Synthesis of protected norcysteines for SPPS compatible with Fmoc strategy

Article abstract of DOI:10.1016/j.tetlet.2007.05.082

We report the synthesis of racemic Alloc-Ncy(Tmob)-OH, the resolution of its methyl ester and demonstrate its application to form a norcystine bridge in octreotide-amide using the Fmoc strategy on solid phase. N-Alloc and S-Tmob protections of norcysteine (Ncy) were found to be a preferred choice for Fmoc strategy over three other protected norcysteines synthesized, that is, Fmoc-Ncy(tBu)-OH, Alloc-Ncy(tBu)-OH, and Alloc-Ncy(Trt)-OH.

Full text of DOI:10.1016/j.tetlet.2007.05.082

Tetrahedron Letters 48 (2007) 5107–5110
Synthesis of protected norcysteines for SPPS compatible
with Fmoc strategy
Manoj P. Samant and Jean E. Rivier*
The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies,
10010 North Torrey Pines Road, La Jolla, CA 92037, USA
Received 12 January 2007; revised 11 May 2007; accepted 14 May 2007
Available online 18 May 2007
Abstract—We report the synthesis of racemic Alloc-Ncy(Tmob)–OH, the resolution of its methyl ester and demonstrate its appli-
cation to form a norcystine bridge in octreotide-amide using the Fmoc strategy on solid phase. N-Alloc and S-Tmob protections of
norcysteine (Ncy) were found to be a preferred choice for Fmoc strategy over three other protected norcysteines synthesized, that is,
Fmoc-Ncy(tBu)–OH, Alloc-Ncy(tBu)–OH, and Alloc-Ncy(Trt)–OH.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Norcysteine (Ncy) or a-thiolglycine (H2N–CH(SH)–
COOH) is an unnatural amino acid possessing an
electronegative sulfur atom attached directly to the
a-carbon atom. Recently, we described 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 cyc-
lic gonadotropin-releasing hormone (GnRH) analogs.1
Boc-Ncy(Mob)–OH is compatible with SPPS using
Boc strategy and can be introduced in peptides as a
bridge head to constrain peptide conformation via nor-
cysteine-containing disulfide bridges that are shorter in
ring size than the cystine bridges by one or two methyl-
ene groups. Herein we describe the synthesis of N- and
S-protected norcysteines for Fmoc strategy.
carbamate (1) and glyoxylic acid monohydrate (2) in
acetone for 5 h or stirring in diethylether at room
temperature (rt) overnight yielded the a-hydroxy inter-
mediate (3). The reaction of tert-butylthiol/tritylthiol/
2,4,6-trimethoxybenzylthiol8 in toluene with 3 in the
presence of PTSA afforded the racemic N- and S-pro-
tected norcysteines (4a–d). We attempted to separate
the enantiomers of 4a–d by stereoselective enzymatic
resolution of their methyl esters 5a–d using papain.1
Methyl esters (5a–d), which are the preferred substrate
for the papain-catalyzed hydrolysis,9 were obtained by
reacting the racemic amino acids (4a–d) with trimethyl-
silyldiazomethane.10 Our attempts to enzymatically re-
solve the racemic methyl esters 5a and 5c using papain
were unsuccessful. The enzymatic resolution of 5a and
5c with a-chymotrypsin and subtilisin also failed, and
may be attributed to the bulkier Fmoc/Trt groups,
which may affect the substrate recognition. However,
we were successful in resolving racemic methyl esters
5b and 5d using papain1 to optically enriched protected
L-norcysteines 6b and 6d11 with enantiomeric excess of
80% and >98%, respectively. The papain catalyzed
hydrolysis was carried out in phosphate buffer (pH
6.2) containing 60% CH3CN at 25 ꢁC and monitored
by chiral RP-HPLC using chiralcelꢂ OD-RHTM column
(Fig. 1). After 24 h, the reaction mixture contained
50% acid according to chiral RP-HPLC and was
quenched by adding acetic acid. The crude products
containing unreacted D-methyl esters and the resolved
L-acids (6b and 6d) were separated by column
chromatography.12
In studies directed toward the protected norcysteines for
Fmoc strategy, the a-amino group of norcysteine was
protected with Fmoc2 and Alloc,3 as both can be easily
removed during SPPS under basic and neutral condi-
tions, respectively. The sulfhydryl function was pro-
tected either with tBu4/Trt5/Tmob6 for their stability
during deprotection of Fmoc/Alloc groups and for their
lability7 after the cleavage of the desired peptide from
the resin or during the cleavage step. The synthesis of
racemic N- and S-protected norcysteines (4a–d) is illus-
trated in Scheme 1. In short, refluxing Fmoc- or Alloc-
Keywords: Norcysteine; Norcystine; SPPS; Somatostatin; Octreotide.
*
Corresponding author. Tel.: +1 858 453 4100; fax: +1 858 552
1546; e-mail: jrivier@salk.edu
0040-4039/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.05.082

5108
M. P. Samant, J. E. Rivier / Tetrahedron Letters 48 (2007) 5107–5110
CH
3
3
;
;
;
1a; 3a-5a R =
(Fmoc)
(Alloc)
R
R
=
=
(tBu)
(tBu)
CH
1
O
CH
3
O
CH
O
O
O
O
O
3
(i)
(ii)
;
;
R =
R =
CH
CH
1b; 3b-6b
1c; 3c-5c
.
3
1
H O
2
OH
R-HN
R-HN
R-NH
2
O
3
H
OH
OH
O
OH
S
R
1
1
3
2
4
O
;
;
(Trt)
O
(Alloc)
(Alloc)
R
=
1
OH
R-HN
O
(iii)
(iv)
R-HN
OCH
3
S
H
S
R
R
1
1
5
6
OCH
3
O
;
1d; 3d-6d R =
R
=
(Tmob)
1
O
H CO
3
OCH
3
Scheme 1. Synthesis of N- and S-protected norcysteines. Reagents and conditions: (i) acetone, reflux, 5 h or diethylether, rt, overnight; (ii) tert-
butylthiol/tritylthiol/2,4,6-trimethoxybenzylthiol, benzene, PTSA, Dean–Stark, reflux, 6 h; (iii) Me3SiCHN2, benzene/methanol (4:1); (iv) papain,
CH3CN/buffer (3:2), pH 6.2.
Figure 1. Chiral HPLC profile of the enzymatic hydrolysis of racemic Boc-Alloc-Ncy(Tmob)-OCH3 (5d). Reaction progress (a) initial, retention time
(tR) for racemic ester is 30.34 min and 30.96 min (b) after 24 h, tR for resolved Alloc-Ncy(Tmob)–OH is 25.32 min and tR for unreacted Alloc-
DNcy(Tmob)-OCH3 is 30.31 min. The HPLC assay conditions for the chiralcelꢂ OD-RHTM reversed-phase column (0.46 cm · 15 cm): buffer A, 0.1%
TFA in H2O, buffer B, CH3CN in A; gradient elution from 10% B to 70% B in 30 min and then 95% B in 2 min at a flow rate of 0.5 mL/min. UV
detection, 0.1 AUFS at 210 nm.
The compatibility of 6b and 6d in SPPS to form a nor-
cystine bridge was investigated in somatostatin analog
octreotide-amide.13 All of the peptides 7–9 (Table 1)
were synthesized manually on a 2,4-dimethoxybenz-
hydrylamine resin (DMBHA-resin,14 substitution 0.25
mmol/g resin) or Rink Amide AM resin (Novabiochem,
substitution, 0.70 mmol/g resin) using the Fmoc
strategy.15
Lys(Boc)5-Thr(tBu)6-Ncy(Tmob)7-Thr(tBu)8-resin] were
cleaved with the cocktail mixture of TFA/H2O/EDT/
TIS (94:2.5:2.5:1) for 3 h to give the crude peptide 8
and disulfide-reduced form of the peptide 9, respectively.
The excess of TFA was then removed in vacuo and the
crude peptides were precipitated by the addition of excess
of tert-butyl methyl ether. Analysis of the crude peptides
by RP-HPLC and mass spectroscopy revealed deprotec-
tion of Tmob, but not tBu protection from the sulfhydryl
function of Ncy. The linear precursor peptide DPhe1-
Ncy2-Phe3-DTrp4-Lys5-Thr6-Ncy7-Thr8-NH2 obtained
from the Boc-DPhe1-Ncy(Tmob)2-Phe3-DTrp(Boc)4-
Lys(Boc)5-Thr(tBu)6-Ncy(Tmob)7-Thr(tBu)8-resin was
Each coupling reaction was achieved using a threefold
excess of amino acid, and utilizing N,N0-diisopropylcar-
bodiimide (DIC)/1-hydroxybenzotriazole(HOBt)-medi-
ated activation of the carboxyl group in DMF.
Piperidine treatment (25% piperidine in DMF) was used
for the Fmoc removal and the Alloc deprotection was
performed under neutral conditions, that is, Pd(PPh3)4/
PhSiH3/DCM16 in the presence of argon. The fully
protected peptido resins [Boc-DPhe1-Ncy(tBu)2-Phe3-
DTrp(Boc)4-Lys(Boc)5-Thr(tBu)6-Ncy(tBu)7-Thr(tBu)8-
resin and Boc-DPhe1-Ncy(Tmob)2-Phe3-DTrp(Boc)4-
17
oxidatively cyclized in the presence of I2 to give the
crude cyclic peptide 9 (Fig. 2).
Our attempts to remove tBu protection from peptide 8
using DMSO/TFA mixtures18 or using Hg(II) acetate19
failed with noticeable side products on RP-HPLC.
Further experiments were directed toward on-resin

M. P. Samant, J. E. Rivier / Tetrahedron Letters 48 (2007) 5107–5110
5109
Table 1. Characterization of peptides
MSd (M+H)+
Calcd. Obsd.
c
No.
Peptides
Ring size
Purity (%)
tR (min)
HPLCa
CZEb
7
8
9
Cyclo(2-7)[DPhe1-Cys2-Phe3-DTrp4-Lys5-Thr6-
Cys7-Thr8-NH2] (Octreotide-amide)
DPhe1-Ncy(tBu)2-Phe3-DTrp4-Lys5-Thr6-
Ncy(tBu)7-Thr8-NH2
Cyclo(2-7)[DPhe1-Ncy2-Phe3-DTrp4-Lys5-
Thr6-Ncy7-Thr8-NH2]
20
—
18
99
98
99
99
17.4
35.8
14.9
1032.436
1118.546
1004.405
1032.404
1118.502
1004.341
99
99
a Percentage purity determined by HPLC using buffer A: TEAP, pH 2.30, buffer B: 60% CH3CN/40% A under gradient conditions (20% to 50% B
˚
over 30 min), at a flow rate of 0.2 mL/min on a Vydac C18 column (0.21 cm · 15 cm, 5 lm particle size, 300 A pore size). Detection at 214 nm.
b 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 an Agilent lSil bare fused-
silica capillary (75 lm i.d. · 40 cm length). Detection at 214 nm.
c Retention times under HPLC conditions described above.
d Mass spectra (MALDI-MS) were measured on an ABI-Voyager DE-STR instrument using saturated solution of a-cyano-4-hydroxycinnamic acid
in 0.3% trifluoroacetic acid and 50% acetonitrile as matrix. The calculated [M+H] of the monoisotope was compared with the observed [M+H]+
monoisotopic mass.
Figure 2. RP-HPLC profile of (a) crude and (b) purified peptide (9). RP-HPLC conditions: buffer A, TEAP pH 2.30; buffer B, 60% CH3CN/40% A;
˚
gradient elution from 20% to 50% buffer B in 30 min at a flow rate of 0.2 mL/min on a Vydac C18 column (0.21 cm · 15 cm, 5 lm particle size, 300 A
pore size). UV detection, 0.1 AUFS at 210 nm.
oxidative deblocking of tBu protection using Tl(tfa)3.20
The fully protected
Boc-DPhe1-Ncy(tBu)2-Phe3-
In conclusion, we have shown that Alloc-Ncy(Tmob)–
OH is compatible with the Fmoc strategy as an alterna-
tive to Boc-Ncy(Mob)–OH (used in the Boc strategy) for
introducing a norcystine bridge in peptides by SPPS.
The optical purity, easy removal of the Na-Alloc protec-
tion (during chain elongation step), and the sulfhydryl
Tmob protection (during the cleavage of peptide from
resin) clearly demonstrate that Alloc-Ncy(TMob)–OH
is a preferred choice for Fmoc-based SPPS.
DTrp(Boc)4-Lys(Boc)5-Thr(tBu)6-Ncy(tBu)7-Thr(tBu)8-
DMBHA resin was treated with Tl(tfa)3 (1.2 equiv), 3 h
in DMF-anisole (19:1) at 0 ꢁC. The cleavage of the pept-
ido resin with a cocktail mixture of TFA/H2O/TIS
(95:2.5:2.5) followed by post cleavage workup did not
show the desired [Ncy2,Ncy7]octreotide-amide (9) on
RP-HPLC.
All of the crude peptides (7–9) were purified21 by RP-
HPLC in at least two different solvent systems (TEAP
pH 2.25 and 0.1% TFA on C18 silica). The analytical
techniques used for the characterization of the analogs
in Table 1 included RP-HPLC with two different solvent
systems (0.1% TFA and TEAP pH 2.30) and capillary
zone electrophoresis (CZE). Mass spectrometric analysis
supported the identity of the intended structures (Table
1). Additionally, the structure of 9 was confirmed by
Acknowledgments
We thank Ron Kaiser and Charleen Miller for technical
1
assistance, Thomas Baiga and Dr. Joseph Noel for H,
13C NMR, and chiral HPLC analyses. We thank Dr.
David Chatenet for critical reading of the manuscript.
We thank Debbie Doan for manuscript preparation.
This work was supported by NIH Grants DK059953
and DK026741. J.R. is the Dr. Frederik Paulsen Chair
in Neurosciences Professor.
coinjection
experiment
on
RP-HPLC
with
[Ncy2,Ncy7]octreotide-amide synthesized in parallel
using resolved Boc-Ncy(Mob)–OH1 and Boc strategy.

5110
M. P. Samant, J. E. Rivier / Tetrahedron Letters 48 (2007) 5107–5110
purification (4a = 65%, 4b = 54%, 4c = 47% and
Supplementary data
4d = 35%). The crude 4d contained approximately 15%
of insoluble polymeric material.
Experimental procedures for the synthesis of amino
acids are given in Scheme 1 and for the synthesis of pep-
tide analog 9 in Table 1. Supplementary data associated
with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2007.05.082.
25
1
11. Compound 6d: oil; ½aꢀD +42.40 (c 1.0, MeOH); H NMR
(300 MHz, DMSO-d6): 7.96 (d, 1H, J = 8.7 Hz), 6.21 (s,
2H), 5.98–5.85 (m, 1H), 5.32 (dd, 1H, J = 17.1, 1.2 Hz),
5.19 (dd, 1H, J = 10.5, 1.2 Hz), 5.12 (d, 1H, J = 8.7 Hz),
4.51 (d, 2H, J = 5.1 Hz), 3.76 (s, 2H), 3.75 (s, 9H); 13C
NMR (75 MHz, DMSO-d6): 170.03, 160.23, 158.34,
158.30, 155.03, 133.34, 133.29, 117.07, 105.77, 90.67,
64.64, 56.17, 55.68, 55.16, 22.46; HRMS Calcd for
C16H21NO7S: 394.0931 (M+Na+). Found: 394.0920
(M+Na+), 2.8 ppm error.
12. The unreacted D-methyl esters were eluted with a mixture
of EtOAc/hexane (25:75) and the resolved Alloc-Ncy(tBu/
Tmob)–OH were eluted with EtOAc/MeOH (85:15).
13. Bauer, W.; Briner, U.; Doepfner, W.; Haller, R.; Hugue-
nin, R.; Marbach, P.; Petcher, T. J.; Pless, J. Life Sci.
1982, 31, 1133–1140.
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10. Compounds 3 and 5 were obtained in quantitative yields
from 1 and 4, respectively. Compounds 4a–d and 5a–d
were purified by column chromatography. The racemic
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21. The RP-HPLC purified peptide analogues 8 and 9 were
obtained in 43% and 32% yields, respectively.