New methods for side-chain protection of cysteine

Article abstract of DOI:10.1021/ol015678d

Matrix presented Cysteine sulfhydryl protection with either the Fmoc or the Fm group was accomplished in one step and in high yield using commercially available FmocCl or FmocOSu, respectively. Mechanisms for the Fmoc to Fm transformations are discussed.

Full text of DOI:10.1021/ol015678d

ORGANIC
LETTERS
2001
Vol. 3, No. 8
1205-1208
New Methods for Side-Chain Protection
of Cysteine
,†,‡
Christopher W. West,† M. Angels Estiarte,†,‡ and Daniel H. Rich*
Department of Chemistry and School of Pharmacy, UniVersity of WisconsinsMadison,
Madison, Wisconsin 53706
dhrich@facstaff.wisc.edu
Received February 7, 2001
ABSTRACT
Cysteine sulfhydryl protection with either the Fmoc or the Fm group was accomplished in one step and in high yield using commercially
available FmocCl or FmocOSu, respectively. Mechanisms for the Fmoc to Fm transformations are discussed. Additionally, Fmoc-Cys(Fmoc)-
OH (7) was synthesized and used in amide bond forming reactions. The S-Fmoc group is cleaved selectively from peptides containing the
N-Fmoc group.
The 9-fluorenylmethyloxycarbonyl (Fmoc) group, introduced
by Carpino and Han in 1970,1 has become one of the most
widely used protecting groups in peptide chemistry and is
gaining popularity in sugar2 and nucleotide3 chemistry. While
Fmoc is used to protect nitrogen and alcohols,4 Fmoc-
protected thiols have not been reported. Instead, the related
9-fluorenylmethyl (Fm) group is used for thiol protection.5
The Fm group is prepared in a two-step process that usually
begins with 9-fluorenylmethanol.6 Here, we report the
synthesis of di-Fmoc-protected cysteine 7 and its use in
peptide coupling reactions. The S-Fmoc protecting group can
be removed preferentially over the N-Fmoc group. Addition-
ally, we developed an efficient, one-step synthesis of the Fm-
protected thiol from either the Fmoc-protected or free thiol.
Reaction of Boc-Cys-OMe7 (1) with FmocCl and TEA in
CH2Cl2 led to the formation of the Fmoc derivative 2 in 98%
yield (Scheme 1). The Fmoc-protected thiol 2 is a stable and
easily handled white solid having a shelf life of at least 6
months. However, when thiol 1 was reacted with FmocOSu
and TEA in CH2Cl2, only Boc-Cys(Fm)-OMe (3) was
isolated in 91% yield. Compound 2 was not detected in the
reaction mixture.
Since the only difference between the reaction conditions
for formation of 2 or 3 is the Fmoc reactant, the reaction
mechanism was investigated in detail. Reaction of 2 with a
1:1 mixture of HOSu and TEA for 4 h led to the loss of
CO2 and the formation of 3 in 92% yield. Additional
† Department of Chemistry.
‡ School of Pharmacy.
(1) Carpino, L. A.; Han, G. Y. J. Am. Chem. Soc. 1970, 92, 5748.
(2) Nicolaou, K. C.; Winssinger, N.; Pastor, J.; DeRoose, F. J. Am. Chem.
Soc. 1997, 119, 449.
(3) (a) Sund, C.; Agback, P.; Koole, L. H.; Sandstroem, A.; Chatto-
padhyaya, J. Tetrahedron 1992, 48, 695. (b) Lin, J.; Shaw, B. R. J. Chem.
Soc., Chem. Commun. 1999, 1517.
(4) Gioeli, C.; Chattopadhyaya, J. B. J. Chem. Soc., Chem. Commun.
1982, 672.
(5) (a) Ruiz-Gayo, M.; Albericio, F.; Pedroso, E.; Giralt, E. J. Chem.
Soc., Chem. Commun. 1986, 1501. (b) Corey, E. J.; Gin, D. Y.; Kania, R.
S. J. Am. Chem. Soc. 1996, 118, 9202. (c) Ponsati, B.; Giralt, E.; Andreu,
D. Tetrahedron 1990, 46, 8255.
(6) (a) Bodanszky, M.; Bednarek, M. A. Int. J. Peptide Protein Res. 1982,
20, 434. (b) Albericio, F.; Nicolas, E.; Rizo, J.; Ruiz-Gayo, M.; Pedroso,
E.; Giralt, E. Synthesis 1990, 119.
(7) Threadgill, M. D.; Gledhill, A. P. J. Org. Chem. 1989, 54, 2940.
10.1021/ol015678d CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/20/2001

Scheme 1
Scheme 2
OMe (4) with TEA in a competition experiment afforded
conversion of S-Fmoc to S-Fm without any N-Fmoc removal
(reaction 1). In addition, reaction of Fmoc-Cys(Fmoc)-OMe
(5) with TEA afforded complete S-Fmoc removal without
loss of the N-Fmoc group (reaction 2), illustrating a remark-
able difference in the chemical reactivity of these two groups.
The yield of Fm 6 could be improved to 82% by changing
the solvent to CH3CN; however, under these conditions a
trace of N-Fmoc cleavage was observed by TLC.
experiments showed that when 2 was exposed to stoichio-
metric TEA, conversion of Fmoc to Fm was complete in 4
h, but when 2 was exposed only to HOSu for 24 h, no
reaction occurred. These results show that the conver-
sion from Fmoc to Fm is dependent upon the tertiary amine
base.
Both the pKa and the steric bulk of the amine affect the
transformation of the S-Fmoc group (2 or 5) to the S-Fm
derivative (3 or 6) as shown by the data in Table 1.8 Weaker
The data obtained are consistent with a mechanism for
conversion of S-Fmoc to S-Fm via the expected elimination/
addition reaction pathway shown in Scheme 3. When
Table 1. Effect of Base and N-Protecting Group on S-Fmoc
Cleavage of R-Cys(Fmoc)-OMe for 2 to 3 (R ) Boc) or 5 to 6
(R ) Fmoc)
Scheme 3
% yield
basea
pKa
3
6
N-hydroxysuccinimide
pyridine
N-methylmorpholine
triethylamine (1.0 equiv)
triethylamine (0.1 equiv)
piperidine
4
5.25
7.38
10.75
10.75
11.12
11.44
0
0
5b
92
11b
69c
<2
0
0
<5c
37
ndd
nd
diisopropylethylamine
<2
a pKa values of the conjugate acid. b Mass balance was starting material
and free thiol. c Mass balance was disulfide formation. d nd ) not deter-
mined.
tertiary bases such as pyridine and N-methylmorpholine do
not efficiently effect S-Fmoc cleavage, and deprotection with
the more hindered diisopropylethylamine is much slower.9
The stability of the S-Fmoc group relative to the N-Fmoc
group was determined in a pair of experiments (Scheme 2).
Reaction of Boc-Cys(Fmoc)-OMe (2) and Fmoc-Thr(OBn)-
FmocOSu is used in the synthesis of 2, free TEA exists in
the reaction mixture, and the reaction proceeds via the
expected elimination-decarboxylation pathway to form the
free thiolate in the presence of the reactive dibenzofulvene.
The thiolate then adds to the dibenzofulvene in a Michael-
type reaction to form the Fm-protected cysteinyl derivative.10
Additional supporting evidence for this pathway includes
(8) The conversion of 2 to 3 took place in CH2Cl2 with only 1 equiv of
the designated base and the cysteine derivative in the reaction mixture. The
1
products were identified by H NMR after a reaction time of 4 h.
(9) At higher concentrations of reactants and temperatures above 40 °C
during workup, the deprotection of the S-Fmoc group is faster.
(10) Addition of nucleophiles to dibenzofulvene: Carpino, L. A.; Han,
G. Y. J. Org. Chem. 1972, 37, 3404.
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Org. Lett., Vol. 3, No. 8, 2001

1
detection of dibenzofulvene in the reaction mixture by H
The protecting groups on dipeptide 8 can be removed
selectively from either the carboxylic acid or sulfur func-
tionality (Scheme 5). For the carboxylic acid, both tert-butyl
NMR and isolation of dibenzofulvene and Boc-Cys-OMe (1)
from the reaction mixture after shorter reaction times.
The surprising result is obtained with FmocCl, which
forms the S-Fmoc derivative exclusively. We believe this
results from the weaker base strength of chloride vs the anion
of N-hydroxysuccinimide formed during the reaction. Reac-
tion of the two Fmoc reagents with the thiol produces two
leaving groups with very different pKa values. The anion of
HOSu is 11 orders of magnitude more basic than that of
chloride (HOSu pKa ) 4 vs HCl pKa ) -7 in water). During
the course of the reaction using FmocCl, the HCl produced
fully protonates TEA, thus preventing abstraction of the
Fmoc â-hydrogen by the amine base. When FmocOSu is
used in the reaction, the HOSu does not fully protonate TEA,
allowing proton abstraction and transformation of 2 to 3 as
demonstrated previously (vide supra). The anion of N-
hydroxysuccinimide also can deprotonate the S-Fmoc group
under specific reaction conditions: DIEA/HOSu slowly
cleaves the S-Fmoc group from esters 2 and 5 but not from
amide 8.11
Scheme 5a
The usefulness of the S-Fmoc derivative was demonstrated
with the synthesis and peptide coupling of N- and S-Fmoc-
protected cysteine derivative 7. Reaction of Fmoc-Cl with
commercially available HCl-Cys-OH by using a protocol
described for di-Boc protection of cysteine (Scheme 4)12 gave
Scheme 4
a (a) 4 N HCl in dioxane, 77%; (b) Pd(PPh3)4, dimedone, THF,
94%; (c) Et3N, I2, MeOH, CH2Cl2, 75%; (d) Et3N, C6H5CH2SH,
CH3CN, CH2CL2 45%.
and allyl were used successfully as orthogonal protecting
groups. Removal of the tert-butyl ester in dipeptide 8 with
4 N HCl in dioxane provided the free acid 9 without any
detectable loss of the S-Fmoc group (reaction 3). Selective
allyl removal was accomplished by reacting fully protected
amino acid Fmoc-Cys(Fmoc)-OAllyl (10) with Pd(PPh3)4 and
dimedone in THF to give free acid 7 in 94% yield (reaction
4).14 Furthermore, the reactivity differences between N- and
S-Fmoc allow selective removal of the S-Fmoc group with
Et3N in the presence of iodine or benzenethiol, to form
disulfides 11 and 12, respectively, without any detectable
loss of the N-Fmoc group (reactions 5 and 6).
Di-Fmoc cysteine 7 was used to produce pseudosymmetri-
cal diamide 15 (Scheme 6). Reaction of 7 and mono-Boc-
protected putrescene15 using EDCI and HOBt gave Fmoc-
Cys(Fmoc)-DAB-Boc 14 in 69% yield. The Boc group was
cleaved with 4 N HCl in dioxane and the acid coupled with
7 to give di-Fmoc-Cys diamide 15 in 71% for both steps.
Attempts to prepare diamide 15 directly from unprotected
putrescene and di-Fmoc cysteine 7 were unsuccessful; only
low yields of 15 were obtained under a variety of reaction
conditions.
di-Fmoc acid 7. Alternate synthetic routes to 7 from Fmoc-
Cys-OH (under various conditions) or by saponification of
Fmoc-Cys(Fmoc)-OMe (5) led to complex mixtures of
products. Incorporation of protected amino acid 7 into
peptides can be readily achieved. Reaction of 7 and HCl-
Leu-OtBu under standard conditions afforded dipeptide 8 in
87% yield.13
(11) In an additional experiment compound 3 was treated with a 1:1
mixture of DIEA:HOSu, yielding a mixture of starting material and disulfide
in a 1:1 ratio.
(12) Schnabel, E.; Stoltefuss, J.; Offe, H. A.; Klaude, E. Justus Liebigs
Ann. Chem. 1971, 743, 57.
(13) The S-Fmoc group in amide derivative 8 is more stable under basic
conditions than esters 2 or 5.
(14) Zhang, H. X.; Guibe, F.; Balavoine, G. Tetrahedron Lett. 1988, 29,
623.
(15) Krapcho, A. P.; Kuell, C. S. Synth. Comm. 1990, 20(16), 2559.
Org. Lett., Vol. 3, No. 8, 2001
1207

Fmoc cleavage rate in the presence of a tertiary amine base.
Interestingly, diisopropylethylamine did not cleave the S-
Fmoc group (Table 1), but N-methylmorpholine did, sug-
gesting that deprotonation with hindered bases is not easily
achieved. It is possible that other systems containing the
S-Fmoc group may be sufficiently hindered to escape
removal with triethylamine.
Scheme 6
Another example of a leaving group altering the normal
N-Fmoc cleavage can be found in a reaction reported by
Vedejs et al. In their work, an N-Fmoc group was rapidly
removed from the carbamate nitrogen in mitosene deriviative
17 in 89% yield by reaction with Et3N in CH3CN.18 Since
Et3N has been shown to be largely ineffective in the removal
of N-Fmoc from simple amines, the observed cleavage may
be due to the enhanced leaving group potential of the
carbamate nitrogen.19
Our results establish that S-Fmoc-protected cysteine is
readily synthesized and can be used to prepare S-Fm-Cys
derivatives in good yield. These reaction conditions give
S-Fm protection more efficiently and in higher yield than
previously reported methods.5 Fmoc-Cys(Fmoc)-OH (7) has
also been shown to be a readily available compound
amenable to peptide coupling reactions in good yield.
Additionally, S-Fmoc was found to be more labile than
N-Fmoc when exposed to a tertiary amine base, and this
differential reactivity may be useful in synthetic strategies
involving selective deprotection of SR groups in the presence
of N-Fmoc groups.
Compound 15 was used to form the 14-membered ring
macrocycle 16 by selective deprotection and oxidation of
sulfur. Synthesis and evaluation of this compound as a
potential parallel â-sheet initiator will be reported separately.
The differences in rate of cleavage of the N- and S-Fmoc
groups can be rationalized by considering the effect of
leaving group pKa on the reaction mechanism. The mecha-
nism for â-elimination in the fulvene system has been
postulated to lie on the borderline between E1cb and E2.16
The leaving group has a large influence on which mechanism
occurs, i.e., the better the leaving group, the more the E2
pathway predominates. When the mechanism is E1cb
(presumably as it is for nitrogen), the rate of reaction
becomes dependent on removal of the Fmoc â-hydrogen.
While typical N-Fmoc deprotection takes place using an
excess of secondary amine base, experimental evidence
shows a tertiary amine base will eventually effect deprotec-
tion, but at a much slower rate.17 However, with the O- and
S-Fmoc cases, better leaving groups are present which shift
the reaction mechanism to the E2 pathway and increase the
Acknowledgment. This work was supported by a re-
search grant from the National Institutes of Health (GM
50113). We also thank the Spanish government for the
postdoctoral fellowship given to M.A.E.
Supporting Information Available: Experimental pro-
cedures and full characterization for compounds 1-14. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL015678D
(16) (a) Meng, Q.; Thibblin, A. J. Am. Chem. Soc. 1997, 119, 4834. (b)
O’Ferrall, R. A. M.; Larkin, F.; Walsh, P. J. Chem. Soc., Perkin Trans. 2
1982, 1573.
(18) Vedejs, E.; Klapars, A.; Naidu, B. N.; Piotrowski, D. W.; Tucci, F.
C. J. Am. Chem. Soc. 2000, 122, 5401.
(17) Bodanszky, M.; Deshmane, S. S.; Martinez, J. J. Org. Chem. 1979,
44, 1622.
(19) Alternatively, the reaction might proceed via an imide anion formed
six atoms away from the Fmoc proton.
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Org. Lett., Vol. 3, No. 8, 2001