Norcystine, a new tool for the study of the structure-activity relationship of peptides

Article abstract of DOI:10.1021/ol0606740

Norcysteine (Ncy) is an unnatural amino acid possessing an electronegative sulfur atom directly attached to the α-carbon atom. We describe the synthesis of Boc-D,L-Ncy(Mob)-OH, the resolution of its methyl ester, and the introduction of both D- and L-Ncy

Full text of DOI:10.1021/ol0606740

ORGANIC
LETTERS
2006
Vol. 8, No. 11
2361-2364
Norcystine, a New Tool for the Study of
the Structure−Activity Relationship of
Peptides
Manoj P. Samant and Jean E. Rivier*
The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute,
La Jolla, California 92037
jriVier@salk.edu
Received March 17, 2006
ABSTRACT
Norcysteine (Ncy) is an unnatural amino acid possessing an electronegative sulfur atom directly attached to the
r
-carbon atom. We describe
the synthesis of Boc-D,L-Ncy(Mob)-OH, the resolution of its methyl ester, and the introduction of both D- and L-Ncy in GnRH analogues.
Cystine is commonly found in biologically active pep-
tides and proteins where it plays the critical role of stabil-
izing their secondary and tertiary conformations. During the
past several decades, chemists have mimicked the intramo-
lecular disulfide bridges in natural or de novo designed
peptides and small proteins with the goal of improving
biological activities. The most common mimics include
lactam (-NH-CO-),1,2 thioether (-CH2-S-CH2-),3 meth-
ylenedithioether (-S-CH2-S-),4,5 dicarba (-CH2-CH2-),6
monoselenide (-S-Se-),7 diselenide (-Se-Se-),8 and tri-
sulfides (-S-S-S-).9 Cystine bridges (CR-CH2-S-S-
CH2-CR) have been extended by one or two methylene
groups (CR-CH2-CH2-S-S-CH2-CH2-CR) by introduc-
ing one or two homocysteines in peptides and proteins.7,10,11
There is no report describing the introduction of one or two
norcysteines to form disulfide bridges such as -CR-S-S-
CH2-CR-, -CR-CH2-S-S-CR-, or -CR-S-S-CR-.
We hypothesized that shorter disulfide bridges than those
resulting from the oxidation of the side chains of two
cysteines would affect the overall conformation of a peptide
or protein and modulate their biological activities.
N-protected R-mercaptoamino acids (or R-thiolamino
acids) are reported in the literature12-14 and are of interest
in relationship to certain antibiotics such as gliotoxin,
sporidesmin, aranotin, chaetocin, and quinomycins.15,16 Our
successful use of R-aminoglycine (Agl)17-19 as a bridge head
(1) Thirumoorthy, R.; Holder, J. R.; Bauzo, R. M.; Richards, N. G. J.;
Edison, A. S.; Haskell-Luevano, C. J. Med. Chem. 2001, 44, 4114-4124.
(2) Felix, A. M.; Wang, C.-T.; Heimer, E. P.; Fournier, A. Int. J. Pept.
Protein Res. 1988, 31, 231-238.
(3) Osapay, G.; Prokai, L.; Kin, H.-S.; Medzihradszky, K. F.; Coy, D.
H.; Liapakis, G.; Reisine, T.; Melacini, G.; Zhu, Q.; Wang, S. H.-H.;
Mattern, R.-H.; Goodman, M. J. Med. Chem. 1997, 40, 2241-2251.
(4) Mosberg, H. I.; Omnaas, J. R.; Medzihradsky, F.; Smith, C. B. Life
Sci. 1988, 43, 1013-1020.
(5) Lindman, S.; Lindeberg, G.; Gogoll, A.; Nyberg, F.; Karlen, A.;
Hallberg, A. Bioorg. Med. Chem. 2001, 9, 763-772.
(6) Miller, S. J.; Blackwell, H. E.; Grubbs, R. H. J. Am. Chem. Soc.
1996, 118, 9606-9614.
(10) Cosmatos, A.; Katsoyannis, P. G. J. Biol. Chem. 1975, 250, 5315-
5321.
(11) Smith, C. W.; Ferger, M. F. J. Med. Chem. 1976, 19, 250-254.
(12) Pojer, P. M.; Rae, I. D. Tetrahedron Lett. 1971, 12, 3077-3079.
(13) Patel, S. M.; Currie, J. O., Jr.; Olsen, R. K. J. Org. Chem. 1973,
38, 126-128.
(14) Zoller, U.; Ben-Ishai, D. Tetrahedron 1975, 31, 863-866.
(15) Ohler, E.; Tataruch, F.; Schmidt, U. Chem. Ber. 1973, 106, 165-
176.
(16) Otsuka, H.; Shoji, J. Tetrahedron 1967, 23, 1535-1542.
(17) Rivier, J. E.; Jiang, G.; Koerber, S. C.; Porter, J.; Simon, L.; Craig,
A. G.; Hoeger, C. A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2031-2036.
(7) Rajarathnam, K.; Sykes, B. D.; Dewald, B.; Baggiolini, M.; Clark-
Lewis, I. Biochemistry 1999, 38, 7653-7658.
(8) Besse, D.; Moroder, L. J. Pept. Sci. 1997, 3, 442-453.
(9) Chen, L.; Zoul´ıkova´, I.; Slaninova´, J.; Barany, G. J. Med. Chem.
1997, 40, 864-876.
10.1021/ol0606740 CCC: $33.50
© 2006 American Chemical Society
Published on Web 04/28/2006

Figure 1. HPLC profile of the enzymatic hydrolysis of racemic Boc-Ncy(Mob)-OCH3 (5). Reaction progress: (a) initial, (b) after 24 h,
and (c) after workup and purification. RP-HPLC conditions: buffer A, 0.1% TFA in H2O; buffer B, 0.1% TFA in 80% CH3CN/20% H2O;
gradient elution from 40 to 70% buffer B in 30 min at a flow rate of 0.2 mL/min; UV detection, 0.1 AUFS at 214 nM.
in some cyclic analogues of the gonadotropin releasing
hormone (GnRH)20 suggested a possible use of norcysteine
(Ncy) to constrain peptide conformations in general and that
of GnRH in particular. In this communication, we report a
synthetic pathway to a chiral derivative of norcysteine
suitable for solid-phase peptide synthesis and demonstrate
its compatibility in analogues of GnRH antagonists.
The racemic R-(4-methoxybenzylthio)-N-(tert-butoxycar-
bonyl)glycine or Boc-D,L-Ncy(Mob)-OH (4) was synthesized
by a modified procedure reported for the synthesis of
R-isopropylthiohyppuric acid by Zoller et al.14 In short,
refluxing tert-butyl carbamate (1) and glyoxylic acid mono-
hydrate (2) in acetone yielded R-hydroxy intermediate (3)
which was immediately reacted with 4-methoxybenzylmer-
captan (Mob) under Dean-Stark conditions to give the
racemic Boc-Ncy(Mob)-OH (4) in 75% yield (Scheme 1).
a sulfhydryl protease, was our first choice because it was
reported to enantioselectively hydrolyze the L-isomer of
N-protected R-alkyloxyglycine esters21 and N-protected
R-aminoglycine esters.22 Boc-D,L-Ncy(Mob)-OCH3 (5), a
preferred substrate for the papain-catalyzed hydrolysis,23 was
obtained in quantitative yield by reacting the racemic amino
acid (4) with trimethylsilyldiazomethane. The papain-
catalyzed hydrolysis was carried out in phosphate buffer (pH
6.2) containing 60% acetonitrile at 25 °C and monitored by
RP-HPLC. After 24 h, the reaction mixture contained 50%
methyl ester and 50% acid according to HPLC (Figure 1)
and was quenched by adding acetic acid. A simple workup
and one purification gave Boc-Ncy(Mob)-OH (6) in 90%
yield with respect to the L-isomer. The enantiomeric excess
was found to be greater than 98% according to chiral
HPLC.24 Our attempts to obtain an optically pure D-isomer
from the unhydrolyzed D-methyl ester failed because of
substantial racemization during chemical saponification. This
observation was analogous to that of Strijtveen et al. for the
synthesis of thiolactic acid by chemical hydrolysis of the
carboxy ester of the enantiomerically pure (R)- or (S)-2-
(acetylsulfanyl)propanoate.25 The coupling efficiency of
Boc-Ncy(Mob)-OH (6) in peptide synthesis was investigated
in GnRH analogues.
Scheme 1. Synthesis of Boc-Ncy(Mob)-OH
GnRH (p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-
NH2) is a highly flexible molecule that exists in solution as
an equilibrium mixture of multiple conformers; the most
favored is a type II â-turn involving residues 5-8 (Tyr5-
Gly6-Leu7-Arg8).26,27 In GnRH antagonists, the overall con-
formational freedom was restricted by cyclization via head-
(20) Rivier, J. E.; Struthers, R. S.; Porter, J.; Lahrichi, S. L.; Jiang, G.;
Cervini, L. A.; Ibea, M.; Kirby, D. A.; Koerber, S. C.; Rivier, C. L. J.
Med. Chem. 2000, 43, 784-796.
To limit the number of possible diastereomeric peptides
containing Ncy, we attempted the stereoselective enzymatic
resolution of its derivative Boc-D,L-Ncy(Mob)-OCH3. Papain,
(21) Kawai, M.; Yamada, K.; Hosoda, K.; Omori, Y.; Kato, S.;
Nagayama, N.; Masui, S.; Kamiya, M.; Yamamura, H.; Araki, S.; Butsugan,
Y. Chirality 1999, 11, 561-568.
(22) Sypniewski, M.; Penke, B.; Simon, L.; Rivier, J. J. Org. Chem.
2000, 65, 6595-6600.
(18) Jiang, G.; Miller, C.; Koerber, S. C.; Porter, J.; Craig, A. G.;
Bhattacharjee, S.; Kraft, P.; Burris, T. P.; Campen, C. A.; Rivier, C. L.;
Rivier, J. E. J. Med. Chem. 1997, 40, 3739-3748.
(19) Rivier, J.; Erchegyi, J.; Hoeger, C.; Miller, C.; Low, W.; Wenger,
S.; Waser, B.; Schaer, J.-C.; Reubi, J. C. J. Med. Chem. 2003, 46, 5579-
5586.
(23) Miyazawa, T.; Iwanaga, H.; Yamada, T.; Kuwata, S. Biotechnol.
Lett. 1994, 16, 373-378.
(24) See Supporting Information.
(25) Strijtveen, B.; Kellogg, R. M. J. Org. Chem. 1986, 51, 3664-3671.
(26) Dutta, A. S. Drugs Future 1988, 13, 43-57.
(27) Karten, M. J.; Rivier, J. E. Endocrine ReV. 1986, 7, 44-66.
2362
Org. Lett., Vol. 8, No. 11, 2006

Table 1. Characterization of Cyclic GnRH Antagonists
% purity
MSe (M + H)+
entry
peptidea
ring size
HPLCb
CZEc
tRd (min)
calcd
obsd
7
8
9
10
11
cyclo(4-10)[Ac-DNal1,DCpa2,DPal3,Ncy4,Arg5,DPal6,Cys10]GnRH
cyclo(4-10)[Ac-DNal1,DCpa2,DPal3,DNcy4,Arg5,DPal6,Cys10]GnRH
cyclo(4-10)[Ac-DNal1,DCpa2,DPal3,Cys4,Arg5,DPal6,Ncy10]GnRH
cyclo(4-10)[Ac-DNal1,DCpa2,DPal3,Cys4,Arg5,DPal6,DNcy10]GnRH
cyclo(4-10)[Ac-DNal1,DCpa2,DPal3,Ncy4,Arg5,DPal6,Ncy10]GnRH
22
22
22
22
21
98
99
99
99
97
98
99
99
99
98
15.74
18.33
16.96
17.67
16.37
1446.6
1446.6
1446.6
1446.6
1432.6
1446.6
1446.6
1446.5
1446.5
1432.5
a IUPAC rules are used for nomenclature except for the following: Ac ) acetyl; Nal ) 3-(2-naphthylalanine); Cpa ) 4-chlorophenylalanine; Pal )
3-(3-pyridyl)alanine; Ncy ) norcysteine. b Percentage purity determined by HPLC using buffer A (TEAP, pH 2.30) and buffer B (60% CH3CN/40% A)
under gradient conditions (25-55% B over 30 min), at a flow rate of 0.2 mL/min on a Vydac C18 column (0.21 × 15 cm, 5 µm particle size, 300 Å pore
size). Detection at 214 nm. c Percentage purity determined by capillary zone electrophoresis (CZE) using a Beckman P/ACE System 2050 controlled by an
IBM Personal system/2 model 50Z; field strength of 15 kV at 30 °C. Buffer, 100 mM sodium phosphate (85:15, H2O/CH3CN), pH 2.50, on a Agilent µSil
bare fused-silica capillary (75 µm i.d. x 40 cm length). Detection at 214 nm. d Retention times under HPLC conditions described above. e Mass spectra
(MALDI-MS) were measured on an ABI-Voyager DE-STR instrument using a saturated solution of R-cyano-4-hydroxycinnamic acid in 0.3% trifluoroacetic
acid and 50% acetonitrile as the matrix. The calculated [M + H]+of the monoisotope was compared with the observed [M + H]+ monoisotopic mass.
to-tail28,29 or through the side chain encompassing the
residues 5-8, with a goal to identify a consensus bioactive
conformation or to improve the antagonist potency. The
compatibility of a number of side chain to side chain bridges
has been systematically explored,20,30,31 and so far, only
two different lactam-bridged constraints, cyclo(4-10) and
cyclo(5-8), were found to be well tolerated both in vivo and
in vitro.32,33 Among the cyclic GnRH antagonists with a
disulfide bridge,30,33-35 the cyclo(4-10)[Ac-DNal1,DCpa2,DPal3,-
Cys4,Arg5,DPal,6Cys10]GnRH32 with a 23-membered ring was
potent in both in vitro and in vivo assays and was chosen
for the present study.
All of the Ncy-containing GnRH analogues shown in
Table 1 were synthesized either manually or automatically
on a p-methylbenzhydrylamine resin (MBHA resin) using
the Boc strategy.36 All of the individual Boc-protected amino
acids were incorporated in a sequential manner utilizing
N, N′-diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole
(HOBt)-mediated activation of the carboxyl group in DMF.
Trifluoroacetic acid (TFA) treatment (60% TFA in DCM)
was used for Boc removal, and the N-terminal acetylation
was performed by using an excess of acetic anhydride in
DCM. The protected peptido resins were cleaved and
deprotected in anhydrous HF (1.5 h at 0-5 °C) in the
presence of a scavenger (anisole, 10% v/v) and methyl sulfide
(5% v/v).24 The cyclization (oxidation) of the fully depro-
tected peptide was carried out with iodine.19 The crude
peptides were purified by RP-HPLC in at least two different
solvent systems (triethylammoniumphosphate (TEAP), pH
2.25, and 0.1% TFA on C18 silica).24 The analytical tech-
niques used for the characterization of the analogues in Table
1 included RP-HPLC with two different solvent systems
(0.1% TFA and TEAP, pH 2.30) and capillary zone elec-
trophoresis (CZE). Mass spectrometric analysis supported
the identity of the intended structures.
Analogues 7, 9, and 11 were synthesized with the resolved
Boc-Ncy(Mob)-OH (6). The RP-HPLC and MALDI analyses
of the crude peptides (obtained after HF cleavage and I2
cyclization) indicated that a single stereoisomer was formed,
and the coupling reaction proceeded without racemization
at the R-carbon. The HPLC profile of the crude peptide 11
is shown in Figure 2. Because the starting amino acid Boc-
DNcy(Mob)-OH for the synthesis of analogues 8 and 10
(28) Baniak, E. L., II; Rivier, J. E.; Struthers, R. S.; Hagler, A. T.;
Gierasch, L. M. Biochemistry 1987, 26, 2642-2656.
(29) Beckers, T.; Bernd, M.; Kutscher, B.; Kuhne, R.; Hoffmann, S.;
Reissmann, T. Biochem. Biophys. Res. Commun. 2001, 289, 653-663.
(30) Rivier, J. E.; Jiang, G.; Struthers, R. S.; Koerber, S. C.; Porter, J.;
Cervini, L. A.; Kirby, D. A.; Craig, A. G.; Rivier, C. L. J. Med. Chem.
2000, 43, 807-818.
(31) Rivier, J. E.; Porter, J.; Cervini, L. A.; Lahrichi, S. L.; Kirby, D.
A.; Struthers, R. S.; Koerber, S. C.; Rivier, C. L. J. Med. Chem. 2000, 43,
797-806.
(32) Rivier, J.; Kupryszewski, G.; Varga, J.; Porter, J.; Rivier, C.; Perrin,
M.; Hagler, A.; Struthers, S.; Corrigan, A.; Vale, W. J. Med. Chem. 1988,
31, 677-682.
(33) Dutta, A. S.; Gormley, J. J.; McLachlan, P. F.; Woodburn, J. R.
Biochem. Biophys. Res. Commun. 1989, 159, 1114-1120.
(34) Rivier, J.; Rivier, C.; Perrin, M.; Porter, J.; Vale, W. In LHRH
Peptides as Female and Male ContraceptiVes; Zatuchni, G. I., Shelton, J.
D., Sciarra, J. J., Eds.; Harper and Row: Philadelphia, PA, 1981; pp 13-
23.
(35) Roeske, R. W.; Anantharamaiah, G. M.; Momany, F. A.; Bowers,
C. Y. In Peptides: Structure and Function; Hruby, V., Rich, D., Eds.; Eighth
American Peptide Symposium; Vol. Pierce Chemical:Rockford, IL, 1983;
pp 333-336.
(36) Rivier, J.; Vale, W.; Burgus, R.; Ling, N.; Amoss, M.; Blackwell,
R.; Guillemin, R. J. Med. Chem. 1973, 16, 545-549.
Figure 2. HPLC profile of crude peptide 11. RP-HPLC condi-
tions: buffer A, TEAP pH 2.30; buffer B, 60% CH3CN/40% A;
gradient elution from 30 to 60% buffer B in 30 min at a flow rate
of 0.2 mL/min; UV detection, 0.1 AUFS at 214 nM.
Org. Lett., Vol. 8, No. 11, 2006
2363

could not be obtained, we synthesized the diastereomeric
mixture of these peptides with the racemic Boc-D,L-Ncy-
(Mob)-OH (4) at positions 4 and 10, respectively. The two
isomers were then separated by RP-HPLC. The stereochem-
istry at the Ncy residue in analogues 8 and 10 was confirmed
by comparison of the HPLC retention times (Table 1) of
each diastereomer with those of the analogues synthesized
with the resolved Boc-Ncy(Mob)-OH (6), i.e., analogues 7
and 9, respectively. Analogues 7 and 9 coeluted on HPLC
with the first eluting diastereomers of the pairs (7 + 8 and
9 + 10) obtained by the incorporation of unresolved Boc-
D,L-Ncy(Mob)-OH (4). This confirmed that analogues 8 and
10 contained the D-enantiomer of Ncy at position 10.
The yields of these peptides (Table 1) containing the
unnatural disulfide bridge were comparable to that of the
homologous peptides with the natural disulfide bridge.24
Peptides were stored as TFA salts at -20 °C and were stable;
no decomposition products were identified on HPLC after
six months.
All of the GnRH analogues (Table 1) were tested in vitro
in a reporter gene assay in HEK-293 cells expressing the
human GnRH receptor and a stably integrated luciferase
reporter gene as previously described.37 The antagonism of
the GnRH-induced response by each analogue was deter-
mined and reported as an IC50 value, the concentration
required to suppress the response in the reporter gene assay
by 50%. In this assay, analogues 7 (IC50 ) 37 nM), 8
(IC50 ) 33 nM), and 11 (IC50 ) 33 nM) are half as potent
In conclusion, we have successfully demonstrated the
applicability of norcysteine (R-thiolglycine) to shorten di-
sulfide bridges in peptides by one or two methylene groups.
This amino acid and its derivatives complement our present
armamentarium of unnatural amino acids to study the
structure-activity relationships of bioactive peptides contain-
ing disulfide bridges and to understand the role of disulfide
bridges in protein structure and function.
Acknowledgment. We thank Ron Kaiser (Salk Institute)
and Charleen Miller (Salk Institute) for technical assistance,
William Low (Salk Institute) and Dr. Wolfgang Fisher (Salk
Institute) for MS analysis, and Thomas Baiga (Salk Institute)
1
and Dr. Joseph Noel (Salk Institute) for H, 13C NMR, and
chiral HPLC analyses. We thank Dr. Judit Ercheygi (Salk
Institute) and Dr. David Chatenet (Salk Institute) for critical
reading of the manuscript. We thank Dr. Glenn Croston
(Ferring Research Institute Inc.) and Doley Hong (Ferring
Research Institute Inc.) for in vitro assay data of GnRH
analogues. We thank Debbie Doan (Salk Institute) for
manuscript preparation. This work was supported by NIH
Grants HD-039899 and DK-059953. J.R. is the Dr. Frederik
Paulsen Chair in Neurosciences Professor.
Supporting Information Available: Detailed experi-
mental procedures and spectral and analytical data. This
material is available free of charge via the Internet at
http://pubs.acs.org.
as the reference cyclic GnRH antagonist cyclo(4-10)
-
OL0606740
[Ac-DNal1,DCpa2,DPal3,Cys4,Arg5,DPal6,Cys10]GnRH32 (IC50
) 13 nM). Analogues 9 (IC50 ) 290 nM) and 10 (IC50
340 nM) were less potent.
)
(37) Samant, M. P.; Gulyas, J.; Hong, D. J.; Croston, G.; Rivier, C.;
Rivier, J. J. Med. Chem. 2005, 48, 4851-4860.
2364
Org. Lett., Vol. 8, No. 11, 2006