U.S. patent application number 11/482638 was filed with the patent office on 2007-05-31 for beta-amino acids.
Invention is credited to Daniel H. Appella, Samuel H. Gellman, Hee-Seung Lee, Paul LePlae, Emilie Porter, Xifang Wang, Matthew Woll.
Application Number | 20070123709 11/482638 |
Document ID | / |
Family ID | 27364677 |
Filed Date | 2007-05-31 |
United States Patent
Application |
20070123709 |
Kind Code |
A1 |
Gellman; Samuel H. ; et
al. |
May 31, 2007 |
Beta-amino acids
Abstract
Disclosed are .beta.-amino acid monomers containing cylcoalkyl,
cycloalkenyl, and heterocylic substituents which encompass the
.alpha. and .beta. carbons of the peptide backbone and
.beta.-polypeptides made from such monomers. Method of generating
combinatorial libraries of polypeptides containing the
.beta.-peptide residues and libraries formed thereby are
disclosed.
Inventors: |
Gellman; Samuel H.;
(Madison, WI) ; Appella; Daniel H.; (Evanston,
IL) ; Lee; Hee-Seung; (Madison, WI) ; LePlae;
Paul; (Madison, WI) ; Porter; Emilie;
(Madison, WI) ; Wang; Xifang; (Madison, WI)
; Woll; Matthew; (Madison, WI) |
Correspondence
Address: |
DEWITT ROSS & STEVENS S.C.;WISCONSIN ALUMNI RESEARCH FOUNDATION
8000 EXCELSIOR DRIVE
# 401
MADISON
WI
53717
US
|
Family ID: |
27364677 |
Appl. No.: |
11/482638 |
Filed: |
July 7, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09833496 |
Apr 11, 2001 |
|
|
|
11482638 |
Jul 7, 2006 |
|
|
|
09464212 |
Dec 15, 1999 |
6613876 |
|
|
09833496 |
Apr 11, 2001 |
|
|
|
09034509 |
Mar 4, 1998 |
6060585 |
|
|
09464212 |
Dec 15, 1999 |
|
|
|
60039905 |
Mar 4, 1997 |
|
|
|
Current U.S.
Class: |
546/223 ;
548/530 |
Current CPC
Class: |
C07D 211/62 20130101;
C07C 2601/14 20170501; C07C 237/24 20130101; C07B 2200/11 20130101;
C07D 211/60 20130101; C07D 207/16 20130101 |
Class at
Publication: |
546/223 ;
548/530 |
International
Class: |
C07D 211/56 20060101
C07D211/56; C07D 207/337 20060101 C07D207/337 |
Claims
1. A .beta.-amino acid selected from the group consisting of
Formula I: ##STR41## wherein X and Y combined, together with the
carbon atoms to which they are bonded, define a substituted or
unsubsituted C.sub.4-C.sub.8 cycloalkyl, cycloalkenyl or
heterocyclic ring having one or more nitrogen atoms as the sole
heteroatom; the substituents on carbon atoms of the rings being
independently selected from the group consisting of linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, or alkynyl; mono- or
bicyclic aryl, mono- or bicyclic heteroaryl having up to 5
heteroatoms selected from N, O, and S; mono- or bicyclic
aryl-C.sub.1-C.sub.6-alkyl, mono- or bicyclic
heteroaryl-C.sub.1-C.sub.6-alkyl, --(CH.sub.2).sub.n+1--OR.sup.4,
--(CH.sub.2).sub.n+1--SR.sup.4,
--(CH.sub.2).sub.n+1--S(.dbd.O)--CH.sub.2--R.sup.4,
--(CH.sub.2).sub.n+1--S(.dbd.O).sub.2--CH.sub.2--R.sup.4,
--(CH.sub.2).sub.n+1--NR.sup.4R.sup.4,
--(CH.sub.2).sub.n+1--NHC(.dbd.O)R.sup.4,
--(CH.sub.2).sub.n+1--NHS(.dbd.O).sub.2--CH.sub.2--R.sup.4,
--(CH.sub.2).sub.n+1--O--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--S--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--S(.dbd.O)--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--S(.dbd.O).sub.2--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--NH--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--N--{(CH.sub.2).sub.m--R.sup.5}.sub.2,
--(CH.sub.2).sub.n+1--NHC(.dbd.O)--(CH.sub.2).sub.n+1--R.sup.5, and
--(CH.sub.2).sub.n+1--NHS(.dbd.O).sub.2--(CH.sub.2).sub.m--R.sup.5;
wherein R.sup.4 is independently selected from the group consisting
of hydrogen, C.sub.1-C.sub.6-alkyl, alkenyl, or alkynyl; mono- or
bicyclic aryl, mono- or bicyclic heteroaryl having up to 5
heteroatoms selected from N, O, and S; mono- or bicyclic
aryl-C.sub.1-C.sub.6-alkyl, mono- or bicyclic
heteroaryl-C.sub.1-C.sub.6-alkyl; and wherein R.sup.5 is selected
from the group consisting of hydroxy, C.sub.1-C.sub.6-alkyloxy,
aryloxy, heteroaryloxy, thio, C.sub.1-C.sub.6-alkylthio,
C.sub.1-C.sub.6-alkylsulfinyl, C.sub.1-C.sub.6-alkylsulfonyl,
arylthio, arylsulfinyl, arylsulfonyl, heteroarylthio,
heteroarylsulfinyl, heteroarylsulfonyl, amino, mono- or
di-C.sub.1-C.sub.6-alkylamino, mono- or diarylamino, mono- or
diheteroarylamino, N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino,
N-aryl-N-heteroarylamino, aryl-C.sub.1-C.sub.6-alkylamino,
carboxylic acid, carboxamide, mono- or
di-C.sub.1-C.sub.6-alkylcarboxamide, mono- or diarylcarboxamide,
mono- or diheteroarylcarboxamide, N-alkyl-N-arylcarboxamide,
N-alkyl-N-heteroarylcarboxamide, N-aryl-N-heteroarylcarboxamide,
sulfonic acid, sulfonamide, mono- or
di-C.sub.1-C.sub.6-alkylsulfonamide, mono- or diarylsulfonamide,
mono- or diheteroarylsulfonamide, N-alkyl-N-arylsulfonamide,
N-alkyl-N-heteroarylsulfonamide, N-aryl-N-heteroarylsulfonamide,
urea; mono- di- or tri-substituted urea, wherein the subsitutent(s)
is selected from the group consisting of C.sub.1-C.sub.6-alkyl,
aryl, heteroaryl; O-alkylurethane, O-arylurethane, and
O-heteroarylurethane; and m is an integer of from 2-6 and n is an
integer of from 0-6; the substituents on heteroatoms of the ring
being independently selected from the group consisting of
--S(.dbd.O).sub.2--CH.sub.2--R.sup.4
--C(.dbd.O)--R.sup.4--S(.dbd.O).sub.2--(CH.sub.2).sub.m--R.sup.5,
and --C(.dbd.O)--(CH.sub.2).sub.n+1--R.sup.5; wherein R.sup.4 and
R.sup.5 are as defined hereinabove, and m is an integer of from 2-6
and n is an integer of from 0-6; provided that when X & Y
together with the carbons to which they are bonded define a five-
or six-membered cycloalkyl or a five-membered heterocyclic ring
having one nitrogen as the sole heteroatom, and the nitrogen is
bonded to a carbon atom adjacent to the carboxy carbon of Formula
I, the cycloalkyl or heterocyclic ring is substituted; R.sup.1 is
selected from the group consisting hydrogen and an amino protecting
group; R.sup.2 is selected from the group consisting of hydrogen
and a carboxy protecting group; racemic mixtures thereof, isolated
or enriched enantiomers thereof; isolated or enriched diastereomers
thereof; and salts thereof.
2. The .beta.-amino acid according to claim 1, wherein X and Y
combined, together with the carbon atoms to which they are bonded,
define a moiety selected from the group consisting of a substituted
cycloalkyl, a substituted or unsubstituted C.sub.4-C.sub.6
cycloalkenyl, and a substituted or unsubstituted heterocyclic ring
having one nitrogen atom as the sole hetero atom.
3. The .beta.-amino acid according to claim 1, wherein X and Y
combined, together with the carbon atoms to which they are bonded,
define a substituted or unsubstituted cyclopentenyl, cyclohexenyl,
pyrrolidinyl, or piperidinyl ring.
4. The .beta.-amino acid according to claim 1, wherein X and Y
combined, together with the carbon atoms to which they are bonded,
define a substituted cyclopentyl, cyclohexyl, cyclopentenyl,
cyclohexenyl, pyrrolidinyl, or piperidinyl ring, wherein the
substituent is selected from the group consisting of amino, mono-
or di-C.sub.1-C.sub.6-alkylamino, carboxamido, sulfonamido, urea,
thio, and C.sub.1-C.sub.6-alkylthio.
5. The .beta.-amino acid according to claim 1, wherein X and Y
combined, together with the carbon atoms to which they are bonded,
define an amino-substituted cyclopentyl, cyclohexyl, cyclopentenyl,
amino-substituted cyclohexenyl, amino-substituted pyrrolidinyl, or
amino-substituted piperidinyl ring.
6. A .beta.-amino acid selected from the group consisting of:
##STR42## R.sup.1 is selected from the group consisting hydrogen
and an amino protecting group; R.sup.2 is selected from the group
consisting of hydrogen and a carboxy protecting group; and when
R.sup.3 is bonded to a carbon atom, R.sup.3 is selected from the
group consisting of hydrogen, hydroxy, linear or branched
C.sub.1-C.sub.6-alkyl, alkenyl, or alkynyl; mono- or bicyclic aryl,
mono- or bicyclic heteroaryl having up to 5 heteroatoms selected
from N, O, and S; mono- or bicyclic aryl-C.sub.1-C.sub.6-alkyl,
mono- or bicyclic heteroaryl-C.sub.1-C.sub.6-alkyl,
--(CH.sub.2).sub.n+1, --OR.sup.4, --(CH.sub.2).sub.n+1--SR.sup.4,
--(CH.sub.2).sub.n+1--S(.dbd.O)--CH.sub.2--R.sup.4,
--(CH.sub.2).sub.n+1--S(.dbd.O).sub.2--CH.sub.2--R.sup.4,
--(CH.sub.2).sub.n+1--NR.sup.4R.sup.4,
--(CH.sub.2).sub.n+1--NHC(.dbd.O)R.sup.4,
--(CH.sub.2).sub.n+1--NHS(.dbd.O).sub.2--CH.sub.2--R.sup.4,
--(CH.sub.2).sub.n+1--O--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--S--(CH.sub.2).sub.mR.sup.5,
--(CH.sub.2).sub.n+1--S(.dbd.O)--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--S(.dbd.O).sub.2--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--NH--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--N--{(CH.sub.2).sub.m--R.sup.5}.sub.2,
--(CH.sub.2).sub.n+1--NHC(.dbd.O)--(CH.sub.2).sub.n+1--R.sup.5, and
--(CH.sub.2).sub.n+1--NHS(.dbd.O).sub.2--(CH.sub.2).sub.m--R.sup.5;
wherein R.sup.4 is independently selected from the group consisting
of hydrogen, C.sub.1-C.sub.6-alkyl, alkenyl, or alkynyl; mono- or
bicyclic aryl, mono- or bicyclic heteroaryl having up to S
heteroatoms selected from N, O, and S; mono- or bicyclic
aryl-C.sub.1-C.sub.6-alkyl, mono- or bicyclic
heteroaryl-C.sub.1-C.sub.6-alkyl; and wherein R.sup.5 is selected
from the group consisting of hydroxy, C.sub.1-C.sub.6-alkyloxy,
aryloxy, heteroaryloxy, thio, C.sub.1-C.sub.6-alkylthio,
C.sub.1-C.sub.6-alkylsulfinyl, C.sub.1-C.sub.6-alkylsulfonyl,
arylthio, arylsulfinyl, arylsulfonyl, heteroarylthio,
heteroarylsulfinyl, heteroarylsulfonyl, amino, mono- or
di-C.sub.1-C.sub.6-alkylamino, mono- or diarylamino, mono- or
diheteroarylamino, N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino,
N-aryl-N-heteroarylamino, aryl-C.sub.1-C.sub.6-alkylamino,
carboxylic acid, carboxamide, mono- or
di-C.sub.1-C.sub.6-alkylcarboxamide, mono- or diarylcarboxamide,
mono- or diheteroarylcarboxamide, N-alkyl-N-arylcarboxamide,
N-alkyl-N-heteroarylcarboxamide, N-aryl-N-heteroarylcarboxamide,
sulfonic acid, sulfonamide, mono- or
di-C.sub.1-C.sub.6-alkylsulfonamide, mono- or diarylsulfonamide,
mono- or diheteroarylsulfonamide, N-alkyl-N-arylsulfonamide,
N-alkyl-N-heteroarylsulfonamide, N-aryl-N-heteroarylsulfonamide,
urea; mono- di- or tri-substituted urea, wherein the subsitutent(s)
is selected from the group consisting of C.sub.1-C.sub.6-alkyl,
aryl, heteroaryl; O-alkylurethane, O-arylurethane, and
O-heteroarylurethane; and m is an integer of from 2-6 and n is an
integer of from 0-6; and when R.sup.3 is bonded to a nitrogen atom,
R.sup.3 is independently selected from the group consisting of
those listed above for when R.sup.3 is attached to a carbon atom,
and further selected from the group consisting of
--S(.dbd.O).sub.2--CH.sub.2--R.sup.4,
--C(.dbd.O)--R.sup.4--S(.dbd.O).sub.2--(CH.sub.2).sub.mR.sup.5, and
--C(.dbd.O)--(CH.sub.2).sub.n+1--R.sup.5; wherein R.sup.4 and
R.sup.5 are as defined hereinabove, and m is an integer of from 2-6
and n is an integer of from 0-6; provided that when the
.beta.-amino acid is of formula ##STR43## R.sup.3 is not hydrogen;
racemic mixtures thereof, isolated or enriched enantiomers thereof;
isolated or enriched diastereomers thereof; and salts thereof.
7. The .beta.-amino acid according to claim 6, wherein R.sup.3 is
selected from the group consisting of hydrogen, hydroxy, linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, alkynyl,
hydroxy-C.sub.1-C.sub.6-alkyl, amino-C.sub.1-C.sub.6-alkyl,
C.sub.1-C.sub.6-alkyloxy,
C.sub.1-C.sub.6-alkyloxy-C.sub.1-C.sub.6-alkyl, amino, and mono- or
di-C.sub.1-C.sub.6-alkylamino.
8. The .beta.-amino acid according to claim 6, wherein R.sup.3 is
selected from the group consisting of hydrogen, hydroxy, linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, alkynyl, and
hydroxy-C.sub.1-C.sub.6-alkyl.
9. The .beta.-amino acid according to claim 6, selected from the
group consisting of: ##STR44## wherein R.sup.1, R.sup.2 and R.sup.3
are as defined in claim 6.
10. The .beta.-amino acid according to claim 9, wherein R.sup.3 is
selected from the group consisting of hydrogen, hydroxy, linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, alkynyl,
hydroxy-C.sub.1-C.sub.6-alkyl, amino-C.sub.1-C.sub.6-alkyl,
C.sub.1-C.sub.6-alkyloxy,
C.sub.1-C.sub.6-alkyloxy-C.sub.1-C.sub.6-alkyl, amino, and mono- or
di-C.sub.1-C.sub.6-alkylamino.
11. The .beta.-amino acid according to claim 9, wherein R.sup.3 is
selected from the group consisting of hydrogen, hydroxy, linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, alkynyl, and
hydroxy-C.sub.1-C.sub.6-alkyl.
12. The .beta.-amino acid according to claim 6, selected from the
group consisting of: ##STR45## wherein R.sup.1, R.sup.2 and R.sup.3
are as defined in claim 6.
13. The .beta.-amino acid according to claim 12, wherein R.sup.3 is
selected from the group consisting of hydrogen, hydroxy, linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, alkynyl,
hydroxy-C.sub.1-C.sub.6-alkyl, amino-C.sub.1-C.sub.6-alkyl,
C.sub.1-C.sub.6-alkyloxy,
C.sub.1-C.sub.6-alkyloxy-C.sub.1-C.sub.6-alkyl, amino, and mono- or
di-C.sub.1-C.sub.6-alkylamino.
14. The .beta.-amino acid according to claim 12, wherein R.sup.3 is
selected from the group consisting of hydrogen, hydroxy, linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, alkynyl, and
hydroxy-C.sub.1-C.sub.6-alkyl.
15. The .beta.-amino acid according to claim 6, selected from the
group consisting of: ##STR46## wherein R.sup.1, R.sup.2 and R.sup.3
are as defined in claim 6.
16. The .beta.-amino acid according to claim 15, wherein R.sup.3 is
selected from the group consisting of hydrogen, hydroxy, linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, alkynyl,
hydroxy-C.sub.1-C.sub.6-alkyl, amino-C.sub.1-C.sub.6-alkyl,
C.sub.1-C.sub.6-alkyloxy,
C.sub.1-C.sub.6-alkyloxy-C.sub.1-C.sub.6-alkyl, amino, and mono- or
di-C.sub.1-C.sub.6-alkylamino.
17. The .beta.-amino acid according to claim 15, wherein R.sup.3 is
selected from the group consisting of hydrogen, hydroxy, linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, alkynyl, and
hydroxy-C.sub.1-C.sub.6-alkyl.
18. The .beta.-amino acid according to claim 6, selected from the
group consisting of: ##STR47## wherein R.sup.1, R.sup.2 and R.sup.3
are as defined in claim 6.
19. The .beta.-amino acid according to claim 18, wherein R.sup.3 is
selected from the group consisting of hydrogen, hydroxy, linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, alkynyl,
hydroxy-C.sub.1-C.sub.6-alkyl, amino-C.sub.1-C.sub.6-alkyl,
C.sub.1-C.sub.6-alkyloxy,
C.sub.1-C.sub.6-alkyloxy-C.sub.1-C.sub.6-alkyl, amino, and mono- or
di-C.sub.1-C.sub.6-alkylamino.
20. The .beta.-amino acid according to claim 18, wherein R.sup.3 is
selected from the group consisting of hydrogen, hydroxy, linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, alkynyl, and
hydroxy-C.sub.1-C.sub.6-alkyl.
21. The .beta.-amino acid according to claim 6, selected from the
group consisting of: ##STR48## wherein R.sup.1, R.sup.2 and R.sup.3
are as defined in claim 6.
22. The .beta.-amino acid according to claim 21, wherein R.sup.3 is
selected from the group consisting of hydrogen, hydroxy, linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, alkynyl,
hydroxy-C.sub.1-C.sub.6-alkyl, amino-C.sub.1-C.sub.6-alkyl,
C.sub.1-C.sub.6-alkyloxy,
C.sub.1-C.sub.6-alkyloxy-C.sub.1-C.sub.6-alkyl, amino, and mono- or
di-C.sub.1-C.sub.6-alkylamino.
23. The .beta.-amino acid according to claim 21, wherein R.sup.3 is
selected from the group consisting of hydrogen, hydroxy, linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, alkynyl, and
hydroxy-C.sub.1-C.sub.6-alkyl.
24. The .beta.-amino acid according to claim 6, selected from the
group consisting of: ##STR49## wherein R.sup.1, R.sup.2 and R.sup.3
are as defined in claim 6.
25. The .beta.-amino acid according to claim 24, wherein R.sup.3 is
selected from the group consisting of hydrogen, hydroxy, linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, alkynyl,
hydroxy-C.sub.1-C.sub.6-alkyl, amino-C.sub.1-C.sub.6-alkyl,
C.sub.1-C.sub.6-alkyloxy,
C.sub.1-C.sub.6-alkyloxy-C.sub.1-C.sub.6-alkyl, amino, and mono- or
di-C.sub.1-C.sub.6-alkylamino.
26. The .beta.-amino acid according to claim 24, wherein R.sup.3 is
selected from the group consisting of hydrogen, hydroxy, linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, alkynyl, and
hydroxy-C.sub.1-C.sub.6-alkyl.
27. The .beta.-amino acid according to claim 6, selected from the
group consisting of: ##STR50## wherein R.sup.1, R.sup.2 and R.sup.3
are as defined in claim 6.
28. The .beta.-amino acid according to claim 27, wherein R.sup.3 is
selected from the group consisting of hydrogen, hydroxy, linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, alkynyl,
hydroxy-C.sub.1-C.sub.6-alkyl, amino-C.sub.1-C.sub.6-alkyl,
C.sub.1-C.sub.6-alkyloxy,
C.sub.1-C.sub.6-alkyloxy-C.sub.1-C.sub.6-alkyl, amino, and mono- or
di-C.sub.1-C.sub.6-alkylamino.
29. The .beta.-amino acid according to claim 27, wherein R.sup.3 is
selected from the group consisting of hydrogen, hydroxy, linear or
branched C.sub.1-C.sub.6-alkyl, alkenyl, alkynyl, and
hydroxy-C.sub.1-C.sub.6-alkyl.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation-in-part of, and priority is hereby
claimed to, co-pending application Ser. No. 09/464,212, filed 15
Dec. 1999, which is a divisional of Ser. No. 09/034,509, filed Mar.
4, 1998 (now U.S. Pat. No. 6,060,585, issued May 9, 2000), which
claims priority to provisional patent application Ser. No.
60/039,905, filed Mar. 4, 1997, the entire contents of all of which
is incorporated herein.
FIELD OF THE INVENTION
[0002] The present invention is directed to .beta.-polypeptide
molecules which are oligomers and polymers of .beta.-amino acids
having stable and well-defined secondary structures, including
helices and sheets, methods of generating combinatorial libraries
using these .beta.-polypeptides, and combinatorial libraries formed
thereby.
DESCRIPTION OF THE PRIOR ART
[0003] Chemists have long sought to extrapolate the power of
biological catalysis and recognition to synthetic systems. These
efforts have focused largely on low molecular weight catalysts and
receptors. Most biological systems, however, rely almost
exclusively on large polymers such as proteins and RNA to perform
complex chemical functions.
[0004] Proteins and RNA are unique in their ability to adopt
compact, well-ordered conformations. These two biopolymers are
unique also because they can perform complex chemical operations
(e.g., catalysis, highly selective recognition, etc.). Folding is
linked to function in both proteins and RNA because the creation of
an "active site" requires proper positioning of reactive groups.
Consequently, there has been a long-felt need to identify synthetic
polymer backbones which display discrete and predictable folding
propensities (hereinafter referred to as "foldamers") to mimic
natural biological systems. Such backbones will provide molecular
"tools" to probe the functionality of large-molecule interactions
(e.g. protein-protein and protein-RNA interactions).
[0005] Much work on .beta.-amino acids and peptides synthesized
therefrom has been performed by a group led by Dieter Seebach in
Zurich, Switzerland. See, for example, Seebaci et al. (1996) Helv.
Chim. Acta. 79:913-941; and Seebach et al. (1996) Helv. Chim. Acta.
79:2043-2066. In the first of these two papers Seebach et al.
describe the synthesis and characterization of a
.beta.-hexapeptide, namely
(H-.beta.-HVal-.beta.-HAla-.beta.-HLeu).sub.2-OH. Interestingly,
this paper specifically notes that prior art reports on the
structure of .beta.-peptides have been contradictory and "partially
controversial." In the second paper, Seebach et al. explore the
secondary structure of the above-noted .beta.-hexapeptide and the
effects of residue variation on the secondary structure.
[0006] Dado and Gellman (1994) J. Am. Chem. Soc. 116:1054-1062
describe intramolecular hydrogen bonding in derivatives of
.beta.-alanine and .gamma.-amino butyric acid. This paper
postulates that .beta.-peptides will fold in manners similar to
.alpha.-amino acid polymers if intramolecular hydrogen bonding
between nearest neighbor amide groups on the polymer backbone is
not favored.
[0007] Suhara et al. (1996) Tetrahedron Lett. 37(10):1575-1578
report a polysaccharide analog of a .beta.-peptide in which
D-glycocylamine derivatives are linked to each other via a C-1
.beta.-carboxylate and a C-2 .alpha.-amino group. This class of
compounds has been given the trivial name "carbopeptoids."
[0008] Regarding methods to generate combinatorial libraries,
several recent reviews are available. See, for instance, Ellman
(1996) Acc. Chem. Res. 29:132-143 and Lam et al. (1997) Chem. Rev.
97:411-448.
SUMMARY OF THE INVENTION
[0009] The present invention is drawn to a genus of
conformationally-restricted .beta.-amino acids and
.beta.-polyamides which strongly favor a helical or sheet secondary
structure and which can serve as building blocks for stable
tertiary structures. These stable secondary structures include
helices analogous to the well-known .alpha.-helical structure seen
in .alpha.-amino acids. Several of the subject compounds assume
helical secondary structures stabilized by hydrogen bonding every
12th or 14th atom of the backbone (12-helix and 14-helix,
respectively). Still other compounds according to the invention
contain a "reverse turn" residue which mimics the reverse turn
often seen in anti-parallel sheet structure seen in conventional
peptides and proteins. These .beta.-peptides according to the
invention exhibit an anti-parallel secondary sheet structure.
[0010] The invention is also directed to the
conformationally-restricted .beta.-amino acid monomers. Here, the
invention is directed to .beta.-amino acids selected from the group
consisting of compounds of Formula I: ##STR1## wherein X and Y
combined, together with the carbon atoms to which they are bonded,
define a substituted or unsubsituted C.sub.4-C.sub.8 cycloalkyl,
cycloalkenyl or heterocyclic ring having one or more nitrogen atoms
as the sole heteroatom; the substituents on carbon atoms of the
rings being independently selected from the group consisting of
linear or branched C.sub.1-C.sub.6-alkyl, alkenyl, or alkynyl;
mono- or bicyclic aryl, mono- or bicyclic heteroaryl having up to 5
heteroatoms selected from N, O, and S; mono- or bicyclic
aryl-C.sub.1-C.sub.6-alkyl, mono- or bicyclic
heteroaryl-C.sub.1-C.sub.6-alkyl, --(CH.sub.2).sub.n+1--OR.sup.4,
--(CH.sub.2).sub.n+1--SR.sup.4,
--(CH.sub.2).sub.n+1--S(.dbd.O)--CH.sub.2--R.sup.4,
--(CH.sub.2).sub.n+1--S(.dbd.O).sub.2--CH.sub.2--R.sup.4,
--(CH.sub.2).sub.n+1--NR.sup.4R.sup.4,
--(CH.sub.2).sub.n+1--NHC(.dbd.O)R.sup.4,
--(CH.sub.2).sub.n+1--NHS(.dbd.O).sub.2--CH.sub.2--R.sup.4,
--(CH.sub.2).sub.n+1--O--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--S--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--S(.dbd.O)--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--S(.dbd.O).sub.2--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--NH--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--N--{(CH.sub.2).sub.m--R.sup.5}.sub.2,
--(CH.sub.2).sub.n+1--NHC(.dbd.O)--(CH.sub.2).sub.n+1--R.sup.5, and
--(CH.sub.2).sub.n+1--NHS(.dbd.O).sub.2--(CH.sub.2).sub.m--R.sup.5;
wherein R.sup.4 is independently selected from the group consisting
of hydrogen, C.sub.1-C.sub.6-alkyl, alkenyl, or alkynyl; mono- or
bicyclic aryl, mono- or bicyclic heteroaryl having up to 5
heteroatoms selected from N, O, and S; mono- or bicyclic
aryl-C.sub.1-C.sub.6-alkyl, mono- or bicyclic
heteroaryl-C.sub.1-C.sub.6-alkyl; and wherein R.sup.5 is selected
from the group consisting of hydroxy, C.sub.1-C.sub.6-alkyloxy,
aryloxy, heteroaryloxy, thio, C.sub.1-C.sub.6-alkylthio,
C.sub.1-C.sub.6-alkylsulfinyl, C.sub.1-C.sub.6-alkylsulfonyl,
arylthio, arylsulfinyl, arylsulfonyl, heteroarylthio,
heteroarylsulfinyl, heteroarylsulfonyl, amino, mono- or
di-C.sub.1-C.sub.6-alkylamino, mono- or diarylamino, mono- or
diheteroarylamino, N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino,
N-aryl-N-heteroarylamino, aryl-C.sub.1-C.sub.6-alkylamino,
carboxylic acid, carboxamide, mono- or
di-C.sub.1-C.sub.6-alkylcarboxamide, mono- or diarylcarboxamide,
mono- or diheteroarylcarboxamide, N-alkyl-N-arylcarboxamide,
N-alkyl-N-heteroarylcarboxamide, N-aryl-N-heteroarylcarboxamide,
sulfonic acid, sulfonamide, mono- or
di-C.sub.1-C.sub.6-alkylsulfonamide, mono- or diarylsulfonamide,
mono- or diheteroarylsulfonamide, N-alkyl-N-arylsulfonamide,
N-alkyl-N-heteroarylsulfonamide, N-aryl-N-heteroarylsulfonamide,
urea; mono- di- or tri-substituted urea, wherein the subsitutent(s)
is selected from the group consisting of C.sub.1-C.sub.6-alkyl,
aryl, heteroaryl; O-alkylurethane, O-arylurethane, and
O-heteroarylurethane; and m is an integer of from 2-6 and n is an
integer of from 0-6; the substituents on heteroatoms of the ring
being independently selected from the group consisting of
--S(.dbd.O).sub.2--CH.sub.2--R.sup.4--C(.dbd.O)--R.sup.4--S(.dbd.O).sub.2-
--(CH.sub.2).sub.m--R.sup.5, and
--C(.dbd.O)--(CH.sub.2).sub.n+1--R.sup.5; wherein R.sup.4 and
R.sup.5 are as defined hereinabove, and m is an integer of from 2-6
and n is an integer of from 0-6; provided that when X & Y
together with the carbons to which they are bonded define a five-
or six-membered cycloalkyl or a five-membered heterocyclic ring
having one nitrogen as the sole heteroatom, and the nitrogen is
bonded to a carbon atom adjacent to the carboxy carbon of Formula
I, the cycloalkyl or heterocyclic ring is substituted; R.sup.1 is
selected from the group consisting hydrogen and an amino protecting
group; R.sup.2 is selected from the group consisting of hydrogen
and a carboxy protecting group; racemic mixtures thereof, isolated
or enriched enantiomers thereof; isolated or enriched diastereomers
thereof; and salts thereof.
[0011] The preferred embodiment of the invention is a .beta.-amino
acid selected from the group consisting of: ##STR2##
[0012] wherein R.sup.1 is selected from the group consisting
hydrogen and an amino protecting group; R.sup.2 is selected from
the group consisting of hydrogen and a carboxy protecting group;
and when R.sup.3 is bonded to a carbon atom, R.sup.3 is selected
from the group consisting of hydrogen, hydroxy, linear or branched
C.sub.1-C.sub.6-alkyl, alkenyl, or alkynyl; mono- or bicyclic aryl,
mono- or bicyclic heteroaryl having up to 5 heteroatoms selected
from N, O, and S; mono- or bicyclic aryl-C.sub.1-C.sub.6-alkyl,
mono- or bicyclic heteroaryl-C.sub.1-C.sub.6-alkyl,
--(CH.sub.2).sub.n+1--OR.sup.4, --(CH.sub.2).sub.n+1--SR.sup.4,
--(CH.sub.2).sub.n+1--S(.dbd.O)--CH.sub.2--R.sup.4,
--(CH.sub.2).sub.n+1--S(.dbd.O).sub.2--CH.sub.2--R.sup.4,
--(CH.sub.2).sub.n+1--NR.sup.4R.sup.4,
--(CH.sub.2).sub.n+1--NHC(.dbd.O)R.sup.4,
--(CH.sub.2).sub.n+1--NHS(.dbd.O).sub.2--CH.sub.2--R.sup.4,
--(CH.sub.2).sub.n+1--O--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--S--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--S(.dbd.O)--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--S(.dbd.O).sub.2--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--NH--(CH.sub.2).sub.m--R.sup.5,
--(CH.sub.2).sub.n+1--N--{(CH.sub.2).sub.m--R.sup.5}.sub.2,
--(CH.sub.2).sub.n+1--NHC(.dbd.O)--(CH.sub.2).sub.n+1--R.sup.5, and
--(CH.sub.2).sub.n+1--NHS(.dbd.O).sub.2--(CH.sub.2).sub.m--R.sup.5;
wherein R.sup.4 is independently selected from the group consisting
of hydrogen, C.sub.1-C.sub.6-alkyl, alkenyl, or alkynyl; mono- or
bicyclic aryl, mono- or bicyclic heteroaryl having up to S
heteroatoms selected from N, O, and S; mono- or bicyclic
aryl-C.sub.1-C.sub.6-alkyl, mono- or bicyclic
heteroaryl-C.sub.1-C.sub.6-alkyl; and wherein R.sup.5 is selected
from the group consisting of hydroxy, C.sub.1-C.sub.6-alkyloxy,
aryloxy, heteroaryloxy, thio, C.sub.1-C.sub.6-alkylthio,
C.sub.1-C.sub.6-alkylsulfinyl, C.sub.1-C.sub.6-alkylsulfonyl,
arylthio, arylsulfinyl, arylsulfonyl, heteroarylthio,
heteroarylsulfinyl, heteroarylsulfonyl, amino, mono- or
di-C.sub.1-C.sub.6-alkylamino, mono- or diarylamino, mono- or
diheteroarylamino, N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino,
N-aryl-N-heteroarylamino, aryl-C.sub.1-C.sub.6-alkylamino,
carboxylic acid, carboxamide, mono- or
di-C.sub.1-C.sub.6-alkylcarboxamide, mono- or diarylcarboxamide,
mono- or diheteroarylcarboxamide, N-alkyl-N-arylcarboxamide,
N-alkyl-N-heteroarylcarboxamide, N-aryl-N-heteroarylcarboxamide,
sulfonic acid, sulfonamide, mono- or
di-C.sub.1-C.sub.6-alkylsulfonamide, mono- or diarylsulfonamide,
mono- or diheteroarylsulfonamide, N-alkyl-N-arylsulfonamide,
N-alkyl-N-heteroarylsulfonamide, N-aryl-N-heteroarylsulfonamide,
urea; mono- di- or tri-substituted urea, wherein the subsitutent(s)
is selected from the group consisting of C.sub.1-C.sub.6-alkyl,
aryl, heteroaryl; O-alkylurethane, O-arylurethane, and
O-heteroarylurethane; and m is an integer of from 2-6 and n is an
integer of from 0-6; and when R.sup.3 is bonded to a nitrogen atom,
R.sup.3 is independently selected from the group consisting of
those listed above for when R.sup.3 is attached to a carbon atom,
and further selected from the group consisting of
--S(.dbd.O).sub.2--CH.sub.2--R.sup.4,
--C(.dbd.O)--R.sup.4--S(.dbd.O).sub.2--(CH.sub.2).sub.m--R.sup.5,
and --C(.dbd.O)--(CH.sub.2).sub.n+1--R.sup.5; wherein R.sup.4 and
R.sup.5 are as defined hereinabove, and m is an integer of from 2-6
and n is an integer of from 0-6; provided that when the
.beta.-amino acid is of formula ##STR3## R.sup.3 is not hydrogen;
racemic mixtures thereof, isolated or enriched enantiomers thereof;
isolated or enriched diastereomers thereof; and salts thereof.
[0013] The invention is further directed to a method for preparing
a combinatorial library of .beta.-polypeptides, the method
comprising at least two successive iterations of first covalently
linking a first subunit via its C terminus to a plurality of
separable solid substrates, the first subunit being an
N-terminus-protected derivative of one of the
conformationally-constrained .beta.-amino acids recited
hereinabove.
[0014] Then randomly dividing the plurality of substrates into at
least two sub-groups and deprotecting the N-termini first subunits
attached to the at least two sub-groups. Then, in separate and
independent reactions, covalently linking to the first subunit of
each of the at least two sub-groups a second subunit independently
selected from the above-listed group, and, in addition thereto, a
residue selected from the group consisting of: ##STR4## wherein
"Pg" is a protecting group. Then combining the at least two
sub-groups into a single plurality. The process is then repeated
one or more times (preferably 5 to 25 times), whereby a
combinatorial library of .beta.-amino acid polypeptides is
assembled. The invention is further drawn to the combinatorial
library of .beta.-polypeptides so formed.
[0015] Another embodiment of the invention is drawn to an array
comprising a plurality of .beta.-polypeptides as described above at
selected, known locations on a substrate or in discrete solutions,
wherein each of the polypeptides is substantially pure within each
of the selected known locations and has a composition which is
different from other polypeptides disposed at other selected and
known locations on the substrate.
[0016] The primary advantage of the present invention is that it
allows the constriction of synthetic peptides of known secondary
structures having high conformational stability. These synthetic
polyamides have utility in investigating the biological
interactions involving biopolymers. The switch in helical hydrogen
bond directionality between the .beta.-peptide 12- and 14-helices
(See FIGS. 2A and 2B) is unprecedented among .alpha.-peptides. The
residue-based conformational control offered by .beta.-peptides,
which was predicted computationally, makes this class of unnatural
foldamers well suited for molecular design efforts, e.g.,
generation of novel tertiary structures, and combinatorial searches
for selective biopolymer ligands.
[0017] The .beta.-amino acid monomers are useful for constructing
combinatorial libraries of compounds that display peptide behavior
(because they are peptides), but resistant to enzymatic degradation
because the compounds do not contain any .alpha.-peptide
linkages.
[0018] As a natural consequence, the invention is further drawn to
the use of these synthetic .beta.-amino acids as base molecules
from which to synthesize large libraries of novel compounds
utilizing the techniques of combinatorial chemistry. In addition to
varying the primary sequence of the .beta.-amino acid residues, the
ring positions of these compounds (and notably the equatorial
positions in the cyclohexyl-rigidified .beta.-amino acids) can be
substituted with a wide variety of substituents, including hydroxy,
linear or branched C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-alkyloxy,
amino, mono- or di-C.sub.1-C.sub.6-alkylamino, carboxamido,
sulfonamido, urea, cyano, fluoro, thio, C.sub.1-C.sub.6-alkylthio,
and the like. The main advantage here is that substituents placed
on the backbone rings do not substantially alter the secondary
structure of the peptide. Consequently, the subject compounds can
be utilized to contruct vast libraries having different
substituents, but all of which share a stabilized secondary
structure in both the solid state and in solution.
[0019] Thus, the .beta.-amino acids described herein are also
useful for fabricating .beta.-polypeptides to model the behavior of
corresponding, naturally-occurring .alpha.-polypeptide. Because the
.beta.-amino acids described herein are not recognized as targets
by enzymes that are specific for naturally-occurring
.alpha.-polypeptides, they can be used in mechanistic studies as
non-enzymatically-degradable models of the corresponding
.alpha.-polypeptide.
[0020] Other aims, objects, and advantages of the invention will
appear more fully from a complete reading of the following Detailed
Description of the Invention and the attached claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows the structure of poly-.beta.-alanine and
further depicts the hydrogen bonds that define the six narrowest
helices available to poly-.beta.-alanine. Poly-.beta.-alanine is
the simplest .beta.-peptide polymer.
[0022] FIG. 2A depicts the crystal structure of oligomer of
trans-ACHC/14-helix. The bottom depiction is the two-dimensional
structure, the middle depiction is a view along the axis of the
helix, and the top depiction is a view perpendicular to the axis of
the helix.
[0023] FIG. 2B depicts the crystal structure of oligomer of
trans-ACPC/12-helix. The views shown are the same as in FIG.
2A.
[0024] FIG. 2C depicts the crystal structure of a standard
.alpha.-helix. The views shown are the same as in FIG. 2A.
[0025] FIG. 3 depicts .sup.1H NMR spectra for a solution containing
2 mM trans-ACHC dimer and 2 mM trans-ACHC hexamer. The bottom
spectrum was obtained in CD.sub.3OH with solvent suppression. The
two NH resonances from the dimer are indicated with an asterisk
(*). All other spectra were obtained in CD.sub.3OD at the times
indicated after dissolution of the sample. Data obtained on a
Bruker 300 MHz spectrometer at 20.degree. C.
[0026] FIG. 4 is a circular dichroism (CD) plot for trans-ACPC
hexamer in CH.sub.3OH.
[0027] FIG. 5 is a CD plot comparing trans-ACPC dimer, trimer,
tetramer and hexamer.
[0028] FIG. 6 depicts .sup.1H NMR spectra for a solution containing
2 mM of a hexamer of alternating amino-substituted-trans-ACHA and
trans-ACHA. All spectra were obtained in D.sub.2O, 100 mM
deuteroacetate buffer, pD 3.9, at the times indicated after
dissolution of the sample. Data obtained on a Bruker 300 MHz
spectrometer at 20.degree. C.
[0029] FIG. 7 is a comparison between the k.sub.obs for amide
proton exchange in a hexamer of alternating
amino-substituted-trans-ACHA and trans-ACHA and a dimer of
alternating amino-substituted-trans-ACHA and trans-ACHA.
[0030] FIG. 8 is a comparison between the CD plot of a hexamer of
alternating amino-substituted-trans-ACHA and trans-ACHA in water
and the CD plot of a hexamer of trans-ACHC in methanol.
[0031] FIG. 9 is a comparison of CD data in methanol for a
.beta.-peptide tetramer, hexamer, and octamer containing
alternating trans-ACPA residues and 4-pyrrolidinyl residues.
[0032] FIG. 10 is a comparison of CD data in water for a
.beta.-peptide tetramer, hexamer, and octamer containing
alternating trans-ACPA residues and 4-pyrrolidinyl residues.
[0033] FIG. 11 is a superimposed plot of the CD data in water and
the CD data in methanol for the octamer containing alternating
trans-ACPA residues and 4-pyrrolidinyl residues.
[0034] FIG. 12 is a CD spectrum in water of an octamer containing
alternating trans-ACPA residues and 3-pyrrolidinyl residues.
[0035] FIG. 13 is a comparison of CD spectra for a hexamer
containing alternating residues of trans-ACHA and
amino-substituted-trans-ACHA and for a "mixed" .beta.-peptide
hexamer comprising alternating residues of trans-ACHA and an
acyclic .beta.-amino acid bearing an aminopropyl substituent on the
.beta.-carbon of the backbone.
[0036] FIG. 14A shows the infrared spectrum of two linked nipecotic
acid residues wherein the two residues have the same absolute
configuration.
[0037] FIG. 14B shows the infrared spectrum of two linked nipecotic
acid residues wherein the two residues have the opposite absolute
configuration. This diastereomer acts as a reverse turn in
.beta.-peptides which adopt a sheet structure.
[0038] FIG. 15 is a schematic representation of the "split and
pool" method of generating combinatorial libraries.
[0039] FIG. 16 is a ball and stick representation of the solid
state conformation of compound 1 described below. For clarity, all
hydrogen atoms, except for those attached to nitrogen, have been
omitted. Hydrogen bonds are indicated with dotted lines.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The first step in creating a foldamer is to identify
polymeric backbones with well-defined secondary structural
preferences.
Helices in .beta.-Peptides:
[0041] Initial molecular modeling studies indicated that
.beta.-amino acid oligomers (".beta.-peptides") are well suited for
adoption of compact secondary structures stabilized by
intramolecular hydrogen bonds. FIG. 1 shows the hydrogen bonds that
define the six narrowest helices available to poly-.beta.-alanine,
the simplest .beta.-peptide polymer. The 12-, 16-, and 20-helices
(nomenclature derived from hydrogen-bonded ring size) contain
hydrogen bonds from carbonyls toward NH groups in the N-terminal
direction, as observed for 3.sub.10- and .alpha.-helices in
proteins, while the 10-, 14-, and 18-helices contain hydrogen bonds
from carbonyls to NH groups in the N-terminal direction. Molecular
mechanics studies of a .beta.-alanine decamer suggested that all
six of these helices constitute local minima on the conformational
energy surface. .beta.-Alanine oligomers, however, have been shown
experimentally to be unordered in solution and to adopt sheetlike
packing patterns in the solid state.
[0042] Incorporation of the two backbone carbons of a .beta.-amino
acid into a small carbocycle or heterocycle provides substantial
rigidity to the backbone. Computational methods were used to
evaluate whether any particular helix/small ring combination would
lead to enhanced conformational stability. For each of the six
minimized deca-.beta.-alanine helices shown in FIG. 1, each residue
was modified by incorporation of the backbone carbons into a
three-, four-, five-, and six-membered cycloalkyl ring. For each
ring size, both cis and trans relationships between the amino and
carboxyl substituents were examined, and for the cis rings, both of
the possible ring orientations relative to the helix were examined.
This process yields 72 helical starting structures (6
helices.times.4 cycloalkyl ring sizes.times.(1 trans+2 cis forms)).
A combination of minimization and dynamics studies predicted that
the 14-helical form of the decamer of
trans-2-aminocyclohexanecarboxylic acid (trans-ACHC) (the
corresponding monomer is refered to herein as trans-ACHA) and the
12-helical form of the decamer of
trans-2-aminocyclopentanecarboxylic acid (trans-ACPC)
(corresponding monomer referred to as trans-ACPA) would be the most
stable among these hypothetical helices. These two structures are
shown below: ##STR5##
[0043] In order to test this computational prediction, optically
active trans-ACHC was prepared by the reported route, Nohira et at.
(1970) Bull. Chem. Soc. Jpn. 43:2230, and polypeptide oligomers
synthesized via standard methods (see below). The crystal
structures of the trans-ACHC tetramer and the trans-ACHC hexamer
reveal that these molecules adopt 14-helical confonrations in the
solid state. The hexamer crystal contains three independent but
very similar molecules, each of which forms the four possible
14-membered ring hydrogen bonds. The regular helix revealed by the
hexamer crystal structure matches the minimum energy conformation
predicted for the decamer.
[0044] An alternatively rigidified .beta.-amino acid,
trans-2-aminocyclopentanecarboxylic acid (trans-ACPC), provides
.beta.-peptides with a dramatically altered secondary structure,
the 12-helix. FIG. 2B depicts the 12-helix trans-ACPC structure, as
compared to a standard .alpha.-helix (FIG. 2C) and
trans-ACHC/14-helix (FIG. 2A). This finding shows that
.beta.-peptides allow profound residue-based control of peptide
conformation.
Sheets:
[0045] There are two types of antiparallel .beta.-peptide sheet
structures: ##STR6##
[0046] The type I sheet has a net dipole due to the parallel
disposition of the carbonyl groups. In contrast, the type II sheet
does not have a net dipole because the carbonyl groups are
anti-parallel. This is distinctly different from .alpha.-peptides,
which can only form sheet structures with anti-parallel carbonyl
groups.
[0047] Creating discrete model systems of sheet formation using
.alpha.-amino acids remains a long-standing challenge in protein
science. The difficulty lies in designing a moiety to mimic the
reverse turn, thereby bringing two attached strands together to
form the sheet structure without inducing uncontrolled aggregation
of the bulk peptide.
[0048] In the present invention, this problem has been addressed
with two moieties which act as the "turn" in the "hairpin" to bring
the two peptide chains into alignment to form sheet structures. The
first moiety, a prolyl-glycolic acid linkage appears as follows:
##STR7## The second moiety contains two residues of nipecotic acid:
##STR8##
[0049] In the di-nipecotic acid residue it is preferred, although
not required, that the two nipecotic acid moieties have opposite
absolute configuration: ##STR9##
[0050] Using these linkages, .beta.-peptide molecules having stable
sheet structures were obtained.
[0051] Analysis of solid state and solution phase conformations of
the class of molecules described herein were established using a
combination of NMR spectroscopy (including COSY, ROESY and NOESY
spectra), IR spectrophotometry, circular dichroism, and X-ray
crystallography. For compound 1 (described below), NOESY
measurements in both CD.sub.2Cl.sub.2 and CD.sub.3OD reveal two
long-range NOEs between the .beta.-amino acid residues (curved
lines in following structure, both of which are consistent with the
solid state conformation depicted in FIG. 16: ##STR10##
[0052] The model compound 1 displays secondary conformation with
the two .beta.-amino acid residues intramolecularly arranged in
type I antiparallel sheet fashion.
Chemistry:
[0053] General. Melting points are uncorrected. CH.sub.2Cl.sub.2
was freshly distilled from CaH.sub.2 under N.sub.2. DMF was
distilled under reduced pressure from ninhydrin and stored over 4
.ANG. molecular sieves. Triethylamine was distilled from CaH.sub.2
before use. Other solvents and reagents were used as obtained from
commercial suppliers. For BOC removal, 4 M HCl in dioxane from was
used. Column chromatography was carried out by using low air
pressure (typically 6 psi) with 230-400 mesh silica gel 60. Routine
.sup.1H-NMR spectra were obtained on a Bruker AC-300 and are
referenced to residual protonated NMR solvent. Routine .sup.13C-NMR
spectra were obtained on a Bruker AC-300 and are referenced to the
NMR solvent. High resolution electron impact mass spectroscopy was
performed on a Kratos MS-80RFA spectrometer with DS55/DS90.
[0054] Infrared Spectroscopy. Spectra were obtained on a Nicolet
Model 740 FT-IR spectrometer. IR samples were prepared under
anhydrous conditions; CH.sub.2Cl.sub.2 was freshly distilled from
CaH.sub.2, compounds and glassware were dried under vacuum for 1-2
days, and solutions were prepared under a nitrogen atmosphere. The
pure solvent spectrum for a particular solution was subtracted from
the sample spectrum prior to analysis. Peaks in the amide NH
stretch region were baseline corrected, and analyzed without
further manipulation.
[0055] NMR Spectroscopy. 1. Aggregation Studies. One-dimensional
spectra for aggregation studies were obtained on a Bruker AC-300
spectrometer. Samples for aggregation studies were prepared by
serial dilution from the most concentrated sample (50 mM or 27 mM).
Dry compounds were dissolved in CD.sub.2Cl.sub.2 previously dried
over 3 .ANG. molecular sieves, and samples were prepared with dry
glassware under a nitrogen atmosphere.
[0056] 2. Conformational Analysis. NMR samples for conformational
analysis were prepared by dissolving the dry compound in dry
deuterated solvent under a nitrogen atmosphere. CD.sub.2Cl.sub.2
samples were then degassed by the freeze-pump-thaw method, and the
NMR tubes were sealed under vacuum. Methanol samples were sealed
with a close fitting cap and parafilm. COSY spectra were obtained
on a Bruker AC-300 spectrometer. TOCSY (Braunschweiler, L.; Ernst,
R. R. (1983) J. Magn. Reson. 53:521), NOESY (Macura, S.; Ernst, R.
R. (1980) Mol. Phys. 41:95), and ROESY (Bothner-By, A. A.;
Stephens, R. L.; Lee, I.; Warren, C. D.; Jeanloz R. W. (1984) J.
Am. Chem. Soc. (1984) 106:811) spectra were squired on a Varian
Unity-500 spectrometer using standard Varian pulse sequences and
hypercomplex phase cycling (States-Haberkorn method), and the data
were processed with Varian "VNMR" version 5.1 software. Proton
signals were assigned via COSY and TOCSY spectra, and NOESY and
ROESY spectra provided the data used in the conformational
analyses. TOCSY spectra were recorded with 2048 points in t.sub.1,
320 or 350 points in t.sub.2, and 8 or 40 scans per t.sub.2
increment. NOESY and ROESY spectra were recorded with a similar
number of t.sub.1 and t.sub.2 points, and 32 and 40 scans per
t.sub.2 increment, depending on the sample concentration. The width
of the spectral window examined was between 2000 and 4000 Hz.
Sample concentrations for two-dimensional spectra were 2 mM in
CD.sub.2Cl.sub.2 and 8 mM in CD.sub.3OD and CD.sub.3OH.
[0057] Far UV Circular Dichroism (CD). Data were obtained on a
Jasco J-715 instrument at 20.degree. C. In all CD plots contained
herein, the mean residue ellipticity is presented on the vertical
axis. Presenting the mean residue ellipticity is a standard
practice in peptide chemistry wherein the intensity of each CD
spectrum is normalized for the number of amide chromophores in the
peptide backbone. Consequently, when the intensities of the maximum
(ca. 205 nm) and minimum (ca. 220 nm) peaks characteristic of helix
formation increase with increasing chain length, this change
represents an increase in the population of the helix structure,
rather than simply an increase in the number of chromophores
present in each molecule.
[0058] Synthesis. The .beta.-amino acids used to assemble the
peptides described herein can be manufactured using several
different literature methods, as well as new methods described
below. For unsubstituted .beta.-amino acids and .beta.-amino acids
containing one or two acyclic substituents on the carbon adjacent
to the amino group in the product .beta.-peptide, the
Arndt-Eisterdt homologation reaction can be used, see Reaction 1.
See also Seebach et al. (1996) Helv. Chim. Acta 79:913. This route
has advantages and disadvantages. A distinct advantage is that the
starting materials, .alpha.-amino acids, are readily available
commercially in enantiomerically pure form. The Arndt-Eisterdt
homologation also results in the simultaneous coupling of two
.beta.-amino residues. A distinct disadvantage is that the reaction
cannot be used to synthesize .beta.-amino acids having rings in the
backbone or .alpha.-carbon substituents. The reaction proceeds via
a Wolff rearrangement of a diazoketone with subsequent trapping of
the reactive intermediate with an amino moiety, as shown in
Reaction 1: ##STR11## (Pg designates a protecting group such as
(t-butoxy)carbonyl (Boc) or an adjacent .beta.-amino residue,
R.sup.1 and R.sup.2 are aliphatic substituents.
[0059] .beta.-Amino acids containing an unsubstituted cycloalkyl
moiety involving the .alpha. and .beta. carbons were synthesized
using literature methods. See, for example, Nohira et al. (1970)
Bull. Chem. Soc. Jpn. 43:2230; Herradon and Seebach (1989) Helv.
Chim. Acta 72:690-714; and Tilley et al. (1992) J. Med. Chem.
35:3774-3783, all threeof which are incorporated herein by
reference.
[0060] In particular, the cyclohexyl-containing .beta.-amino acids
can be synthesized via Reaction 2: ##STR12##
[0061] (1R,6S)-6-Methoxycarbonyl-3-cyclohexene-1-carboxylic acid
(23): 4600 u of PLE was suspended in pH 8.01 aqueous buffer
solution (0.17 M KH.sub.2PO.sub.4). The diester 22 (10.1 g, 0.05
mol) was dissolved in 30 mL of acetone and added to the buffer
solution. Reaction was allowed to stir at rt overnight. The enzyme
was filtered off through a well-packed celite pad, the solution was
then acidified to pH 1 with 1M HCl and the product was extracted
with ethyl acetate (5.times.400 mL). The combined organic extracts
were dried over anhydrous magnesium sulfate and concentrated to
yield 9.00 g yellow oil. Product taken on without further
purification.
[0062] Methyl (1S,6R)-6-benzyloxycarbonylaminocyclohex-3-ene
carboxylate (24): Ethylchloroforamate (4 mL, 0.042 mol) was added
to a mixture of 23 (5.14 g, 0.028 mol) and triethylamine (6 mL,
0.043 mol) in acetone (100 mL) at O.degree. C. and vigorously
stirred for 10 min. An aqueous solution of NaN.sub.3 (3.04 g, 0.047
mol, in 25 mL water) was added in one portion. The resulting
mixture was stirred for 30 min at O.degree. C. The reaction mixture
was diluted with water and extracted with diethyl ether. The
organic extracts were dried over anhydrous magnesium sulfate and
concentrated without heat to yield a viscous yellow liquid. The
liquid was dissolved in 100 mL of benzene and refluxed under
nitrogen atmosphere for 30 min. Benzyl alcohol (12 mL, 0.116 mol)
was added and solution was refluxed for an additional 16 h. The
reaction was cooled to rt and concentrated to yield 17.12 g of a
yellow liquid (mixture of benzyl alcohol and desired product in a
5.4:1 ratio, respectively by .sup.1H NMR, .about.5.67 g product).
Mixture taken on without further purification.
[0063] Methyl (1S,6R)-6-tert-butoxycarbonylaminocyclohexane
carboxylate (25): The yellow oil from the previous reaction, which
contains compound 24 (5.6 g, 0.020 mol) and benzyl alcohol, was
dissolved in methanol. 0.525 g of 10% Pd on carbon was added to the
methanol solution, and the heterogenous mixture was placed under 50
psi H.sub.2 and shaken at rt for 24 h. The mixture was filtered
through celite, and the filtrate was concentrated to yield 13.74 g
of dark golden yellow liquid. 25 mL of 1M HCl was added to the
filtrate, and the benzyl alcohol was extracted with diethyl ether
(3.times.25 mL). The pH of the aqueous solution was adjusted to 9
using K.sub.2CO.sub.3. 25 mL of dioxane and Boc.sub.2O (5 g, 0.023
mol) were added to the solution, and the reaction was stirred at rt
for 20 h. 15 mL of water was added and the solution was extracted
with ethyl acetate (3.times.50 mL). The combined organic extracts
were dried over anhydrous magnesium sulfate and concentrated.
Residue was purified via column chromatography (SiO.sub.2, eluting
with 6:1 Hex:EtOAc), to yield 2.00 g viscous clear oil.
[0064] Methyl (1R,6R)-6-tert-butoxycarbonylaminocyclohexane
carboxylate (26): Sodium metal (0.14 g, 6.1 mmol) was placed into a
flame dried flask under nitrogen atmosphere and cooled to O.degree.
C. 10 mL of freshly distilled methanol was added and the mixture
stirred until all the sodium dissolved. An amount of 25 (2.00 g,
7.7 mmol) was dissolved in 10 mL of freshly distilled methanol and
transferred to NaOMe solution via cannula. The solution was
refluxed under nitrogen for 5.5 h, cooled to rt and acidified with
0.5 M aqueous 0.5 M ammonium chloride (18 mL, 9 mmol). The methanol
was removed under reduced pressure, and the resulting solid
collected by filtration to yield 1.27 g of desired product.
[0065] .beta.-Amino acids containing a substituted cycloalkyl
moiety were synthesized using the following illustrative protocol,
the first four steps of which are described in Kobayashi et al.
(1990) Chem. Pharm. Bull. (1990) 38:350. The remaining steps to
yield a cyclohexyl ring having two differentially protected amino
substituents were developed in furtherance of the present invention
and have not heretofore been described in the literature and are
shown in Reaction 3: ##STR13## As depicted in Reaction 3, the
4-position amino substituent is protected by a Boc group and the
I-position amino substituent is protected by a Cbz group. The
starting material is available commercially (Aldrich Chemical Co.,
Milwaukee, Wis.).
[0066] Synthesis of .beta.-amino acids containing a heterocylic
ring moiety encompassing the .alpha. and .beta. carbons were
synthesized using Reactions 4 and 5, below. Reaction 4 details an
illustrative synthesis of a .beta.-proline wherein the exocyclic
amino substituent is in the 3-position relative to the ring
nitrogen.
[0067] Compound 42: Tap water (200 ml) and baker's yeast (25 g)
were mixed, and were shaken on an orbital shaker for 1 hour.
Compound 41 (1.0 g) was then added. The mixture was shaken at room
temperature for 24 hours. The mixture was filtered through a bed of
Celite. The Celite was washed with water (20 ml). The filtrate was
extracted with diethyl ether (5.times.100 ml). The extracts were
washed with water (2.times.50 ml), dried over MgSO.sub.4, and
concentrated to yield a slightly yellow oil. The crude product was
purified by column chromatography with ethyl acetate/hexane (1/1,
v/v) as eluent to give a colorless oil (0.5 g) in 50% yield.
[0068] Compound 43: Compound 42 (228 mg) and Ph.sub.3P (346 mg)
were dissolved in benzene (anhydrous, 4 ml) under nitrogen.
HN.sub.3 (1.64 M in benzene, 0.8 ml) was then added. A solution of
diethyl azodicarboxylate (0.18 ml) in benzene (1.0 ml) was
subsequently introduced via syringe over 5 minutes. The reaction
mixture turned cloudy towards the end of the addition. The reaction
mixture was stirred under nitrogen at room temperature for 3.0
hours. The reaction mixture was then taken up in ethyl acetate (50
ml), washed with 1N NaOH (10 ml), saturated NaRCO.sub.3 (10 ml),
and finally dilute brine (5 ml). The organic was dried over
MgSO.sub.4, and concentrated to give a slightly yellow oil. The
crude oil was purified by ##STR14## column chromatography with
ethyl acetate/hexane (1/1, v/v) as eluent to afford a colorless oil
(190 mg) in 76% yield.
[0069] Compound 44: Compound 43 (1.1 g) was dissolved in methanol
(50 ml). SnCl.sub.2 (2.2 g) was then added. The mixture was stirred
at room temperature for 30 hours. The methanol was then removed
under reduced pressure. The residue was dissolved in methylene
chloride (50 ml). The resulting cloudy solution was filtered
through Celite. The methylene chloride was then removed under
reduced pressure. The residual white solid was dissolved in
acetone/water (2/1, v/v, 50 ml). NaHCO.sub.3 (3.3 g) was added,
followed by Cbz-OSU (1.16 g). The reaction mixture was stirred at
room temperature for 24 hours. Water (50 ml) was added. The acetone
was removed under reduced pressure. The aqueous mixture was
extracted with ethyl acetate (3.times.100 ml). The extracts were
washed with dilute brine (30 ml), dried over MgSO.sub.4, and
concentrated to give a colorless oil. The crude product was
purified by column chromatography with ethyl acetate/hexane (3/7,
v/v) as eluent to give the clean product as a colorless oil (1.35
g) in 89% yield.
[0070] Compound 45: Compound 44 (1.35 g) was dissolved in
methanol/water (3/1, v/v, 80 ml), cooled to O.degree. C. LiOH.H2O
(1.68 g) was added. The mixture was stirred at O.degree. C. for 24
hours, by which time TLC indicated that the hydrolysis was
complete. Saturated ammonium hydroxide (20 ml) was added. The
methanol was removed under reduced pressure. The aqueous was washed
with diethyl ether (50 ml), acidified with 1N HCl to pH 3,
extracted with methylene chloride (3.times.150 ml). The extracts
were washed with dilute brine (50 ml), dried over MgSO.sub.4,
concentrated to give a sticky colorless residue (1.25 g, 99%),
which was used directly without further purification.
[0071] Compound 46: Compound 45 (1.25 g) was dissolved in methanol
(50 ml) in a hydrogenation flask. 5% Palladium on activated carbon
(190 mg) was added. The flask was pressurized with hydrogen to 35
psi, rocked at room temperature for 7 hours, by which time TLC
indicated that the hydrogenolysis was complete. The Pd/C was
removed by filtration. The filtrate was concentrated to give a
white solid. The white solid was dissolved in acetone/water (2/1,
v/v, 70 ml), cooled to 0.degree. C. NaHCO.sub.3 (1.7 g) was added,
followed by FMOC-OSU (1.39 g). The reaction mixture was stirred at
room temperature for 16 hours. Water (50 ml) was added. The acetone
was removed under reduced pressure. The aqueous was washed with
diethyl ether (50 ml), acidified with 1N HCl to pH 3, extracted
with methylene chloride (3.times.150 ml). The extracts were washed
with dilute brine (50 ml), dried over MgSO.sub.4, concentrated to
give a foamy white solid. The crude white solid was purified by
column chromatography with methanol/ethyl acetate (3/7, v/v) as
eluent to give the clean product as a white solid (1.3 g) in 86%
yield.
[0072] Reaction 5 illustrates the synthesis of a .beta.-amino acid
wherein the exocyclic amino substituent the nitrogen heteroatom is
in the 4-position relative to the ring nitrogen.
[0073] Compound 52: Compound 51 (2.0 g) and NaBH.sub.3CN (0.54 g)
were dissolved in methanol (40 ml), 1N HCl (aqueous) was added
dropwise to maintain pH 3-4. After 15-20 minutes, pH change slowed.
The mixture was stirred for an additional 1.0 hour, while 1N HCl
was added occasionally to keep pH 3-4. Water (100 ml) was added.
The mixture was extracted diethyl ether (3.times.150 ml). The
extracts were washed with IN NaHCO3 (100 ml) and dilute brine (100
ml), dried over MgSO.sub.4, and concentrated to give a colorless
oil (1.9 g) in 95% yield. The product was used directly without
further purification.
[0074] Compound 53: Compound 52 (1.9 g) and Ph.sub.3P (2.8 g) were
dissolved in toluene (anhydrous, 30 ml) under nitrogen. A solution
of diethyl azodicarboxylate (1.5 ml) in toluene (10 ml) was
subsequently introduced via syringe over 15 minutes. The reaction
mixture was stirred under nitrogen at room temperature for 12
hours. The toluene was removed under reduced pressure. The residue
was purified by column chromatography with ethyl acetate/hexane
(3/7, v/v) as eluent to afford a colorless oil (1.6 g) in 91%
yield.
[0075] Compound 54: Compound 53 (1.0 g) and
R-(+)-.alpha.-methylbenzylamine (1.1 ml) were mixed with water (15
ml). The mixture was stirred at 55.degree. C. for 67 hours. The
mixture was taken up in diethyl ether (300 ml), and the aqueous
layer was separated. The ether solution was washed with water
(3.times.50 ml), dried over MgSO.sub.4, and concentrated to give a
slight yellow oil. The diastereometic isomers were separated by
column chromatography with ethyl acetate/hexane (2/8, v/v) as
eluent to give RSS (0.2 g) and RRR (0.34 g) in 51% overall
yield.
[0076] Compound 55: Compound 54 (4.2 g) was dissolved in ethyl
acetate (200 ml). 4N HCl in dioxane (4.35 ml) was added dropwise
while stirring. A white precipitate resulted. The ethyl acetate was
removed under reduced pressure, and the resulting white solid (4.6
g, 100%) was dried ini vacuo. ##STR15## ##STR16##
[0077] Compound 56: Compound 55 (4.6 g) was dissolved in 95%
ethanol (150 ml) in a hydrogenation flask. 10% Palladium on
activated carbon (0.5 g) was added. The flask was pressurized with
hydrogen to 50 psi, rocked at room temperature for 22 hours, by
which time NMR spectroscopy indicated that the hydrogenolysis was
complete. The Pd/C was removed by filtration. The filtrate was
concentrated to give a white solid. The white solid was dissolved
in acetone/water (2/1, v/v, 150 ml). NaHCO.sub.3 (9.7 g) was added,
followed by Cbz-OSU (3.4 g). The reaction mixture was stirred at
room temperature for 14 hours. Water (100 ml) was added. The
acetone was removed under reduced pressure. The aqueous mixture was
extracted with ethyl acetate (3.times.200 ml). The extracts were
washed with 1N HCl (3.times.100 ml) and saturated NaHCO.sub.3
(aqueous), dried over MgSO.sub.4, and concentrated to give a
colorless oil. The crude product was purified by column
chromatography with ethyl acetate/hexane (3/7, v/v) as eluent lo
give the clean product as a colorless sticky oil (4.0 g) in 90%
yield.
[0078] Compound 57: Compound 56 (2.0 g) was dissolved in
methanol/water (3/1, v/v, 115 ml), cooled to O.degree. C., LiOH.H20
(2.4 g) was added. The mixture was stirred at O.degree. C. for 15
hours, by which time TLC indicated that the hydrolysis was
complete. Saturated ammonium hydroxide (aqueous, 100 ml) was added.
The methanol was removed under reduced pressure. The aqueous was
acidified with 1N HCl to pH 3, extracted with ethyl acetate
(3.times.200 ml). The extracts were washed with dilute brine (100
ml), dried over MgSO.sub.4, concentrated to give a foamy solid
(1.63 g, 88%), which was used directly without further
purification).
[0079] Compound 58: Compound 57 (1.63 g) was dissolved in methanol
(70 ml) in a hydrogenation flask. 5% Palladium on activated carbon
(250 mg) was added. The flask was pressurized with hydrogen to 35
psi, rocked at room temperature for 15 hours, by which time NMR
spectroscopy indicated that the hydrogenolysis was complete. The
Pd/C was removed by filtration. The filtrate was concentrated to
ive a white solid. The white solid was dissolved in acetone/water
(2/1, v/v, 90 ml), cooled to O.degree. C. NaHCO.sub.3 (2.27 g) was
added, followed by FMOC-OSU (1.83 g). The reaction mixture was
stirred at O.degree. C. for 2 hours, then at room temperature for
28 hours. Water (50 ml) was added. The acetone was removed under
reduced pressure. The aqueous was acidified with 1N HCl to pH 3,
extracted with ethyl acetate (3.times.200 ml). The extracts were
washed with dilute brine (100 ml), dried over MgSO.sub.4,
concentrated to give a foamy white solid. The crude white solid was
purified by column chromatography with methanolfethyl acetate (3/7,
v/v) as eluent to give the clean product as a white solid (1.68 g)
in 84% yield.
[0080] Reaction 5a illustrates an alternative synthesis of the
.beta.-amino acid wherein the exocyclic amino substituent the
nitrogen heteroatom is in the 4-position relative to the ring
nitrogen. This synthesis is the preferred route. ##STR17##
[0081] As compared to Reaction 5, Reaction 5a is streamlined in
that the number of chemical operations is reduced and the need for
chromatographic separations is eliminated.
[0082] Ketoester 51a is allowed to react with
(R)-.alpha.-methylbenzylamine in the presence of acetic acid, and
the resulting enamine is reduced in situ with NaBH.sub.3CN. This
reduction produces a mixture of four diastereomeric
.beta.-aminoesters in which 52a is the major product. A two-step
crystallization protocol that allows isolation of hydrochloride
salt 52a in diastereomerically pure form. The crude
.beta.-aminoester mixture is dissolved in ethyl acetate and
converted to a mixture of ammonium salts by treatment with HCl. A
single trans isomer crystallizes in relatively pure form after this
treatment, although there is contamination from the other trans
isomer. Recrystallization from acetonitrile yields a very pure form
of 52a, in 38% overall yield from 51a. When
(R)-.alpha.-methylbenzylamine is used, the purified
.beta.-aminoester hydrochloride is spectroscopically identical to
material previously identified by crystal structure determination
as the diastereomer shown in Reaction 5a. Thus, use of
(R)-.alpha.-methylbenzylamine leads ultimately to a protected form
of (3S,4R)-trans-3-aminopyrrolidine-4-carboxylic acid.
[0083] The route is completed by alkaline ester hydrolysis,
hydrogenolytic removal of the .alpha.-methylbenzyl group and Fmoc
protection of the resulting amino group. These three steps can be
performed in rapid succession, and the final product can be
purified by crystallization from n-heptane/ethyl acetate. The
protection pattern of diamino acid derivative 54a is suitable for
Fmoc-based synthesis of the .beta.-peptide backbone on a solid
support, with deprotection of the pyrrolidine ring nitrogens upon
acidolytic cleavage from the resin. The synthetic route outlined in
Reaction 5a is amenable to multi-gram synthesis of .beta.-peptide
building block 54a from 51a in one week.
[0084] Compound 52a. To a stirred solution of .beta.-ketoester 51a
(16.0 g, 62.3 mmol) in absolute ethanol (250 mL) under N.sub.2 was
added (R)-(+)-.alpha.-methylbenzylamine (16.0 mL, 124.5 mmol) and
glacial acetic acid (7.1 mL, 124.5 mmol) to obtain a cloudy
solution. The reaction mixture was stirred at room temperature
until the formation of the enamine was complete (3 h, monitored by
TLC, product R.sub.f=0.55, 7:3 hexane:ethyl acetate). Sodium
cyanoborohydride (16.5 g, 249.2 mmol) was then added to the
reaction mixture at room temperature and the resulting solution was
heated to 75.degree. C. and stirred for 14 h under N.sub.2. (This
reaction must be carefully monitored by TLC; disappearance of the
enamine indicates completion of reaction. Higher temperature and/or
longer reaction time leads to formation of an
.alpha.,.beta.-unsaturated ester side product.) The ethanol was
removed via rotary evaporation. Water (250 mL) was added. The
mixture was extracted three times with diethyl ether. The combined
organic extracts were washed with brine, dried over MgSO.sub.4, and
concentrated to give a colorless oil. The oil was applied to a plug
of silica gel and washed with 2:1 hexane:ethyl acetate. The
filtrate was concentrated to obtain a colorless oil. The oil was
dissolved in ethyl acetate (250 mL), and 4 N HCl in dioxane (15.6
mL) was added dropwise at room temperature. The resulting solution
was cooled to 0.degree. C. and allowed to stand for 3 h at
0.degree. C. A precipitate formed during this time. The solid was
filtered and washed two times with 100 mL portions of ethyl acetate
(.gtoreq.98% de, 42% crude yield from 51a; the diastereomeric
excess was determined by GC-MS). This solid could be purified by
recrystallization from acetonitrile. The solid was suspended in
acetonitrile (200 mL) and heated to reflux for 1 h. The solution
was then cooled to 0.degree. C. for 3 h. The resulting solid was
filtered and washed two times with 30 mL portions of acetonitrile.
The solid was further dried under vacuum to give 9.4 g of 52a as a
white crystalline solid (>99.0% de, 38% yield from 51a; the
diastereomeric excess was determined by GC-MS): mp 190-191.degree.
C., [.alpha.].sup.23.sub.D=4.8 (c 1.05, MeOH); .sup.1H NMR
(DMSO-d.sub.6, 300 Mhz, 24.degree. C.) .delta. 10.12-9.75 (br, 2H),
7.70-7.28 (m, 5H), 4.54 (m, 1H), 4.05 (q, J.sub.HH=7.2 Hz, 2H),
3.80-3.59 (m, 3H), 3.54-3.22 (m, 3H), 1.62 (d, J.sub.HH 6.3 Hz,
3H), 1.37 (s, 9H, 1.13 (t, J.sub.HH=7.2 Hz, 3H); .sup.1H NMR
(DMSO-d.sub.6, 500 Mhz, 60.degree. C.) .delta. 10.22 (br s, 2H),
7.66-7.65 (m, 2H), 7.42-7.83 (m, 3H), 4.48 (m, 1H), 4.05 (q,
J.sub.HH=7.0 Hz, 2H), 3.78-3.68 (m, 3H), 3.54-3.35 (m, 3H), 1.67
(d, J.sub.HH=7.0 Hz, 3H), 1.36 (s, 9H), 1.13 (t, J.sub.HH=7.0 Hz,
3H); .sup.13C NMR (DMSO-d.sub.6, 125.7 MHz, 60.degree. C.) .delta.
170.20, 152.70, 136.54, 128.68, 128,58, 127.90, 78.95, 60.92,
56.61, 56.00, 47.86, 46.69, 44.70, 27.82, 19.76, 13.50.
[0085] Compound 53a. A sample of compound 52a was mixed with excess
of saturated Na.sub.2CO.sub.3 solution, extracted into ethyl
acetate, dried over MgSO.sub.4 and concentrated in vacua. .sup.1H
NMR (CDCl.sub.3, 300 MHz) .delta. 7.34-7.16 (m, 5H), 4.22-4.07 (m,
2H), 3.80 (q, J.sub.HH=6.0 Hz, 1H), 3.71-3.53 (m, 1H), 3.53-3.22
(m, 3H), 2.94-2.78 (m, 2H), 1.39 (d, rotamer, 9H), 1.32 (d,
J.sub.HH=6.3 Hz, 3H), 1.29-1.18 (m, 3H); H NMR (DMSO-d.sub.6, 500
MHz, 60.degree. C.) .delta. 7.35-7.25 (m, 3H), 7.22-7.18 (m, 2H),
4.12-4.05 (m, 2H), 3.78 (q, J.sub.HH=7.0 Hz, 1H), 3.54 (m, 1H),
3.36 (m, 1H), 3.24 (m, 2H), 3.00-2.90 (m, 2H), 1.36 (s, 9H), 1.24
(d, J.sub.HH=6.5 Hz, 3H), 1.54 (t, J.sub.HH=7.0 Hz, 3H); .sup.13C
NMR (DMSO-d.sub.6, 125.7 MHz, 24.degree. C.) .delta. 172.57,
172.43, 153.25, 153.14, 145.90, 128.11, 126.60, 126.51, 78.31,
60.28, 58.84, 58.02, 55.61, 51.39, 51.17, 48.53, 47.79, 46.70,
39.33, 24.56, 13.86; .sup.13C NMR (DMSO-d.sub.6, 125.7 MHz,
60.degree. C.) .delta. 172.09, 153.02, 145.72, 127.79, 126.27,
126.21, 78.06, 59.94, 58.43, 55.50, 51.19, 48.28, 46.52, 27.83,
24.12, 13.57; MSMALDI z/e; 363.3 (M.+-.H)
[0086] Compound 54a. Compound 52a (1.39 g, 3.49 mmol) was dissolved
in THF/MeOH/H.sub.2O (6/3/1, v/v/v, 40 mL), and the solution was
cooled to 0.degree. C. LiOH.H.sub.2O (732 mg, 17.4 mmol) was added.
The mixture was stirred at 0.degree. C. for 3 h. Aqueous HCl (1 N,
18 mL) was added at 0.degree. C. The solvent was then removed on a
vacuum rotary evaporator to give a white solid (R.sub.f=0.32, 1:9
MeOH/CH.sub.2Cl.sub.2). The white solid was dissolved in 150 mL of
95% ethanol in a hydrogenation flask. Pd--C (10%, 1.1 g) was added.
The resulting mixture was shaken under H.sub.2 (45 psi) for 24 h.
After the reaction was complete (disappearance of starting
material, as monitored by TLC), the mixture was filtered through
celite, and the filtrate was concentrated to obtain a white solid.
This solid was dissolved in acetone/H.sub.2O (2/1, v/v, 150 mL),
cooled to 0.degree. C., and Fmoc-Osu (1.53 g, 4.54 mmol) and
NaHCO.sub.3 (2.93 g, 34.9 mmol) were added. The reaction mixture
was stirred at 0.degree. C. for 1 h, and then allowed to stir at
room temperature overnight. Water (50 mL) was added. The acetone
was removed under reduced pressure. The aqueous layer was stirred
for 1 h with diethyl ether (50 mL), the layers were separated and
the aqueous layer was acidified with 1 N aqueous HCl, extracted
with ethyl acetate, dried over MgSO.sub.4, and concentrated to give
a foamy solid. The crude product was purified by crystallization
from n-heptane/ethyl acetate to afford 1.13 g (72%) of 54a as a
white solid: mp 113-115.degree. C., [.alpha.].sup.23.sub.D=-18.3 (c
1.2, MeOH); .sup.1H NMR (CD.sub.3OD, 300 Mhz) .delta. 7.77 (d,
J.sub.HH=7.2 Hz, 2H), 7.62 (d, J.sub.HH=7.5 Hz, 2H), 7.37 (t,
J.sub.HH=7.2 Hz, 2H), 7.28 (t, J.sub.HH=7.2 Hz, 2H), 4.48-4.27 (m,
3H), 4.18 (t, J.sub.HH=7.2 Hz, 1H), 3.72-3.46 (m, 3H), 3.14 (m,
1H), 2.88 (m, 1H), 1.44 (s, 9H); .sup.13C NMR(CD.sub.3OD, 75.4 MHz,
24.degree. C.) .delta. 174.63, 158.13, 156.02, 145.24, 145.16,
142.56, 128.74, 128.11, 126.17, 126.10, 120.90, 81.29, 67.77,
54.73, 54.06, 51.73, 51.20, 28.70; .sup.13C NMR (CD.sub.3OD, 125.7
MHz, 50.degree. C.) .delta. 158.20, 156.11, 145.28, 145.22, 142.56,
128.71, 128.09, 126.06, 120.87, 81.12, 67.81, 55.10, 51.61, 28.77;
MS-MALDI z/e; 475.3 (M+Na), 491.2 (M+K)
[0087] An alternative route to prepare cycloalkyl-constrained
.beta.-amino acids is depicted in Reaction Schemes 5b and 5c,
below. The initial six steps of Scheme 5b (up to the
1-MeOBn-2,3-epoxycyclopentane) is taken directly from the
literature: Colombini et al. (1995), Tetrahedron 51:8089. ##STR18##
##STR19##
[0088] To synthesize the nipecotic reverse turn moiety, Reaction 6
was used. ##STR20##
[0089] To synthesize .beta.-peptides having reverse turn moiety
which is a prolyl-glycolic acid residue, the following protocols
are preferred:
[0090] (2S,3R)-3-Amino-2-methylpentanoic acid was prepared
according to the procedures given by Jefford and McNulty (1994), J.
Helv. Chim. Acta 77:2142. However, unlike the description in this
paper, the synthesized
(2S,3S)-2-methyl-3-(tosylamino)butano-4-lactone contained up to 8%
(2R,3S)-2-methyl-3-(tosylamino)butano-4-lactone as a byproduct,
which could be removed by recrystallization from toluene.
(2S,3S)-3-Amino-2-benzyl-4-phenylthiobutanoic acid was prepared in
a synthetic sequence derived from the one by Jefford and McNulty.
This synthesis is described below. Homo-.alpha.-amino acids were
prepared according to the procedures by Podlech and Seebach (1995),
Liebigs Ann. 1217. Depsi-.beta.-peptides were synthesized by
conventional dicyclohexylcarbodiimide/N-hydroxysuccinimide
(DCC/HOSu) or 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide
hydrochloride/N,N-dimethyl-4-aminopyridine (EDCI/DMAP)
solution-phase coupling procedures (see, for example, Bodanszky,
M.; Bodanszky, A. The Practice of Peptide Synthesis; Springer
Verlag: New York, 1984). Illustrative procedures are given below.
##STR21##
[0091] (2S,3S)-2-Benzyl-3-(tosylamino)butano-4-lactone (4). A
solution of lithium diisopropylamine (LDA) in THF was generated by
adding 1.5 M methyllithium in diethyl ether (30 mL, 45.0 mmol) to a
solution of diisopropylamine (6.4 mL, 45.7 mmol) in 100 mL THF at
O.degree. C. under nitrogen and stirring for 10 min. The solution
was then cooled to -78.degree. C., and a solution of
(3S)-3-(tosylamino)butano4-lactone (5.36 g, 21.1 mmol) in 30 mL THF
was added dropwise. The resulting yellow solution was stirred for 1
hour at -78.degree. C., and then benzyl bromide (10 mL, 84.1 mmol)
was added rapidly. Stirring at -78.degree. C. was continued for 2
hours, and the reaction was quenched with 20 mL sat. aq. NH.sub.4Cl
solution and allowed to warm to room temperature. The mixture was
acidified with 1 M HCl and extracted three times with methylene
chloride. The combined organic extracts were dried over
Na.sub.2SO.sub.4 and evaporated to give an orange semisolid that
was purified by chromatography (silica gel, hexanelethyl acetate
3:2) to yield 2.22 g (8.70 mmol, 41%) recovered starting material
and 3.37 g (9.76 mmol; 46%) of 4. No diastereomeric addition
product could be detected. For further purification 4 can be
recrystallized from toluene to give colorless needles. mp.
108.5-109.degree. C., .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta.
7.58 (d, J=8.5 Hz, 2 H), 7.31 (d, J=8.2 Hz, 2 H), 7.20 (m, 3 H),
6.92 (dd, J=7.7, 1.7 Hz, 2H), 4.97 (d, J=5.5 Hz, 1 H), 4.27 (dd,
J=7.2, 9.8 Hz, 1 H), 3.98 (dd, J=7.2, 9.8 Hz, 1 H), 3.65 (m, 1 H),
3.00 (m, 1 H), 2.77 (m, 2 H), 2.46 (s, 3 H), .sup.13C-NMR (75.5
MHz, CDCl.sub.3) .delta. 144.27 (C), 138.00 (C), 135.83 (C), 129.89
(CH), 128.97 (CH), 128.88 (CH), 127.10 (CH) 71.25 (CH.sub.2) 53.21
(CH), 46.54 (CH), 33.54 (CH.sub.2) 21.50 (CH.sub.3), EI MS m/e
345.1027 calc. for C.sub.18H.sub.19NO.sub.4S 345.1035.
##STR22##
[0092] (2S,3S)-2-Benzyl-4-phenylthio-3-(tosylamino)butanoic acid
(7). (2S,3S)-2-Benzyl-3-(tosylamino)butano-4-lactone (4) (0.91 g,
2.64 mmol) was dissolved in 10 mL methylene chloride. At O.degree.
C. trimethylsilyliodide (1 mL, 7.03 mmol) and anhydrous ethanol
(0.72 mL, 12.2 mmol) were added under nitrogen. The solution was
stirred 30 min. at O.degree. C., allowed to warm to room
temperature and stirred for 1 day. Then the addition of
trimethylsilyliodide and ethanol was repeated and stirring at room
temperature was continued for 12 hours. The reaction was quenched
by the addition of 3 mL ethanol and stirring for 30 min. To the
solution 20 mL of water were added, the layers were separated, and
the aqueous layer was extracted five times with methylene chloride.
The combined organic extracts were washed with 5% aq.
Na.sub.2S.sub.2O.sub.3 solution, dried over Na.sub.2SO.sub.4 and
concentrated in vacuo to give 1.78 g of crude 5 as an orange solid,
which was used in the next step without further purification.
[0093] At O.degree. C., thiophenol (0.73 ml, 7.11 mmol) was added
to a suspension of NaH (289.7 mg, 7.24 mmol) in 6 mL DMF under
nitrogen, warmed to room temperature and stirred for 15 min. A
solution of crude 5 (1.78 g) in 10 mL DMF was added to the
thiophenolate solution at O.degree. C. After warming to room
temperature the solution was stirred for 1 hour. The reaction was
quenched with 50 ml water and extracted three times with methylene
chloride. The combined organic extracts were washed with brine,
dried over Na.sub.2SO.sub.4 and concentrated in vacuo to give 2.43
g of 6 as a colorless oil, which was used in the next step without
further purification.
[0094] To a solution of 6 (2.43 g) in 18 mL methanol a 1.5 M aq.
NaOH solution was added and the mixture heated to 60.degree. C. for
2 hours. After evaporation of methanol in vacuo, 20 mL water was
added and the mixture extracted two times with diethyl ether. The
aqueous layer was acidified with conc. HCl and extracted four times
with diethyl ether. The organic extracts were dried over
Na.sub.2SO.sub.4 and evaporated to yield 1.04 g (2.28 mmol, 86%) of
7. .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 7.45 (d, J=8.3 Hz, 2
H), 7.24-7.17 (m, 6 H), 7.09-7.00 (m, 6 H), 5.54 (d, J=8.3 Hz, NH),
3.46 (m, 1 H), 3.28 (m, 1 H), 3.00 (m, 3 H), 2.67 (dd, J=7.1, 14.0
Hz, 1H), 2.34 (s, 3H). ##STR23##
[0095] (2S ,3S)-3-Amino-2-Benzyl-4-phenylthiobutanoic acid (8).
Compound 7 and phenol (0.77 g) were dissolved in 50 mL 48% HBr and
heated to reflux for 1.5 hours under nitrogen. After cooling to
room temperature 150 mL water was added and the solution extracted
two times with diethyl ether. The yellow aqueous layer was
evaporated to give 0.58 g of
(2S,3S)-3-amino-2-benzyl-4-phenylthiobutanoic acid hydrobromide as
an orange solid. .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 7.69 (b,
3 NH), 7.43 (m, 2 H), 7.34-7.01 (m 8 H), 3.60 (m, 1H), 3.35 (m, 3
H), 3.08 (dd, J=8.2, 14.2 Hz, 1 H), 2.87 (dd, J=7.5, 14.2 Hz, 1
H).
[0096] The hydrobromide was dissolved in 140 mL anhydrous ethanol,
and 28 mL methyloxirane was added. The solution was heated to
reflux for 1 hour under nitrogen. The solvent was evaporated to
yield 0.45 g (1.45 mmol, 65%) of 8. ##STR24##
[0097] (2S,
3S)-3-(t-Butoxycarbonylamino)-2-benzyl-4-phenylthiobutanoic acid.
To a solution of 8 (0.18 g, 0.597 mmol) in 1 mL water and 2 mL
dioxane was added K.sub.2CO.sub.3 (167.9 mg, 1.21 mmol). After
cooling to O.degree. C., di-t-butyl-dicarbonate (153.2 mg, 0.681
mmol) was added, the solution warmed to room temperature and
stirred for 1 day. The solution was concentrated in vacuo, and the
residue dissolved in 20 mL water. The solution was acidified to pH
2-3 (congo red) with 1 M HCl and extracted five times with ethyl
acetate. The combined organic extracts were dried over MgSO.sub.4
and evaporated to give an orange oil that was purified by
chromatography (silica gel, hexane/ethyl acetate 1:2) to yield 63.4
mg (0.159 mmol, 27%) of 9. .sup.1H-NMR (300 MHz, CDCl.sub.3)
.delta. 7.37-7.13 (m, 10 H), 5.47 (d, J=8.5 Hz, NH), 3.88 (m, 1 H),
3.20 (m, 1 H), 3.00 (m, 1 H), 2.84 (m, 3 H), 1.39 (s, 9 H),
.sup.13C-NMR (75.5 MHz, CDCl.sub.3) .delta. 174.82 (C), 156.49 (C),
140.14 (C), 136.80 (C), 130.44 (C), 130.02 (C), 129.81 (C), 129.36
(C), 127.33 (C), 127.27 (C), 79.68 (C), 52.46 (CH), 52.33 (CH),
37.37 (CH.sub.2), 35.25 (CH.sub.2), 28.55 (3 CH.sub.3).
##STR25##
[0098] Methyl-(2S,3R)-3-(t-butoxycarbonylamino)-2-methylpentanoic
amide (10). (2S ,3R)-3-(t-Butoxycarbonylamino)-2-methylpentanoic
acid (149.1 mg, 0.645 mmol) was dissolved in 1 mL DMF. At O.degree.
C. methylamine hydrochloride (88.6 mg, 1.31 mmol) and DMAP (195.7
mg, 1.60 mmol) were added, followed by EDCI
(1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride)
(376.9 mg, 1.97 mmol). After stirring at room temperature for 2
days, the solvent was removed in a stream of nitrogen and the
residue dried in vacuo. The residue was titurated with 1 mL 1 M HCl
and 4 mL water, and the white precipitate was collected by suction
filtration to yield 121.0 mg (0.495 mmol, 66%) of the amide 10 mp.
206-207.degree. C., .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 5.92
(b, NH), 4.72 (b, NH), 3.58 (m, 1 H), 2.77 (d, J=4.8 Hz, 3 H), 2.45
(m, 1 H), 1.45 (m, 1 H), 1.41 (s, 9 H), 1.40 (m, 1 H), 1.13 (d,
J=7.2 Hz, 3 H), 0.90 (t, J=7.4 Hz, 3 H), .sup.13C-NMR (75.5 MHz,
CDCl.sub.3) .delta. 174.83 (C), 156.15 (C), 79.35 (C), 54.62 (CH),
45.02 (CH), 28.35 (3 CH.sub.3), 26.24 (CH.sub.3), 25.18 (CH.sub.2),
13.71 (CH.sub.3), 10.85 (CH.sub.3), EI MS m/e 244.1789 calc. for
C.sub.12H.sub.24N.sub.2O.sub.3 244.1787. ##STR26##
[0099] Compound 12. Compound 10 (121.0 mg, 0.495 mmol) was
dissolved in 2 mL of 4 M HCl/dioxane, and the resulting solution
was stirred 1 hour at room temperature. HCl/dioxane was then
removed in a stream of nitrogen and the deprotected amide dried in
vacuo. The activated glycolic ester was prepared by adding EDCI
(188.7 mg, 0.635 mmol) to a solution of glycolic acid (45.5 mg,
0.598 mmol) and HOSu (N-hydroxysuccinimide) (72.7 mg, 0.632 mmol)
in 1 mL DMF and stirring of the solution at room temperature for 2
hours. The deprotected amide and triethylamine (85 .mu.l, 0.610
mmol) were dissolved in 1 mL DMF and transferred into the activated
ester solution. After stirring the resulting solution for 2 days at
room temperature, the solvent was removed in a stream of nitrogen
and the residue dried in vacuo. The residue was separated by
chromatography (silica gel, CHCl.sub.3/MeOH 4:1) to yield impure 11
(192.7 mg), which was used in the next step without further
purification.
[0100] Compound 11 (192.7 mg) and BOC-L-proline (213.3 mg, 0.991
mmol) were dissolved in 3 mL DMF. DMAP (15.6 mg, 0.128 mmol) was
added, followed by DCC (dicyclohexylcarbodiimide) (248.3 mg, 1.20
mmol), and the resulting solution was stirred overnight at room
temperature. The white precipitate was filtered off by suction
filtration, and the filtrate was concentrated in vacuo. The residue
was separated by chromatography (silica gel, CHCl.sub.3/MeOH 19:1)
to yield 145.7 mg (0.365 mmol, 74% based on 10) of 12. .sup.1H-NMR
(300 MHz, CDCl.sub.3) .delta. 7.02 (d, J=8.6 Hz, NH major rotamer
89%), 6.91 (d, J=9.0 Hz, NH minor rotamer 11%), 6.10 (M, NH), 4.78
(AB, A part, J=15.3 Hz, 1 H), 4.49 (AB, B part, J=15.3 Hz, 1 H)
4.26 (m, 1 H), 3.90 (m, 1 H), 3.44 (m, 2 H), 2.74 (d,J=4.6 Hz, 3
H), 2.45 (quint., J=7.0 Hz, 1 H), 2.22 (m, 1 H), 1.98 (m, 2 H),
1.88 (m, 1 H), 1.56 (m, 1 H), 1.43 (s, 9 H), 1.43 (m, 1 H), 1.08
(d, J=7.0 Hz, 3 H), 0.86 (t, J=7.4 Hz), .sup.13C-NMR (75.5 MHz,
CDCl.sub.3) .delta. 174.80 (C), 172.24 (C), 167.47 (C), 154.78 (C),
80.33 (C), 62.79 (CH.sub.2), 58.77 (CH), 53.74 (CH), 46.75
(CH.sub.2), 45.58 (CH), 29.91 (CH.sub.2), 28.26 (3 CH.sub.3), 26.05
(CH.sub.3), 24.97 (CH.sub.2), 24.49 (CH.sub.2), 14.43 (CH.sub.3),
10.62 (CH.sub.3). ##STR27##
[0101] Compound 13. Compound 12 (12.3 mg, 30.8 .mu.mol) was
dissolved in 1 mmol 4 M HCl/dioxane and the solution was stirred
for 1 hour at room temperature. HCl/dioxane was removed in a stream
of nitrogen and the residue dried in vacuo. The deprotected
depsipeptide and (2S,3
S)-2-benzyl-3-(t-butoxycarbonylamino)-4-phenylthiobutanoic acid (9)
(14.3 mg, 35.6 .mu.mol) were dissolved in 0.5 mL methylene
chloride. DMAP (5.0 mg, 40.9 .mu.mol) was added, followed by EDCI
(13.7 mg, 71.5 .mu.mol). After stirring at room temperature for 2
days, the solvent was removed in a stream of nitrogen and the
residue dried in vacuo. The residue was titurated with 1 mL water,
which was acidified to pH 2. The resulting solid was collected and
purified by chromatography (silica gel, CHCl.sub.3/MeOH 19:1) to
yield 14.1 mg (20.6 .mu.mol, 67%) of 13. .sup.1H-NMR (300 MHz,
CDCl.sub.3) .delta. 7.41 (d, J=10.1 Hz, NH), 7.38-7.13 (m, 10
H+NH), 5.06 (AB, A part, J=15.3 Hz, 1 H), 5.03 (d, J=10.5 Hz, NH),
4.45 (m, 1 H), 4.32 (AB, B part, J=15.5 Hz, 1 H), 4.26 (m, 1 H),
4.02 (t, J=7.6 Hz, 1 H), 3.31 (m, 1 H), 3.08 (m, 3 H), 2.97 (m, 1
H), 2.83 (m, 1 H), 2.78 (d, J=4.6 Hz, 3 H), 2.53 (m, 1 H), 2.39
(dq, J=10.1 Hz, 6.9 Hz, 1 H), 1.98 (m, 1 H), 1.73 (m, 2H), 1.50 (m,
1 H), 1.44 (m, 1 H), 1.41 (s, 9 H), 1.30 (m, 1 H), 1.07 (d, J=6.9
Hz, 3 H), 0.98 (t, J=7.4 Hz, 3 H), .sup.13C-NMR (75.5 MHz,
CDCl.sub.3) .delta. 175.44 (C), 171.63 (C), 167.03 (C), 155.54 (C),
138.29 (C), 135,46 (C), 129.43 (CH), 129.06 (CH), 128.68 (CH),
128.30 (CH), 126.60 (CH), 126.47 (CH), 80.24 (C), 62.69 (CH.sub.2),
59.27 (CH), 52.71 (CH), 52.46 (CH), 49.39 (CH), 46.75 (CH.sub.2),
46.66 (CH), 38.16 (CH.sub.2), 36.32 (CH.sub.2), 28.58 (CH), 28.11
(3 CH.sub.3), 26.26 (CH.sub.2), 25.85 (CH.sub.3), 25.05 (CH.sub.2),
16.22 (CH.sub.3), 10.47 (CH.sub.3). ##STR28##
[0102] Compound 1. Compound 13 (14.1 mg, 20.6 .mu.mol) was
dissolved in 1 mL 4 M HCl/dioxane and the solution was stirred for
1 hour at room temperature. HCl/dioxane was removed in a stream of
nitrogen and the residue dried in vacuo. The deprotected
depsipeptide and triethylamine (5.8 .mu.L, 41.6 .mu.mol) were
dissolved in 0.41 mL methylene chloride, and acetic anhydride (2.4
.mu.L, 25.4 .mu.mol) was added. After stirring the solution at room
temperature overnight the solvent was removed in a stream of
nitrogen and the residue dried in vacuo. The residue was purified
by chromatography (silica gel, CHCl.sub.3/MeOH 19:1) to yield 9.2
mg (14.7 .mu.mol, 71%) of 1. mp. 196.5-197.degree. C., .sup.1H-NMR
(300 MHz, CDCl.sub.3) .delta. 7.40 (d, J=9.0 Hz, NH), 7.39-7.11 (m,
10 H+NH), 5.99 (d, J=10.1 Hz, NH), 5.03 (AB, A part, J=15.3 Hz, 1
H), 4.78 (tt, J=10.3 Hz, 3.6 Hz, 1 H), 4.34 (AB, B part, J=15.3 Hz,
1 H), 4.25 (dq, J=10.0 Hz, 1 H), 4.02 (t, J=7.4 Hz, 1 H), 3.36 (m,
1 H), 3.20-3.00 (m, 3 H), 2.85-2.75 (m, 2 H), 2.79 (d, J=4.6 Hz, 3
H), 2.60 (m, 1 H), 2.42 (dq, J=10.1 Hz, 6.9 Hz, 1 H), 2.00 (m, 1
H), 1.86 (s, 3 H), 1.85-1.62 (m, 3 H), 1.52 (m, 1 H), 1.31 (m, 1
H), 1.07 (d, J=6.8 Hz, 3 H), 0.97 (t, J=7.4 Hz, 3 H), .sup.13C-NMR
(75.5 MHz, CDCl.sub.3) .delta. 175.46 (C), 171.59 (C), 170.17 (C),
167.08 (C), 138.14 (C), 136.87 (C), 135,36 (C), 129.49 (CH), 129.15
(CH), 128.60 (CH), 128.34 (CH), 126.88 (CH), 126.64 (CH), 62.69
(CH.sub.2), 59.30 (CH), 52.80 (CH), 51.08 (CH), 48.69 (CH), 46.83
(CH.sub.2), 46.28 (CH), 37.37 (CH.sub.2), 36.30 (CH.sub.2), 34.45
(CH2), 28.59 (CH.sub.2), 26.03 (CH.sub.3), 25.07 (CH.sub.2), 23.00
(CH.sub.3), 16.09 (CH.sub.3), 10.46 (CH.sub.3), IR (1 mM in
CH.sub.2Cl.sub.2) 3423, 3367, 1753, 1669, 1626 cm.sup.-1, EI MS m/e
624.2989 calc. for C.sub.33H.sub.44N.sub.4O.sub.6S 624.2981.
##STR29##
[0103] Methyl-3-(t-butoxycarbonylamino)propionic amide (14).
BOC-.beta.-alanine (0.50 g, 2.64 mmol) was dissolved in 4 mL DMF.
Methylamine hydrochloride (198 mg, 2.93 mmol) and DMAP (427.2 mg,
3.50 mmol) were added, followed by EDCI (1.06 g, 5.53 mmol). After
stirring at room temperature for 2 days the solvent was removed in
a stream of nitrogen and the residue dried in vacuo. It was
dissolved in 5 mL 1 M HCl, and the solution was extracted five
times with ethyl acetate. The combined organic extracts were dried
over MgSO.sub.4 and concentrated to yield 0.43 g (2.13 mmol, 81%)
of BOC-.beta.-alanine methylamide (14) as a white solid. mp.
117-118.degree. C., .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 5.78
(b, NH), 5.15 (b, NH), 3.38 (q, J=6.1 Hz, 2 H), 2.78 (d, J=4.8 Hz,
3 H), 2.36 (t, J=6.1 Hz, 2 H), 1.40 (s, 9 H) .sup.13C-NMR (75.5
MHz, CDCl.sub.3) .delta. 171.74 (C), 79.15 (C), 36.41 (CH.sub.2),
36.03 (CH), 28.17 (3 CH.sub.3), 26.04 (CH.sub.3). ##STR30##
[0104] Compound 16. Compound 14 (0.33 g, 1.63 mmol) was dissolved
in 5 mL of 4 M HCl/dioxane, and the solution was stirred at
12.degree. C. for 1 hour. The HCl/dioxane was removed in a stream
of nitrogen and the residue dried in vacuo. An activated ester
solution was prepared by adding DCC (509.9 mg, 2.47 mmol) to a
solution of glycolic acid (145.7 mg, 1.92 mmol) and HOSu (326.4 mg,
2.84 mmol) in 10 mL methylene chloride. A white precipitate formed
after a few minutes. The suspension was stirred at 12.degree. C.
for 6 hours. The deprotected amide and triethylamine (0.27 mL, 1.94
mmol) were dissolved in 10 mL methylene chloride and transferred
into the activated ester solution. After stirring the resulting
solution overnight at room temperature, the white precipitate was
filtered off by suction filtration and the filtrate concentrated to
give a white solid, which was purified by chromatography (silica
gel, CHCl.sub.3/MEOH 19:1) to yield 0.30 g of impure 15, which was
used in the next step without further purification.
[0105] Compound 15 (0.30 g) and BOC-L-proline (371.5 mg, 1.73 mmol)
were dissolved in 50 mL methylene chloride. At O.degree. C. DMAP
(25.6 mg, 0.210 mmol) was added, followed by DCC (402.9 mg, 1.95
mmol). After stirring 1 hour at O.degree. C. the suspension was
allowed to warm to room temperature and stirred overnight. The
white precipitate was filtered off by suction filtration and the
filtrate concentrated. The residue was subjected to chromatography
(silica gel, CHCl.sub.3/MEOH 19:1) to yield 0.23 g (0.644 mmol, 40%
based on 14) of 15 as a colorless glass. .sup.1H-NMR (300 MHz,
CDCl.sub.3) .delta. 7.55 (b, NH major rotamer 84%), 7.05 (b, NH
minor rotamer 16%), 6.25 (b, NH major rotamer 83%), 6.04 (b, NH,
minor rotamer 17%), 4.59 (s, 2 H), 4.25 (m, 1 H), 3.59 (m, 1 H),
3.42 (m, 3 H), 2.73 (d, J=4.8 Hz, 3 H), 2.41 (t, J=6.5 Hz, 2 H),
2.22 (m, 1 H), 1.96 (m, 2 H), 1.88 (m, 1 H), 1.42 (s, 9 H).
##STR31##
[0106] Compound 17. Compound 16 (0.23 g, 0.644 mmol) was dissolved
in 2 mL 4 M HCl/dioxane, and the solution was stirred for 1 hour at
room temperature. HCl/dioxane was removed in a stream of nitrogen
and the residue dried in vacuo. The deprotected depsipeptide and
BOC-.beta.-alanine (133.3 mg, 0.705 mmol) were dissolved in 5 mL
methylene chloride. DMAP (96.9 mg, 0.793 mmol) was added, followed
by EDCI (258.7 mg, 1.349 mmol). After stirring at room temperature
for 2 days the solvent was removed in a stream of nitrogen. The
residue was dissolved in 0.1 M HCl and the solution was extracted
four times with methylene chloride. The combined organic extracts
were dried over MgSO.sub.4 and concentrated to give a white solid
that was purified by chromatography (silica gel, CHCl.sub.3/MeOH
19:1) to yield 0.18 g (0.420 mmol, 66%) of 17 as a white solid.
.sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 7.54 (b, NH), 6.30 (b,
NH), 5.58 (b, NH), 4.66 (AB, A part, J=15.4 Hz, 1 H), 4.47 (AB, B
part, J=15.5 Hz, 1 H), 4.35 (m, 1 H), 3.51 (m, 4 H), 3.35 (m, 2 H),
2.72 (d, J=4.8 Hz, 3 H), 2.51 (m, 2 H), 2.41 (m, 2 H), 2.20 (m, 1
H), 2.10 (m, 1 H), 1.98 (m, 2 H), 1.37 (s, 9 H). ##STR32##
[0107] Compound 2. Compound 17 (0.18 g, 0.420 mmol) was dissolved
in 2 mL 4 M HCl dioxane and the solution was stirred for 1 hour at
room temperature. HCl/dioxane was removed in a stream of nitrogen
and the residue dried in vacuo. The deprotected depsipeptide and
triethylamine (0.12 mL, 0.861 mmol) were dissolved in 5 mL
methylene chloride. At O.degree. C. acetic anhydride (50 .mu.L,
0.530 mmol) was added and the solution was stirred 1 hour at
O.degree. C. and then allowed to warm to room temperature with
stirring overnight. The solvent was removed in a stream of nitrogen
and the residue dried in vacuo. The remaining white solid was
purified by chromatography (silica gel, CHCl.sub.3/MeOH 19:1) to
yield 0.12 g (0.324 mmol, 77%) of 2 as a white solid. mp.
153.5-154.degree. C., .sup.1H-NMR (300 MHz, CDCl.sub.3) 7.79 (d,
J=4.4 Hz, NH), 7.32 (d, J=3.9 Hz, NH), 6.08 (b, NH), 4.75 (AB, A
part, J 15.4 Hz, 1 H), 4.44 (AB, B part, J=15.3 Hz, 1 H), 4.32 (m,
1 H), 3.62-3.40 (m, 5 H), 2.74 (d, J=4.8 Hz, 3 H), 2.59-2.34 (m, 4
H), 2.25-1.91 (m, 3 H), 1.97 (s, 3 H), .sup.13C-NMR (75.5 MHz,
CDCl.sub.3) .delta. 171.57 (C), 171.30 (C), 170.63 (C), 167.42 (C),
62.78 (CH.sub.2), 59.10 (CH), 47.02 (CH.sub.2), 35.83 (CH.sub.2),
35.54 (CH.sub.2), 34.69 (CH.sub.2), 33.75 (CH.sub.2), 29.00
(CH.sub.2), 26.17 (CH.sub.3), 25.05 (CH2), 22.83 (CH.sub.3), IR (1
mM in CH.sub.2Cl.sub.2) 3452, 3334, 1757, 1669, 1635, 1539
cm.sup.-1, EI MS m/e 370.1868 calc. for
C.sub.16H.sub.26N.sub.4O.sub.6 370.1852. ##STR33##
[0108] Methyl-(S)-3-(t-butoxycarbonylamino)butanoic amide (18).
BOC-homoalanine (Podlech, J.; Seebach, D, (1995) Liebigs Ann. 1217)
(0.44 g, 2.17 mmol) was dissolved in 5 mL methylene chloride. HOSu
(376.4 mg, 3.27 mmol) was added and the solution cooled to
O.degree. C. After addition of DCC (587.8 mg, 2.85 mmol) the
solution was stirred 1 hour at O.degree. C., warmed to room
temperature and stirred for an additional 2 hours. A stream of
methylamine was bubbled through the suspension for 10 minutes, and
stirring was continued overnight. The white precipitate was
filtered off by suction filtration and the filtrate concentrated to
give a pale yellow solid that was purified by chromatography
(silica gel, CHCl.sub.3/MeOH 19:1) to yield 0.41 g (1.90 mmol, 88%)
Of BOC-homoalanine methylamide (18) as a white solid. .sup.1H-NMR
(300 MHz, CDCl.sub.3) .delta. 6.11 (b, NH), 5.23 (b, NH), 3.92 (m,
1 H), 2.75 (d, J=4.8 Hz, 3 H), 2.35 (m, 2 H), 1.39 (s, 9 H), 1.17
(d, J=6.6 Hz, 3 H). ##STR34##
[0109] Compound 20. Compound 18 (0.41 g, 1.90 mmol) was dissolved
in 2 mL of 4 M HCl/dioxane, and the solution was stirred at room
temperature for 1 hour. HCl/dioxane was removed in a stream of
nitrogen and the residue dried in vacuo. An activated ester
solution was prepared by adding DCC (0.59 g, 2.86 mmol) to a
solution of glycolic acid (175.5 mg, 2.31 mmol) and HOSu (421.9 mg,
3.67 mmol) in 5 mL DMF at O.degree. C. The suspension was stirred
at O.degree. C. for 1 hour and then 2 hours at room temperature.
The deprotected amide and triethylamine (0.32 mL, 2.30 mmol) were
dissolved in 5 mL DMF and transferred into the activated ester
solution. After stirring the resulting solution overnight at room
temperature the white precipitate was filtered off by suction
filtration and the filtrate concentrated to give a semisolid that
was chromatographed (silica gel, CHCl.sub.3/MeOH 9:1) to yield 0.42
g of impure 19, which was used in the next step without further
purification.
[0110] Compound 19 (55 mg, 0.317 mmol, impure) and BOC-D-proline
(148 mg, 0.688 mmol) were dissolved in 2 mL DMF. DMAP (10.0 mg,
0.082 mmol) was added, followed by DCC (171.4 mg, 0.831 mmol).
After stirring the resulting suspension for 1 day at room
temperature the white precipitate was filtered off by suction
filtration and the filtrate concentrated. The remaining semisolid
was purified by chromatography (silica gel, CHCl.sub.3/MeOH 19:1)
to yield 52.1 mg (0.140 mmol, 44%) of 20. .sup.1H-NMR (300 MHz,
CDCl.sub.3) .delta. 7.53 (d, J=6.3 Hz, NH minor rotamer 21%), 7.30
(d, J=7.4 Hz, NH major rotamer 79%), 6.35 (b, NH major rotamer
84%), 6.13 (b, NH minor rotamer 16%), 4.74 (AB, A Part, J=15.4 Hz,
1 H), 4.44 (AB, B part, J=15.4 Hz, 1 H), 4.28 (m, 2 H), 3.45 (m, 2
H), 2.72 (d, J=4.8 Hz, 3 H), 2.41 (dA-B, A part, J=7.4 Hz, 14.3 Hz,
1 H), 2.31 (dAB, B part, J=5.3 Hz, 14.3 Hz, 1 H), 2.23 (m, 1 H),
1.98 (m, 2 H), 1.88 (m, 1 H), 1.43 (s, 9 H), 1.25 (d, J=6.8 Hz, 3
H). ##STR35##
[0111] Compound 21. Compound 20 (52.1 mg, 0.140 mmol) was dissolved
in 1 mL4 M HCl/dioxane and the solution was stirred for 1 hour at
room temperature. HCl/dioxane was removed in a stream of nitrogen
and the residue dried in vacuo. The deprotected depsipeptide and
BOC-homophenylalanine (42.5 mg, 0.152 mmol) were dissolved in 5 mL
methylene chloride. DMAP (32.4 mg, 0.265 mmol) was added, followed
by EDCI (59.4 mg, 0.310 mmol). After stirring at room temperature
for 2 days the solvent was removed in a stream of nitrogen. The
residue was dissolved in 0.1 M HCl, and the solution was extracted
three times with methylene chloride. The combined organic extracts
were dried over MgSO.sub.4 and concentrated to give a colorless
glass that was purified by chromatography (silica gel,
CHCl.sub.3/MeOH 19:1) to yield 62.8 mg (0.118 mmol, 84%) of 21.
.sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 7.36 (b, NH), 7.31-7.12
(m, 5 H), 6.43 (b, NH), 5.22 (b, NH), 4.82 (AB, A part, J=14.9 Hz,
1H), 4.49 (m, 2 H), 4.41 (AB, B part, J=15.6 Hz, 1 H), 4.21 (m,
1H), 3.52 (m, 1 H), 3.32 (m, 1 H), 2.89 (m, 1 H), 2.78 (m, 1 H),
2.71 (d, J=4.8 Hz, 3 H), 2.46 (m, 3 H), 2.40 (m, 1 H), 2.24 (m, 1
H), 2.12-1.89 (m, 3 H), 1.38 (s, 9 H), 1.25 (d, J=6.8 Hz, 3 H).
##STR36##
[0112] Compound 3. Compound 21 (62.8 mg, 0.118 mmol) was dissolved
in 1 mL 4 M HCl/dioxane and the solution was stirred for 1 hour at
room temperature. HCl/dioxane was removed in a stream of nitrogen
and the residue dried in vacuo. The deprotected depsipeptide and
triethylamine (90 .mu.L, 0.646 mmol) were dissolved in 1 mL
methylene chloride. At O.degree. C. acetic anhydride (35 .mu.L,
0.371 mmol) was added and the solution was stirred 1 hour at
O.degree. C. and then allowed to warm to room temperature with
stirring overnight. The solvent was removed in a stream of nitrogen
and the residue dried in vacuo. The residue was purified by
chromatography (silica gel, CHCl.sub.3/MeOH 19:1) to yield 52.1 mg
(0.110 mmol, 93%) of 3. mp. 128-129.degree. C., .sup.1H-NMR (300
MHz, CDCl.sub.3) .delta. 7.49 (d, J=8.5 Hz, NH), 7.31-7.16 (m, 5
H), 6.80 (d, J=8.5 Hz, NH), 6.35 (m, NH), 4.73 (AB, A part, J=15.1
Hz, 1 H), 4.49 (m, 1 H), 4.45 (AB, B part, J=15.4 Hz, 1 H), 4.38
(m, 2 H), 3.55 (m, 1 H), 3.28 (m, 1 H), 2.99 (dAB, A part, J=6.2
Hz, 13.4 Hz, 1 H), 2.88 (dAB, B part, J=8.5 Hz, 13.6 Hz, 1 H), 2.72
(d, J=4.8 Hz, 3 H), 2.55 (dAB, A part, J=5.2 Hz, 15.6 Hz, 1 H),
2.43 (dAB, B part, J=5.9 Hz, 15.5 Hz, 1 H), 2.45 (d, J=6.3 Hz, 2
H), 2.25 (m, 1 H), 2.21-1.89 (m, 3 H), 1.93 (s, 3 H), 1.26 (d,
J=6.6 Hz, 3 H), .sup.13C-NMR (75.5 MHz CDCl.sub.3) .delta. 170.90
(C), 170.31 (C), 169.75 (C), 128.93 (CH), 128.32 (CH), 126.40 (CH),
62.60 (CH.sub.2), 58.89 (CH), 47.31 (CH.sub.2), 42.44 (CH,
CH.sub.2), 39.57 (CH.sub.2), 36.22 (CH.sub.2),28.92 (CH.sub.2),
25.96 (CH.sub.3), 24.94 (CH.sub.2), 23.02 (CH.sub.3), 20.21
(CH.sub.3), IR(1 mM in CH.sub.2Cl.sub.2) 3452, 3433, 3346
cm.sup.-1, EI MS m/e 474.2474 calc. for
C.sub.24H.sub.34N.sub.4O.sub.6 474.2478.
[0113] Construction of polypeptides using any type of .beta.-amino
acid can be accomplished using conventional and widely recognized
solid-phase or solution-phase synthesis. Very briefly, in
solid-phase synthesis, the desired C-terminal amino acid residue is
linked to a polystyrene support as a benzyl ester. The amino group
of each subsequent amino acid to be added to the N-terminus of the
growing peptide chain is protected with Boc, Fmoc, or another
suitable protecting group. Likewise, the carboxylic acid group of
each subsequent amino acid to be added to the chain is activated
with DCC and reacted so that the N-terminus of the growing chain
always bears a removable protecting group. The process is repeated
(with much rinsing of the beads between each step) until the
desired polypeptide is completed. In the classic route, the
N-terminus of the growing chain is protected with a Boc group,
which is removed using trifluoracetic acid, leaving behind a
protonated amino group. Triethylamine is used to remove the proton
from the N-terminus of the chain, leaving a the free amino group,
which is then reacted with the activated carboxylic acid group from
a new protected amino acid. When the desired chain length is
reached, a strong acid, such as hydrogen bromide in trifluoracetic
acid, is used to both cleave the C-terminus from the polystyrene
support and to remove the N-terminus protecting group.
[0114] The preferred solid-phase synthesis used herein is shown in
Reaction 7: ##STR37##
[0115] Solid-phase peptide synthesis is widely employed and well
know. Consequently, it will not be described in any further detail
here.
[0116] Solution phase synthesis, noted above, can also be used with
equal success. For example, solution-phase synthesis of a
.beta.-peptide chain containing alternating residues of
unsubstituted cyclohexane rings and amino-substituted cyclohexane
rings proceeds in conventional fashion as outlined in Reaction 8:
##STR38## Reaction 8 works with equal success to build peptides
wherein the residues are the same or different.
[0117] Reaction 9 is an illustration of a homologation reaction
combined with conventional solution-phase peptide synthesis which
yields a .beta.-peptide having acyclic-substituted residues
alternating with ring-constrained residues: ##STR39##
[0118] As noted above, the .beta.-peptides of the present invention
can be substituted with any number of substituents, including
hydroxy, linear or branched C.sub.1-C.sub.6-alkyl, alkenyl,
alkynyl; hydroxy-C.sub.1-C.sub.6-alkyl,
amino-C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-alkyloxy,
C.sub.1-C.sub.6-alkyloxy-C.sub.1-C.sub.6-alkyl, amino, mono- or
di-C.sub.1-C.sub.6-alkylamino, carboxamido,
carboxamido-C.sub.1-C.sub.6-alkyl, sulfonamido,
sulfonamido-C.sub.1-C.sub.6-alkyl, urea, cyano, fluoro, thio,
C.sub.1-C.sub.6-alkylthio, mono- or bicyclic aryl, mono- or
bicyclic heteroaryl having up to 5 heteroatoms selected from N, O,
and S; mono- or bicyclic aryl-C.sub.1-C.sub.6-alkyl,
heteroaryl-C.sub.1-C.sub.6-alkyl, and combinations thereof.
Effecting such substitutions is well within the set of skills
possessed by a synthetic peptide chemist.
[0119] For example, appending a sulfonamido moiety to the cylic
backbone substituent can be accomplished in conventional fashion
using Reaction 10.
[0120] Compound 63: Compound 61 (90 mg) was dissolved in 4 N HCl in
dioxane (2.0 ml). The reaction mixture was stirred for 1.5 hours.
The dioxane was then removed in vacuo. The residue was dissolved in
pyridine (2.0 ml), then cooled to O.degree. C. in an ice-bath.
[0121] Methanesulfonylchloride (71 .mu.I) was added dropwise. After
the addition, the reaction mixture was stirred at room temperature
for 12 hours. The pyridine was then removed in vacuo. The residue
was taken up in ethyl acetate (50 ml). The mixture was washed with
dilute brine (2.times.10 ml), dried over MgSO.sub.4, and
concentrated to give the clean product as a colorless oil (70 mg)
in 82% yield.
[0122] Compound 64: Compound 62 (30 mg) was dissolved in 4 N HCl in
dioxane (2.0 ml). The reaction mixture was stirred for 1.5 hours.
The dioxane was then removed in vacuo. The residue was dissolved in
pyridine (1.0 ml), then cooled to O.degree. C. in an ice-bath.
Toluenesulfonylchloride (63 mg) was added in portions. After the
addition, the reaction mixture was stirred at room temperature for
12 tours. The pyridine was then removed in vacuo. The residue was
taken up in methylene chloride/dithyl ether (1/1, v/v, 100 ml). The
mixture was washed with dilute brine (3.times.20 ml), dried over
MgSO.sub.4, and concentrated to give a liquid residue. The crude
product was purified by column chromatography with ethyl
acetate/hexane (4/6, v/v) as eluent to give the clean product as a
colorless oil (25 g) in 74% yield.
[0123] Analogous reactions will append a carboxyamido group.
##STR40##
Conformational Analysis
EXAMPLES
[0124] The following Examples are included herein solely to provide
a more complete understanding of the invention. The Examples do not
limit the scope of the invention disclosed and claimed herein in
any fashion.
Example 1
Amide Proton Exchange in Trans-ACHC Dimer and Hexamer
[0125] Amide proton exchange is one of the most powerful methods
for assessing conformational stability of peptides and proteins;
adoption of a stable intramolecularly hydrogen-bonded conformation
leads to diminution of the rate of exchange. NH/ND exchange
behavior of the trans-ACHC hexamer relative to the corresponding
dimer (which is too small to form a favorable internal hydrogen
bond) shows that the hexamer adopts a very stable intramolecularly
hydrogen-bonded folding pattern in methanol solution.
[0126] To ensure a direct comparison, this Example was conducted
with solutions containing 2 mM of the trans-ACHC dimer and 2 mM of
the trans-ACHC hexamer. The .sup.1H NMR results are shown in FIG.
3. Upon dissolution of the 1:1 dimer:hexamer mixture in CD.sub.3OD,
the amide proton and the urethane proton of the dimer (marked with
asterisks) are completely exchanged within 6 min, according to
.sup.1H NMR (FIG. 3). In contrast, three of the six amide protons
of the trans-ACHC hexamer show strong resonances at this point.
[0127] One of these protected protons exchanges within a ca. 20 h,
but the other two require >2 days for complete exchange. Thus,
two of the amide protons of hexamer display >100-fold protection
from NH/ND exchange with CD.sub.3OD, which indicates remarkable
conformational stability for this six-residue foldamer in a
hydrogen-bonding solvent. The two most protected protons of the
hexamer are believed to be the amide groups of residues 2 and 3
(numbering from the N-terminus), although Applicants do not wish to
be limited to this interpretation. The amide protons of residues 5
and 6 should exchange rapidly because they cannot be involved in
14-helical hydrogen bonds. The protons of residues 1 and 4 occur at
the ends of the 14-helix in the hexamer crystal. The ends of
.alpha.-helices in short .alpha.-peptides are "frayed" in solution,
and similar fraying in the trans-ACHC hexamer would presumably
enhance the NH/ND exchange rate. This conclusion is supported by
the observation that only one of the NH resonances of the
corresponding tetramer can be detected by .sup.1H NMR within a few
minutes of dissolution in CD.sub.3OD, and this proton exchanges
completely in less than an hour. Adoption of a stable folding
pattern in solution requires the intrinsic rigidity of the
trans-ACHC residue; dissolution of an analogous .beta.-alanine
hexamer in CD.sub.3OD causes all NH groups to exchange within 6
min. This Example shows that trans .beta.-peptide oligomers
constructed from an appropriately rigidified residue are highly
predisposed to form a specific helix.
Example 2
Circular Dichroism and NOE's in Trans-ACPC
[0128] As noted above, computational comparisons involving
alternative cycloalkane-based .beta.-amino acids and alternative
.beta.-peptide helices suggested that the trans-ACHC/14-helix
combination would generate a stable .beta.-peptide secondary
structure. Using the same technique, the trans-ACPC/ 12-helix
combination was predicted to be almost as favorable. This latter
prediction is intriguing because there is no precedent for the
12-helix in the contradictory literature on polymers constructed
from optically active .beta.-amino acids. Among these polymers,
poly(.alpha.-isobutyl-L-aspartate) has been particularly
intensively studied, and proposed secondary structures include 14-,
16-, 18- and 20-helix, as well as sheet. Since the computational
predictions regarding the trans-ACHC/14-helix relationship
described above proved to be correct, the trans-ACPC/12-helix
prediction was then explored.
[0129] Optically active trans-ACPC was prepared using the protocols
described above, and .beta.-peptides were generated via standard
coupling methods. A trans-ACPC octamer displays the predicted
12-helical conformation in the solid state; all six of the possible
12-membered ring hydrogen bonds are formed. A trans-ACPC hexamer
also displays the predicted 12-helical conformation in the solid
state, with all four of the possible 12-membered ring hydrogen
bonds formed. In both cases, the regular helix frays at the
C-terminus, perhaps because the C-terminal ester group cannot serve
as a hydrogen bond donor.
[0130] Circular dichroism data for trans-ACPC hexamer in CH.sub.3OH
(FIG. 4) indicates the adoption of a distinctive secondary
structure. The main graph in FIG. 4 depicts two virtually
superimposable CD plots: one at a hexamer concentration of 2.0 mM,
the other at hexamer concentration of 0.1 mM. Data were obtained on
a Jasco J-715 instrument at 20.degree. C. using a 1 mm pathlength.
The inset graph shows the CD data at 0.1 mM and 0.02 mM using a 5
mm pathlength. The 14-helical conformation in .beta.-peptides
composed of acyclic residues has a far-UV CD signature comprising a
maximum at ca. 197 nm, a zero crossing at ca. 207 nm, and minimum
at ca. 215 nm. In contrast, the CD signature for the trans-ACPC
hexamer is clearly different: maximum at ca. 204 nm, zero crossing
at ca. 214 nm, and minimum at ca. 221 nm. Since the CD signature of
trans-ACPC hexamer in CH.sub.3OH does not vary significantly
between 2.0 mM and 0.02 mM, it is unlikely that aggregation occurs
under these conditions.
[0131] Two-dimensional .sup.1H NMR data obtained for the trans-ACPC
hexamer in pyridine-d.sub.5 and in CD.sub.3OH indicate that the
12-helix is highly populated in these solvents. Resonances of all
NH groups, all protons at C.sub..beta. of each trans-ACPC residue,
and nearly all protons at C.sub..alpha. of each residue were
resolved in pyridine-d.sub.5, and could be assigned via a
combination of TOCSY and ROESY data. The N-terminal NH (residue 1)
could be identified because it was the only one of the six amidic
protons that did not show an NOE to C.sub..alpha.H of another
residue (this NH also showed an NOE to the Boc methyl groups). The
C-terminal residue could be identified because it had the only
C.sub..alpha.H that did not show an NOE to an NH of another
residue. Assignment of these terminal resonances allowed us to
"walk through" the remaining backbone resonances by virtue of
short-range C.sub..alpha.H.sub.i.fwdarw.NH.sub.i+1 NOEs.
[0132] The secondary structure of the trans-ACPC hexamer in
pyridine-d.sub.5 is defined by the long-range NOEs summarized in
Table 1, below. C.sub..beta.H.sub.i.fwdarw.NH.sub.i+2 and
C.sub..beta.H.sub.i.fwdarw.C.sub..alpha.H.sub.i+2 NOEs are expected
for the 12-helix. All four possible
C.sub..beta.H.sub.i.fwdarw.NH.sub.i+2 NOEs were observed for the
ACPC hexamer, as were two of the four
C.sub..alpha.Hi.fwdarw.C.sub..alpha.H.sub.i+2 NOEs; NOEs consistent
with the other two
C.sub..beta.H.sub.i.fwdarw.C.sub..alpha.H.sub.i+2 interactions were
observed but could not be unambiguously assigned because of overlap
of the C.sub..alpha.H resonances of residues 3 and 4. An NOE
between NH of residue 3 and the Boc methyl groups provided further
evidence for 12-helical folding. In CD.sub.3OH, NOEs consistent
with all of the long range NOEs detected in pyridine-d.sub.5 were
observed, and there did not appear to be any additional NOEs in
CD.sub.3OH. Interpretation of the CD.sub.3OH data was less
definitive, however, because of greater overlap among proton
resonances (especially for C.sub..beta.H of the trans-ACPC
residues). The pattern of NOEs in CD.sub.3OH was identical for 25
mM and 2.5 mM samples of the trans-ACPC hexamer; chemical shifts
and line widths were also invariant between these two
concentrations, suggesting that aggregation does not occur under
these conditions. TABLE-US-00001 TABLE 1 Inter-residue NOEs of
hexamer 2 in pyridine-d.sub.5 Residue H-atom Residue H-atom NOE* 1
C.sub..alpha.H 2 NH Strong 1 C.sub..beta.H 2 NH Weak 1
C.sub..beta.H 3 NH Medium 1 C.sub..beta.H 3 C.sub..alpha.H
Medium.dagger. 1 CH.sub.3(Boc) 3 NH Strong 1 C.sub..beta.H 4 NH
Weak 2 C.sub..alpha.H 3 NH Strong 2 C.sub..beta.H 4 NH Medium 2
C.sub..beta.H 4 C.sub..alpha.H Medium.dagger. 3 C.sub..alpha.H 4 NH
Strong 3 C.sub..beta.H 5 NH Medium 3 C.sub..beta.H 5 C.sub..alpha.H
Medium 4 C.sub..alpha.H 5 NH Strong 4 C.sub..beta.H 6 NH Medium 4
C.sub..beta.H 6 C.sub..alpha.H Weak 5 C.sub..alpha.H 6 NH Strong 5
C.sub..beta.H 6 NH Weak *Strong, 2.3 .ANG.; medium, 3.0 .ANG.;
weak, 4.0 .ANG.. .dagger.NOE present, but overlap of C.sub..alpha.H
of residues 3 and 4 prevents definite assignment.
[0133] NOE-restrained molecular dynamics simulations of the
trans-ACPC hexamer generated 10 low-energy conformations.
Superposition of these conformers shows a high degree of order
(RMSD of backbone atoms <0.2 .ANG.), with fraying at the
C-terminus). The conformer from this set that is closest to the
average agrees quite well with the crystal structure of the
hexamer, although there is variation at the C-terminus and in
individual cyclopentane ring conformations.
[0134] FIG. 5 shows CD data for the dimer, trimer, tetramer, and
hexamer of trans-ACPC in methanol. A comparison between the dimer
and the hexamer indicates the profound change in secondary
structure which takes place as the peptide chain increases in
length. The dimer is essentially unstructured. However, the
tetramer clearly displays a CD curve indicative of 12-helical
secondary structure.
[0135] This Example shows that short .beta.-peptides of trans-ACPC
have a high propensity to adopt the 12-helical folding pattern. In
combination with the 14-helix formation by short oligomers of
trans-ACHC, the present results indicate that .beta.-peptide
secondary structure can be profoundly and rationally altered by
manipulating torsional preferences about the
C.sub..alpha.-C.sub..beta. bonds of individual residues.
Example 3
Amide Proton Exchange in Amino-Substituted Trans-ACHA/Trans ACHA
Dimers and Hexamers
[0136] In this Example, amino-substituted-trans-ACHA (i.e., ACHA
containing an exocyclic amino substituent) was synthesized
according to Reaction 3, above. The amino-substituted-trans-ACHA
was then coupled with unsubstituted trans-ACHA as shown in Reaction
7 to yield .beta.-peptides wherein the residues alternate between
unsubstituted-trans-ACHA and amino-substituted-trans-ACHA. These
molecules were synthesized because it was anticipated that the
amino group would be protonated in water and that the resulting
positive charge would render these .beta.-peptides water-soluble.
They are indeed water-soluble.
[0137] The amide exchange of the dimer and the hexamer were then
compared in the same fashion as described in Example 1 in order to
probe for 14-helix formation in water. FIG. 6 depicts the
two-dimensional .sup.1H NMR data obtained for the alternating
unsubstituted-trans-ACHA/amino-substituted-trans-ACHA hexamer in
D.sub.2O, 100 mM deuteroacetate buffer, pD 3.9. Hydrogen/deuterium
exchange can be examined at five of the six backbone NH groups in
this acidic D.sub.2O solution. The spectra were taken at room
temperature. Five NH peaks are observed (marked with asterisks) in
the 8 minute plot. Each peak disappears at a different rate over
the course of two days as the NH groups become ND groups. The
protons slowest to exchange are those hydrogen bonded and buried in
a folded secondary conformation.
[0138] As shown in FIG. 7, the rate of amide proton exchange in the
dimer had a k.sub.obs of 6.6.times.10.sup.-4/sec. In contrast, the
slowest amide proton exchange in the hexamer had a k.sub.obs of
1.times.10.sup.-4/sec, more than 6-fold slower (protection
factor=6.6). In other words, the slowest exchanging NH of the
hexamer takes 6.6 times longer to exchange than the NH of the dimer
(which is too small to adopt a folded conformation). This Example
shows that there is substantial peptide folding of the alternating
unsubstituted-trans-ACHA/amino-substituted-trans-ACHA hexamer in
aqueous solution. The data strongly suggest that the hexamer is
forming a 14-helix.
Example 4
Comparison of CD Data from Amino-Substituted Trans-ACHA/Trans-ACHA
Hexamers and Trans-ACHC
[0139] In this Example, the CD data from the alternating hexamer
described in Example 3 was compared with a hexamer of unsubstituted
trans-ACHC. The trans-ACRC hexamer adopts a 14-helical conformation
in methanol solution. Therefore, the CD plot for trans-ACHC should
be representational of the 14-helix structure and can serve as a
means of comparison for other peptides. FIG. 8 compares the CD data
of trans-ACHC hexamer in methanol with the CD data of the
alternating unsubstituted-trans-ACHA/amino-substituted-trans-ACHA
described in Example 3. The similarity of the two plots provides
strong evidence that the alternating hexamer adopts a 14-helical
conformation in aqueous solution. In contrast, conventional
peptides made from .alpha.-amino acids never show evidence of
.alpha.-helicity where there are fewer than 10 residues.
[0140] Examples 3 and 4 in combination show that in addition to the
substantial peptide folding indicated by the slow amide proton
exchange rate of the alternating hexamer, the CD data strongly
indicates that the alternating hexamer adopts a 14-helix secondary
structure in aqueous solution.
Example 5
CD Data of .beta.-Peptides Containing Alternating Trans-ACPA
Residues and 3-Amino-4-Carboxy-Pyrrolidinyl Residues
[0141] In this Example, a .beta.-amino acid containing a
3-amino-4-carboxy-pyrrolidinyl residue was synthesized according to
Reaction 5, and coupled into a series of peptides with alternating
trans-ACPA residues using conventional coupling methods (Reaction
7). CD data for the tetramer, hexamer, and octamer in this series
were then gathered. The results, in methanol, are shown in FIG. 9.
Note the distinct and increasing intensity of the characteristic
maximum at about 205 nm and minimum at about 221 nm as the chain
length increases.
[0142] FIG. 10 shows another plot of CD data, in this instance
using water as the solvent. Again, the maximum and minimum
characteristic peaks increase in intensity with increasing chain
length.
[0143] FIG. 11 is a direct comparison of the CD data in water
versus the CD data in methanol for the alternating
4-pyrrolidinyl/trans-ACPA octamer. These data indicate that the
12-helix formation is only slightly less stable in water than it is
in methanol.
[0144] Taken together, the CD data presented in this Example
strongly indicate that the pyrrolidinyl .beta.-peptides assume a
12-helix conformation in both methanol and aqueous solutions.
Example 6
CD Data of Octamer Containing Alternating Trans-ACPA Residues and
3-Amino-2-Carboxy-Pyrrolidinyl Residues
[0145] In this Example, a .beta.-amino acid containing a
3-amino-2-carboxy-pyrrolidinyl residue was synthesized according to
Reaction 4, and coupled into a peptide with alternating trans-ACPA
residues using conventional coupling methods (Reaction 7). CD data
for the octamer of this peptide was then gathered. The results, in
aqueous solution, are shown in FIG. 12. This .beta.-peptide clearly
adopts the 12-helix conformation in water as evidenced by the
characteristic maximum at about 205 nm and the characteristic
minimum at about 221 nm.
[0146] This Example shows that the nitrogen heteroatom introduced
in the pyrrolidinyl moiety can be located in different postions
along the heterocyclic ring without adversely affecting the
formation of the 12-helical structure in solution.
Example 7
Comparison of Secondary Structure in Amino-Subsituted-Trans-ACHC
and .beta.-Peptides Containing Aliphatic-Substituted Residues
[0147] In this Example, the CD spectrum of a .beta.-peptide
containing alternating residues of trans-ACHA and
amino-substituted-trans-ACHA was compared to the CD spectrum of a
"mixed" .beta.-peptide comprising alternating residues of
trans-ACHA and an acyclic .beta.-amino acid bearing an aminopropyl
substituent on the .beta.-carbon of the backbone. The acyclic
.beta.-amino acid was synthesized and coupled to the trans-ACHA
residue as detailed in Reaction 9, described above.
[0148] The CD data are presented in FIG. 13. While the removal of 3
cyclohexyl units clearly diminishes the extent of helix formation,
the extent of helix formation in the mixed .beta.-peptide is still
significant. The data presented here indicate that .beta.-peptides
which include acyclic residues along with ring-constrained residues
will adopt moderately stable secondary structures in solution.
Example 8
Infrared Analysis of Reverse Turn Using Linked Nipecotic Acid
Moieties
[0149] In this Example, two nipecotic acid residues were linked to
each other to act as a reverse turn moiety. This was done using
Reaction 6. FIG. 14A shows the IR spectrum for the diastereomer in
which the two nipecotic acid residues have the same absolute
configuration and FIG. 14B shows the IR spectrum for the
diastereomer in which the two nipecotic acid residues have opposite
absolute configuration. Both samples were taken in dilute solution
(to minimize intermolecular hydrogen bonding) with solvent
subtraction. As is clear from the diastereomer shown in FIG. 14A,
there is little intramolecular hydrogen bonding as evidenced by the
large peak at 3454 cm.sup.-1 which is due to N--H units not
involved in hydrogen bonding. This was as predicted from computer
modeling studies.
[0150] In contrast, the spectrum shown in FIG. 14B for the
heterochiral diastereomer exhibits a very large peak at 3350
cm.sup.-1, indicative of hydrogen-bonded N--H groups.
[0151] This Example shows that synthetic reverse turn moieties can
be constructed from two nipecotic .beta.-amino acid residues having
opposite configuration.
Example 9
X-ray Crystallography of Synthetic Reverse Turn
[0152] Compound 1 was synthesized as described above. Crystals of
compound 1 suitable for X-ray analysis were obtained by vapor
diffusion (over 2 weeks) of n-heptane into a solution of the sample
in ethyl acetate. The data were collected on a Siemens P4/CCD
diffractometer running software provided by the manufacturer. The
ball and stick schematic of the crystal structure in the solid
state is presented in FIG. 16. All hydrogens except those bonded to
nitrogen have been removed for clarity. Hydrogen bonds are shown in
dotted lines.
Combinatorial Chemistry:
[0153] The defined conformation conferred by the
.beta.-polypeptides described herein makes these polyamide
compounds highly useful for constructing large libraries of
potentially useful compounds via combinatorial chemistry.
Combinatorial exploration of functionalized oligomers of, for
instance, trans-ACHC, trans-ACPC, or .beta.-peptides wherein the
.alpha. and .beta. carbons are part of a cycloalkenyl ring or a
heterocyclic ring such a pyrrolidinyl or piperidinyl moiety, or
"mixed" .beta.-peptides containing acyclic residues in addition to
cyclic residues, has a potential yield of literally millions of
novel polypeptide molecules, all of which display a well-defined
helical or sheet secondary structure.
[0154] Of particular note here is that the equatorial positions of
the cyclohexyl .beta.-peptides can be substituted with virtually
any substituent, including very large substituents, without
disrupting the helical secondary structure. At least in helical
structures, this is because any equatorial substituent extends
essentially perpendicular from the axis of rotation of the helix,
thereby leaving the hydrogen bonds of the helix undisturbed.
[0155] The amino acids which comprise the finished peptides can be
functionalized prior to being incorporated into a polypeptide, or
an unfunctionalized polypeptide can be constructed and then the
entire oligomer functionalized. Neither method is preferred over
the other as they are complementary depending upon the types of
compounds which are desired.
[0156] Combinatorial libraries utilizing the present compounds may
be constructed using any means now known to the art or developed in
the future. The preferred methods, however, are the "split and
pool" method using solid-phase polypeptide synthesis on inert solid
substrates and parallel synthesis, also referred to as multipin
synthesis.
[0157] The "split and pool" concept is based on the fact that
combinatorial bead libraries contain single beads which display
only one type of compound, although there may be up to 10.sup.13
copies of the same compound on a single 100 .mu.m diameter bead.
The process proceeds as follows, utilizing standard solid-phase
peptide synthesis as described above:
[0158] Several suitable solid substrates are available
commercially. The substrates are generally small diameter beads,
e.g. about 100 .mu.m, formed from inert polymeric materials such as
polyoxyethylene-grafted polystyrene or polydimethylacrylamide. An
illustrative substrate, marketed under the trademark "ARGOGEL" is
available from Argonaut Technologies, Washington, D.C.
[0159] Referring now to FIG. 15, which is a schematic depicting the
split and pool method, a plurality of inert substrates are divided
into two or more groups and then a first set of subunits is
covalently linked to the inert support. As depicted in FIG. 15, the
initial plurality of substrates is divided into three subgroups.
The appearance of the three groups of beads after the first round
of coupling is shown at I of FIG. 15. The three groups of beads are
then pooled together to randomize the beads. The beads are then
again split into a number of subgroups. Another round of coupling
then takes place wherein a second subunit is bonded to the first
subunit already present on each bead. The process is then repeated
(theoretically ad infinitum) until the desired chain length is
attained.
[0160] The split and pool process is highly flexible and has the
capability of generating literally millions of different compounds
which, in certain applications, can be assayed for activity while
still attached to the inert substrate.
[0161] A critical aspect of the split and pool methodology is that
each reaction be driven to completion to prior to initiating a
subsequent round of coupling. So long as each coupling reaction is
driven to completion, each substrate bead will only display a
single compound. Because the rate of reaction will differ from bead
to bead as the library construction progresses, the beads can be
monitored using conventional dyes to ensure that coupling is
completed prior to initiating another round of synthesis. The
presence of only a single compound per bead comes about because
each individual bead encounters only one amino acid at each
coupling cycle. So long as the coupling cycle is driven to
completion, all available coupling sites on each bead will be
reacted during each cycle and therefore only one type of peptide
will be displayed on each bead.
[0162] The resulting combinatorial library is comprised of a
plurality of inert substrates, each having covalently linked
thereto a different .beta.-polypeptide. The polypeptides can be
screened for activity while still attached to the inert support, if
so desired and feasible for the activity being investigated. Beads
which display the desired activity are then isolated and the
polypeptide contained thereon characterized via conventional
peptide chemistry, such as the Edman degradation. Where a
solution-phase assay is to be used to screen the library, the
polypeptides are cleaved from the solid substrate and tested in
solution.
[0163] As applied in the present invention, one or more of the
subunits coupled to the inert substrate are selected from the
.beta.-amino acids described herein. In this fashion, large
libraries of .beta.-polypeptides can be assembled, all of compounds
contained therein which display predictable secondary
structure.
[0164] An alternative approach to generating combinatorial
libraries uses parallel synthesis. In this approach, a known set of
first subunits is covalently linked to a known location on a inert
substrate, one subunit type to each location. The substrate may be
a series of spots on a suitable divisible substrate such as filter
paper or cotton. A substrate commonly used is an array of pins,
each pin being manufactured from a suitable resin, described
above.
[0165] After the initial round of coupling, each pin of the array
bears a first subunit covalently linked thereto. The array is then
reacted with a known set of second subunits, generally different
from the first, followed by reactions with a third set of subunits,
and so on. During each reiteration, each individual pin (or
location) is coupled with a incoming subunit selected from a
distinct set of subunits, with the order of the subunits being
recorded at each step. The final result is an array of
polypeptides, with a different polypeptide bonded to each solid
substrate. Because the ordering of the subunits is recorded, the
identity of the primary sequence of the polypeptide at any given
location on the substrate (i.e., any given pin) is known. As in the
split and pool method, each coupling reaction must be driven to
completion in order to ensure that each location on the substrate
contains only a single type of polypeptide.
Large Molecule Interactions:
[0166] Another use for the present compounds is as molecular probes
to investigate the interactions between biological macromolecules
to identify antagonists, agonists, and inhibitors of selected
biological reactions. As noted above, many biological reactions
take place between very large macromolecules. The surface areas in
which these reactions take place are thought by many to be far too
large to be disrupted, altered, or mimicked by a small molecule.
Until the present invention, it has been difficult, if not
impossible, to manufacture molecular probes of modest size that
display a well-defined conformation. Because the compounds
described herein assume a highly predictable helical or sheet
conformation, even when functionalized, they find use as reagents
to probe the interaction between large biomolecules.
[0167] Employing the combinatorial methods described herein greatly
expands the medicinal application of the compounds as vast
libraries of compounds can be screened for specific activities,
such as inhibitory and antagonist activity in a selected biological
reaction.
[0168] It is understood that the invention is not confined to the
particular reagents, reactions, and methodologies described above,
but embraces all modified and equivalent forms thereof as come
within the scope of the following claims.
Sequence CWU 1
1
17 1 4 PRT Artificial Sequence Beta-Polypeptide MISC_FEATURE
(1)..(1) 2-aminocyclopentanecarboxylic acid MISC_FEATURE (2)..(2)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (3)..(3)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (4)..(4)
2-aminocyclopentanecarboxylic acid 1 Xaa Xaa Xaa Xaa 1 2 6 PRT
Artificial Sequence Beta-Polypeptide MISC_FEATURE (1)..(1)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (2)..(2)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (3)..(3)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (4)..(4)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (5)..(5)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (6)..(6)
2-aminocyclopentanecarboxylic acid 2 Xaa Xaa Xaa Xaa Xaa Xaa 1 5 3
6 PRT Artificial Sequence Beta-Polypeptide MISC_FEATURE (1)..(1)
2,5-diaminocyclohexanecarboxylic acid MISC_FEATURE (2)..(2)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (3)..(3)
2,5-diaminocyclohexanecarboxylic acid MISC_FEATURE (4)..(4)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (5)..(5)
2,5-diaminocyclohexanecarboxylic acid MISC_FEATURE (6)..(6)
2-aminocyclohexanecarboxylic acid 3 Xaa Xaa Xaa Xaa Xaa Xaa 1 5 4 6
PRT Artificial Sequence Beta-Polypeptide MISC_FEATURE (1)..(1)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (2)..(2)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (3)..(3)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (4)..(4)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (5)..(5)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (6)..(6)
2-aminocyclohexanecarboxylic acid 4 Xaa Xaa Xaa Xaa Xaa Xaa 1 5 5 4
PRT Artificial Sequence Beta-Polypeptide MISC_FEATURE (1)..(1)
2-amino-4-pyrrolidinecarboxylic acid MISC_FEATURE (2)..(2)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (3)..(3)
2-amino-4-pyrrolidinecarboxylic acid MISC_FEATURE (4)..(4)
2-aminocyclopentanecarboxylic acid 5 Xaa Xaa Xaa Xaa 1 6 6 PRT
Artificial Sequence Beta-Polypeptide MISC_FEATURE (1)..(1)
2-amino-4-pyrrolidinecarboxylic acid MISC_FEATURE (2)..(2)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (3)..(3)
2-amino-4-pyrrolidinecarboxylic acid MISC_FEATURE (4)..(4)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (5)..(5)
2-amino-4-pyrrolidinecarboxylic acid MISC_FEATURE (6)..(6)
2-aminocyclopentanecarboxylic acid 6 Xaa Xaa Xaa Xaa Xaa Xaa 1 5 7
8 PRT Artificial Sequence Beta-Polypeptide MISC_FEATURE (1)..(1)
2-amino-4-pyrrolidinecarboxylic acid MISC_FEATURE (2)..(2)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (3)..(3)
2-amino-4-pyrrolidinecarboxylic acid MISC_FEATURE (4)..(4)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (5)..(5)
2-amino-4-pyrrolidinecarboxylic acid MISC_FEATURE (6)..(6)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (7)..(7)
2-amino-4-pyrrolidinecarboxylic acid MISC_FEATURE (8)..(8)
2-aminocyclopentanecarboxylic acid 7 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 8 8 PRT Artificial Sequence Beta-Polypeptide MISC_FEATURE
(1)..(1) 2-amino-5-pyrrolidinecarboxylic acid MISC_FEATURE (2)..(2)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (3)..(3)
2-amino-5-pyrrolidinecarboxylic acid MISC_FEATURE (4)..(4)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (5)..(5)
2-amino-5-pyrrolidinecarboxylic acid MISC_FEATURE (6)..(6)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (7)..(7)
2-amino-5-pyrrolidinecarboxylic acid MISC_FEATURE (8)..(8)
2-aminocyclopentanecarboxylic acid 8 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 9 6 PRT Artificial Sequence Beta-Polypeptide MISC_FEATURE
(1)..(1) 3-amino-3-(3-aminopropyl)propionic acid MISC_FEATURE
(2)..(2) 2-aminocyclohexanecarboxylic acid MISC_FEATURE (3)..(3)
3-amino-3-(3-aminopropyl)propionic acid MISC_FEATURE (4)..(4)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (5)..(5)
3-amino-3-(3-aminopropyl)propionic acid MISC_FEATURE (6)..(6)
2-aminocyclohexanecarboxylic acid 9 Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10
4 PRT Artificial Sequence Beta-Polypeptide MISC_FEATURE (1)..(1)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (2)..(2)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (3)..(3)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (4)..(4)
2-aminocyclohexanecarboxylic acid 10 Xaa Xaa Xaa Xaa 1 11 4 PRT
Artificial Sequence Beta-Polypeptide MISC_FEATURE (1)..(1)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (2)..(2)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (3)..(3)
2-aminocyclopentanecarboxylic acid MISC_FEATURE (4)..(4)
2-aminocyclopentanecarboxylic acid 11 Xaa Xaa Xaa Xaa 1 12 4 PRT
Artificial Sequence Beta-Polypeptide MISC_FEATURE (1)..(1)
1-phenylthio-2-phenylmethyl-beta-amino acid MISC_FEATURE (3)..(3)
glycolic acid MISC_FEATURE (4)..(4) 1-ethyl-2-methyl-beta-amino
acid 12 Xaa Pro Xaa Xaa 1 13 6 PRT Artificial Sequence
Beta-Polypeptide MISC_FEATURE (1)..(1) 1-(3-aminopropyl)-beta amino
acid MISC_FEATURE (2)..(2) 2-aminocyclohexanecarboxylic acid
MISC_FEATURE (3)..(3) 1-(3-aminopropyl)-beta amino acid
MISC_FEATURE (4)..(4) 2-aminocyclohexanecarboxylic acid
MISC_FEATURE (5)..(5) 1-(3-aminopropyl)-beta amino acid
MISC_FEATURE (6)..(6) 2-aminocyclohexanecarboxylic acid 13 Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 14 8 PRT Artificial Sequence Beta-Polypeptide
MISC_FEATURE (1)..(1) 3-aminopropanoic acid MISC_FEATURE (1)..(2)
3-aminopropanoic acid MISC_FEATURE (3)..(3) 3-aminopropanoic acid
MISC_FEATURE (4)..(4) 3-aminopropanoic acid MISC_FEATURE (5)..(5)
3-aminopropanoic acid MISC_FEATURE (6)..(6) 3-aminopropanoic acid
MISC_FEATURE (7)..(7) 3-aminopropanoic acid MISC_FEATURE (8)..(8)
3-aminopropanoic acid 14 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 15 10
PRT Artificial Sequence Beta-Polypeptide MISC_FEATURE (1)..(1)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (2)..(2)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (3)..(3)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (4)..(4)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (5)..(5)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (6)..(6)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (7)..(7)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (8)..(8)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (9)..(9)
2-aminocyclohexanecarboxylic acid MISC_FEATURE (10)..(10)
2-aminocyclohexanecarboxylic acid 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 10 16 10 PRT Artificial Sequence Beta-Polypeptide
MISC_FEATURE (1)..(1) 2-aminocyclopentanecarboxylic acid
MISC_FEATURE (2)..(2) 2-aminocyclopentanecarboxylic acid
MISC_FEATURE (3)..(3) 2-aminocyclopentanecarboxylic acid
MISC_FEATURE (4)..(4) 2-aminocyclopentanecarboxylic acid
MISC_FEATURE (5)..(5) 2-aminocyclopentanecarboxylic acid
MISC_FEATURE (6)..(6) 2-aminocyclopentanecarboxylic acid
MISC_FEATURE (7)..(7) 2-aminocyclopentanecarboxylic acid
MISC_FEATURE (8)..(8) 2-aminocyclopentanecarboxylic acid
MISC_FEATURE (9)..(9) 2-aminocyclopentanecarboxylic acid
MISC_FEATURE (10)..(10) 2-aminocyclopentanecarboxylic acid 16 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 17 10 PRT Artificial
Sequence Alpha-Polypeptide 17 Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala 1 5 10
* * * * *