Synthesis of (3R)-carboxy pyrrolidine (a β-proline analogue) and its oligomer

Article abstract of DOI:10.1016/S0960-894X(00)00486-8

A decamer of a β-amino acid analogue of L-proline, (3R)-carboxy pyrrolidine (β-proline), was synthesized from a readily available (R)-glycidol. It was found to possess a rigid secondary structure, as evidenced by its CD spectrum. The β-proline decamer, however, failed to bind to profilin, whereas the corresponding α-L-proline decamer bound tightly to this protein. (C) 2000 Published by Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00486-8

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2417±2419
Synthesis of (3R)-Carboxy Pyrrolidine (a ꢀ-Proline Analogue)
and its Oligomer
Yong Jip Kim,a Donald A. Kaiser,b Thomas D. Pollardb and Yoshitaka Ichikawaa,*
aDepartment of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
bStructural Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA
Received 29 February 2000; accepted 14 August 2000
AbstractÐA decamer of a b-amino acid analogue of l-proline, (3R)-carboxy pyrrolidine (b-proline), was synthesized from a readily
available (R)-glycidol. It was found to possess a rigid secondary structure, as evidenced by its CD spectrum. The b-proline decamer,
however, failed to bind to pro®lin, whereas the corresponding a-l-proline decamer bound tightly to this protein. # 2000 Published
by Elsevier Science Ltd.
Since naturally occurring peptides are prone to being
hydrolyzed by cellular proteases, their usefulness as
therapeutic agents is very limited. Therefore, a new class
of peptide analogues has been actively studied with the
expectation that they would be resistant to proteolytic
degradation and yet retain biological activity similar to
that of their natural counterparts.1 b-Amino acids possess
an additional methylene unit between the amino and
carboxyl groups, and their oligomers have been found
to form an a-helical-like conformation, the l+2 helix,
as evidenced by NMR and X-ray crystallographic
studies.2 4 However, only a few of these designed oli-
gomers have yet been shown to have biological activities
similar to those of the naturally occurring peptides
composed of a-amino acids.5 7
Yuki and co-workers have previously prepared poly-
mers of b-proline analogue (3-carboxy pyrrolidine) and
indicated that these polymers exist in random con-
formations.8 Seebach et al. have prepared oligomers of
b2- and b3-homoprolines (2-carboxymethyl- and 2-car-
boxyethyl-pyrrolidine, respectively) and suggested that
they form rigid secondary structures.9 Recently, Gell-
man et al. have reported that oligomers of a b-proline
analogue, (3S)-carboxy pyrrolidine, form a rigid sec-
ondary structure, as indicated by their CD spectra.10
The report from Gellman's group has prompted us to
report our preliminary work on the synthesis and bio-
logical evaluation of an oligomer of a di€erent b-proline
analogue, (3R)-carboxy pyrrolidine 1, which is an
enantiomer of Gellman's compound. In this commu-
nication, we will describe a facile synthesis of 1 and the
biological evaluation of its decamer I as a potential
ligand of pro®lin.
In the course of our ongoing research program of
developing a new class of peptide analogues, we decided
to simply replace the a-amino acid of a peptide with the
corresponding b-amino acid and to examine its con-
formation and biological activity, and we chose a proline
oligomer as our target peptide.
Pro®lin interacts with a variety of biological molecules,
including actin monomers and poly-proline rich sequen-
ces of proteins, and it plays a key role in regulating the
*Corresponding author. Tel.: +1-410-614-2892; fax: +1-410-955-3023; e-mail: ichikawa@jhmi.edu
0960-894X/00/$ - see front matter # 2000 Published by Elsevier Science Ltd.
PII: S0960-894X(00)00486-8

2418
Y. J. Kim et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2417±2419
Scheme 1. Reagents and conditions: (a) ref 18; (b) (1) H2/Raney1 Ni/EtOH; (2) Boc2O/Et3N/MeOH (70% in two steps); (c) MsCl/Et3N/CH2Cl2
(93%); (d) LiCN/DMF (84%); (e) concd HCl; (f) FmocCl/Na2CO3/H2O±dioxane (72% in two steps).
dynamics of actin polymerization, which is responsible
for many motile process in live cells.11 Although the
physiological signi®cance of pro®lin binding to poly-
proline is still unknown, the binding site on pro®lin was
found to lie in a shallow groove between the N- and C-
terminal helices and the underlying b-sheet.12 In water,
poly-proline forms a left-handed helix with a trans-peptide
bond and three residues per turn (a type II helix),
whereas in organic solvents it is known to form a right-
handed cis helix (type I).13 A recent X-ray crystal-
lographic study of pro®lin complexed with an l-a-pro-
line decamer has shown that the binding is due to
speci®c hydrogen bonding between the amido carbonyl
group of the proline oligomer and the NH group of
pro®lin as well as the hydrophobic interaction between
the pyrrolidine framework of a-l-prolines and the tryp-
tophane residues of pro®lin.14 We therefore planned to
synthesize decamers of a b-proline analogue, (3R)-car-
boxyl pyrrolidine, as a b-peptide and of a-proline as a
control and to evaluate the binding anities of these
decamers for a recombinant amoeba pro®lin in order to
examine the possibility that b-peptide can substitute for
the natural peptide.
acid16 or by microbial optical resolution of a cyclobute-
none derivative,17 we decided to develop a new and facile
synthetic route because we were interested in oligomers
of a b-proline analogue with (3R) stereochemistry (see 1
and 2).
Our synthesis of 1 started with a commercially available
(R)-glycidol 3 (Scheme 1). According to the Sharpless
procedure,18 3 was converted to the 3-nitrile derivative 4,
which was then reductively cyclized19 to form a pyrro-
lidine derivative 5.20 The 3-OH group of 5 was mesy-
lated and subsequently displaced with a nitrile group to
give 7.20 The nitrile group of 7 was hydrolyzed with
concentrated HCl to give 1, which was isolated as the
N-Fmoc derivative 8.20 Oligomers of 1 and 2 were pre-
pared by standard solid-phase Fmoc chemistry,21 and
the l-tyrosine residue was incorporated into the C-ter-
minus of each peptide for future tracer experiments.
Binding Study of Pro®lin and the Decamers I and II
Because the binding of poly(a-l-proline) enhances the
intrinsic tryptophan ¯uorescence of pro®lin, we used
¯uorescence spectroscopy to evaluate the anity of the
decamers I and II for the puri®ed Acanthamoeba pro®lin-
I.22 As expected, the decamer of a-proline II showed an
increased ¯uorescence (excitation wavelength=295 nm;
emission wavelength=300±380 nm) that is consistent with
previous observations of the l-proline decamer. How-
ever, no signi®cant increase in ¯uorescence was
observed when the decamer of b-proline I was mixed
Synthesis of ꢀ-Proline: (3R)-Carboxy Pyrrolidine
Although several synthetic procedures have been repor-
ted for the preparation of the optically active b-proline
analogue 3-carboxylic pyrrolidine: by optical resolution
of the racemic compounds,8 from trans (4R)-hydroxy-l-
proline15 as used by Gellman et al.,9 from l-aspartic
Figure 1. CD spectral data of decamers I and II (0.5 mM) in 10 mM potassium phosphate bu€er (pH 7.0).

Y. J. Kim et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2417±2419
2419
with pro®lin at concentrations as high as 100 mM,
whereas the maximum ¯uorescence of pro®lin was
achieved in the presence of a-proline decamer II at
318 nM.
7. Porter, E. A.; Wang, X.; Lee, H.-S.; Weisblum, B.; Gell-
man, S. H. Nature 2000, 404, 565.
8. Yuki, H.; Okamoto, Y.; Kobayashi, Y. J. Polym. Sci.,
Polym. Chem. Ed. 1979, 17, 3867.
9. Abele, S.; Vogtli, K.; Seebach, D. Helv. Chim. Acta 1999,
82, 1539.
10. Huck, B. R.; Langenhan, J. M.; Gellman, S. H. Org. Lett.
1999, 1, 1717.
CD Spectra of Decamers I and II
11. Kaiser, D. A.; Vinson, V. K.; Murphy, D. B.; Pollard, T.
D. J. Cell Sci. 1999, 112, 3779 and references therein.
12. (a) Archer, S. J.; Vinson, V.; Pollard, T. D.; Torchia, D.
FEBS Lett. 1994, 337, 1451. (b) Metzler, W. J.; Bell, A. J. Ernst,
E.; Lavoie, T. B.; Mueller, L. J. Biol. Chem. 1994, 269, 4620.
13. Rabanal, F.; Ludevid, M. D.; Pons, M.; Giralt, E. Bio-
polymers 1993, 33, 1019.
14. (a) Mahoney, N. M.; Janmay, P. A.; Almo, S. C. Nature
Struct. Biol. 1997, 4, 953. (b) Mahoney, N. M.; Rozwarski, D.
A.; Fedorov, E.; Fedorov, A. A.; Almo, S. C. Nature Struct.
Biol. 1999, 6, 6661.
The decamer of a-l-proline II showed a CD spectrum
typical of a type II poly (l-proline) helix, with a small
positive band at 228 nm and a large negative band at
208 nm, in 10mM potassium phosphate bu€er (pH 7.0);
these values are in good agreement with the reported22
®gures (Fig. 1b). In contrast, the other decamer of b-pro-
line I had a large positive band at 215 nm and a negative
band at 198 nm in the same bu€er solution (Fig. 1a),
yielding a curve opposite to that reported by Gellman et al.
for the enantiomer of 1,10 indicating that the b-proline
decamer I may have a rigid, ordered conformation.23
15. Klein, S. I.; Czekaj, M.; Molino, B. F.; Chu, V. Bioorg.
Med. Chem. Lett. 1997, 7, 1773.
16. (a) Gmeiner, P.; Orecher, F.; Thomas, C.; Weber, K. Tet-
rahedron Lett. 1995, 36, 381. (b) Thomas, C.; Hubner, H.;
Gmeiner, P. Bioorg. Med. Chem. Lett. 1999, 9, 841.
17. Mazzini, C.; Lebreton, J.; Alphand, V.; Furstoss, R. J.
Org. Chem. 1997, 62, 5215.
Summary
We have developed an ecient synthetic route for a
b-proline analogue, (3R)-carboxy pyrrolidine, from an
optically active (R)-glycidol employing a CN as a facile
source of aminomethyl and carboxylate groups. The
resulting decameric peptide indicated a rigid secondary
structure based on its CD spectrum; however, it failed to
bind to pro®lin, which shows a tight hydrogen bonding
to the amido backbone of the a-proline decamer II.
18. Klunder, J. M.; Onami, T.; Sharpless, K. B. J. Org. Chem.
1989, 54, 1295.
19. Makino, K.; Ichikawa, Y. Tetrahedron Lett. 1998, 39, 8245.
20. Compound 5: 1H NMR (300 MHz, CDCl3) d 1.31 (s, 9H),
1.80 (br, 2H), 3.15±3.38 (m, 4H), 4.21 (br d, 2H); 13C NMR
(75 MHz, CDCl3) d 28.2, 33.1, 33.6, 43.3, 53.7, 53.9, 69.4,
70.2, 79.1, 154.6; HRMS FAB calcd for C9H17NO3 (M+H)+
188.1287, found 188.1288. Coumpound 6: 1H NMR (300
MHz, CDCl3) d 1.34 (s, 9H), 2.05 (br, 2H), 2.94 (s, 3H), 3.32±
3.54 (m, 4H), 5.13 (br, 1H); 13C NMR (75 MHz, CDCl3) d
28.3, 31.5, 32.4, 38.4, 43.1, 43. 5, 51.6, 52.0, 79.5, 80.1, 154.5;
HRMS FAB calcd for C10H20NO5S (M+H)+ 266.1062,
Acknowledgements
1
found 266.1060. Compound 7: H NMR (300 MHz, CDCl3) d
The NMR studies were performed in the Biochemistry
NMR Facility at Johns Hopkins University, which was
established by a grant from the National Institutes of
Health (GM 27512) and a Biomedical Shared Instru-
mentation Grant (1S10-RR06262-0). This research was
partly supported by the National Institutes of Health
(GM 52324).
1.40 (s, 9H), 2.06±2.26 (m, 2H), 3.06 (dt, J=6.6, 13.5 Hz),
3.32±3.64 (m, 4H), 5.13 (br, 1H); 13C NMR (75 MHz, CDCl3)
d 27.4, 28.2, 29.1, 29.9, 44.2, 48.6, 79.9, 119.8, 153.6; HRMS
FAB calcd for C10H17N2O2 (M+H)+ 197.1290, found
1
197.1289. Compound 8: H NMR (300 MHz, CDCl3) d 2.23
(m, 2H), 3.15 (dt, J=7.2, 14.1 Hz), 3.48±3.58 (m, 2H), 3.72 (d,
2H, J=6.9 Hz), 4.23±4.27 (m, 1H), 4.33±4.43 (m, 2H), 7.31
(dd, 2H, J=7.2, 9.0 Hz), 7.40 (dd, 2H, J=7.5 Hz), 7.60 (d, 2H,
J=7.5 Hz), 7.76 (d, 2H, J=7.5 Hz); 13C NMR (75 MHz,
CDCl3) d 47.4, 61.3, 65.95, 65.99, 67.5, 99.0, 119.9, 125.0,
127.0, 127.6, 141.3, 144.0, 154.9, 177.3; HRMS FAB calcd for
C20H20NO4 (M+H)+ 338.1392, found 338.1392.
References and Notes
21. Both peptides I and II were prepared by the Core Facility
at Johns Hopkins University School of Medicine using a
standard Fmoc chemistry on a solid phase and were HPLC-
puri®ed (>98%).
1. Hintermann, T.; Seebach, D. Chimia 1997, 51, 244.
2. Gellman, S. H. Acc. Chem. Res. 1998, 31, 173.
3. Seebach, D.; Matthews, J. L. J. Chem. Soc., Chem. Com-
mun. 1997, 2015.
22. The bu€er solution for intrinsic ¯uorescence assay con-
tained: 5 mM pro®lin-1, 75 mM KCl, 10 mM Tris, pH 7.5,
1 mM NaN3; for the detailed experimental procedure, see Pet-
rella, E. C.; Machesky, L. M.; Kaiser, D. A.; Pollard, T. D.
Biochemistry 1996, 35, 16535.
23. Further study of the conformation by NMR is in progress
in order to determine the conformational similarity or di€er-
ence between the oligomers of b-proline I and the corre-
sponding a-proline counterpart II.
4. DeGrado, W. F.; Schnerider, J. P.; Hamuro, Y. J. Pep.
Res. 1999, 54, 206.
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tro, J. A.; Seebach, D.; Rognan, D. J. Med. Chem. 1999, 42,
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H.; Abele, S.; Seebach, D. Helv. Chim. Acta 1999, 82, 1774.
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