U.S. patent application number 09/883069 was filed with the patent office on 2002-01-31 for metallopeptide combinatorial libraries and applications.
Invention is credited to Sharma, Shubh D., Shi, Yiqun.
Application Number | 20020012948 09/883069 |
Document ID | / |
Family ID | 22342804 |
Filed Date | 2002-01-31 |
United States Patent
Application |
20020012948 |
Kind Code |
A1 |
Sharma, Shubh D. ; et
al. |
January 31, 2002 |
Metallopeptide combinatorial libraries and applications
Abstract
Metallopeptide combinatorial libraries and methods of making
libraries and metallopeptides are provided, for use in biological,
pharmaceutical and related applications. The combinatorial
libraries are made of peptides, peptidomimetics and peptide-like
constructs, and include a metal ion-binding region thereof which
includes at least one orthogonal sulfur-protecting group, in which
the peptide, peptidomimetic or construct is conformationally fixed
on deprotection of the sulfur and complexation of the metal
ion-binding region with a metal ion. Thereafter the library members
may be screening to select those with the desired specificity and
affinity.
Inventors: |
Sharma, Shubh D.;
(Plainsboro, NJ) ; Shi, Yiqun; (East Brunswick,
NJ) |
Correspondence
Address: |
PEACOCK MYERS AND ADAMS P C
P O BOX 26927
ALBUQUERQUE
NM
871256927
|
Family ID: |
22342804 |
Appl. No.: |
09/883069 |
Filed: |
June 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60112235 |
Dec 14, 1998 |
|
|
|
Current U.S.
Class: |
435/7.1 ;
530/320; 530/331 |
Current CPC
Class: |
Y02P 20/55 20151101;
C07K 7/06 20130101; C07K 1/047 20130101; C07K 5/0827 20130101; C07K
5/1027 20130101 |
Class at
Publication: |
435/7.1 ;
530/320; 530/331 |
International
Class: |
G01N 033/53; C07K
007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 14, 1999 |
US |
PCT/US99/29743 |
Claims
What is claimed is:
1. A combinatorial library of different sequence peptide members
synthesized on solid phase, where each constituent library member
comprises: (a) a peptide sequence of three or more amino acid
residues bound to solid phase characterized by (i) a sequence of
two or more amino acid residues forming a metal ion-binding domain
and including at least one amino acid residue containing at least
one S wherein the said S is protected by an orthogonal S-protecting
group, (ii) a sequence of one or more amino acid residues at the N-
or C-terminus of the metal ion-binding domain, or at both the N-
and C-terminus of the metal ion-binding domain, and (iii) a
cleavable bond attaching the peptide sequence to solid phase; and
(b) a unique selection or sequence of amino acid residues in the
peptide sequence of at least one of the constituent members of the
library; wherein the orthogonal S-protecting group may be removed
without cleaving the peptide sequence from the solid phase.
2. A combinatorial library of different sequence peptidomimetic
members synthesized on solid phase, where each constituent library
member comprises: (a) a peptidomimetic sequence of a combination of
three or more amino acid residues and mimics of amino acid residues
bound to solid phase characterized by (i) a sequence of two or more
amino acid residues, mimics of amino acid residues or combinations
thereof forming a metal ion-binding domain and including at least
one amino acid residue or mimic of an amino acid residue containing
at least one S wherein the said S is protected by an orthogonal
S-protecting group, (ii) a sequence of one or more amino acid
residues, mimics of amino acid residues or combinations thereof at
the N- or C-terminus of the metal ion-binding domain, or at both
the N- and C-terminus of the metal ion-binding domain, and (iii) a
cleavable bond attaching the peptidomimetic sequence to solid
phase; and (b) a unique selection or sequence of amino acid
residues, mimics of amino acid residues or combinations thereof in
the peptidomimetic sequence of at least one of the constituent
members of the library; wherein the orthogonal S-protecting group
may be removed without cleaving the peptidomimetic sequence from
the solid phase.
3. A combinatorial library of different sequence peptide or
peptidomimetic members synthesized in solution, where each
constituent library member comprises: (a) a peptidomimetic sequence
of a combination of three or more amino acid residues and mimics of
amino acid residues bound to solid phase characterized by (i) a
sequence of two or more amino acid residues, mimics of amino acid
residues or combinations thereof forming a metal ion-binding domain
and including at least one amino acid residue or mimic of an amino
acid residue containing at least one S wherein the said S is
protected by an orthogonal S-protecting group, (ii) a sequence of
one or more amino acid residues, mimics of amino acid residues or
combinations thereof at the N- or C-terminus of the metal
ion-binding domain, or at both the N- and C-terminus of the metal
ion-binding domain; and (b) a unique selection or sequence of amino
acid residues, mimics of amino acid residues or combinations
thereof in the peptidomimetic sequence of at least one of the
constituent members of the library.
4. The combinatorial library of claim 1, 2 or 3 wherein the metal
ion-binding domain further comprises at least one N available for
binding to a metal ion upon removal of the orthogonal S-protecting
group.
5. The combinatorial library of claim 1, 2 or 3 wherein the metal
ion-binding domain comprises three residues forming an
N.sub.3S.sub.1 ligand.
6. The combinatorial library of claim 1, 2 or 3 wherein the
orthogonal S-protecting group is S-thio-butyl, acetamidomethyl,
4-methoxytrityl, S-sulfonate or 3-nitro-2-pyridinesulfenyl.
7. The combinatorial library of claim 1, 2 or 3 wherein the
orthogonal S-protecting group may be removed from constituent
library members thereof without otherwise altering the constituent
library members or any amino acid side chain protecting group
therein.
8. The combinatorial library of claim 1, 2 or 3 wherein the
structural diversity occurs in the metal ion-binding domain.
9. The combinatorial library of claim 1, 2 or 3 wherein the
structural diversity occurs outside the metal ion-binding
domain.
10. The combinatorial library of claim 1, 2 or 3 wherein one or
more constituent library members include at least one amino acid
residue or mimic of an amino acid residue in the sequence at the N-
or C-terminus of the metal ion-binding domain containing at least
one S wherein the said S is protected by a non-orthogonal
S-protecting group, whereby the orthogonal S-protecting group may
be removed without removing the non-orthogonal S-protecting
group.
11. The solid phase combinatorial library of claim 1 wherein the at
least one amino acid residue containing at least one S wherein the
said S is protected by an orthogonal S-protecting group is an L- or
D-3-mercapto amino acid, including but not limited to L- or
D-cysteine or L- or D-penicillamine.
12. The combinatorial library of claim 2 or 3 wherein the at least
one amino acid residue or mimic of an amino acid residue containing
at least one S wherein the said S is protected by an orthogonal
S-protecting group is an L- or D-3-mercapto amino acid, including
but not limited to L- or D-cysteine or L- or D-penicillamine;
3-mercapto phenylananine; 2-mercaptoacetic acid;
3-mercaptopropionic acid; 2-mercaptopropionic acid;
3-mercapto-3,3,-dimethyl propionic acid; 3-mercapto-3,3,-diethyl
proprionic acid; 3-mercapto,3-methyl propionic acid;
2-mercapto,2-methyl acetic acid;
3-cyclopentamethlene,3-mercaptopropionic acid; or
2-cyclopentamethlene,2-mercaptoacetic acid.
13. A method for generating a metallopeptide or
metallopeptidomimetic combinatorial library, comprising the steps
of: (a) constructing a library containing a plurality of sequences
of the formula Aaa-MBD-Baa cleavably bound to solid phase, wherein
(i) MBD comprises at least two amino acid residues, mimics of amino
acid residues or combinations thereof, with at least one of said
residues comprising at least one nitrogen atom available to complex
with the coordination sphere of a metal ion, the metal ion to be
provided, and with at least one of said residues comprising at
least one sulfur atom protected by an orthogonal S-protecting
group; (ii) Aaa and Baa each comprise from 0 to about 20 amino acid
residues, mimics of amino acid residues or combinations thereof,
provided that Aaa and Baa comprise at least 1 amino acid residue or
mimic of an amino acid residue, and provided that between at least
two of the plurality of sequences of the formula Aaa-MBD-Baa at
least either Aaa or Baa differ in at least either the sequence of
residues or the selection of residues; (b) deprotecting the sulfur
atom protected by an orthogonal S-protecting group by cleaving the
said orthogonal S-protecting group without cleaving the sequence
from the solid phase; and (c) complexing a metal ion to the MBD;
wherein the resulting metal ion-complexed sequences form a
metallopeptide or metallopeptidomimetic combinatorial library.
14. A method for producing substantially pure metallopeptides or
metallopeptidomimetics without a solution purification step,
comprising the steps of: (a) synthesizing a sequence of the formula
Aaa-MBD-Baa cleavably bound to solid phase, wherein (i) MBD
comprises at least two amino acid residues, mimics of amino acid
residues or combinations thereof, with at least one of said
residues comprising at least one nitrogen atom available to complex
with the coordination sphere of a metal ion, the metal ion to be
provided, and with at least one of said residues comprising at
least one sulfur atom protected by an orthogonal S-protecting
group; (ii) Aaa and Baa each comprise from 0 to about 20 amino acid
residues, mimics of amino acid residues or combinations thereof;
(b) deprotecting the sulfur atom protected by an orthogonal
S-protecting group by cleaving the said orthogonal S-protecting
group without cleaving the sequence from the solid phase; (c)
complexing a metal ion to the MBD; (d) cleaving the metal
ion-complexed sequence from the solid phase; and (e) recovering the
resulting substantially pure metal ion-complexed sequence.
15. The method of claim 13 or 14 wherein the step of deprotecting
the sulfur atom protected by an orthogonal S-protecting group is
performed concurrent with the step of complexing a metal ion to the
MBD.
16. The method of claim 13 or 14 wherein the step of deprotecting
the sulfur atom protected by an orthogonal S-protecting group is
performed prior to the step of complexing a metal ion to the
MBD.
17. The method of claim 13 or 14 wherein at least one of said
residues comprising at least one sulfur atom protected by an
orthogonal S-protecting group is an L- or D-3-mercapto amino acid,
including but not limited to L- or D-cysteine or L- or
D-penicillamine; 3-mercapto phenylananine; 2-mercaptoacetic acid;
3-mercaptopropionic acid; 2-mercaptopropionic acid;
3-mercapto-3,3,-dimethyl propionic acid; 3-mercapto-3,3,-diethyl
proprionic acid; 3-mercapto,3-methyl propionic acid;
2-mercapto,2-methyl acetic acid,
3-cyclopentamethlene,3-mercaptopro- pionic acid; or
2-cyclopentamethlene,2-mercaptoacetic acid.
18. The method of claim 13 or 14 wherein MBD comprises three
residues forming an N.sub.3S.sub.1 ligand.
19. The method of claim 13 or 14 wherein the orthogonal
S-protecting group is S-thio-butyl, acetamidomethyl,
4-methoxytrityl, S-sulfonate or 3-nitro-2-pyridinesulfenyl.
20. The method of claim 13 or 14 wherein the metal ion is V, Mn,
Fe, Co, Ni, Cu, Zn, Ga, As, Se, Y, Mo, Tc, Ru, Rh, Pd, Ag, Cd, In,
Sn, W, Re, Os, Ir, Pt, Au, Hg, TI, Pb, Bi, Po, At, Sm, Eu and
Gd.
21. The method of claim 13 or 14 wherein the sulfur atom protected
by an orthogonal S-protecting group is cleaved without cleaving any
other amino acid side chain protecting group.
22. The method of claim 13 further comprising the step (d) of
cleaving the sequence from the solid phase.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing of U.S.
Provisional Patent Application Serial No. 60/112,235, entitled
Metallopeptide Combinatorial Libraries and Application, filed on
Dec. 14, 1998, and the specifications thereof is incorporated
herein by reference.
[0002] This application is related to U.S. patent application Ser.
No. 08/660,697, entitled Structurally Determined Metallo-Constructs
and Applications, filed Jun. 5, 1996, and U.S. Patent No.
5,891,418, entitled Peptide--Metal Ion Pharmaceutical Constructs
and Applications, issued Apr. 6, 1999, the teachings of both of
which are incorporated herein by reference as if set forth in
full.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention (Technical Field)
[0004] The present invention relates to solid and solution phase
metallopeptide combinatorial libraries, metal ion-complexed
peptidomimetic and peptide-like combinatorial libraries and
metallo-construct combinatorial libraries, wherein at least a
portion of each library constituent is conformationally constrained
upon complexation with a metal ion, and methods for use and making
of the same. The invention also relates to methods for synthesizing
and assembling such libraries, and methods for identification and
characterization of library constituents which are capable of
binding a target molecule of interest, or mediating a biological
activity of interest.
[0005] 2. Background Art
[0006] Peptide Libraries and Combinatorial Chemistry. U.S. patent
application Ser. No. 08/660,697 (the "'697 Application") teaches
general combinatorial chemistry techniques for metallopeptides,
including prior art methods now well recognized tools for rapid
drug discovery. A library of peptides and other small molecules,
with its enormous pool of structurally diverse molecules, is well
suited for both lead generation as well as lead optimization.
Libraries of a variety of molecular species have been described in
literature and screened for drug discovery, including peptides,
peptoids, peptidomimetics, oligonucleotides, benzodiazepines, and
other libraries of small organic molecules.
[0007] Various approaches have been used to construct libraries of
structurally diverse chemical compounds, include chemical synthesis
and genetic engineering methods. Chemically synthesized libraries
have been synthesized by general solution chemical means and by
solid-phase methods. The prior art on designing, synthesizing,
screening, and evaluation of peptide-based libraries has been
reviewed in numerous articles, including the following,
incorporated herein by reference: Pinilla C et al., Biopolymers
(Peptide Sci) 37:221-240, 1995; Lebl M et al, Biopolymers (Peptide
Sci) 37:177-198, 1995; Lam K S et at, Chemical Reviews
97:411-448,1997; Smith G P and Petrenko G P, Chemical Reviews
97:391-410,1997; Nefzi A et al, Chemical Reviews 97:449-472,1997;
Holmes C P et al, Biopolymers (Peptide Sci) 37:199-211, 1995; and,
Moran E J et at, Biopolymers (Peptide Sci) 37:213-219, 1995.
[0008] Spatially Addressable Parallel Synthesis of Solid Phase
Bound Libraries. Various strategies for chemical construction of a
library of peptides or other small molecules are also well
established. One strategy involves spatially separate synthesis of
compounds in parallel on solid phase or on a solid surface in a
predetermined fashion so that the location of one compound or a
subset of compounds on the solid surface is known. The first such
method was developed by Geysen for peptide epitope mapping (Geysen
H M, Meloen R H, Barteling S J: Proc Natl Acad Sci USA
81:3998-4002, 1984). This method involves synthesis of various sets
and subsets of a library of peptides on a multiple number of
polypropylene pin tips in a predetermined fashion. The assembly of
a library of greater than 10,000 molecules by this method is,
however, cumbersome and time consuming. The light-directed
spatially addressable parallel chemical synthesis technique (Fodor
SPA et al: Science 251:767-773, 1991), based upon use of
photolithographic techniques in peptide synthesis on a solid
surface, such as a borosilicate glass microscope slide, is a better
method of constructing libraries containing more than 100,000
spatially separated compounds in a pre-determined fashion. However,
synthesis of libraries that are structurally more diverse than
simple peptides requires the development of orthogonal photolabile
protecting groups that can be cleaved at different wavelengths of
light. In addition, the solid surface bearing these libraries also
has been reported to cause a pronounced effect on binding
affinities in library screening assays (Cho C Y et at: Science
261:1303-1305, 1993; Holmes C P et al: Biopolymers 37:199-211,
1995).
[0009] The DIVERSOMER.RTM. apparatus designed by DeWitt and
coworkers at Parke-Davis Pharmaceutical Research Division of
Warner-Lambert Company, Ann Arbor, Mich., USA, offers a convenient
and automated method of parallel synthesis of small organic
molecule libraries on a solid phase (DeWitt S H et al: Proc Natl
Acad Sci USA 90:6909-6913, 1993; U.S. Pat. No. 5,324,483; DeWitt S
H et al: Acc Chem Res 29:114-122, 1996). Another conceptually
similar apparatus for the solid phase synthesis of small organic
molecule libraries has been reported by Meyers and coworkers
(Meyers H V et at: Molecular Diversity 1:13-20, 1995).
[0010] Pooling and Split Synthesis Strategies. Large libraries of
compounds can be assembled by a pooling strategy that employs
equimolar mixtures of reactants in each synthetic step (Geysen H M
et at: Mol Immunol 23:709-715, 1986) or preferably by adjusting the
relative concentration of various reactants in the mixture
according to their reactivities in each of the coupling reactions
(Ostresh J M et al: Biopolymers 34:1681-1689, 1994; U.S. Pat. No.
5,010,175 to Rytter W J and Santi D V). In one approach equimolar
mixtures of compounds are obtained by splitting the resin in equal
portions, each of which is separately reacted with each of the
various monomeric reagents. The resin is mixed, processed for the
next coupling, and again split into equal portions for separate
reaction with Individual reagents. The process is repeated as
required to obtain a library of desired oligomeric length and size.
This approach is also the basis of the "one-bead one-peptide"
strategy of Furka et al. and Lam et al. (Furka et al: Int. J.
Peptide Protein Res. 37:487, 1991; Lam K S et al: Nature 354:82-84,
1991; Lam K S et al: Nature 360:768, 1992) which employs amino acid
sequencing to ascertain the primary structure of the peptide on a
hit bead in a bioassay. Automated systems have been developed for
carrying out split synthesis of these libraries with rather more
efficiency (Zukermann R N et al: Peptide Res 5:169-174, 1992;
Zukermann R N et al: Int J Peptide Protein Res 40:497-506, 1992). A
common artifact occasionally seen with all these resin bound
libraries is altered target-specific affinity by some solid phase
bound compounds in bioassays, which can result in totally
misleading results.
[0011] Another strategy involves construction of soluble libraries
(Houghten R A et al: Proc Natl Acad Sci USA 82:5131-5135, 1985;
Berg et al: J Am Chem Soc 11 1:8024-8026, 1989; Dooley C T et al:
Science 266:2019-2022, 1994; Blondelle S E: Antimicrob Agents
Chemother 38:2280-2286, 1994; Panilla C: Biopolymers 37:221-240,
1995). This strategy involves a deconvolution process of iterative
re-synthesis and bioassaying until all the initially randomized
amino acid positions are defined. Several modifications to this
strategy have been developed, including co-synthesis of two
libraries containing orthogonal pools, as demonstrated by Tartar
and coworkers, which eliminates the need of iterative re-synthesis
and evaluation (Deprez B et al: J Am Chem Soc 117: 5405-5406,
1995). The positional scanning method devised by Houghton and
coworkers eliminates iterative re-synthesis (Dooley C T et al: Life
Sci 52:1509-1517, 1993; Pinilla C et al: Biotechniques 13:901-905,
1992; Pinilla C et al: Drug Dev Res 33:133-145, 1992). A
combination of this strategy with the split synthesis methods
described above has also been described (Erb E et al: Proc Natl
Acad Sci USA, 91:11422-11426, 1994). A major limitation of the
soluble library approach is its applicability to high affinity
systems. The abundance of each compound in solution can be
influenced by the total number of compounds in a library which can
affect the biological activity. For this reason, a highly active
compound in any pool may not in fact be the most potent molecule.
Lack of reasonable solubilities of certain members in a library may
further influence this phenomenon. In fact, for several libraries
the most active peptide was not even identified in the most active
library pool (Dooley C T et al: Life Sci 52:1509-1517, 1993;
Eichler J, in Proc. 23rd Eur. Peptide Symp., Berga, September.
1994, Poster 198; Wyatt J R: Proc Natl Acad Sci USA, 91:1356-1360,
1994).
[0012] Various strategies for determination of the structure for a
positive hit in a random library have been developed. See, e.g.,
U.S. Pat. No. 5,698,301. For a solid-phase library, direct
analytical modalities include Edman degradation for peptide
libraries, DNA sequencing of oligonucleotide libraries, and various
mass spectrometry techniques on matrix bound compounds. The
technique of creating a series of partially end-capped compounds at
each of the synthetic steps during library assembly helps their
unambiguous identification by mass spectrometry (Youngquist R S et
al: J Am Chem Soc 117:3900-3906, 1995; Youngquist R S et al: Rapid
Commun Mass Spectr 8:77-81, 1994). Direct mass spectrometric
analysis of compounds covalently bound to a solid phase matrix of
particles is also now possible by the use of matrix-assisted laser
desorption/ionization (MALDI) techniques (Siuzdak G et al: Bioorg
Med Chem Left 6:979, 1996; Brown B B et al: Molecular Diversity
1:4-12, 1995). In addition to these analytical techniques, various
encoding strategies have been devised for structure elucidation in
organic molecule-based libraries, including both non-peptide and
non-nucleotide libraries, such as DNA encoding, peptide coding,
haloaromatic tag encoding, and encoding based on radiofrequency
transponders. See, e.g., U.S. Pat. No. 5,747,334.
[0013] Most of the libraries described above are termed "random"
libraries because of their enormous structural and conformational
diversity. Libraries of relatively restricted and biased structures
have also been reported. Examples of libraries of conformationally
rigid compounds built on a structurally common template include
benzodiazepine, .beta.-lactam, .beta.-turn mimetics,
diketopiperazines, isoquinolines, dihydro- and
tetrahydroisoquinolines, 1,4 dihydropyridines, hydantoins,
pyrrolidines, thiazolidine-4-carboxylic acids, 4-thiazolidines and
related 4-metathiazanones and imidazoles.
[0014] Among the various classes of libraries of small molecules,
peptide libraries remain the most versatile because of the
structural diversity offered by the use of naturally occurring
amino acids, incorporation of a variety of "designer" amino acids,
and the high efficiency and ease with which peptide synthesis can
be accomplished. In addition, another level of structural diversity
in peptide-based libraries has been added by post-synthesis
modification of the libraries. These modifications include
permethylation, acylation, functionalization of the side chain
functionality, and reductive amination of the N-terminus.
[0015] Many libraries specifically customized for one particular
biological target have also been reported. These libraries are
generally assembled by incorporating only a set of structural
elements that might be essential for eliciting a target-specific
response. Some of the reported libraries of this class include
aspartic acid protease, zinc proteases, carbonic anhydrase
inhibitors, tyrosine kinase inhibitors, estrogen receptor ligands,
and antioxidants.
SUMMARY OF THE INVENTION (DISCLOSURE OF THE INVENTION)
[0016] The present invention provides a combinatorial library of
different sequence peptide members synthesized on solid phase,
where each constituent library member includes a) a peptide
sequence of three or more amino acid residues bound to solid phase
characterized by (i) a sequence of two or more amino acid residues
forming a metal ion-binding domain and including at least one amino
acid residue containing at least one S wherein the said S is
protected by an orthogonal S-protecting group, (ii) a sequence of
one or more amino acid residues at the N- or C-terminus of the
metal ion-binding domain, or at both the N- and C-terminus of the
metal ion-binding domain, and (iii) a cleavable bond attaching the
peptide sequence to solid phase; and (b) a unique selection or
sequence of amino acid residues in the peptide sequence of at least
one of the constituent members of the library. In this library, the
orthogonal S-protecting group may be removed without cleaving the
peptide sequence from the solid phase.
[0017] The invention further provides a combinatorial library of
different sequence peptidomimetic members synthesized on solid
phase, where each constituent library member includes (a) a
peptidomimetic sequence of a combination of three or more amino
acid residues and mimics of amino acid residues bound to solid
phase characterized by (i) a sequence of two or more amino acid
residues, mimics of amino acid residues or combinations thereof
forming a metal ion-binding domain and including at least one amino
acid residue or mimic of an amino acid residue containing at least
one S wherein the said S is protected by an orthogonal S-protecting
group, (ii) a sequence of one or more amino acid residues, mimics
of amino acid residues or combinations thereof at the N- or
C-terminus of the metal ion-binding domain, or at both the N- and
C-terminus of the metal ion-binding domain, and (iii) a cleavable
bond attaching the peptidomimetic sequence to solid phase; and (b)
a unique selection or sequence of amino acid residues, mimics of
amino acid residues or combinations thereof in the peptidomimetic
sequence of at least one of the constituent members of the library.
In this library, the orthogonal S-protecting group may be removed
without cleaving the peptidomimetic sequence from the solid
phase.
[0018] The invention further provides a combinatorial library of
different sequence peptide or peptidomimetic members synthesized in
solution, where each constituent library member includes (a) a
peptidomimetic sequence of a combination of three or more amino
acid residues and mimics of amino acid residues bound to solid
phase characterized by (i) a sequence of two or more amino acid
residues, mimics of amino acid residues or combinations thereof
forming a metal ion-binding domain and including at least one amino
acid residue or mimic of an amino acid residue containing at least
one S wherein the said S is protected by an orthogonal S-protecting
group, (ii) a sequence of one or more amino acid residues, mimics
of amino acid residues or combinations thereof at the N- or
C-terminus of the metal ion-binding domain, or at both the N- and
C-terminus of the metal ion-binding domain; and (b) a unique
selection or sequence of amino acid residues, mimics of amino acid
residues or combinations thereof in the peptidomimetic sequence of
at least one of the constituent members of the library. In this
library and the other libraries provided above, the members may
include a sequence with at least one amino acid residue or mimic of
an amino acid residue containing at least one sulfur atom in which
the sulfur atom is protected by a non-orthogonal S-protecting
group. In this library and the other libraries provided above, the
orthogonal S-protecting group may be removed without removing the
non-orthogonal S-protecting group.
[0019] In any of the foregoing combinatorial libraries, the metal
ion-binding domain can further include at least one N available for
binding to a metal ion upon removal of the orthogonal S-protecting
group. In one embodiment, the metal ion-binding domain consists of
three residues forming an N.sub.3S.sub.1 ligand. The orthogonal
S-protecting group may be S-thio-butyl, acetamidomethyl,
4-methoxytrityl, S-sulfonate or 3-nitro-2-pyridinesulfenyl, and the
orthogonal S-protecting group may further be removed without
otherwise altering the constituent library member. Structural
diversity may occur in the metal ion-binding domain, or the
residues and sequence of residues comprising the metal ion-binding
domain can be fixed, with structural diversity occurring elsewhere
in the sequence.
[0020] In the case of peptide libraries, the amino acid residue
containing at least one S protected by an orthogonal S-protecting
group can be an L- or D-3-mercapto amino acid, including but not
limited to L- or D-cysteine or L- or D-penicillamine. In the
combinatorial libraries that include peptidomimetic members, the
amino acid residue or mimic of an amino acid residue containing at
least one S protected by an orthogonal S-protecting group can be an
L- or D-3-mercapto amino acid, including but not limited to L- or
D-cysteine or L- or D-penicillamine; 3-mercapto phenylananine;
2-mercaptoacetic acid; 3-mercaptopropionic acid;
2-mercaptopropionic acid; 3-mercapto-3,3,-dimethyl propionic acid;
3-mercapto-3,3,-diethyl proprionic acid; 3-mercapto,3-methyl
propionic acid; 2-mercapto,2-methyl acetic acid;
3-cyclopentamethlene,3-mercaptopropionic acid; or
2-cyclopentamethlene,2-mercaptoacetic acid.
[0021] The invention further includes a method for generating a
metallopeptide or metallopeptidomimetic combinatorial library,
including the steps of (a) constructing a library containing a
plurality of sequences of the formula Aaa-MBD-Baa cleavably bound
to solid phase, wherein (i) MBD includes at least two amino acid
residues, mimics of amino acid residues or combinations thereof,
with at least one of said residues comprising at least one nitrogen
atom available to complex with the coordination sphere of a metal
ion, the metal ion to be provided, and with at least one of said
residues comprising at least one sulfur atom protected by an
orthogonal S-protecting group; (ii) Aaa and Baa each include from 0
to about 20 amino acid residues, mimics of amino acid residues or
combinations thereof, provided that the combination of Aaa and Baa
contain at least 1 amino acid residue or mimic of an amino acid
residue, and provided that between at least two of the plurality of
sequences of the formula Aaa-MBD-Baa at least either Aaa or Baa
differ in at least either the sequence of residues or the selection
of residues; (b) deprotecting the sulfur atom protected by an
orthogonal S-protecting group by cleaving the orthogonal
S-protecting group without cleaving the sequence from the solid
phase; and (c) complexing a metal ion to the MBD. The resulting
metal ion-complexed sequences form a metallopeptide or
metallopeptidomimetic combinatorial library. The method can
optionally include the step (d) of cleaving the sequence from the
solid phase.
[0022] The invention further includes a method for producing
substantially pure metallopeptides or metallopeptidomimetics
without a solution purification step, including the steps of: (a)
synthesizing a sequence of the formula Aaa-MBD-Baa cleavably bound
to solid phase, wherein (i) MBD includes at least two amino acid
residues, mimics of amino acid residues or combinations thereof,
with at least one residues including at least one nitrogen atom
available to complex with the coordination sphere of a metal ion,
the metal ion to be provided, and with at least one residue
including at least one sulfur atom protected by an orthogonal
S-protecting group; (ii) Aaa and Baa each contain from 0 to about
20 amino acid residues, mimics of amino acid residues or
combinations thereof; (b)deprotecting the sulfur atom protected by
an orthogonal S-protecting group by cleaving the orthogonal
S-protecting group without cleaving the sequence from the solid
phase; (c) complexing a metal ion to the MBD; (d) cleaving the
metal ion-complexed sequence from the solid phase; and (e)
recovering the resulting substantially pure metal ion-complexed
sequence.
[0023] In either of the foregoing methods, the step of deprotecting
the sulfur atom protected by an orthogonal S-protecting group can
be performed concurrent with the step of complexing a metal ion to
the MBD. It is also possible and contemplated that the step of
deprotecting the sulfur atom protected by an orthogonal
S-protecting group can be performed prior to the step of complexing
a metal ion to the MBD. In both methods, the step of deprotecting
the sulfur atom protected by an orthogonal S-protecting group can
be performed without cleaving any other amino acid side chain
protecting group. In both methods, the residue including a sulfur
atom protected by an orthogonal S-protecting group can be an L- or
D-3-mercapto amino acid, including but not limited to L- or
D-cysteine or L- or D-penicillamine; 3-mercapto phenylananine;
2-mercaptoacetic acid; 3-mercaptopropionic acid;
2-mercaptopropionic acid; 3-mercapto-3,3,-dimethyl propionic acid;
3-mercapto,3-methyl propionic acid; 3-mercapto-3,3,-diethyl
proprionic acid; 2-mercapto,2-methyl acetic acid;
3-cyclopentamethlene,3-mercaptopropionic acid; or
2-cyclopentamethlene,2-mercaptoacetic acid. In the methods, the MBD
can be three residues forming an N.sub.3S.sub.1 ligand. The
orthogonal S-protecting group can be S-thio-butyl, acetamidomethyl,
4-methoxytrityl, S-sulfonate or 3-nitro-2-pyridinesulfenyl. Any
metal ion may be employed, including V, Mn, Fe, Co, Ni, Cu, Zn, Ga,
As, Se, Y, Mo, Tc, Ru, Rh, Pd, Ag, Cd, In, Sn, W, Re, Os, Ir, Pt,
Au, Hg, TI, Pb, Bi, Po, At, Sm, Eu and Gd.
[0024] In each of the methods and libraries provided, a specific
conformational restriction is obtained upon complexing the peptides
or amino acid sequences with a metal ion, such that the
conformationally constrained peptide-metal ion complexes can serve
as surrogates for reverse turn structures, such as beta turns and
gamma turns commonly found in naturally occurring peptides and
proteins.
[0025] Accordingly, it is an object of this invention to provide
libraries of conformationally constrained peptide-metal ion
complexes as surrogates for reverse turn structures, such as beta
turns and gamma turns commonly found in naturally occurring
peptides and proteins. The turns formed as a consequence of metal
ion complexation are more stable than the naturally occurring turn
structures, which are stabilized only by weaker interactions such
as van der Waals' interactions and hydrogen bonds.
[0026] Another,object of this invention is to provide combinatorial
peptide libraries of peptide-metal ion complexes, wherein the
peptides include a metal ion-binding domain, such that a specific
conformational structure is obtained upon metal complexation.
[0027] Another object of this invention to provide combinatorial
peptide libraries of peptide-metal ion complexes, wherein the amino
acids comprising the peptides may be naturally occurring amino
acids, isomers and modifications of such amino acids, non-protein
amino acids, post-translationally modified amino acids,
enzymatically modified amino acids, constructs or structures
designed to mimic amino acids, and the like, so that the library
includes pseudopeptides and peptidomimetics.
[0028] Another object of this invention is to provide
metallopeptide libraries, wherein the metallopeptides include a
metal ion-binding domain, such that a specific conformational
structure is obtained upon metal complexation, and the
metallopeptides further include distinct, unique and different
amino acid sequences.
[0029] Another object of this invention is to provide
metallopeptide libraries, wherein the metallopeptides include a
metal ion-binding domain and distinct, unique and different amino
acid sequences, wherein the metallopeptides may be exposed to a
substance to which one or more metallopeptides will exhibit
specificity and affinity for the substance of interest.
[0030] Another object of this invention is to provide
metallopeptide libraries, wherein the metallopeptides include a
metal ion-binding domain and distinct, unique and different amino
acid sequences, wherein the metallopeptides may be exposed to a
substance to which one or more determinable metallopeptides will
preferentially bind.
[0031] Another object of this invention is to provide
metallopeptide libraries, wherein the metallopeptides include a
metal ion-binding domain, which may be either soluble or solid
phase libraries.
[0032] Another object of this invention is to provide methods for
synthesis of peptides wherein the peptides contain one or more
reactive SH groups forming a part of a metal ion-binding domain,
whereby the reactive SH groups are protected during synthesis, and
are deprotected only upon complexing the peptides with a metal ion.
For solid phase libraries, such deprotection, and subsequent metal
ion complexation occurs in solid phase, with the peptides being
optionally subsequently cleaved from solid phase.
[0033] Another object of this invention is to provide combinatorial
metallopeptide libraries wherein each of the peptides forming the
library contain a reverse turn structure as a consequence of metal
ion complexation.
[0034] Another object of this invention is to provide combinatorial
metallopeptide libraries containing metallopeptides with high
specificity and affinity for the target molecule of interest, such
high specificity and affinity resulting from each of the peptides
forming the library containing a reverse turn structure as a
consequence of metal ion complexation.
[0035] Another object of this invention is to provide a method for
rapid and efficient complexation of a pool of diverse peptides with
a metal ion, including a rhenium metal ion.
[0036] Another object of this invention is to provide a method for
the identification of specific metallopeptides through internal
signatures resulting from use of metal ions with two or more
isotopic peaks, such as through use of rhenium containing two
isotopes in fixed relative abundance that differ in mass by 2
units.
[0037] Other objects, advantages and novel features, and the
further scope of applicability of the present invention, will be
set forth in part in the detailed description to follow, taken in
conjunction with the accompanying drawings, and in part will become
apparent to those skilled in the art upon examination of the
following, or may be learned by practice of this invention. The
objects and advantages of this invention may be realized and
attained by means of the instrumentalities and combinations
particularly pointed out in the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] The accompanying drawings, which are incorporated into and
form a part of the specification, illustrate several embodiments of
the present invention and, together with the description, serve to
explain the principles of the invention. The drawings are only for
the purpose of illustrating a preferred embodiment of the invention
and are not to be construed as limiting the invention. In the
drawings:
[0039] FIGS. 1A-1J Examples of molecular templates which may be
employed in the libraries and structures of this invention. R.sub.1
through R.sub.7 are variable functional groups which may be
employed to obtain the desired specificity, affinity and potency
for a target molecule upon complexation of the metal ion, and M is
a metal ion, or a metal-oxo (M.dbd.O) group, or a metal-nitrido
(M.ident.N) group, or a nitrido-N-substituted metal-nitrido
(M.dbd.N--R.sub.8) group where R.sub.8 is a functional group which
may contribute to the desired specificity and affinity for the
target molecule, and where M is further employed to fix the
structure in a specific conformational restriction upon
complexation of the metal ion.
[0040] FIG. 2 Mass spectrum of a library pool of 25 metallopeptides
synthesized according to Example 6.
[0041] FIG. 3 Mass spectrum of a library pool of 4 metallopeptides
synthesized according to Example 10.
[0042] FIGS. 4A-4E Reversed phased HPLC profiles of a library pool
of 4 metallopeptides synthesized according to Example 10.
[0043] FIG. 5 Flow chart of a split pool and combination synthesis
method according to Example 6.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Best Modes for Carrying Out the Invention
[0044] Definitions. Certain terms as used throughout the
specification and claims are defined as follows:
[0045] The terms "bind," "binding," "complex," and "complexing," as
used throughout the specification and claims, are generally
intended to cover all types of physical and chemical binding,
reactions, complexing, attraction, chelating and the like.
[0046] The "peptides" of this invention can be a)
naturally-occurring, b) produced by chemical synthesis, c) produced
by recombinant DNA technology, d) produced by biochemical or
enzymatic fragmentation of larger molecules, e) produced by methods
resulting from a combination of methods a through d listed above,
or f) produced by any other means for producing peptides.
[0047] By employing chemical synthesis, a preferred means of
production, it is possible to introduce various amino acids which
do not naturally occur along the chain, modify the N- or
C-terminus, and the like, thereby providing for improved stability
and formulation, resistance to protease degradation, and the
like.
[0048] The term "peptide" as used throughout the specification and
claims is intended to include any structure comprised of two or
more amino acids, including chemical modifications and derivatives
of amino acids. For the most part, the peptides of this invention
comprise fewer than 100 amino acids, and preferably fewer than 60
amino acids, and most preferably ranging from about 2 to 20 amino
acids. The amino acids forming all or a part of a peptide may be
naturally occurring amino acids, stereoisomers and modifications of
such amino acids, non-protein amino acids, post-translationally
modified amino acids, enzymatically modified amino acids,
constructs or structures designed to mimic amino acids, and the
like, so that the term "peptide" includes pseudopeptides and
peptidomimetics, including structures which have a non-peptidic
backbone. The term "peptide" also includes dimers or multimers of
peptides. A "manufactured" peptide includes a peptide produced by
chemical synthesis, recombinant DNA technology, biochemical or
enzymatic fragmentation of larger molecules, combinations of the
foregoing or, in general, made by any other method.
[0049] The "amino acids" used in this invention, and the term as
used in the specification and claims, include the known naturally
occurring protein amino acids, which are referred to by both their
common three letter abbreviation and single letter abbreviation.
See generally Synthetic Peptides: A User's Guide, G A Grant,
editor, W. H. Freeman & Co., New York, 1992, the teachings of
which are incorporated herein by reference, including the text and
table set forth at pages 11 through 24. As set forth above, the
term "amino acid" also includes stereoisomers and modifications of
naturally occurring protein amino acids, non-protein amino acids,
post-translationally modified amino acids, enzymatically
synthesized amino acids, derivatized amino acids, constructs or
structures designed to mimic amino acids, and the like. Modified
and unusual amino acids are described generally in Synthetic
Peptides: A User's Guide, cited above; Hruby V J, Al-obeidi F and
Kazmierski W: Biochem J 268:249-262, 1990; and Toniolo C: Int J
Peptide Protein Res 35:287-300, 1990; the teachings of all of which
are incorporated herein by reference. A single amino acid is
sometimes referred to herein as a "residue."
[0050] The library constructs of this invention also include a
metal ion, which may be an ionic form of any element in metallic
form, including but not limited to metals and metalloids. The metal
ion may, but need not, be radioactive, paramagnetic or
superparamagnetic. The metal ion can be of any oxidation state of
any metal, including oxidation states of vanadium (V), manganese
(Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn),
gallium (Ga), arsenic (As), selenium (Se), yttrium (Y), molybdenum
(Mo), technetium (Tc), ruthenium (Ru), rhodium (Rh), palladium
(Pd), silver (Ag), cadmium (Cd), indium (In), tin (Sn), tungsten
(W), rhenium (Re), osmium (Os), iridium (Ir), platinum (Pt), gold
(Au), mercury (Hg), thallium (TI), lead (Pb), bismuth (Bi),
polonium (Po), astatine (At), samarium (Sm), europium (Eu), and
gadolinium (Gd). The metal ion can also be a radionuclide of any of
the foregoing, including In, Au, Ag, Hg, Tc, Re, Sn, At, Y and Cu.
A preferred metal ion with a tetradentate coordination sphere is
Re. For radiopharmaceutical applications, or applications wherein a
radioisotope is desirable for screening, an alpha-, gamma- or
beta-emitting radionuclide may be employed.
[0051] The coordination sphere of various common metal ions, in
general, is tetradentate to hexadentate. In one embodiment
according to this invention, an amino acid or amino acid mimetic
sequence is included within each library member such that it
contains the desired number of groups (4 to 6 in most cases) for
complexing with the metal. The molecule is designed so that, upon
complexing with a metal, it forms a mimic of a reverse turn
structure about the site of metal complexation. A metal with
coordination number 4, 5 or 6, and complexing respectively with an
amino acid sequence forming a tetra, penta, or hexadentate ligand,
will fold and constrain the ligand. The amino acid or amino acid
mimetic sequence forming a ligand is defined as the metal
ion-binding domain ("MBD") of the peptide or peptidomimetic. A
highly flexible molecule like a peptide, in other words, is folded
to form a kind of reverse turn upon its complexation with a metal.
This resulting turn is a highly constrained structure in the
conformational sense.
[0052] The biological-binding domain ("BBD") of the peptide or
peptidomimetic is defined in the specification and claims as a
sequence of one or more amino acids which constitute a biologically
active sequence, exhibiting binding to a biological receptor found
on cells, tissues, organs and other biological materials, thereby
constituting the peptide as a member of a specific binding pair.
The BBD also includes any sequence, which may be consecutive amino
acids or mimetics (sychnological) or non-consecutive amino acids or
mimetics (rhegnylogical) which forms a ligand, which ligand is
capable of forming a specific interaction with its acceptor or
receptor. The term "receptor" is intended to include both acceptors
and receptors. The receptor may be a biological receptor. The
sequence or BBD may transmit a signal to the cells, tissues or
other materials associated with the biological receptor after
binding, but such is not required. Examples include, but are not
limited to, BBDs specific for hormone receptors, neurotransmitter
receptors, cell surface receptors, enzyme receptors and
antibody-antigen systems. The BBD may thus be either an agonist or
antagonist, or a mixed agonist-antagonist. The BBD may also include
any ligand for site-specific RNA or DNA binding, such as sequences
which may be employed as mimics of transcription and other gene
regulatory proteins. The BBD may also include any sequence of one
or more amino acids or mimetics, or other constrained molecular
regions, which exhibit binding to a biological receptor found on
other peptides, on enzymes, antibodies, or other compositions,
including proteinaceous compositions, which may themselves exhibit
binding to another biological receptor. A peptide or peptidomimetic
complexed to a metal ion with such a BBD constitutes a member of a
"specific binding pair," which specific binding pair is made up of
at least two different molecules, where one molecule has an area on
the surface or in a cavity which specifically binds to a particular
spatial and polar organization of the other molecule. Frequently,
the members of a specific binding pair are referred to as ligand
and receptor or anti-ligand. Examples of specific binding pairs
include antibody-antigen pairs, hormone-receptor pairs,
peptide-receptor pairs, enzyme-receptor pairs, carbohydrate-protein
pairs (glycoproteins), carbohydrate-fat pairs (glycolipids),
lectin-carbohydrate pairs and the like.
[0053] The BBD is further defined to include the portion of a
construct, wherein the construct is a peptidomimetic, peptide-like,
or metallo-construct molecule, which upon binding of the construct
with a metal ion, is biologically active, exhibiting binding to a
biological receptor found on cells, tissues, organs and other
biological materials. The BBD may, in this instance, be
sychnological or rhegnylogical, and generally has the attributes
and functions of a BBD of a peptide. The BBD may be coextensive
with all or a portion of the MBD, so that the same amino acids or
other residues which constitute the MBD also constitute all or a
part of the BBD. In some instances, one or amino acids of the MBD
will form a part of the BBD, and one or more additional amino
acids, which are not part of the MBD, form the remainder of the
BBD.
[0054] Conformational constraint refers to the stability and
preferred conformation of the three-dimensional shape assumed by a
peptide or other construct. Conformational constraints include
local constraints, involving restricting the conformational
mobility of a single residue in a peptide; regional constraints,
involving restricting the conformational mobility of a group of
residues, which residues may form some secondary structural unit;
and global constraints, involving the entire peptide structure. See
generally Synthetic Peptides: A User's Guide, cited above.
[0055] The primary structure of a peptide is its amino acid
sequence. The secondary structure deals with the conformation of
the peptide backbone and the folding up of the segments of the
peptide into regular structures such as .alpha.-helices,
.beta.-sheets, turns and the like. Thus, the three-dimensional
shape assumed by a peptide is directly related to its secondary
structure. See generally Synthetic Peptides: A User's Guide, cited
above, including the text, figures and tables set forth at pages
24-33, 39-41 and 58-67. A global structure refers to a peptide
structure which exhibits a preference for adopting a
conformationally constrained three-dimensional shape.
[0056] The product resulting from the methods set forth herein can
be used for both medical applications and veterinary applications.
Typically, the product is used in humans, but may also be used in
other mammals. The term "patient" is intended to denote a mammalian
individual, and is so used throughout the specification and in the
claims. The primary applications of this invention involve human
patients, but this invention may be applied to laboratory, farm,
zoo, wildlife, pet, sport or other animals. The products of this
invention may optionally employ radionuclide ions, which may be
used for diagnostic imaging purposes or for radiotherapeutic
purposes.
[0057] Peptide and Metallo-Construct Molecule Libraries and
Combinatorial Chemistries. Using the methods of this invention,
libraries of peptides and peptidomimetics are designed wherein each
constituent library member includes an MBD sequence necessary for
providing a coordination site for complexation with a metal, it
being understood that such sequence may differ among the
constituent members of the library. Upon complexing the MBD with a
metal, a specific structure results, forming a mimic of a reverse
turn structure. The specific stereochemical features of this
complex are due to the stereochemistry of the coordination sphere
of the complexing metal ion. Thus the preferred geometry of the
coordination sphere of the metal dictates and defines the nature
and extent of conformational restriction.
[0058] Libraries of this invention contain constituents which are
either locally or globally constrained structures. Libraries may
include molecules with either local conformation restrictions or
global conformation restrictions, or some combination thereof. This
aspect of the invention includes a variety of methods of synthesis,
screening and structural elucidation of positive hits in screening
systems. The importance of these aspects are well known to those
skilled in the art and will also become evident from the following
description and examples.
[0059] In general, most of the metals that may prove useful in this
invention have a coordination number of 4 to 6, and rarely as high
as 8, which implies that the putative MBD must be made of residues
with reactive groups located in a stereocompatible manner so as to
establish a bond with a metal ion of given geometry and
coordination sphere. Coordinating groups in the peptide chain
include nitrogen atoms of amine, amide, imidazole, or guanidino
functionalities; sulfur atoms of thiols or disulfides; and oxygen
atoms of hydroxy, phenolic, carbonyl, or carboxyl functionalities.
In addition, the peptide chain or individual amino acids can be
chemically altered to include a coordinating group, such as oxime,
hydrazino, sulfhydryl, phosphate, cyano, pyridino, piperidino, or
morpholino groups. For a metal with a coordination number of 4, a
tetrapeptide amino acid sequence may be employed (such as
Gly-Gly-Gly-Gly), or a tripeptide amino acid sequence in which at
least one of the amino acids has a side chain with a coordinating
group can similarly be employed (such as Gly-Gly-Cys). The side
chain can have a nitrogen, oxygen or sulfur-based coordination
group. Thus, an amino acid sequence can provide an N.sub.4,
N.sub.3S, N.sub.2S.sub.2, NS.sub.3, N.sub.2SO or similar ligand,
yielding tetradentate coordination of a metal ion utilizing
nitrogen, sulfur and oxygen atoms.
[0060] In another embodiment of the invention, the MBD includes one
or more amino acid residues and one or more derivatized amino acids
or spacer sequences, with the derivatized amino acid or spacer
sequence having a nitrogen, sulfur or oxygen atom available for
complexing with the various oxidation states of the metal Examples
of derivatized amino acids include amide, primary alkyl or aryl
amide, 1,2,3,4-tetrahydroisoqu- inoline-2-carboxylic acid and its
corresponding 7-hydroxy derivative, N-carboxymethylated amino
acids, 2'-mercapto-Trp, N.sup..beta.-(2
mercaptoethane)-.alpha.,.beta.-diaminopropionic acid and similar
higher homologs of other homologous amino acids, N.sup..beta.-(2
aminoethane)-.alpha.,.beta.-diaminopropionic acid and similar
higher homologs of other homologous amino acids,
N.sup..beta.-(picolinoyl)-.alph- a.,.beta.-diaminopropionic acid
and similar higher homologs of other homologous amino acids,
.beta.-(picolylamide)-Asp and similar homologs of other homologous
amino acids, N.sup..beta.-(2-amino-benzoyl)-.alpha.,.bet-
a.-diaminopropionic acid and similar higher homologs of other
homologous amino acids, .beta.-(2-amidomethylpyridine)-Asp and
similar homologs of other homologous amino acids,
N-benzyloxycarbonyl amino acid, N-tert butyloxycarbonyl amino acid,
N-fluorenylmethyloxycarbonyl amino acid and other similar
urethane-protected amino acid derivatives, and other derivatized or
synthetic amino acids relating to any of the foregoing. Examples of
spacer sequences which may be employed in this invention include
2-mercaptoethylamine, succinic acid, glutaric acid,
2-mercaptosuccinic acid, ethylenediamine, diethylenetriamine,
triethylenetetraamine, tetraethylenepentaamine, glycol,
polyethylene glycol, thioglycolic acid, mercaptopropionic acid,
pyridine-2-carboxylate, picolylamine, 2-mercaptoaniline,
2-aminobenzoic acid, and 2-aminomethylpyridine. In general, any
sequence which may be linked, directly or indirectly, to one or two
amino acids so as to form a continuous sequence, and which has a
nitrogen, sulfur or oxygen atom available for complexing with the
valences of the metal ion, may be employed as an element of the
MBD.
[0061] S-Protected Thiol Group Compounds in Metallo-Libraries. A
free thiol (SH) group is preferred for complexation of most metal
ions to the peptides and peptidomimetics of this invention, and in
many cases an SH group is necessary in order to form a stable
exchange-inert complex with a metal. Peptides and other organic
molecules with free SH groups, however, are easily oxidized in air
and in solution, and can often form a disulfide-linked dimer. If
more than one free SH group is present in a molecule, oxidation may
lead to a complex polymer. Similarly, if a mixture of different
peptides or organic molecules with free SH groups are prepared,
oxidation generally leads to a complex mixture of polymers of
unknown composition. This is of serious concern in preparing
libraries of metallopeptides or other organic molecules where one
or more SH group is intended for use in metal complexation.
[0062] A variety of SH protecting groups have been employed for a
variety of purposes, including radiopharmaceutical manufacture and
formulation. For example, in its protected form
S-Benzoyl-mercaptoacetyl-glycyl-glycyl- -glycine (Bz-MAG.sub.3) has
been used to complex .sup.99mTc under conditions where the S-Bz
group splits during .sup.99mTc complexation. The use of S-Bz
protection, however, is not compatible with the methods of peptide
synthesis. See, e.g., European Patent Applications 0,250,013 and
0,173,424.
[0063] In order to construct metallopeptide libraries of this
invention which incorporate an SH group, if mixed pool synthesis is
employed the peptides must be S-protected derivatives. The SH
protecting group is chosen such that (a) the synthesis of peptide
derivatives with S-protecting group is compatible with methods of
solution and solid phase peptide synthesis, so that the
S-protecting group is stable during synthetic procedures, and (b)
the S-protecting group can be deprotected in situ, without cleavage
from the resin in the case of solid phase synthesis, during the
metal complexation step. Many prior art methods, such as
Bz-MAG.sub.3, meet at most only one of the two criteria specified
above (Bz-MAG.sub.3 meets only criterion (a) above.
[0064] Use of orthogonally S-protected thiol groups permits
synthesis of metallo-compounds in a single pot. A mixture of
compounds, each compound containing an orthogonal S-protected group
("OSPG"), is used for complexation with a metal ion, and it is only
during metal ion complexation that the S-protected group is
deprotected, and accordingly polymerization and cross-linking is
avoided. This procedure thus provides homogenous libraries of
metallo-compounds.
[0065] One OSPG meeting the criteria specified above, and which can
be used in this invention, employs an S.sup.tBu (S-thio-butyl or
S-t-butyl) group to protect the SH group. The S.sup.tBu group is
stable under both the acidic and basic conditions typically
employed in peptide synthesis. Further, the S.sup.tBu group may be
cleaved by reduction using a suitable phosphine reagent, which
reduction step may be employed immediately prior to or in
conjunction with complexation of a metal ion to the peptide. Such
OSPG cleavage does not cleave the peptide from the resin, or
otherwise alter the structure of the peptide.
[0066] Another OSPG meeting the criteria specified above and
suitable for this invention employs an S-Acm (S-acetamidomethyl)
group to protect the SH group. The Acm group is also stable under
the acid and base conditions usually employed during peptide
synthesis. The S-Acm group may be removed by treatment of
S-Acm-protected peptide or peptide resin with mercury (II) acetate
or silver (I) tertrafluoroborate, which liberates the thiol peptide
in its mercury or silver ion-complexed state. Free thiol-containing
peptide can then be recovered by treating the mercury or silver ion
and thiol complexed salts with an excess of a thiol-containing
reagent, such as beta-mercaptoethanol or dithiothreitol. The
resulting peptide is then used for metal complexation.
Alternatively, the mercury or silver ion and thiol complexed
peptide may be directly treated with a metal ion complexing reagent
to form the desired metallopeptide.
[0067] Other examples of OSPGs for metallopeptides include
4-methoxytrityl (Mmt), 3-nitro-2-pyridinesulfenyl (Npys) and
S-sulfonate (SO.sub.3H). Mmt is selectively removed upon treatment
with 1% TFA in dichloromethane. Npys and S-sulfonate are
selectively removed by treatment with a thiol-containing reagent
such as beta-mercaptoethanol or dithiothreitol or a phosphine
reagent such as tributyl phosphine. The Npys group (R. G. Simmonds
R G et al: Int J Peptide Protein Res, 43:363, 1994) is compatible
with Boc chemistry for peptide synthesis and the S-sulfonate
(Maugras I et al: Int J Peptide Protein Res, 45:152, 1995) is
compatible with both Fmoc and Boc chemistries. Similar OSPGs
derived from homologous series of S-alkyl, or S-aryl, or S-aralkyl
may also be used in this invention. A primary characterization of
the OSPG is that its use results in the formation of a disulfide
(S--S) bond utilizing one sulfur atom each from the
thiol-containing amino acid and the protecting group. In addition,
the resulting disulfide (S--S) bond is cleavable by the use of any
of a variety of disulfide cleaving agents, including but not
limited to phosphine- and thiol-containing reagents.
[0068] The method employing S.sup.tBu protected SH groups, or other
OSPGs, may be employed for the generation of either solid phase or
soluble libraries. For solid phase libraries, peptides may be
synthesized by use of conventional Fmoc chemistry. In the case of
conventional Fmoc chemistry, Fmoc-L-Cys-(S.sup.tBu) is coupled to
an appropriate resin, via one or more intermediate amino acids, and
additional amino acids are thereafter coupled to the
L-Cys-(S.sup.tBu) residue. S.sup.tBu may be employed with either L-
or D-Cys, and any of a variety of other amino acids, including
designer or unnatural amino acids and mimics thereof, characterized
by an SH group available for binding to a metal ion, including, but
not limited to, 3-mercapto phenylananine and other related
3-mercapto amino acids such as 3-mercapto valine (penicillamine);
2-mercaptoacetic acid; 3-mercaptopropionic acid;
2-mercaptopropionic acid; 3-mercapto-3,3,-dimethyl propionic acid;
3-mercapto,3-methyl propionic acid; 3-mercapto-3,3,-diethyl
proprionic acid; 2-mercapto,2-methyl acetic acid;
3-cyclopentamethlene,3-mercaptopropionic acid;
2-cyclopentamethlene,2-mercaptoacetic acid and related amino acids.
In all these cases, S-protection can be by S-Bu.sup.t, S-Acm, Mmt,
Npys, S-sulfonate and related groups, as described above.
[0069] Metal Ion Complexation to MBD. The complexation of metal
ions to the sequences in a library, and specifically to the MBD, is
achieved by mixing the sequences with the metal ion. This is
conveniently done in solution, with the solution including an
appropriate buffer. In one approach, the metal ion is, when mixed
with the peptide or peptidomimetic constituents, already in the
oxidation state most preferred for complexing to the MBD. Some
metal ions are complexed in their most stable oxidation state, such
as calcium (II), potassium (I), indium (III), manganese (II),
copper (II), zinc (II) and other metals. In other instances, the
metal must be reduced to a lower oxidation state in order to be
complexed to the MBD. This is true of ferrous, ferric, stannous,
stannic, technetiumoxo[V], pertechnetate, rheniumoxo[V], perrhenate
and other similar metal ions. Reduction may be performed prior to
mixing with the sequences, simultaneously with mixing with the
sequences, or subsequent to mixing with the sequences. Any means of
reduction of metal ions to the desired oxidation state known to the
art may be employed.
[0070] For tetradentate coordination with a metal ion, rhenium is a
preferred ion. Solid phase resin bound peptide or peptidomimetic
sequences may be labeled with rhenium ion by treatment with the
rhenium transfer agent ReOCl.sub.3(PPh.sub.3).sub.2 in the presence
of 1,8-Diazabicyclo[5,4,0]undec-7-ene as a base. The sequences may
then be cleaved from the resin. Alternatively, peptide or
peptidomimetic sequences in a soluble library may similarly be
labeled by treatment with the rhenium transfer agent
ReOCl.sub.3(PPh.sub.3).sub.2 in the presence of
1,8-Diazabicyclo[5,4,0]undec-7-ene as a base. Metal complexation in
the presence of 1,8-Diazabicyclo[5,4,0]undec-7-ene (DBU) as a base
can conveniently be accomplished at ambient room temperature.
[0071] In an alternative method of metal complexation a mild base,
such as sodium acetate, can be used. In this case the
thiol-containing sequence, either in solution or bound to solid
phase, is taken in a suitable solvent, such as DMF, NMP, MeOH, DCM
or a mixture thereof, and heated to 60-70.degree. C. with the
rhenium transfer agent ReOCl.sub.3(PPh.sub.3).s- ub.2 in the
presence of sodium acetate for 15 minutes. Similarly, other bases
such as triethylamine, ammonium hydroxide and so on, may be
employed. According to this invention, MeOH is a preferred choice
of solvent for rhenium complexation in the case of S-deprotected
peptides in solution. The solvent choice for S-deprotected peptides
still attached to the solid phase is guided mainly by
considerations of superior solvation (swelling) of the solid phase.
DMF and NMP may be employed. Various mixtures of these solvents,
also in combination with MeOH, and DCM, CHCl.sub.3 and so on, may
also be employed to yield optimized complexation results.
[0072] In one embodiment of this invention, an S-Bu.sup.t protected
peptide is treated in situ with rhenium transfer agent in the
presence of DBU and tributylphosphine to effect S-deprotection and
rhenium complexation in one pot. Alternately, complexation of
rhenium to the S-Bu.sup.t protected peptide in the presence of
rhenium perrhenate may be accomplished by treatment with
Sn[II]Cl.sub.2. This reagent effects S-deprotection as well as
conversion of ReO.sub.4 state to ReO state in situ to cause
complexation of the rhenium to the S-deprotected peptide. A
preferred procedure in this invention is the use of S-Bu.sup.t
protected peptide with S-deprotection by treatment with
tributylphosphine, and metal complexation of the resulting peptide
utilizing ReOCl.sub.3(PPh.sub.3).sub.2 in the presence of DBU at
room temperature.
[0073] In the libraries of this invention, the MBD forms a reverse
turn structure upon complexation with a metal ion, with the library
constructed such that side chains of amino acids within the MBD are
varied, and similarly amino acids not forming a part of the MBD are
also varied. Various compounds in a library of metallopeptides can
be obtained by varying the sequence of amino acids in a set of
peptides that are all optimized to form a complex of nearly similar
geometry when coordinated with a metal ion. This optimization can
be obtained, for example, by appropriate positioning of amino acids
having high affinity to complex a metal ion. Examples of naturally
occurring amino acids with high affinity for metal complexation
include Cys and His. A library of such peptides, therefore, would
have at least one of these amino acids that is suitably placed in
the sequence, with this amino acid being common to all the
molecules in the library, with this amino acid thus
non-randomized.
[0074] A conceptual, generalized view of a solid phase library of
metallopeptides that is constructed using local conformational
restriction is: 1
[0075] where M is a metal ion, R.sub.1 and R.sub.2 are randomly
selected amino acid side chains forming parts of the reverse turn
structure which is the BBD, and "Peptide Chain" denotes one or more
amino acids. A similar library can also be constructed in which the
components are soluble, and thus not bound to a resin.
[0076] One illustration of a locally restricted metallopeptide
library, in which the members are conformationally constrained upon
metal ion complexation, is a library directed towards the family of
various integrin receptors that recognize the RGD sequence. In this
library, individual amino acid positions are degenerated by
selecting a set of cationic amino acids for one position, a second
set of anionic amino acids for another position and a third set of
selected amino acids with strong metal complexation properties.
Other positions in the peptides are randomized. The common rigid
structure of the metal-peptide complex in this library allows for
various forms of presentation of the cationic and anionic centers
for interaction with the integrin receptors. A library of these
structures can be employed to identify metallopeptides specific to
individual integrin receptors. The general structure of this
library, which can either be soluble or solid-phase library, prior
to metal ion complexation is:
[0077] R.sub.1,-Aaa-Bbb-Ccc-Ddd-R.sub.2, where R.sub.2 is coupled
to a resin for solid phase bound libraries, and
[0078] where
[0079] Aaa is an L- or D-configuration residue providing an N for
metal ion complexation, such as Arg, Lys, Orn, homoArg, 2,3-diamino
propionic acid, 2,4,-diaminobutyric acid, S-aminoethyl cysteine,
3(O-aminotheyl) Ser, or another synthetic basic amino acids;
[0080] Bbb is an L- or D-configuration residue providing an N for
metal ion complexation, such as Gly, Ala, Aib, Val, Nle, Leu or
similar amino acids with uncharged side chains;
[0081] Ccc is an L- or D-configuration residue providing both an N
and S, or alternatively two Ns, available for metal ion
complexation, such as Cys, HomoCys, Pen, or other amino acids,
natural or unnatural, containing both an N and S, together with an
OSPG, available for metal ion binding;
[0082] Ddd is an L- or D- configuration residue with a negatively
charged side chain functional group, such as Asp, Glu, or synthetic
amino acids with a negatively charged side chain functional group;
and
[0083] R.sub.1 and R.sub.2 are H, Alkyl, aryl, alkylcarbonyl,
arylcarbonyl, alkyloxycarbonyl, aryloxycarbonyl, or a polymer such
as PEG, PVA, or polyamino acid, attached directly or through a
carbonyl group.
[0084] Other forms of this library include sets of structures with
general formulas shown below, wherein the spatial distances between
the cationic and anionic centers can be different than those shown
above, such as of the formulas:
R.sub.1-Bbb-Aaa-Ccc-Ddd-R.sub.2,
R.sub.1-Bbb-Ddd-Ccc-Aaa-R.sub.2, or
R.sub.1-Ddd-Bbb-Ccc-Aaa-R.sub.2.
[0085] For each of the foregoing, the definitions of R.sub.1,
R.sub.2, Aaa, Bbb, Ccc and Ddd are as described above. All four
classes of libraries described above can either be synthesized
individually or prepared as one library.
[0086] Another embodiment of this invention provides for
construction of a library with global conformational restriction.
In this embodiment, the MBD can be held constant, and a randomized
or selected series of sequences of amino acids or mimetics varied
to form the library. This type of library encompasses
metallopeptides in which a MBD is an isosteric replacement for a
disulfide, lactam, lactone, thioether or thioester moiety in cyclic
peptides. In these constructs a set MBD is introduced between two
pre-selected ends of a linear peptide or peptidomimetic that
contains the randomized or selected series of sequences of amino
acids or mimetics under investigation. The general structure of a
metallopeptide library of this type is: 2
[0087] where M is a metal ion and R.sub.1 and R.sub.2 are
structural elements that may provide additional stability to metal
complexation, or may modulate biological activity, such as
determining the organ of clearance, or altering biodistribution
patterns or pharmacokinetics. The "Peptide Chain" sequence may be
randomly varied, thereby resulting in a random library, or may be
directed in a predetermined fashion, based upon known
characteristics of the target molecule.
[0088] One illustration of a globally-constrained metallopeptide
library is a library of peptides wherein all the individual members
of the library include a metal ion-binding domain and the library
is directed specifically towards a family of peptide hormones, such
as somatostatin, cholecystokinin, opioid peptides, melanotropins,
luteinizing hormone releasing hormone, tachykinins and similar
peptide hormones. The general formula of this library of peptides,
before complexation to a metal ion, is: 3
[0089] where X is a fixed MBD including a plurality of amino acids,
so that all of the valences of the metal ion are satisfied upon
complexation of the metal ion with X, R.sub.1 and R.sub.2 each
comprise from 0 to about 20 amino acids, and Aaa, Baa and Caa each
comprise one or more amino acids connected to X through an amide,
thioether, thioester, ester, carbamate, or urethane bond, wherein
each of Aaa, Baa, and Caa is varied. In this example, the MBD may
include an OSPG. Other thiols in the sequence may optionally
include S-protecting groups that are not orthogonal, such that the
OSPG may be removed without removal of other S-protecting groups in
the sequence.
[0090] For solid phase libraries the peptide constructs are
attached to a resin, and the resin is omitted for soluble
libraries. This library of globally constrained metallopeptides can
also be screened for detecting compounds that are mimetics for
various peptides that are known to exist in a reversed turn
structure as their hypothesized biologically active structure. The
examples of these include various peptide hormones such as
somatostatin, cholecystokinin, opioid peptides, melanotropins,
luteinizing hormone releasing hormone, tachykinins and various
antibody epitopes.
[0091] The functional equivalent of each these peptide libraries
may also be obtained through the development of a library of
non-amino acid building blocks so as to result in structural mimics
of these peptides. The peptide bonds may be replaced by
pseudopeptide bonds, such as thioamides, thioethers, substituted
amines, carbanate, urethane, aliphatic moieties, and functionally
similar constructs.
[0092] A peptide library is first assembled according to the
sequence specification and degeneration, as described above, by
well-known methods of peptide synthesis. These libraries can be
synthesized as discreet, spatially addressable compounds in
parallel synthesis, using split synthesis approaches, or by
deconvolution techniques of soluble libraries. Using similar
methods, a pseudopeptide, peptidomimetic or non-peptide library can
be obtained. The non-peptide libraries may also optionally
incorporate one of various tagging approaches that are well known
to those skilled in the art. Both solid-phase and soluble libraries
can be obtained in this manner. The entire library is then reacted
with an appropriate metal-complexing agent to obtain the
corresponding metal-coordinated library, comprising a similar class
of predetermined structures. For example, to complex a peptide
library with rheniumoxo metal ion, the peptide library can be
treated with Re(O)Cl.sub.3(PPh.sub.3).sub.2 in the presence of
sodium acetate. This procedure results in quantitative complexation
of ReO with the peptide. In order to complex Zn, Co, Mn, Fe or Cu
ions, the peptide library is treated with chloride or other
suitable salts of these metal ions to yield the library of
corresponding metal ions. Essentially, a variety of metal ions can
be used to construct different metallopeptide libraries. One
limiting factor in selection of the appropriate metal ion is the
relative stability of a particular metal-peptide complex, related
in large part to the metal-peptide binding constant or constants.
It is well known in the art that some metal-peptide constructs are
stable only within specified pH or other special conditions, or are
easily oxidized in air. Some peptide-metal ion complexes, such as
those with ReO, are stable in pure form and can be isolated and
stored under normal storage conditions for a long period of
time.
[0093] A metallopeptide library constructed according to this
invention can be screened to identify one or more receptor-binding
or pharmacologically-active candidates by various techniques that
have been reported in the prior art. Both soluble and solid phase
libraries may be directly employed in these assays. These
techniques include direct target binding approaches as described by
Lam and coworkers (Lam K S et al: Nature 354:82-84, 1991; Lam K S
et al: Nature 360:768, 1992), deconvolution and iterative
re-synthesis approaches (Houghten R A et al: Proc Natl Acad Sci USA
82:5131-5135, 1985; Berg et al: J Am Chem Soc 111:8024-8026, 1989;
Dooley C T et al: Science 266:2019-2022, 1994; Blondelle S E:
Antimicrob Agents Chemother 38:2280-2286, 1994; Panilla C:
Biopolymers 37:221-240, 1995), approaches using orthogonal pools of
two co-synthesized libraries according to Tartar and coworkers (
Deprez B et al: J Am Chem Soc 117: 5405-5406, 1995), positional
scanning methods devised by Houghton and coworkers that eliminate
iterative re-synthesis (Dooley C T et al: Life Sci 52:1509-1517,
1993; Pinilla C et al: Biotechniques 13:901-905, 1992; Pinilla C et
al: Drug Dev Res 33:133-145, 1992), and a combination of the
positional scanning method with split synthesis methods (Erb E et
al: Proc Natl Acad Sci USA, 91:11422-11426, 1994).
[0094] Among these techniques, the deconvolution and iterative
resynthesis approach, the approach involving orthogonal pools of
two co-synthesized libraries, and the positional scanning method
may be directly applied to soluble metallopeptide libraries to
elucidate the structure of a "hit," or peptide identified as a
receptor-binding or pharmacologically-active candidate in the
screening process. For solid phase libraries, other than spatially
addressable parallel synthesis libraries, the structure of hits can
be directly determined by various strategies well known to those
skilled in the art. These include direct mass spectrometric
analysis of compounds covalently bound to solid phase matrix of
particles by the use of matrix-assisted laser desorption/ionization
(MALDI) techniques (Siuzdak G et al: Bioorg Med Chem Lett 6:979,
1996; Brown B B et al: Molecular Diversity 1:4-12, 1995). The
technique of creating a series of partially end-capped compounds at
each of the synthetic steps during library assembly also helps in
unambiguous identification by mass spectrometry (Youngquist R S et
al: J Am Chem Soc, 117:3900-3906, 1995; Youngquist R S et al: Rapid
Commun Mass Spectr 8:77-81, 1994). In addition to these analytical
techniques, various encoding strategies that have been devised for
structure elucidation in organic molecule-based libraries,
including non-peptide and non-nucleotide libraries, may be
utilized. Various encoding strategies, such as DNA encoding,
peptide encoding, haloaromatic tag encoding, and encoding based on
radiofrequency transponders, are now well known in the art and can
be used directly in combination with metallopeptide libraries.
These tagging strategies require the incorporation of the tags
during the course of synthesis of libraries, which can be
accomplished during the construction of a metallopeptide libraries,
since metal complexation is a final, post-synthesis step.
[0095] Structural Diversity of Library Members. Examples of some of
the molecular templates which may be employed in this invention are
shown in FIG. 1. These templates show the appropriate location of R
groups required to make a molecule selective and potent for a given
biological target. The templates of FIGS. 1A, 1H and 1J allow
introduction of as many as 7 to 8 different functional groups. The
templates of FIGS. 1B and 1C allow introduction of as few as four
functional groups. The templates are derived from both peptide and
non-peptide polyamine backbones, and may be partly peptidic and
partly non-peptidic in nature. The metal coordination sphere
illustrated in these templates is either an N.sub.3S or an
N.sub.2S.sub.2 configuration where N and S are the metal
coordinating atoms.
[0096] Using the methods of this invention, that portion of the
template forming a constrained sequence upon metal ion complexation
can have attached thereto a wide variety of functional groups,
which groups may be attached by any means known. The ten structures
depicted at FIGS. 1A through 1J are for illustration purposes only,
and in no way limit the scope of this invention. Other structures
may be employed, including a variety of linking groups. In
addition, different structures are employed with metal ions
requiring other than tetradentate coordination.
[0097] The templates shown in FIGS. 1A through 1J, and other
templates which may be created based upon the methods of this
invention, allow for inclusion of as many as seven or eight
different functional groups. These different functional groups may
either individually or in combination impart desired affinity,
specificity, selectivity, potency, and pharmacological and
pharmacokinetic profiles to the molecule towards a biological
target. For example, the structure depicted in FIG. 1D is obtained
by complexation of a metal ion "M" with a linear peptide. The
sulfur "S" atom for complexation to "M" is provided by the side
chain of a Cys amino acid residue. The functional groups R.sub.2,
R.sub.3, and R.sub.4 may be side chains of amino acids. R.sub.1 may
also be an amino acid or an organic group, while R.sub.5 may be a
beta-substitution in the Cys residue.
[0098] The structure depicted in FIG. 1B is derived from a linear
peptide chain in which .beta.-substituted .beta.-mercaptoacetic
acid has been substituted at the N-terminus. The structure depicted
in FIG. 1G is similar to that of FIG. 1B, except that
mercaptoacetic acid is substituted for Cys. The structure depicted
in FIG. 1G offers a different topographic structural relationship
of the side chain functionalities R.sub.1, R.sub.2, R.sub.3 and
R.sub.4 from that of the structure depicted in FIG. 1B. These kinds
of minor, yet potentially biologically important, differences in
structure may result in different affinities, specificities,
selectivities or potencies. The five structures depicted in FIGS.
1B, 1C, 1D, 1G and 1I can be obtained from the corresponding linear
peptide derivatives synthesized by common methods well known to
those skilled in the art. Complexation of metal ion to these linear
peptides can be accomplished on solid phase or solution phase using
the methods discussed above.
[0099] The structures of FIGS. 1A, 1F, 1H, 1J and 1E are derived
from the structures of FIGS. 1B, 1C, 1D, 1G and 1I by reducing one
or more "M" complexing amide bonds (CO--NH group) to a
pseudopeptide bond (CH.sub.2--NH group), followed by the
functionalization of the resulting amine nitrogen with an "R"
group. These reduced amide bond molecules, therefore, allow the
introduction of additional "R" groups in the molecule to enhance
the structural diversity and complexity of the molecules. The "N"
complexation to the "M" in these cases is through the formation of
a coordinate bond. Such molecules have different charge
characteristics than the amide "N" complexed molecules. Differences
of this type in the core charge of metal complexes are known to
alter the passage of metal complexes across the biological
compartment barriers. For example, .sup.99mTc-MAG.sub.3
(mercaptoacetylglycylglycylglycine), with an ionic core charge of
-1, is not compartmentalized and is excreted through the kidneys,
while .sup.99mTc-MIBI (methoxyisobutylisonitrile), with an ionic
core charge of +1, is compartmentalized in heart muscles.
.sup.99mTc-HMPAO (hexamethylpropyleneamine oxime) and
.sup.99mTc-ECD (ethyl cysteinate dimer), each with no charge, pass
through the blood-brain barrier and are used as brain perfusion
imaging agents. Thus in one aspect of this invention libraries may
be constructed with differences in ionic charges, as is generally
shown in FIG. 1, by providing either an amide "N" or an amine "N"
to modulate the pharmacokinetic profile of library members.
Synthesis of reduced amide bond molecules capable of providing an
amine "N" may be accomplished by employing methods of making linear
peptide derivatives with pesudopeptide bonds that are well known to
those skilled in the art. See, for example, Wen J J and Spatola A
F: J Peptide Res 49:3-14, 1997.
[0100] In yet another aspect of this invention, libraries may be
constructed wherein a variety of metal ions "M" are used for
complexation. In the structures depicted in FIG. 1, metal ions
requiring tetradentate coordination are most applicable, so that
"M" may be a metal ion such as Tc or Re. However, use of all other
metal ions is contemplated and possible, and in such case, it is
also contemplated and possible that other structures would be
employed, providing for hexadentate or other coordination spheres.
Further, "M" may be a metal ion (M), a metal-oxo group (M.dbd.O), a
metal-nitride group (M.ident.N), or an N-nitrido substituted
metal-nitride or organoimido (M.dbd.N--R'). The organoimido
M.dbd.N--R' species,allows for inclusion of an R' which may act as
a biologically significant functional group similar to the various
R groups described in the structures of FIG. 1. In this case, the
size of the library may be expanded by substitution of different R'
groups.
[0101] The metal-oxo compounds may be obtained from a suitable
pre-formed metal-oxo transfer agent such as
Re(O)Cl.sub.3(PPh.sub.3).sub.2 (Johnson N P et al: Inorg Syntheses
9:145-148, 1967; Parshall G: Inorg Synth 17:110, 1977; and Sullivan
B P et al: Inorg Syntheses 29:146-150, 1992). Metal-oxo compounds
may also be obtained by direct reduction of rhenium perrhenate with
stannous or another suitable reducing agent in the presence of the
complexing molecule, thereby yielding a metal-oxo complex
(Rouschias G: Chemical Review 74:531-566, 1974; and Vites J C and
Lynam M M: Coordination Chemistry Review 142:1-20, 1995).
[0102] Nitrido complexes can be prepared by several methods. See,
for example, the method taught in U.S. Pat. No. 5,288,476; U.S.
Pat. No. 5,300,278; Sullivan B P et al: cited above; Rouschias G:
cited above; Vites J C and Lynam M M: cited above; Dehnicke K and
Strahle J: Angew Chem Int Ed Engl, 31:955-978, 1992; Baldas J:
Inorganic Chem 25:150-153, 1986; and Marchi A et al: Inorganic Chem
29:2091-2096, 1986. In general, the nitrido complexes can be
obtained by treating a rhenium oxo complex with a hydrazine or by
the use of Re(N)Cl.sub.2(PPh.sub.3).sub.2 as a transfer agent.
[0103] The organoimido M.dbd.N--R' species may be prepared by
condensing a rhenium oxo complex with a primary amine, or with 1,2
disubstituted hydrazines, or with phosphinimines or with phenyl
isocyanate (Rouschias G: Chemical Review, 74:531-566, 1974). The
rhenium oxo, rhenium nitrido and rhenium organoimido complexes are
extremely stable and are reported to persist through fairly drastic
substitution reactions.
[0104] The functionalized sulfydryl-containing templates may be
synthesized from corresponding halogenated compounds by treating
them with sodium thiosulfate to form S-sulfonate salts. These
salts, on treatment with a reducing agent such as tributyl
phosphine or sodium borohyd ride, yield a functionalized
sulfhydryl. Alternatively, S-sulfonate derivatives can directly be
used in the synthesis of metallo-compounds as discussed above. In
this case, the S-sulfonate group also acts as a OSPG in both the
solution phase and the solid phase synthesis of metallo-constructs
using either Boc or Fmoc chemistries. The S-sulfonate OSPG may be
selectively removed by treatment with a tributyl or triisopropyl
phosphine immediately prior to the complexation of the metal
ion.
[0105] The structures of FIGS. 1A through 1J, and similar
structures, may be used in libraries by selective substitution of
any or all of R.sub.1 through R.sub.7. It is also possible and
contemplated that different related structures of any of FIGS. 1A
through 1J, or similar structures, may be used in libraries, while
keeping the functionalities of R.sub.1 through R.sub.7
constant.
[0106] The invention is further illustrated by the following
non-limiting examples.
EXAMPLE 1
Single-pot Synthesis of Four-member N.sub.3S.sub.1 Type
Metallopeptide Compound Library
[0107] A SynPep multiple peptide synthesizer was used for synthesis
of the peptides on solid phasing using convention Fmoc chemistry.
Fmoc-L-Glu-(O.sup.tBu)-Wang resin (1.2 g, 0.54 mM) was swollen in
NMP and the Fmoc-group removed by adding 20% piperidine in NMP and
mixing by bubbling nitrogen for 20 min. The solvent was drained by
suction and the resin washed. Four equivalent of
Fmoc-L-Cys-(S.sup.tBu)-OH (0.93 g), four equivalent of
2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium (TBTU) and six
equivalent of diisopropylethylamine (DIEA) were added with NMP and
bubbled with nitrogen for 30 min. The resin was again washed and
deprotection of the Fmoc-group repeated, yielding
NH.sub.2-L-Cys-(S.sup.t- Bu)-L-Glu-(O.sup.tBu)-Wang resin. The
resin was then split into two pools, and using similar methods
Fmoc-L-Gln-(Trt)-OH was added to one pool and Fmoc-D-Gln-(Trt)-OH
added to the other pool. The two pools were mixed and the
Fmoc-group deprotected, yielding
NH.sub.2-(L,D)-Gln-(Trt)-L-Cys-(S.su- p.tBu)-L-Glu-(O.sup.tBu)-Wang
resin. The resin was again split into two pools, and
Fmoc-L-Asn-(Trt)-OH added to one pool and Fmoc-D-Asn-(Trt)-OH added
to the other pool. The two pools were mixed and the Fmoc-group
deprotected, yielding
NH.sub.2-(L,D)-Asn-(Trt)-(L,D)-Gln-(Trt)-L-Cys-(S.s-
up.tBu)-L-Glu-(O.sup.tBu)-Wang resin. To this resin was added 0.072
g of succinic anhydride in pyridine, yielding
HOOC(CH.sub.2).sub.2CONH-(L,D)-A-
sn-(Trt)-(L,D)-Gln-(Trt)-L-Cys-(S.sup.tBu)-L-Glu-(O.sup.tBu)-Wang
resin. After washing, the S.sup.tBu group was removed by adding
13.8 mL of DMF/Tributylphosphine (20/3, v/v; 0.52 M ) and bubbling
for 3 hours. The resulting resin was again washed, yielding
HOOC(CH.sub.2).sub.2CONH-(L,D)-
-Asn-(Trt)-(L,D)-Gln-(Trt)-L-Cys-L-Glu-(O.sup.tBu)-Wang resin. 0.6
g of ReO(PPh.sub.3).sub.2Cl.sub.3 (8 eq.) and 0.32 g of sodium
acetate (final 1 M) to the resin solution, and the solution heated
at 70.degree. C. for 2 hours. After cooling to room temperature,
the resin was washed and dried, and 3 mL of a TFA "cocktail" ( 5%
water, 5% TIPS, 5% thioanisole and 85% TFA) was added. The solution
was allowed to stand for 3 hours. The resin was then filtered and
washed once with 1 mL of TFA. Cold ether was added to the collected
TFA solution, and the resulting precipitate was washed with cold
ether and dried under high vacuum. The resulting mixture was a gray
colored solid that weighed 40 mg, a yield of approximately 56 %,
and which contained equal quantities of
HOOC(CH.sub.2).sub.2CONH-L-Asn-(Trt)-L-Gin-(Trt)-L-Cys-L-Glu;
HOOC(CH.sub.2).sub.2CONH-L-Asn-(Trt)-D-Gln-(Trt)-L-Cys-L-Glu;
HOOC(CH.sub.2).sub.2CONH-D-Asn-(Trt)-L-Gln-(Trt)-L-Cys-L-Glu; and
HOOC(CH.sub.2).sub.2CONH-D-Asn-(Trt)-D-Gln-(Trt)-L-Cys-L-Glu.
EXAMPLE 2
Alternate Single-pot Synthesis of Four Member N.sub.3S.sub.1 Type
Metallopeptide Compound Library
[0108] 1,8-diazabicyclo[5,4,]undec-7-ene (DBU) as alternative base,
replacing sodium acetate. The peptide
HOOC(CH.sub.2).sub.2CONH-L-Asn-(Trt-
)-L-Gln-(Trt)-L-Cys-L-Glu-O.sup.tBu attached to Wang resin was
mixed with 1,8-diazabicyclo(5,4,0]undec-7-ene (DBU) (8 eq.) and
ReO(PPh.sub.3).sub.2Cl.sub.3 (8 eq.) in DMF. The reaction was
carried out at room temperature for 4 hours. The subsequent
cleavage of product from the resin, washing and precipitation was
as described for Example 1 above.
EXAMPLE 3
In Situ Formation of Metallo-complexes in the Presence of Reducing
Agent and ReO(PPh.sub.3).sub.2Cl.sub.3
[0109] The resin
HOOC(CH.sub.2).sub.2CONH-(L,D)-Asn-(Trt)-(L,D)-Gln-(Trt)--
L-Cys-(S.sup.tBu)-L-Glu-(O.sup.tBu)-Wang obtained from Example 1
above was mixed with tributylphosphine (0.52 M) and
ReO(PPh.sub.3).sub.2Cl.sub.3 (8 eq.) in DMF. The bases used in the
reaction were either sodium acetate (0.1 M) or
1,8-diazabicyclo(5,4,0]undec-7-ene (DBU) (8 eq.). In the case of
sodium acetate, the reaction was conducted at about 70.degree. C.
for 4 hours. In the case of DBU, the reaction container was shaken
at room temperature for 4 hours. The subsequent cleavage of product
from the resin, washing and precipitation was as described for
Example 1 above.
EXAMPLE 4
Synthesis of Ac-His-X-Cys-Trp-NH.sub.2--Rhenium Complexes with PEG
Resin (Where X=Trp, Homophe, 2-Nal, or Phenylglycine)
[0110] The procedures were similar to those described for Examples
1, 2 and 3. A NovaSyn TGR resin was used. Histidine, Cysteine and
Tryptophan were protected by trityl, thio-t-butyl and Boc groups,
respectively. The cleavage cocktail was TFA/TIS (95/5). After three
hours the resin was filtered and washed with TFA. To the TFA
solution was added cold ether, and the resulting precipitate was
spun down by centrifugation. The resulting pellets were washed with
ether twice, and 0.5 mL of 95% acetic acid was added. Five mL of
water was added after one hour. The resulting product was then
lyophilized under high vacuum.
EXAMPLE 5
Development of a Prototype Metallopeptide Library for the
Melanocortin Receptor.
[0111] The library design was based on the tetrapeptide message
sequence, His-Phe-Arg-Trp (6-9 sequence), of A-MSH. This sequence
exists as a reverse turn, making it suitable for conversion into a
metallopeptide format of this invention. In this approach
metallopeptides were designed around a tripeptide N.sub.3S.sub.1
MBD designed for a rhenium metal ion. The MBD was derivatized to
yield the pentapeptide Ac-His-Phe-Arg-Cys-Trp-- NH.sub.2 as a
putative candidate for melanocortin ("MC") receptors. Further
refinements in the structure were made in response to other
considerations, including the chirality of amino acid side chains,
yielding a template structure Ac-His-D-Phe-Arg-Cys-Trp-NH.sub.2.
The structure of this peptide after binding to rhenium is: 4
[0112] The template structure was used to define a small
combinatorial library utilizing split synthesis methodologies. The
final template selected for the combinatorial library was
Ac-D-His-Xaa-D-Cys-Trp-NH.sub.- 2, where Xaa was D-(2')
Naphthylalanine, D-Trp, D-HomoPhe, or D-Phenylglycine. For this
library, the peptide resin, Cys(S.sup.tBu)-Trp(Boc)-Resin was split
in four equal parts. Each part was reacted with one of the four Xaa
types. After coupling, the resin pools were mixed and synthesis
continued in a single pool to couple the His residue. The final
result was four separate peptides in a single pool, each peptide
varying by one amino acid, in the Xaa position.
[0113] An S.sup.tBu OSPG group was used to protect the SH group
during synthesis. After solid-phase assembly of the peptide chain
using Fmoc chemistry with acid labile side chain protecting groups,
the S.sup.tBu group was split using tributylphosphine. The
resulting free SH-containing peptide-resin was treated with the
rhenium transfer agent Re(O)Cl.sub.3(PPh.sub.3).sub.2 in the
presence of 1,8-Diazabicyclo[5,4,0]- undec-7as base. The resulting
metallopeptide resin was then treated with TFA to cleave it from
the resin and de-protect all the side chain protecting groups. The
products were analyzed by mass spectrometry. HPLC analysis was
performed and individual peaks collected and subjected to mass
analysis. The resulting peptides were analyzed by electron spray
mass spectrometry, yielding the predicted mass, including the
rhenium complexed to the peptide.
EXAMPLE 6
Design and Synthesis of Melanocortin Receptor-specific
Metallopeptide Library
[0114] The library was rationally designed based upon data relating
to melanocortin receptors and peptide sequences specific to the
melanocortin receptors, including melanotropin side-chain
pharmacophores, D-Phe.sup.7 and Trp.sup.9, that interact with a
hydrophobic network of receptor aromatic residues in transmembrane
regions 4, 5, 6, and 7. Based on this design criterions, a
pharmacophore for the melanocortin receptor was preliminarily
defined, and a combinatorial library designed for identification of
potent and receptor-selective agonists.
[0115] Based on the design criteria, the putative structure
R-Aaa-Baa-L-Cys-Caa-NH.sub.2 was selected, in which each of Aaa,
Baa and Caa are selected from L- or D-isomers of 2-Nal (1), Phe
(2), Trp (3), Tyr (4) and Ala (5), so that any one of the foregoing
can be substituted for any one of Aaa, Baa or Caa. In the
nomenclature adopted for the library design, the five amino acids
were designated 1 through 5, with the isomerism conventionally
notated, so that, for example, Baa.sub.2L refers to L-Phe in the
Baa position.
[0116] The terminal R group was selected from Ac,
C.sub.6H.sub.5OOH, CH.sub.3(CH.sub.2).sub.5--COOH,
C.sub.6H.sub.5CH.dbd.CH--COOH (trans) and Pyridine-3-carboxylate.
The terminal R group represents a truncated amino acid, and offers
additional structural diversity.
[0117] A pool and split library synthesis scheme was employed such
that 5,000 separate compounds were synthesized, resulting in 200
final pools each containing 25 different compounds, with the
compounds differing solely by the amino acids in the Aaa and Baa
position. Using this methodology, binding characteristics relating
to the Caa amino acid or R terminal group can be identified through
inter-group comparison, thereby simplifying the deconvolution
strategy.
[0118] The library synthesis steps are set forth in FIG. 5. The
resin of step I was divided into 10 groups. At step 2 each of
Caa.sub.1L through Caa.sub.5D were coupled to an individual resin
group, and L-Cys was coupled to each resin group, resulting in 10
groups and 20 couplings. Each of the resin groups of step 2 was
then divided into 10 sub-groups as shown at step 3 (with only one
subgroup illustrated at step 3, and for each subgroup of step 3,
each of Baa.sub.1L through Baa.sub.5D were coupled to one group
within the subgroup, resulting in 100 groups in 10 subgroups and
100 couplings. For each subgroup of step 3, the five Baa.sub.xL
members and the five Baa.sub.xD members were separately pooled in
step 4, resulting in 20 subgroups, with each subgroup containing
five different sequences differing by the Baa.sub.x member. Each of
the 20 subgroups of step 4 were then in step 5 divided into 10
groups (with only one shown for illustration purposes in FIG. 5),
and for each subgroup, each of Aaa.sub.1L through Aaa.sub.5D were
coupled to one group within the subgroup, resulting in 200 groups
in 20 subgroups and 200 couplings. For each subgroup of step 5, the
five Aaa.sub.xL members and the five Aaa.sub.xD members were
separately pooled in step 6, resulting in 40 subgroups, with each
subgroup containing twenty-five different sequences differing by
the Baa.sub.x and Aaa.sub.x member. In step 7, each of the 40
subgroups of step 6 were divided into five groups, and each of
R.sub.1 through R.sub.5 were coupled to one group within the
subgroup, resulting in 200 groups in 40 subgroups, with each group
containing 25 different sequences differing by the Baa.sub.x and
Aaa.sub.x member.
[0119] Peptides were synthesized using Fmoc chemistry, with side
chain functionalities protected using add labile groups. The SH
group of the Cys residue was protected by a S.sup.tBu OSPG
cleavable in presence of both base and acid labile groups using
tributylphosphine as the reducing agent. The peptide chain was
assembled on the solid phase using
1-(1H-benzotriazole-1-yl)-1,1,3,3,-tetra-methyluronium
tetrafluoroborate (TBTU) as a coupling agent. The SH group was then
selectively unprotected and rhenium metal ion complexed using the
rhenium transfer agent Re(O)Cl.sub.3(PPh.sub.3).sub.2 in the
presence of 1,8-Diazabicyclo[5,4,0]- undec-7-ene (DBU) as base. In
this manner, the metal-peptide complex was formed with the peptide
chain still tethered to the solid support. The metallopeptide was
then liberated from the solid support by treatment with TFA. This
solid phase approach to metal ion complexation is fully compatible
with split synthesis methodologies employed in combinatorial
libraries.
[0120] The synthesis process was performed using commercial
automated synthesizers. Multiple manual synthesizers (such as those
commercially available from SynPep Corporation, Dublin, Calif.)
allow parallel synthesis of ten peptides simultaneously.
[0121] Quality control protocols were employed as required, and
include HPLC, mass spectral analysis, and amino acid analysis on
each individual pool of 25 compounds. The presence of each of pool
constituent is established by molecular ion mass spectral analysis.
Negative ion mode electron spray (ES) and matrix-assisted laser
desorption (MALDI) techniques were employed. Using mass spectral
analysis, three different measures were made: (a) the presence of
up to 25 individual compounds by molecular ion peak measurement
(assuming different masses for each compound), (b) confirmation
that the molecular ion peaks show complexation to a rhenium metal
ion, and (c) absence of peaks with molecular masses corresponding
to peptides uncomplexed with metal ion. Rhenium is a mixture of two
isotopes that differ in mass by 2 units (186 and 188) with a
relative abundance of these isotopes of 1:2. The molecular ion
profile of a metallopeptide appears as two peaks that differ by 2
mass units with integrated area ratios of 1:2. Rhenium thus acts as
an internal mass spectral reference for these metallopeptides. A
spectral analysis of one such pool of 25 compounds synthesized by
the methods of this claim is shown at FIG. 2. Five sets of two
metallopeptides in this pool have similar masses due to the
presence of the same amino acids assembled in different sequences.
The relative intensities of the peaks is due to differential
ionization of individual compounds in the pool and does not reflect
the relative amounts in the mixture. Each pair of peaks with mass
unit differences of 2 and relative ratios of 1:2 are due to the
relative abundance of two stable isotopes of rhenium (Re-185 and
Re-187). The spectral analysis did not reveal any free uncomplexed
linear peptides, which would be approximately 197 to 199 mass units
less than the corresponding metallopeptide, due to the absence of
the rhenium-oxo core.
[0122] Amino acid analysis of each pool of 25 metallopeptides was
also employed, and was used to determine the relative equimolar
ratio of each of 25 compounds in a pool. The synthetic protocols of
split synthesis were designed to assure equimolar amounts of pool
constituents.
EXAMPLE 7
Screening of Melanocortin Receptor-specific Library
[0123] Metallopeptide library pools are screened for MC-4 receptor
and MC1-R receptor binding activity in high throughput screening
assays. The MC receptor-binding assay uses membrane preparations
from B16-F1 melanoma cells as the source of MC receptor. Cell
membranes prepared from MC-4 receptor-expressing 293 cells and
negative control, untransfected 293 cells, are substituted for
B16-F1 melanoma cell membranes in MC-4 receptor specific binding
assays. The MC receptor-binding assays use the Millipore
Multi-Screen System and are performed in 96-well Millipore filter
plates (Durapore, 0.45 mm porosity) pre-blocked with 0.5% bovine
serum albumin in phosphate buffered saline. Cell membrane
preparations (12.5 .mu.g/well) are incubated with 0.4 nM
.sup.125I-NDP-MSH in HEPES Buffer containing 0.2% bovine serum
albumin. Non-specific binding is determined by addition of
10.sup.-6M .alpha.-MSH or 10.sup.-7M NDP-MSH. Metallopeptides to be
tested are added to reaction wells at a final concentration of 1
mM. After incubation for 90 min at room temperature, the binding
reaction is rapidly terminated by filtration to capture the
membranes. Filters are washed 3 times with ice-cold PBS and
air-dried. Individual filters are then punched from the plates and
distributed into gamma counter tubes. Radioactivity associated with
the membranes is determined in a Packard Cobra gamma counter.
Specific binding is determined as the radioactivity in wells
containing .sup.1251-NDP-MSH alone minus the radioactivity in wells
containing 10.sup.-6M .alpha.-MSH. Test compounds are screened in
duplicate wells and are considered to be active where 1 .mu.M
concentrations inhibit >50% of the specific binding. Standard
curves of unlabeled NDP-MSH will be included on each plate as an
internal assay control.
[0124] A commercially available cAMP kit (R&D Systems, DE0350,
low pH) is employed to evaluate agonist potential of
metallopeptides that bind to MC-4 receptor. 293 cells stably
transfected with hMC-4 receptor, or B16-F1 melanoma cells, are
grown to confluence in 96-well dishes. Cells are washed and fresh
RPMI containing 0.2 mM isobutylmethylxanthine (CAMP
phosphodiesterase) and varying concentrations of metallopeptides,
or .alpha.-MSH as a positive control, are added, and the cells are
incubated for 1 hr at 37.degree. C. Medium is aspirated, and cell
layers extracted with 150 .mu.l of 0.1 M HCl. Total CAMP
accumulation in 100 .mu.l of cell extract is quantitated in 96-well
plates by competitive immunoassay with the CAMP kit, using an
acetylation modification. EC.sub.50 values for test compounds will
be calculated based on CAMP accumulation in cells treated with
10.sup.-6M .alpha.-MSH. The capabilities of both of these cell
types to accumulate cAMP in the presence of .alpha.-MSH and MSH
analog peptides are documented in the scientific literature cited
in Example 1 and Oilman MM et al: Science 278:135-138, 1997.
EXAMPLE 8
Deconvolution of Melanocortin Receptor-specific Library
[0125] Deconvolution of a positive pool is done by iterative
re-synthesis and screening deconvolution approaches. The individual
25 constituents are synthesized separately, or alternatively in 5
smaller pools of 5 compounds each, with each pool screened in
receptor binding assays. The latter approach is preferred where
there is a high hit frequency in the preliminary screen. The
compounds in pools with the best results (closest to receptor
affinity in the nanomolar range and MC-4 to MC-1 receptor
selectivity of at least 100) are individually synthesized and
screened.
EXAMPLE 9
Alternative Method of Deconvolution of Melanocortin
Receptor-specific Library
[0126] In this example, an alternative method of mass spectral
deconvolution of metallopeptide libraries is employed. The method
is based on the internal signature of rhenium-complexed peptides
(two isotopic peaks in 1:2 ratios differing by 2 mass units), which
generally permits metallopeptide identification even in mixed
solutions. A positive pool is incubated with receptor-bearing
cells, the excess unbound compounds washed away under controlled
conditions, and the cells treated with a solvent to disrupt
metallopeptide binding and extract the metallopeptide in the
solvent. Mass spectral analysis of the solvent reveals the
metallopeptide or metallopeptides which are bound to the
receptor-bearing cells, and through comparison to the quality
control data it is possible to ascertain the specific
metallopeptide or metallopeptides which are bound. This process
provides high throughput of metallopeptide library screening.
EXAMPLE 10
Single Pot Synthesis of a Library of Four Metallopeptides of the
General Structure Ac-His-Xaa-Cys-Trp-NH.sub.2
[0127] A synthesis procedure similar to that described in Example 1
was used in making this library. A NovaSyn TGR resin for making
peptide amides (substitution 0.2 mM/gm) was used. Fmoc synthetic
strategy was employed using the following protected amino acids:
Fmoc-Trp(Boc), Fmoc-Cys(SBu.sup.t), Fmoc-Xxx, and Fmoc-His(Trityl).
The Xaa amino acids were Trp, HomoPhe, 2'-Naphthylalanine, and
Phenylglycine. The peptide resin Cys(Bu.sup.t)-Trp-NH.sub.2 was
split into four equal pools and one of the Xaa amino acids was
coupled to one individual pool. After completion of the coupling
reaction, the four resin pools were mixed again. The synthesis
proceeded with the coupling of His followed by acetylation of the
N-terminus. After the complete assembly of the peptide chain
Ac-His(Trt)-Xaa-Cys(S-Bu.sup.t)-Trp(Boc)-NH.sub.2, the S-Bu.sup.t
OSPG group was removed by treatment with DMF/tributylphosphine and
rhenium-oxo metal ion was complexed as generally described in
Example 1. The fully protected metallopeptide was deblocked and
liberated from the solid support by treatment with a cleavage
cocktail (95:5 mixture of trifluoroacetic acid--triisopropylsilane)
for three hours. The metallopeptide library was recovered by
precipitation using cold ether. The resulting pellet was washed
twice and 0.5 ml of 95% acetic acid was added. After one-half hour
5 ml of water was added and the solution was freeze-dried yielding
the desired library in solid form. Mass spectrometric analysis of
the library pool confirmed the correct masses for all four members
of the library:
1 Compound Structure Calculated Mass Mass (M + 1) found 1
Ac-His-Phg- 815.7 and 817.6 815.2 and 816.7 Cys-Trp-NH.sub.2 2
Ac-His-Trp- 868.8 and 870.7 868.0 and 870.1 Cys-Trp-NH.sub.2 3
Ac-His-HPhe- 843.8 and 845.7 842.8 and 845.2 Cys-Trp-NH.sub.2 4
Ac-His-2'Nal- 880.0 and 881.9 879.1 and 880.9 Cys-Trp-NH.sub.2
[0128] As noted in the table, two molecular ion peaks differing in
mass units of 2 were calculated and observed for each structure;
this difference is presumptively due to the presence of two natural
isotopes of rhenium, Re-185 and Re-187, in the complexation step.
In addition, the area under the observed peaks in the spectrometric
analysis showed that for each structure the area was in a 1:2
ratio, which is identical to and presumptively related to the
relative abundance of Re-185 and Re-187 isotopes. These results
confirmed the complexation of rhenium to the peptides. FIG. 3
depicts the mass spectrum of a library pool of 4 metallopeptides.
The relative intensities of these peaks is due to the differential
ionization of individual compounds in the pool and does not reflect
the relative amounts in the pool. The spectral analysis did not
reveal any free uncomplexed linear peptides, which would be
approximately 197 to 199 mass units less than the corresponding
metallopeptide, due to the absence of the rhenium-oxo core. FIG. 3
depicts reversed phased HPLC profiles of a library pool of 4
metallopeptides. The pool is shown in FIG. 4A, and each of the
individual peptides is shown in FIGS. 4B through 4E. Each
individual peak in FIGS. 4B through 4E matched HPLC profiles in the
pool in FIG. 3A, showing the presence of each of the four compounds
in the pool. Each individual peptide is resolved into two isomeric
peaks (syn- and anti-isomers) that are due to two alternate
orientations of the oxygen atom in the Re.dbd.O core. All four
compounds used for this comparison were individually prepared using
the methods described above for synthesis of the library. The HPLC
profiles of the individual compounds are shown as FIGS. 4A to
4E.
EXAMPLE 11
Synthesis of a Small Multi-pool Library Targeted for Human
Neutrophil Elastase of the General Structure
R-Aaa-Bbb-Cys-Val-NH.sub.2
[0129] The library was synthesized as a 60-member library in 12
pools of 5 metallopeptides each. In each of the 12 pools only Aaa
was randomized using five different structural variants, while both
R and Bbb were constant for that pool. The R group was a N-terminal
capping group selected from benzyloxycarbonyl, acetyl and succinyl
groups. Aaa was selected from five different residues, Ala, Leu,
lie, Phe, and Lys(N.sup..epsilon.-Z). Bbb was selected from Gly,
Ala, Ser, and Asn. The structure of the metallopeptides in these
pools is described in the Example 12.
[0130] All 12 metallopeptide pools were synthesized using the
general strategy and methodology described in Examples 1, 6, and 9.
Each of the pools was characterized by mass spectrometry, and shown
to have the five correct masses for rhenium-complexed peptides
(with two isotopic peaks in ratio of 1:2 differing by mass units of
2). Additionally the mass spectra did not show any peaks related to
linear peptides not complexed to a metal ion, signifying all or
substantially all of the peptide was complexed to rhenium ions.
EXAMPLE 12
Screening of 60-membered Multi-pool Library of the General
Structure R-Aaa-Bbb-Cys-Val-NH.sub.2 Targeted for Human Neutrophil
Elastase
[0131] Purified human neutrophil elastase (HNE) was purchased from
Sigma (St. Louis, Mo.). The assay was performed
spectrophotometrically using a chromogenic p-nitroanilide
substrate, MeOSuc-Ala-Ala-Pro-Val-pNA, also purchased from Sigma.
The assay protocols were similar to those described by others
(e.g., Nakajima K et al: J Biol Chem, 254:4027-4032, 1979).
Briefly, 0.05 units of HNE were treated with 1 mM concentration of
the library pool in the presence of p-nitroanilide substrate.
Spectrophotometric measurement were made at a wavelength of 410 nm.
The results of the assay are shown in the following table. The
screening results revealed that only three out of the 12 pools
displayed inhibition below an assigned assay cutoff of 1 mM. Also,
among this limited series of compounds, benzyloxycarbonyl group was
identified as a preferred R group where Bbb was a Gly, Ala, or Ser
residue.
[0132] The following shows the general structure ReO
--[R-Aaa-Bbb-Cys-Val-NH.sub.2]:
2 5 The following table shows the structure of the 60 members of
the 12 pool library, where Z is benzyloxycarbonyl, Ac is acetyl and
Suc is succinyl: Compounds per Pool R Aaa is one of: Bbb IC.sub.50
(.mu.M) 5 Z Ala, Leu, Ile, Phe, Lys(N.epsilon.-Z) Ser 385 5 Z Ala,
Leu, Ile, Phe, Lys(N.epsilon.-Z) Ala 625 5 Z Ala, Leu, Ile, Phe,
Lys(N.epsilon.-Z) Asn >1000 5 Z Ala, Leu, Ile, Phe,
Lys(N.epsilon.-Z) Gly 769 5 Ac Ala, Leu, Ile, Phe,
Lys(N.epsilon.-Z) Ser >1000 5 Suc Ala, Leu, Ile, Phe,
Lys(N.epsilon.-Z) Ser >1000 5 Ac Ala, Leu, Ile, Phe,
Lys(N.epsilon.-Z) Ala >1000 5 Suc Ala, Leu, Ile, Phe,
Lys(N.epsilon.-Z) Ala >1000 5 Ac Ala, Leu, Ile, Phe,
Lys(N.epsilon.-Z) Asn >1000 5 Suc Ala, Leu, Ile, Phe,
Lys(N.epsilon.-Z) Asn >1000 5 Ac Ala, Leu, Ile, Phe,
Lys(N.epsilon.-Z) Gly >1000 5 Suc Ala, Leu, Ile, Phe,
Lys(N.epsilon.-Z) Gly 1000
EXAMPLE 13
Deconvolution of Library Pool of the General Structure
Z-Aaa-Ser-Cys-Val-NH2 Targeted for Human Neutrophil Elastase
[0133] pool with highest activity, Z-Aaa-Ser-Cys-Val-NH.sub.2, was
deconvoluted by an iterative process. All five individual peptides
in this pool were synthesized by the methodologies and Example 10.
Each individual peptide was characterized by mass spectrometry and
HPLC. assay was performed as generally described in Example 11. The
best results were obtained with Leu or lie in the position Aaa in
the peptide chain. The peptide Z-Leu-Ser-Cys-Val-NH.sub.2 displayed
an IC.sub.50 value of 139 .mu.M and the peptide
Z-lle-Ser-Cys-Val-NH.sub.2 displayed an IC.sub.50 value of 179
.mu.M. Substitution of the three residues Ala, Phe, and
Lys(NE.sup..epsilon.-Z) at Aaa yielded IC.sub.50 values in excess
of 1,000 .mu.M. Based on these results, the two peptides
Z-Leu-Ser-Cys-Val-NH.sub.2 and Z-lle-Ser-Cys-Val-NH.sub.2 appeared
to meet stereochemical requirements as an HNE inhibitor. The
results of this assay, where Z is benzyloxycarbonyl, are shown in
the following table.
3 R Aaa Bbb IC.sub.50 (.mu.M) Z Ala Ser >1000 Z Leu Ser 139 Z
Ile Ser 179 Z Phe Ser >1000 Z Lys(N.epsilon.-Z) Ser >1000
EXAMPLE 13
Synthesis of --SH Containing Building Blocks from Halogenated
Compounds
[0134] A variety of "S" containing building blocks (other than
amino acids) for use in the synthesis of libraries according to
this invention can be synthesized from corresponding halogenated
congeners. The process relies on the treatment of a halogenated
compound with sodium thiosulfate at slightly basic pH to obtain the
corresponding Bunte salt (the S-sulfonate derivative) as described
by Wunderlin R et al: HeIv Chim Acta 68:12-22, 1985. The Bunte salt
S-sulfonate derivative is treated with a reducing agent such as
2-mercaptoethane, sodium borohydride or tributyl phosphine to yield
a free thiol-containing product. This product can then be
S-protected with conventional S-protecting groups such as Trt, mmt,
Npys, Bu.sup.t, S-Bu.sup.t known in the art of peptide synthesis.
Alternatively, and preferably in this invention, the Bunte salt
S-sulfonate derivative may be directly used in the synthesis of
peptides as described by Maugras I et al: Int J Peptide Protein Res
45:152, 1995. The S-sulfonate derivative is stable using either
Fmoc or Boc synthetic peptide synthesis methods, and following
peptide synthesis the resulting peptide may be treated with
tributyl phosphine to liberate the free --SH group. In either
method, the resulting peptide has an available free thiol for
complexation with a metal ion, which complexation may be according
to either the solid phase or solution phase methods described
above.
EXAMPLE 14
Synthesis of Reduced Peptide Bond Metallopeptide Library
[0135] Metallopeptide libraries may be synthesized wherein the
library members contain either a reduced peptide bond or an
N-substituted reduced peptide bond, such that the library members
contain a CH.sub.2--NH group or CH.sub.2--NR group rather than a
CO--NH peptide bond. Examples of these structures are given at
FIGS. 1A, 1D, 1E, 1H and 1J. The synthetic methods employed are
similar to those for metallopeptides containing solely peptide
bonds. The compounds are produced by the methods of this invention
using either solution phase or solid phase synthesis. In general,
the synthetic strategy involves assembling a linear peptide
derivative incorporating a reduced peptide bond, as is generally
discussed in Wen J and Spatola A F: J Peptide Res 49:3-14, 1997. In
the synthesis strategy, an orthogonal S-protecting group is
employed that is compatible with the reductive alkylation step
during the introduction of a pseudopeptide bond. For example, a Mmt
(4-methoxytritryl) group may be used for S-protection during Fmoc
synthetic strategy. The Mmt group is released upon treatment with
1% TFA in dichloromethane without cleaving the peptide from the
resin, such that solid phase metal ion complexation may be
employed. As necessary, means to provide orthogonal protection of
the amine functionality of the reduced peptide bond (CH.sub.2NH)
generated during the introduction of the reduced peptide bond may
also be employed. One group that may be employed to provided
orthogonal protection of the amine functionality is the Dde group
[1-(4,4-Dimethylaminophenylazo)benzoyl] which is stable during both
Fmoc and Boc cleavage conditions and is selectively removed by
treatment with 2% hydrazine in DMF for 2-5 minutes. Using such
protecting groups, a fully assembled peptide is treated first with
hydrazine to remove Dde groups and then with 1% TFA in
dichloromethane to remove Mmt group from the peptide on the resin.
The resulting sequence may then be complexed to a metal ion while
on solid phase, and thereafter cleaved from the resin as described
above.
[0136] Alternatively, the amine protection on the reduced peptide
bond CH.sub.2NH group is a Boc group and the fully assembled
peptide is deprotected and liberated from the resin in a single
step upon treatment with TFA. Metal complexation can then be
accomplished in solution as described above.
[0137] Library members containing an N-derivatized functionality
such as CH.sub.2--NR group rather than a CO--NH peptide bond, as
shown at FIGS 1A, 1D, 1E, 1H and 1J, may be synthesized by
assembling a linear peptide with S-Mmt protection and a CH.sub.2NH
amine which is N protected using a Dde group. The Ddd is then
selectively removed by the treatment with hydrazine in DMF. The
liberated amine is then functionalized by attachment of an R group
through treatment with the corresponding halogenated derivative of
the R group in the presence of a base. Following removal of the
S-Mmt group by treatment with 1% TFA in dichloromethane, the
resulting library member may be complexed with a metal ion as
described above. Alternatively, after N-functionalization, the
resulting library member can be deprotected and released cleaved
from the resin in one step and the metal complexation accomplished
in solution as described in above.
EXAMPLE 15
Synthesis of Library Members Providing S.sub.2N.sub.2 Metal Ion
Complexation
[0138] Libraries containing members with S.sub.2N.sub.2 metal ion
coordination, as shown in FIGS. 1C, 1E, 1I and 1J, may be
synthesized by means similar to those employed with libraries
containing members with S.sub.1N.sub.3 metal ion coordination. In
one method, a second "S" is introduced in the structure by
incorporating into the linear assembly of the peptide an
S-protected derivative of 2-mercaptoacetic acid or Cys. The choice
of S-protecting groups is guided by selection of orthogonal
protection criteria described above. After the assembly of the
linear peptide, the protecting groups from both of the "S" atoms is
removed and metal ion complexation is accomplished as generally
described above.
[0139] Alternatively, S-deprotection, and removal of any other
protecting group, may be accomplished concomitantly with the
cleavage of the peptide from the resin in a single step. The metal
ion complexation is then achieved in solution as described
above.
EXAMPLE 16
Synthesis of Rhenium[V] Nitrido Complexes
[0140] The synthesis of rhenium nitrido compounds is similar to
that for corresponding rhenium oxo complexes. The use of
Re(N)Cl.sub.2(PPh.sub.3).- sub.2 transfer agent (which may be
synthesized as described by Sullivan B P et al: Inorg Syntheses
29:146-150, 1992) instead of Re(O)Cl.sub.3(PPh.sub.3).sub.2 yields
the corresponding nitrido complexes. As described above for rhenium
oxo complexes, the corresponding nitrido compounds can be prepared
either on solid phase or in solution phase. Since metal
complexation is the last step in the synthesis, the assembly of
linear peptide chain library members is identical to the methods
described for rhenium oxo complexes. Nitrido compounds can
alternatively be prepared directly from a rhenium oxo compound by
treating the rhenium oxo compound with substituted hydrazines.
EXAMPLE 17
Synthesis of Rhenium Organoimido Complexes
[0141] The synthesis of rhenium organoimido complexes is achieved
in several different methods. In one method, treatment of a rhenium
oxo complex with a primary amine converts it to an organoimido
complex. Primarily aromatic organoimido derivatives may prepared by
this method. In an alternative method, treatment of rhenium oxo
complexes with 1,2 disubstituted hydrazine results in the
corresponding organoimido complex. Both aromatic and aliphatic
organoimido complexes can be prepared in this manner. In yet
another alternative method, reaction of rhenium oxo complexes with
phosphinimines (Ph.sub.3P.dbd.NR) yields the corresponding
organoimido complexes. Besides aliphatic and aromatic organoimido
complexes, this method is effective in producing aroylimido
complexes (R.dbd.PhCO). In yet another alternative method,
treatment of rhenium oxo complexes with phenyl ioscyanate yields
the corresponding phenyl organoimido complex.
EXAMPLE 18
Site Specific Complexation of Metal Ion to a Peptide Chain by
Orthogonal Protection of Sulfydryl Groups in a Peptide Sequence
[0142] Where two or more sulfhydryl groups are desired in a
peptide, OSPGs may be employed, together with other S-protecting
groups, such that the desired thiol group may be selectively
deprotected while leaving the other thiols protected. The metal ion
is complexed to the deprotected thiol and subsequent to metal ion
complexation the remaining protecting groups are liberated to yield
the desired metallo-peptide complex.
[0143] A fully protected peptide-resin containing two S-protected
thiol groups (Cys residues),
Fmoc-Tyr(Bu.sup.t)-Cys(Trt)-Gly-Phe-Cys(S.sup.tBu)- -Wang resin,
was prepared by standard solid-phase peptide synthesis means. The
N-terminal Fmoc-group was removed by treatment with 20% piperidine
in DMF to give the resin
Tyr(Bu.sup.t)-Cys(Trt)-Gly-Phe-Cys(S.sup.tBu)-Wang resin. In this
case, the Cys(Trt) group and the Cys(S.sup.tBu) protecting groups
are orthogonal to each other. While the Cys(Trt) is an acid labile
group, the Cys(S.sup.tBu) group may be removed under reductive
conditions, thereby allowing selective deprotection of one thiol
group. The peptide-resin was treated with tributylphosphine (0.52
M) in DMF to remove the S-S.sup.tBu group from the Cys residue. The
resin was then treated with Re(O)Cl.sub.3(PPh.sub.3).sub.2 (8 eq.)
in the presence of DBU as base for 4 hours at room temperature to
complete the formation of the ReOM complex with the peptide. The
peptide resin was washed extensively, dried and treated with
TFA/TIS (95:5) cleavage cocktail to yield the metallo-peptide. The
metallo-peptide was precipitated using MeOH-ether, and the product
was dried and purified by HPLC. The purified peptide was analyzed
by electron spray mass spectrometry, yielding predicted mass for
the metallo-peptide complex.
EXAMPLE 19
Synthesis of a Metallo-peptide Library Containing Two "S" Groups
but Utilizing only One "S" For Site Specific Complexation of Metal
Ion to a Peptide Chain by Orthogonal Protection of Sulfydryl Group
a in the Peptide Sequences. Synthesis of a Library with General
Structure Tyr-Cys-[Aaa-Phe-Cys]-ReO[V]
[0144] A library of metallo-peptides containing two "S" capable of
complexing with ReO[V] core, but directing this bond formation with
only one of these two "S" atoms can be synthesized as set forth
above. Fully protected peptide resin
Fmoc-Tyr(Bu.sup.t)-Cys(Trt)-Aaa-Phe-Cys(S.sup.tBu- )-Wang resin is
prepared by solid-phase methods of peptide synthesis. The "Aaa" is
an alpha amino amino acid, synthetic or naturally occurring in
either L- or D-isomeric form, as may be desired for the composition
of individual library members. Individual compounds with different
Aaa group may be synthesized in parallel for the synthesis of a
parallel library of compounds. Alternatively, all compounds may be
synthesized as a mixture in one pot using different "Aaa" building
blocks at the coupling step for Aaa. Alternatively, the library
mixture may also be synthesized by a split and pool approach as
described above using various Aaa groups.
[0145] The finished peptide-resin is treated with 20% piperidine to
remove the terminal Fmoc group. Treatment of the
Tyr(Bu.sup.t)-Cys(Trt)-Gly-Phe-- Cys(S.sup.tBu)-Wang resin thus
obtained with tributylphosphine and Re(O)Cl.sub.3(PPh.sub.3).sub.2
in the presence of DBU can be accomplished as described above to
complex the ReO metal ion specifically at the C-terminal Cys
residue while leaving the Cys(Trt) residue inert. Cleavage of the
metallo-peptide resin may be accomplished as described above, and
the Cys(Trt) group may be thereafter deprotected.
[0146] Each of the foregoing is merely illustrative, and other
equivalent embodiments are possible and contemplated.
[0147] Although this invention has been described with reference to
these preferred embodiments, other embodiments can achieve the same
results. Variations and modifications of the present invention will
be obvious to those skilled in the art and it is intended to cover
in the appended claims all such modifications and equivalents. The
entire disclosures of all applications, patents, and publications
cited above are hereby incorporated by reference.
[0148] The preceding examples can be repeated with similar success
by substituting the generically or specifically described reactants
and/or operating conditions of this invention for those used in the
preceding examples.
* * * * *