthrich, K. (1986) NMR of Proteins and Nucleic Acids., Wiley,
N.Y.), and are listed in Table 2 below.
7TABLE 2 Chemical Shifts.sup.a (backbone coupling constants.sup.b)
of the apamin-sequence peptides 1 and 3 Residue 1b 1c 1d 1e 3a 3b
Acetyl CH.sub.3 2.02 2.01 2.02 2.02 2.02 2.02 Thr1 H.sup.N 8.34
8.47 8.46 8.46 8.33 8.34 H.sup..alpha.(.sup.3J.sub.HN-H.alpha.)
4.22(7.2) 4.22(6.9) 4.21(6.9) 4.22(6.9) 4.22(7.5) 4.22(7.3)
H.sup..beta. 4.17 4.30 4.28 4.30 4.15 4.16 H.sup..gamma. 1.13 1.18
1.17 1.19 1.12 1.14 Asn2 H.sup.N 8.69 8.78 8.78 8.79 8.55 8.6
H.sup..alpha.(.sup.3J.sub.HN-H.alpha.) 4.54(6.6) 4.47(5.0)
4.49(5.4) 4.47(5.0) 4.60(6.9) 4.54(6.3) H.sup..beta. 2.75* 2.77*
2.77* 2.78* 2.73* 2.73* Gln3 (D-Thiolysine) H.sup.N 8.49 8.53 8.56
8.46 8.28 8.42 H.sup..alpha.(.sup.3J.sub.HN-H.alpha.) 4.12(6.5)
4.11(4.9) 3.93(4.7) 4.03(4.9) 4.11(6.5) 4.01(6.4) H.sup..beta.
2.01, 1.87 2.11, 1.75 2.11, 1.81 2.09, 1.83 1.72, 1.64 1.84*
H.sup..gamma. 2.22* 2.42, 2.21 2.33, 2.26 2.33, 2.24 1,32* 1.29*
H.sup..delta. n.a. n.a. n.a. n.a. 1.52* 1.63, 1.47 H.sup..epsilon.
8.01 7.99 2.52* 2.53, 2.59 Asp4 H.sup.N 8.32 8.05 8.09 8.03 8.29
8.07 H.sup..alpha.(.sup.3J.sub.HN-H.alpha.) 4.46(6.6) 4.31(4.8)
4.32(4.7) 4.32(4.8) 4.50(6.7) 4.37(5.5) H.sup..beta. 2.65* 2.77,
2.61 2.76, 2.60 2.76, 2.61 2.71, 2.60 2.67* Leu5 H.sup.N 8.20 7.92
7.93 8.03 8.24 8.15 H.sup..alpha.(.sup.3J.sub.HN-H.alpha.)
4.05(5.7) 3.95(5.3) 3.97(5.1) 3.94(4.8) 4.07(5.5) 4.06(5.5)
H.sup..beta. 1.60* 1.69, 1.50 1.67, 1.50 1.66, 1.50 1.61, 1.51
1.64, 1.51 H.sup..gamma. 1.50 1.61 1.60 1.59 1.58 1.61
H.sup..delta. 0.84, 0.76 0.83, 0.79 0.83, 0.79 0.83, 0.79 0.85,
0.77 0.85, 0.79 Ala6 H.sup.N 8.14 8.01 7.93 8.02 8.18 8.12
H.sup..alpha.(.sup.3J.sub.HN-H.alpha.) 4.07(5.1) 3.96(4.6) 3.97(nd)
3.96(5.0) 4.07(5.2) 4.11(5.8) H.sup..beta. 1.33 1.37 1.33 1.36 1.33
1.38 Ala7 H.sup.N 7.94 8.67 8.59 8.35 8.00 8.21
H.sup..alpha.(.sup.3J.sub.HN-H.alpha- .) 4.13(5.4) 3.77(4.4)
3.94(4.8) 3.87(4.5) 4.11(5.2) 4.09(5.5) H.sup..beta. 1.34 1.46 1.47
1.46 1.33 1.40 Arg8 H.sup.N 8.01 7.65 7.91 7.82 8.08 7.89
H.sup..alpha.(.sup.3J.sub.HN-H.alpha- .) 4.14(6.3) 4.01(4.9)
4.03(4.6) 4.02(4.9) 4.14(6.3) 4.17(6.1) H.sup..beta. 1.77* 1.86*
1.85* 1.86* 1.75* 1.83, 1.75 H.sup..gamma. 1.64, 1.56 1.76, 1.60
1.73, 1.59 1.77, 1.59 1.62, 1.55 1.54, 1.49 H.sup..delta. 3.11*
3.15, 3.08 3.14, 3.05 3.15, 3.06 3.11* 3.10* H.sup..epsilon. 7.21
7.23 7.23 Arg9 H.sup.N 8.17 7.78 7.89 7.94 8.19 7.97
H.sup..alpha.(.sup.3J.sub.HN-H.alpha.) 4.17(6.1) 4.10(5.5)
4.11(5.2) 4.08(5.1) 4.18(6.3) 4.21(6.6) H.sup..beta. 1.75* 1.87*
1.83* 1.83* 1.74* 1.85, 1.77 H.sup..gamma. 1.61, 1.54 1.71, 1.56
1.71, 1.57 1.72, 1.56 1.61, 1.54 1.64, 1.56 H.sup..delta. 3.11*
3.13* 3.12* 3.11* 3.11* 3.11* H.sup..epsilon. 7.24 7.23 7.23 Glu10
(L-Thiolysine) H.sup.N 8.30 7.92 7.71 7.73 8.18 8.09
H.sup..alpha.(.sup.3J.sub.HN-H.alpha.) 4.14(6.3) 4.02(5.4)
4.17(6.3) 4.07(5.6) 4.14(6.4) 4.16(6.3) H.sup..beta. 2.03, 1.95
2.11* 2.08* 2.12, 2.06 1.71* 1.75, 1.65 H.sup..gamma. 2.26* 2.47,
2.41 2.49, 2.36 2.40, 2.27 1.34* 1.32* H.sup..delta. n.a. n.a. n.a.
n.a. 1.64* 1.54, 1.49 H.sup..epsilon. 7.71 7.85 2.53* Gln11 H.sup.N
8.36 7.79 7.82 7.87 8.39 8.19
H.sup..alpha.(.sup.3J.sub.HN-H.alpha.) 4.18(6.9) 4.15(6.1)
4.17(6.3) 4.15(6.2) 4.19(6.6) 4.20(6.6) H.sup..beta. 2.04, 1.95
2.13, 2.04 2.09, 2.04 2.12, 2.05 2.04, 1.95 2.05, 1.96
H.sup..gamma. 2.32* 2.44, 2.38 2.38* 2.39* 2.32* 2.31* Gln12
H.sup.N 8.40 7.97 8.11 7.87 8.34 8.34
H.sup..alpha.(.sup.3J.sub.HN-H.alpha.) 4.16(7.0) 4.15(6.7)
4.17(6.7) 4.15(6.7) 4.18(7.0) 4.18(6.9) H.sup..beta. 2.04, 1.93
2.08, 1.99 2.07, 1.96 2.08, 1.98 2.05, 1.94 2.06, 1.93
H.sup..gamma. 2.32* 2.41, 2.37 2.36* 2.39, 2.35 2.31* 2.32* .sup.a
Chemical shifts obtained at pH 4.5 and 5.degree. C. Shifts are
relative to the internal H.sub.2O resonance at 4.96 parts per
million (p.p.m.), and are accurate to .+-. 0.02 p.p.m. .sup.b
3J.sub.HN-H.alpha. are listed in parentheses in units of hertz
(Hz).
[0702] Representative TOCSY and ROESY spectra of a
diamide-constrained peptide (1c) are shown in FIGS. 10 and 3. A
summary of the H.sup.N-H.sup.N and H.sup..alpha.-H.sup.N ROEs
between neighboring residues used to make these assignments are
depicted in FIG. 4 for 1b and 1c. The degree of helicity of each
peptide was judged from these spectra by the presence of intense
sequential H.sup.N-H.sup.N ROE cross-peaks, the presence of I, I+3
H.sup..alpha.-H.sup.N or H.sup..alpha.-H.sup..beta- . ROE
cross-peaks and .sup.3J.sub.HN-H.alpha. less than 6.0 Hz. The data
summarized in FIG. 4 indicate that peptide 1c is helical between
residues Asn2 and Gln10. Beyond Gln1, .sup.3J.sub.HN-H.alpha. rises
above 6.0 Hz but some medium range ROEs are still present,
indicating partial or transient helical character. Such fraying at
helix termini is commonly observed in NMR studies of peptides and
proteins. The .sup.1H chemical shifts of 1c change by less than
0.02 ppm over the concentration range 8.0-0.06 mM; this indicated
that the helical conformation was not stabilized by a
self-association event.
[0703] The incorporation of the diamide cross-link in peptides 1
and 2 clearly reduced the mean value of .sup.3J.sub.HN-H.alpha. in
the restrained region, increased the number of observable (I, I+3)
ROEs and increased the percent helicity observed by circular
dichroism (CD) as shown in Table 3 below. Thus, peptides 1c-1e and
2c-2e were significantly more helical than the control peptides 1b
and 2b. The results for peptide 4 indicated that formation of the
disulfide bond constrained the peptide to be helical. However, a
number of medium-range ROEs could not be observed and
.sup.3J.sub.HN-H.alpha. values were greater than 6.0 Hz for the two
thiolysine residues and Leu9 in 4b; this indicated a distortion
from an ideal helical structure in the region of the D-thiolysine
residue, as expected from simple structural considerations. The
data in Table 3 below indicated that incorporation of the disulfide
bond in peptide 3b did not impart helical character, suggesting
that the thiolysine method may have a dependence on primary
sequence and is therefore not generally applicable.
8TABLE 3 Evaluation of peptide helicity. Fraction of Percent Mean
.sup.3J.sub.NH--N.alpha. in medium-range Helicity by Peptide
Description constrained region ROEs obs. CD 1b Apamin, N-methyl Gln
Control 6.00 0.14 20 1c Apamin, 3 carbon linker 4.98 0.69 84 1d
Apamin, 4 carbon linker 5.18 0.56 63 1e Apamin, 5 carbon linker
4.96 0.69 100 2b C-tide, N-methyl Gln Control 5.89 0.08 32 2c
C-tide, 3 carbon linker 4.81 0.75 60 2d C-tide, 4 carbon linker
4.83 0.43 82 2e C-tide, 5 carbon linker 4.90 0.80 63 3a Apamin,
S-Acm thiolys control 6.01 0.03 10 3b Apamin, thiolys disulfide
5.96 0.08 35 4a C-tide S-Acm thiolys control 5.96 0.05 19 4b
C-tide, thiolys disulfide 5.65 0.48 27
[0704] Values below 6 for the mean 3-bond NH-Na coupling constant
indicate helicity. Medium range I-I+3 ROEs are expressed as the
observed fraction of the total number of such ROEs possible, with
very weak ROEs counted as one half. Percent helicity as determined
by CD is derived as by Lyu et al.,; Sherman, J. C.; Chen, A.;
Kallenbach, N. R., Proc. Natl. Acad. Sci. U.S.A. 88:5317-5320
(1991), and Johnson, W. C.; Tinoco Jr., I., J. Am. Chem. Soc., 94:
4389-4390 (1972).
[0705] Peptide 1c was chosen for a more detailed analysis by NMR.
ROESY spectra with higher sensitivity (increased total acquisition
time and peptide concentration) and NOESY spectra were acquired and
analyzed to provide input restraints to structure calculations. In
addition to the ROEs described above, H.sup..alpha.-H.sup.N (I,
I+4) interactions were observed, indicating that the helical
conformation adopted is not of the 3.sub.10 type, but rather is of
the regular .alpha. helical variety (Wuthrich, K. (1986) NMR of
Proteins and Nucleic Acids., Wiley, New York). Interproton distance
restraints were generated from the ROESY and NOESY data, and used
as a basis for calculating a structure for 1c using distance
geometry (DG) and restrained molecular dynamics (rMD). Nearly half
(66) of the 141 restraints were between amino acids two to four
residues apart in the primary sequence, as expected for a helical
conformation. Dihedral angle restraints, based on observed
.sup.3J.sub.HN-H.alpha. and .sup.3J.sub.H.alpha.-H.beta., were also
used in these calculations, but explicit hydrogen bond restraints
were not utilized.
[0706] The final ensemble of 20 structures is depicted in FIG. 5.
The structures agreed with the input data very well, with no
distance restraint violations above 0.1 angstroms (.ANG.), no
dihedral angle violations above 1.0.degree., and a mean restraint
violation energy term of 0.10.+-.0.09 kilocalories/mole
(kcal.multidot.mol.sup.-1). The available NMR data define well the
backbone atoms of residues Thr1 to Gln10 (average root mean squared
deviation from the mean structure=0.38.+-.0.08 .ANG.), but the two
C-terminal glutamine residues are not well defined. The side chains
of Thr1, Gln3, Asp4, Leu6 and Gln10 have well defined .chi..sub.1
values, but only Gln10 has a consistent value of .chi..sub.2 in all
structures.
[0707] H.sup.N(I)-I(I-4) hydrogen bonds are were to the amide
protons of Leu5, Ala6, and Gln10 in greater than 90% of the
structures, indicating that these residues adopted a predominantly
.alpha.-helical conformation. Although (i,i4) hydrogen bonds were
observed to the amide protons of Ala7, Arg8 and Arg9 in
approximately 50% of the structures, H.sup.N(I)-O(I-3) hydrogen
bonds were present in 25-35% of the structures, indicating that
there was a slight distortion of the helix in this region. The data
presented in Table 4 below indicated that the amide hydrogens of
Leu5 to Gln10 were all protected from exchange with solvent in
peptide 1c compared to the control peptide 1b by factors of up to
25. This observation is also consistent with the amide hydrogens of
these residues participating in hydrogen bonds. Interestingly,
hydrogen bonds from Asp4 H.sup.N to Thr10.sup..gamma.2 were present
in 80% of the structures, indicating that an N-cap hydrogen bonding
interaction (Harper, E. T.; Rose, G. D. Biochemistry 1993,
32:7605-7609) was present even in this short peptide. However, the
amide proton of Asp4 was not noticeably protected from exchange
(Table 4), hence this hydrogen bond may be more transient.
9TABLE 4 Amide hydrogen exchange rates constants.sup.a and
protection factors.sup.b for peptide 1b and 1c Residue log k (1b)
log k (1c) Protection Factor Thr1 -2.44 -2.48 approx. 1 Asn2 n.d.
n.d. -- Gln3 n.d. n.d. -- Asp4 -2.72 -2.84 1.3 Leu5 -2.69 -3.51 6.7
Ala6 -2.73 -3.77 10.9 Ala7 -2.51 -3.55 11.1 Arg8 n.d. -3.33 >26
Arg9 n.d. -3.21 >20 Gln10 n.d. -3.29 >25 Gln11 n.d. -1.83
approx. 1.0 Gln12 n.d. n.d. -- .sup.aThe rate constants are
expressed in units of seconds.sup.-1 (s.sup.-1). n.d. indicates
that the exchange was sufficiently fast than no peak was observed
in the NMR spectrum acquired 300 seconds (s) after addition of
D.sub.2O. In these cases, assuming the greater than 90% of the
hydrogens have exchanged in 300 s allows a lower limit of 0.013
s.sup.-1 (log k = -1.89) to be calculated for the rate constant.
.sup.bProtection factors are calculated as the rate constant for
peptide 1c divided by that of peptide 1b.
[0708] With the exception of the .psi. angles of Ala6 and Gln10,
the backbone dihedral angles throughout the tethered region were
close to those expected for an ideal a helix (mean
.phi.=-63.degree..+-.8.degree., mean
.psi.=-42.degree..+-.8.degree.) indicating that any deviation from
ideality was very slight. The .psi. of Ala6 is was 15.degree. lower
than expected for an .alpha. helix and was more similar to that
expected for a 31 0 helix; the higher value of .psi. for Gln10
reflected the fraying beyond the tethered region. The slight
distortion at Ala6 could be the result of the short tether present
in this peptide (only three methylene groups). Although the diamide
linkage was not well defined by the NMR data, the side-chains of
Gln3 and Gln10 adopted conformations close to those predicted by
the modeling experiments described above (Gln3
.chi..sub.1=-173.degree..+-.17.degree.,
.chi..sub.2=34.degree..+-.47.degr- ee.; Gln10
.chi..sub.1=-71.degree..+-.7.degree., .chi..sub.2=174.degree..+-
-.22.degree.). The overall conclusion was that in solution, 1c
adopted an .alpha. helical structure from Asp2 to Gln10 with an
N-terminal capping box and a very slight distortion in the central
turn of the helix.
[0709] Circular Dichroism
[0710] CD spectra were acquired on aqueous solutions of 1-4 between
20 and 120 micromoles/liter (.mu.M) at 280K, pH 5. Spectra of
peptides 1 and 3 (apamin sequence) are shown in FIG. 8 and those of
peptides 2 and 4 (C-peptide sequence) in FIG. 8. Numerical values
for percent helicity, calculated from the per-residue molar
ellipticity of the peptides at 222 nanometers (nm), are shown above
in Table 3.
[0711] The CD data supported the conclusions derived from the NMR
studies. Both tethering methods substantially enhanced the helicity
of the C-peptide sequence (FIG. 8). However, only the diamide
method was capable of rendering the apamin sequence helical under
the conditions used; the thiolysine-constrained peptide 3b did not
appear to be helical (FIG. 7). The CD spectrum of 4b, in spite of
substantial negative ellipticity at 222 nm, showed several features
which indicated a lesser degree of helicity than those of 2c-2e:
the short-wavelength minimum in 4b was shifted from 208 nm (a
typical value for an .alpha. helix) to 204 nm, and the observable
shoulder of the 190 nm maximum was much smaller than those of
2c-2e.
[0712] Thermal denaturation experiments were performed on the
apamin-based peptides 1b-1e. In the initial experiment, CD spectra
of peptides 1b-1e were taken at 10.degree. C. intervals from
7.degree. C. (280K) to 57.degree. C.(330 K). Given that 1c-1e
showed good retention of helicity in this temperature range,
spectra of 1c were taken up to 97.degree. C. (370 K), where some
loss of helicity was observed (FIG. 6). The molar ellipticity of 1c
at 97.degree. C. and 222 nm was still substantially more negative
than that of the non-helical control peptide 1b at 7.degree. C. and
222 nm.
[0713] Experiments to examine the effects of heating and recooling
the peptides were complicated by several factors: the CD
spectrometer showed a baseline drift over long experiments; the
concentration of the samples changed because of evaporation at
higher operating temperatures; and there appeared to be some
variation in sample behavior depending on the rates of heating and
cooling. A set of CD spectra of 1c was acquired before, during, and
after heating at 87.degree. C. for one day. The effect of baseline
drift was reduced by linear normalization of the spectra based on
.THETA..sub.245. The effect of concentration change due to sample
evaporation was corrected by normalizing the post-heating spectrum
to the same amplitude as the pre-heating spectrum at wavelength
(.lambda.) of 204 nm. This wavelength was chosen as the point where
an .alpha. helix and a random coil have equal contributions to the
ellipticity, and hence interconversion of a peptide between these
conformations will not affect the magnitude of the ellipticity. The
resulting spectra are shown in FIG. 9. The close match in curve
shape between the pre- and post-heating spectra indicated that most
or all of the helical structure was regained on cooling after the
partial denaturation induced by heating at 87.degree. C. The small
difference in overall amplitude could be due to a small amount of
permanent denaturation or could be an artifact of the normalization
procedure. This experiment demonstrated that the .alpha. helix of
1c was stable to relatively harsh conditions, a feature which
improves its general utility.
[0714] Conclusion
[0715] A new method for constraining small peptides to an .alpha.
helical conformation has been devised:. This I to I+7 amide-based
tether is successful as a general method for inducing a helicity in
small peptides and possesses several desirable features. First, it
allows the maximum possible sequence variability. Any residue
except the two tethering residues themselves may be changed.
Second, the helicity induced by this method approaches 100% in
aqueous solution at room temperature (RT). The comparison of
helical peptides 1c-1e with non-helical peptide 1b shows that the
helicity is achieved by introduction of the linker rather than
being a property of the primary sequence. Third, these tethered
peptides are synthesized by standard solid-phase (Merrifield)
chemistry and require only inexpensive, commercially available
reagents. Fourth, the method can be used for peptides as short as
eight residues. Fifth, it poses no chemical requirements as to
environment and has been shown to induce good helicity despite
changes in temperature and buffer conditions. This method is
generally useful for studies of biologically active helical regions
of proteins, for the experimental study of helix formation,
propagation, and stability, and for physical organic experiments on
the interactions of helical peptides with their environments.
Example 2
[0716] The peptide cyclized peptide FNM(5)QQRRFY(6)ALH (FIG. 11)
was synthesized using Fmoc chemistry with standard solid phase
protocols in which Fmoc-glutamic acid,
.delta.-(5-allyloxycarbonyl-1,5-diaminopentane) (5) (synthesized as
described below) and Fmoc glutamic acid .delta.-allyl ester (6)
(commercially available from Millipore) are incorporated as
standard amino acids in peptide synthesis, followed by cyclization
as shown in FIG. 11. Fmoc-glutamic acid,
.delta.-(5-allyloxycarbonyl-1,5-dia- minopentane) (5) was
synthesized as shown in Scheme 1 below. 169
[0717] Mono-t-butyloxycarbonyl (BOC) 1,5-pentanediamine was
synthesized by using 1,5-diaminopentane (12.5 g, 122 mmol) in place
of 1,3-diaminopropane in the synthesis of
mono-allyloxycarbonyl-1,3-diaminop- ropanedescribed in Example 1
above, yielding 10 g (49 mmol) of
mono-tert-butyloxycarbonyl-1,5-diaminopentane(l). The
mono-tert-butyloxycarbonyl-1,5-diaminopentane (1) (5.8 g, 28.7
mmol) was dissolved in 75 mL of dichloromethane with 7.5 mL of
diisopropylethylamine and cooled to 0 C. A solution of allyl
chloroformate (3.3 mL) in dichloromethane (25 mL) was added over
five minutes. The reaction was allowed to warm to room temperature
for one hour and then solvent was removed by rotary evaporation.
The residue was dissolved in 100 mL of ethyl acetate and washed
with three 100 mL portions of 10% citric acid, once with 100 mL
saturated aqueous sodium bicarbonate and once with 100 mL of
saturated aqueous sodium chloride. The organic phase was dried over
magnesium sulfate and solvent was removed by rotary evaporation.
The resulting oil (2) was treated with 25 mL of trifluoroacetic
acid for 30 minutes. The trifluoroacetic acid was removed by rotary
evaporation and the resulting reside was twice dissolved in
dichloromethane and then evaporated to remove residual solvent. The
residue was dissolved in 50 mL of 3N hydrochloric acid and washed
with two 50 mL portions of dichloromethane. The aqueous phase was
cooled in an ice bath and the pH was adjusted to approximately 13
with 50% aqueous sodium hydroxide. The basic aqueous phase was
extracted with three 100 mL portions of dichloromethane, the
combined organics were washed with 100 mL of saturated aqueous
sodium chloride and then dried over potassium carbonate. The
mixture was filtered, the solvent removed first by rotary
evaporation and then by high vacuum to yield 3.95 g of
mono-allyloxycarbonyl-1,5-diaminopentane (3) as a colorless
oil.
[0718] Fmoc-Glutamic acid, .alpha.-tert-butyl ester, 9.0 g (21.1
mmol, Bachem Calif.) was dissolved in 100 mL of dichloromethane.
Dicyclohexyl carbodiimide (4.4g, 21.3 mol) and
N-hydroxybenzotriazole (0.3 g, 2.1 mmol) was added to this
solution, followed by the mono-allyloxycarbonyl-1-
,5-diaminopentane (3) (3.95 g, 21.2 mmol). The reaction was stirred
at 25.degree. C. for 14 hours, then cooled to 0.degree. C. for one
hour. Insoluble material was removed by filtration, and the
filtrate was concentrated by rotary evaporation. The residue was
dissolved in 150 mL of ethyl acetate and washed twice with 100 mL
of 10% aqueous citric acid, twice with 100 mL of saturated aqueous
sodium bicarbonate and once with 100 mL brine. After drying over
magnesium sulfate and filtering the solvent was removed by rotary
evaporation. The residue was dissolved in approximately 75 mL of
ethyl acetate with heating and 2:1 hexanes:ethyl acetate was added
until the solution became cloudy. After standing for several hours
the crystalline precipitate was removed by filtration, the white
crystals were washed with 2:1 hexanes:ethyl acetate and dried under
vacuum to yield 11.4 g of (4) (90%).
[0719] The tert-butyl ester (4), 11 g, 18.5 mmol) was dissolved in
50 mL of trifluoroacetic acid with stirring. After 45 minutes, the
trifluoroacetic acid was removed by rotary evaporation; residual
trifluoroacetic acid was removed by evaporation from 50 mL of
dichloromethane three times. The residue was dissolved in 75 mL of
ethyl acetate with heating, filtered through celite, and 3:1
hexanes:ethyl acetate was added until a haze developed. Crystals
were allowed to grow at 25.degree. C. for three hours, then cooled
to 0.degree. C. for one hour. The crystals were isolated by
filtration and washed with 3:1 hexanes:ethyl acetate, then dried
under vacuum to yield 9.5 g (95%) of (5) as off white crystals.
[0720] Following peptide synthesis of FNM(5)QQRRFY(6)ALH, the
N-terminus of the solid phase peptide was coupled to mono
tert-butyl-succinic acid the allyl and allyloxycarbonyl protecting
groups were removed using 500 mg Pd(PPh.sub.3).sub.2Cl.sub.2 in 20
mL of 20% piperidine in dimethyl acetamide for 1.5 hours at room
temperature. The resin was then washed with 20% piperidine in
dimethyl acetamide, dimethyl acetamide, dichloromethane and finally
with 0.5% trifluoroacetic acid in dichloromethane. The resin was
suspended in dichloromethane and 1.5 equivalents of HATU with 3 eq
N,N-diisopropylethylamine in 5 mL of dimethyl acetamide was added.
After two hours the resin was checked for free amines by ninhydrin
test and found to be negative. The peptide was cleaved from the
resin with 95% trifluoroacetic acid 5% triethylsilane and purified
using reverse phase HPLC.
[0721] The helical structure of the cyclized peptide shown in FIG.
11 was confirmed by circular dichroism (CD) and nuclear magnetic
resonance (NMR). Both of these methods indicated that the locked
helix peptide displayed predominantly .alpha.-helical character.
The locked helix peptide was determined to bind IgG with an
affinity (Kd) of approximately 100 .mu.M both by microcalorimetry
and surface plasmon resonance. A control peptide lacking the
locking portion of the molecule did not exhibit IgG binding
detectable by microcalorimetry.
Example 3
[0722] To confirm that the covalent locking mechanism is fully
functional and that peptides constrained by this technique are able
to bind ligand with high affinity, a 33 amino acid peptide based on
helix I of the Z domain of protein A was synthesized with the i to
i+7 linkage as shown in Scheme 2 below: 170
[0723] where (5) and (6) are the allyloxycarbonyl and allyl
protected amino acids described in Example 2 above. The peptide was
synthesized and cyclized as described in Example 2 above. The
helicity of the peptide was verified by CD and NMR, and thermal
denaturation of the peptide as monitored by CD indicated that the
peptide only partly unfolds at 90.degree. C., consistent with the
stability of the covalent linkage. The IgG binding affinity (Kd) of
this peptide (as measured by surface plasmon resonance) was
determined to be approximately 20 nM.
Example 4
[0724] Linear peptides derived from the ectodomain of the HIV-1
envelope protein gp41 are known to inhibit viral fusion events. The
most potent of these (DP178) corresponds to a membrane proximal
region of gp41, which is predicted to be .alpha.-helical. However,
DP178 itself lacks discernable structure in solution, rendering
mechanistic interpretation of its activity difficult. By applying
the helix locking chemistry taught herin, constrained versions of
DP178 were made to determine whether helicity is necessary or
sufficient for its infectivity inhibition activity and to define a
likely mode of action for this molecule in primary infection (as
measured using viral infectivity assays).
[0725] By constraining DP178 analogs into a helical conformation we
show that helicity is necessary, but not sufficient, for inhibitory
potency. The correct face of the helix must also be exposed. Two
recent crystal structures of gp41 indicate that this face is buried
in a groove formed by a coiled-coil trimer. Taken together, these
results indicate that DP178 inhibits infectivity by blocking this
groove, and that the conformation of gp41 observed by
crystallography represents the fusogenic state.
[0726] A series of analogs of DP178 in which segments of the amide
backbone were constrained to be helical FIG. 12) were prepared.
Because short .alpha.-helices are usually unstructured in solution
(Marqusee et al. Proc. Natl. Acad. Sci. USA 86:5286-5290 (1989)), a
covalent crosslink between amino acid side chains at positions i
and i+7 of the polypeptide chain as taught herein (see also Phelan
et al., J. Am. Chem. Soc. 119:455-460 (1997), which is
incorportated herein by reference) which lock the intervening
residues into a stable .alpha.-helical conformation.
[0727] A truncated form of DP178, designated HIV35 (FIG. 12) was
used as a reference. In the absence of detailed information
regarding the associaton of DP178 with DP107, the coiled-coil
propensities (Lupas et al., Science 252:1162-1164 (1991)) for 29
distinct gp160 sequences were computed in order to determine
whether the region corresponding to DP178 scored as a coiled-coil.
The N-terminal 27 residues of DP178, selected for the reference
peptide HIV35, maintained a high overall score with a consistent
heptad register. The "a-d" face predicted by the scoring algorithm
corresponded to the face seen to pack against the trimer core. This
corresponding region is entirely helical in the x-ray structures,
and packs against the trimer core using a 4-3 heptad repeat akin to
that found in coiled-coils. Using the helical locking chemistry and
methods taught herin we enforced the exposure of this repeat
(positions "a" and "d" of the heptad) by introducing crosslinks
between pairs of adjacent residues on the opposite face of the
helix (position "f"). Thus, "f" to "f," (tethers) locks were made
to constrain the potential helix. Analogs of HIV35 (FIG. 12) were
prepared containing either one (HIV24) or two (HIV31) tethers to
impart increasing helicity. A control peptide (HIV30) was prepared
in which a tether was introduced between successive "d" residues to
stabilize helicity while blocking potential binding interactions
across the "a-d" face.
[0728] Linear peptides were synthesized according to standard solid
phase techniques using Fmoc chemistry (Fields et al., Int. J.
Peptide Protein Res. 35:161-214 (1990)) as taught herein. In
particular, helix dipole effects were minimized by blocking the
C-termini as amides and the N-termini as succinate groups. After
formation of the lactam bridges as taught herein (see also Phelan
et al., J. Am. Chem. Soc. 119:455-460 (1997)), the peptides were
cleaved from the resin and purified to homogeneity using
preparative reversed phase HPLC with water/acetonitrile/0.1% TFA
gradients in the mobile phase. The identity of each peptide was
confirmed by electrospray mass spectrometry: HIV 24, calculated
mass 3396.8, observed, 3396.0; HIV 30, calculated mass 3413.7,
observed, 3413.8; HIV 31, calculated mass 3520.0, observed, 3520.7;
HIV 35, calculated mass 3330.8, observed, 3330.5.
[0729] Circular dichroism analysis (FIG. 13) confirms that the
locking strategy markedly increases the helicity of the DP178
truncations. CD spectra were recorded on an AVIV 62DS CD
spectrometer using 0.05 cm pathlength cuvettes. Spectra were
gathered by averaging data from three runs spanning 250 run to 190
nm in 1.0 nm increments, with 2 second averaging time at each
wavelength. Peptide concentrations were approximately 200 .mu.M in
a solution of 10 mM Tris HCl pH 7.5 with 6% acetonitrile (v/v). For
conversion of raw data to molar ellipticity values, precise
concentrations were determined by measuring A.sub.276 and A.sub.280
(Edelhoch, Biochemistry 6:1948-1954 (1967)); these values were
confirmed by quantitative amino acid analysis.
[0730] The unconstrained peptide HIV35 has an almost featureless
spectrum, similar to that reported for DP178 (Lawless, et al.,
Biochemistry 35:13697-13708 (1996)). The CD spectra of peptides
containing a single constraint (HIV24 and HIV30) display minima at
209 and 222 run characteristic of .alpha.-helices. The intensity
ratios of these two regions are skewed from ideality, suggesting
that regions of the peptide backbone outside the constrained
segment are disordered. By constrast, the doubly-constrained analog
HIV31 appears to be largely helical by CD, giving the shape and
intensity profile of a typical .alpha.-helix.
[0731] Viral infectivity assays were used to characterize the
locked-helix constructs. Normal human peripheral blood mononuclear
cells (PBMCs) were stimulated with phytohemagglutinin (PHA) in RPMI
1640 medium containing interleukin 2 for 24 hours. The PHA medium
was removed and the cells grown overnight in RPMI 1640 with
glutamine, 20% heat inactivated fetal calf serum, and gentamicin.
At the start of the assay, pre-titered virus stocks were
equilibrated with peptides for one hour before adding to the PBMCs
(2.5.times.10.sup.5 cells per well). Cells were grown for three
days, rinsed to remove extracellular virus and peptides, then
supplemented with fresh medium and grown for an additional four
days. After seven days the cells were lysed and p24 antigen was
determined by ELISA. Peptides were run in triplicate at each
concentration. Viral titers were determined in duplicate for each
run. Each assay also included the following controls, in
triplicate: Uninfected cells as a negative control, infected cells
without peptide as a positive control, and virus innoculum without
cells to establish a baseline p24 level. Peptides were tested for
cytotoxicity by incubating them at the highest assay concentration
(approximately 100 .mu.M) with uninfected cells and then growing
the cells as described above. After 7 days the cell counts were
estimated by microscopy and compared to an identical batch of cells
which were not treated with the peptides; none of the peptides
inhibited normal cell growth under these conditions.
[0732] When tested in viral infectivity assays, the peptides
displayed a striking pattern of relative potency that extended
across both syncitium inducing (SI) and non-syncitium inducing
(NSI) strains of HIV-1 (Zhang et al., Nature 383:768 (1996)). As
shown in FIGS. 14A and 14B, truncating the hydrophobic C-terminus
of DP178 (HIV35) caused a dramatic drop in activity, which was
partially restored when a single restraint, i.e. constrained
helical peptide, (and partial .alpha.-helical character) was
introduced (HIV24). Adding a second restraint (HIV31) imparted
strong helical character and enhanced the potency of the peptide to
levels comparable to DP178. Thus, the additional stabilization
afforded by preorganizing HIV31 into an active helical conformation
offset the loss of binding energy caused by deleting the
C-terminus. By contrast, a single restraint that induced helicity
while blocking the "a-d" face (HIV30) completely ablated
activity.
[0733] A series of shorter constrained peptides spanning positions
631-644,643-656, 649-662, 656-669, and 663-678 of HIV-1.sub.LAI,
tethered between adjacent residues at the "f" positions of the
heptad, were prepared to determine whether a subset of HIV35 or its
N- and C-terminal flanking regions was sufficient to block
infectivity. All peptides, whether constrained or unconstrained,
failed to show significant activity. Peptide 631-644 contains the
hydrophobic cluster observed in the x-ray structure to pack into a
cavity in the trimer core (Chan et al., Cell 89:263-273
(1997)).
[0734] The relative activities of HIV35, HIV24, and HIV31
demonstrate a clear correlation between helicity and inhibitory
potency. The widely disparate activities of HIV30 and HIV24
indicate that peptide inhibition also requires exposure of the face
of the helix seen by crystallography to pack against the N-terminal
trimer core of gp41.
[0735] The data presented herin, combined with prior model studies
on isolated peptides and the recently published crystal structures,
strongly support the hypothesis that the peptides inhibit viral
infectivity by binding to the resting state of gp41 and preventing
formation of the fusogenic state. Peptide HIV31 is conformationally
constrained to be largely helical, and is likely to interact as
such with an accessible cognate surface in the resting state of
gp41. Because x-ray analysis shows that the face of HIV31 required
for inhibiting viral fusion is buried in the groove formed by the
N-terminal trimer core, we believe (without being bound to any
particular theory) that this groove represents the cognate surface
for the peptides.
[0736] FIG. 15 outlines schematically a current model for assembly
of the fusgenic state of gp41, and the mechanism by which the
constrained helices inhibit this process. The model is presented
without meaning to be limiting to the invention and without binding
the inventors to any particular theory of operation of the
invention. The resting state of gp41 (upper left) is presumed to be
constitutively trimerized, featuring a coiled-coil bundle near the
N-terminal fusion peptide (arrow). The region corresponding to the
C-terminus of the ectodomain (dark lines) is not initially bound to
the trimer bundle, and has an unknown conformation. A
conformational shift resulting from the binding of gp120 to either
CD4, a co-receptor, or both, may then allow association of the
C-terminal portion of gp41 with the N-terminal bundle. The
resulting antiparallel helical array (top right) observed in the
x-ray structures is presumably the fusogenic state of gp41.
Rearrangement to this state can be blocked if the trimer grooves
are occupied by inhibitory peptides (bottom left). Once blocked in
this manner, a subsequent conformational shift in the gp41 cluster
would sequester the protein off-pathway (bottom right). Peptides
DP178 and HIV24 effectively inhibit the infectivity of genetically
distant and phenotypically distinct subtypes of HIV-1 (Gao et al.,
Journal of Virology 70:1651-1667 (1996)). Moreover, the surface to
which they are proposed to bind is one of the most highly conserved
regions in the HIV-1 genome. We have assayed DP178 against other
strains and found it to have similar inhibitory potency against the
laboratory-adapted strain MN/H9 and primary isolates 301660 and
Th009. Strain Th009 is from subtype E and is genetically distant
from the predominant North American subtype B (e.g. JRCSF) (Zhang
et al., Nature 383:768 (1996)). These results are in accord with
observations from other labs (Wild et al., Proc. Natl. Acad. Sci.
USA 89:10537-10541 (1992); Wild et al., Proc. Natl. Acad. Sci. USA
91: 9770-9774 (1994); Jiang et al., Nature 365:113 (1993)). In
addition to JRCSF and BZ167, we tested HIV24 against Th009 and
found it to have comparable potency, suggesting that the membrane
fusion mechanism proposed extends to widely disparate strains of
HIV-1.
[0737] Other agents, such as antibodies, which target this surface
may thus hold promise for the therapeutic treatment of AIDS.
Example 5
[0738] To prepare a vaccine that would be effective against HIV
infection, either as a prophylactic or post-infection therapeutic
(optionally in combination with anti-HIV drugs or other subunit
vaccines), constrained .alpha.-helical peptides from the 633-678
region of gp41 were prepared and used as immunogens.
[0739] Variants of HIV 24 were prepared with the sequence "Gly Gly
Cys" at the C-terminus or "Cys Gly Gly" at the N-terminus. These
peptides were conjugated to KLH using a heterobifuinctional
crosslinker such as 4(N-Maleimidomethyl)-cyclohexane-1-carboxylic
acid 3-sulfo-N-hydroxysuccinimide ester, available from Sigma, or
its equivalent (e.g. "Sulfo-MBS" from Pierce). Immunizations were
performed as described below.
[0740] Polyclonal antibodies were generated in female guinea pigs
(Hartley Strain from Simonson Labs) against KLH-conjugated HIV
peptides. Fifty .mu.g peptide in 250 .mu.L PBS was emulsified with
250 .mu.L Freund's adjuvant (complete adjuvant for the primary
injection and incomplete adjuvant for all boosts). Injections of
70-100 .mu.g peptide/kg body weight were administered with a
combination of subcutaneous and intramuscular sites in a three-week
cycle. Bleeds were taken on the second and third weeks following
each boost.
[0741] Sera from immunized animals was loaded on a Protein A column
to provide, on elution, purified total Ig. Antibodies selective for
the locked helices were obtained by passing the total Ig pool over
an affinity column containing support loaded with immobilized
locked helices. This support was prepared by first reacting the
cysteine-containing peptides described above (HIV 26, 27, 28, and
29) with biotin-maleimide (also from Sigma;
N-biotinyl-N'-[6-maleimidohexanoy- l]-hydrazide) to afford peptides
biotinylated at either terminus. These peptides were loaded onto a
resin pre-loaded with streptavidin (Pierce, "Ultralink Avidin") to
provide the affinity gel described.
[0742] The total Ig pool from the protein A column was passed over
the appropriate affinity column (i.e. the one with the matching
hapten immobilized). Nonspecific antibodies were eluted in the
flow-through and saved as negative controls. Specific antibodies
were eluted as from the Protein A column, dialyzed into assay
buffer, and stored.
[0743] Surprisingly, the antibody titers observed were quite high
for gp41 subunit peptides. This is particularly surprising since
this region of gp41 (633-678) is not known in the art to generate
HIV neutralizing antibodies.
[0744] The affinity purified polyclonal antibodies are tested in
the viral infectivity assays used to evaluate the peptides. The
haptens used to generate polyclonal antibody preparations that
inhibit infectivity are desirable immunogenic agents for use in a
vaccine. Most preferred are candidates that elicit broadly
cross-reactive antibodies able to neutralize a variety of diverse
HIV-1 isolates in vitro.
[0745] Candidate HIV-1 vaccines can be tested in available animal
models, for example, in chimpanzees as described by Berman et al.,
J. Virol. 7:4464-9 (1992); Haigwood et al., J. Virol. 66:172-82
(1992) and Salmon-Ceron et al., AIDS Res. and Human Retroviruses
11: 1479-86 (1995) for gp120 subunit vaccines. Most preferred are
candidates that elicit broadly cross-reactive antibodies able to
neutralize a variety of diverse HIV-1 isolates in these animal
studies, providing protection from challenge by homologous and
heterologous strains of HIV-1. Successful protection of chimpanzees
is encouraging and has historically proved to be a reliable
indicator of vaccine efficacy.
Sequence CWU 1
1
* * * * *