An improved method for cysteine alkylation

Article abstract of DOI:10.1016/S0040-4039(01)00098-3

An improved method for the synthesis of S-alkylated cysteine derivatives with branched alkyl chains is reported. These compounds can be obtained in good yield and high purity by refluxing the cysteine thiol with the appropriate alkyl bromide in a solution of sodium ethoxide in ethanol.

Full text of DOI:10.1016/S0040-4039(01)00098-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 1859–1861
An improved method for cysteine alkylation
David A. Perrey and Fatih M. Uckun*
Parker Hughes Cancer Center, Parker Hughes Institute, St. Paul, MN 55113, USA
Received 27 October 2000; revised 12 January 2001; accepted 15 January 2001
Abstract—An improved method for the synthesis of S-alkylated cysteine derivatives with branched alkyl chains is reported. These
compounds can be obtained in good yield and high purity by refluxing the cysteine thiol with the appropriate alkyl bromide in
a solution of sodium ethoxide in ethanol. © 2001 Elsevier Science Ltd. All rights reserved.
Posttranslational modification of cysteine residues by
addition of an isoprenyl side chain to the sulfhydryl
group is an important step for several proteins involved
in signal transduction, such as yeast a-factor mating
hormones, nuclear lamins, ras proteins and the g-sub-
unit of transducin. The majority of these alterations
occur at a C-terminal cysteine with a ‘CAAX’ motif
(C=cysteine, A=an aliphatic amino acid, X=any
amino acid). In ras proteins, this modification allows
the proteins to associate with the cellular membrane, a
necessary step in the activation of these proteins for
signal transduction.1
S-alkylated cysteine derivatives. While our initial work
in this area was successful using the method of Brown
et al., attempts to synthesize 2-methylpropyl derivatives
and other similarly b-branched aliphatic analogues
failed. This prompted us to examine other conditions
for cysteine alkylation. The results of this study and a
comparison of various methods of cysteine alkylation
are discussed in detail below.
The S-alkylation of N-acetyl-L-cysteine with 1-bromo-
2-methylpropane was examined under various condi-
tions (Table 1). The procedure developed by Brown et
al. (Table 1, entry 1), which uses a solution of ammonia
in methanol as the base, has been shown to selectively
alkylate thiols even in the presence of other potential
Kamiya et al. synthesized prenylated proteins via alky-
lation of the sulfhydryl group with farnesyl bromide in
an alcohol/water mixture.2 More recently, Yang et al.
alkylated polypeptides in high yield by using an excess
of farnesyl bromide in a mixed solvent system of
DMSO/DMF/ACN containing diisopropylethylamine
(DIEA).3 Brown et al. prepared prenylated cysteine
derivatives using farnesyl chloride in an ammonia/
methanol solution.4 A pentapeptide substrate, Lys-Cys-
Val-Leu-Ser (which contains free amino, hydroxy and
acid groups in addition to the cysteine thiol), was
S-farnesylated using farnesyl chloride in refluxing liq-
uid ammonia. Or et al. alkylated a model tetrapeptide,
Ser-Leu-Cys-Phe, with a variety of alkyl substituents
using the method4 of Brown et al., demonstrating that
this method is not restricted to allylic substrates.5 Fur-
ther, Xue et al. reported the regioselective isoprenyla-
tion of peptides in acidic conditions using zinc acetate
as a catalyst.6
Table 1. Comparison of various reaction conditions in the
S-alkylation of N-acetyl-L-cysteine with 1-bromo-2-methyl-
propane
Entry
Method
Yield (%)
1
2
3
4
5
6
7
8
NH3 in MeOH/16 h
ꢀ10a
Pyridine (cat. DMAP)/16 h
Et3N in THF (cat. DMAP)/16 h
Et3N in THF at reflux/4 h
NaOEt/EtOH at reflux/4 h
NaOMe/MeOH at reflux/4 h
NaH in THF/16 h
0
0
0
74
81
0
The anti-leukemia drug discovery research7 undertaken
in our laboratories has involved the synthesis of various
NaH in DMF/16 h
0
* Corresponding author.
a Estimated by 1H NMR, highly contaminated product.
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00098-3

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D. A. Perrey, F. M. Uckun / Tetrahedron Letters 42 (2001) 1859–1861
Table 2. Comparison of the yields obtained using the sodium ethoxide/ethanol versus the ammonia/methanol reaction
conditions for the S-alkylation of various cysteine thiol derivatives with several different alkyl bromides
Entry
Thiol
Bromide
Yield
NH3/MeOH
NaOEt
1
2
3
4
5
6
7
8
9
Acetyl-cysteine
Acetyl-cysteine
Acetyl-cysteine
3-Mercaptopropionic acid
Acetyl-cysteine
Acetyl-cysteine
Acetyl-cysteine
Acetyl-cysteine
Acetyl-cysteine
(Bromomethyl)cyclohexane
1-Bromo-2-ethylbutane
1-Bromododecane
1-Bromododecane
1-Bromo-2,2-dimethylpropane
2-Bromopropane
73
90
83
68
28a
58
45
96
99
48
0
0
98
21
0
0
Bromoethane
35
99
97
73
trans, trans-Farnesyl bromide
trans-Geranyl bromide
1-Bromododecane
10
Cysteine methyl ester
a Yield obtained after 12 hour reflux.
nucleophiles. In this case, however, the reaction did not
proceed satisfactorily, giving poor yields of highly
impure product. Using 1-iodo-2-methylpropane instead
of its bromo counterpart did produce pure product in a
33% yield, although the lack of commercially available
iodides does limit this approach. Most of the other
methods provided no improvement and simply yielded
starting material upon work-up. In contrast, the reac-
tions which employed a sodium alkoxide (Table 1,
entries 5 and 6) gave a pure product with no trace of
either the alkyl bromide or the cysteine starting materi-
als.8 Additionally, the reaction involving sodium ethox-
ide produced a precipitate of NaBr, which indicated
that the reaction was proceeding. This precipitate only
formed while the reaction mixture was refluxing, how-
ever; at room temperature, no precipitate was produced
and no appreciable reaction occurred.
and 1-bromo-2,2-dimethylpropane under the new or the
standard conditions; only after prolonged reflux was
any significant material produced using the new
method. This result suggests that while the method
involving sodium ethoxide gives greatly improved yields
for many branched alkyl bromides, reactions involving
alkyl bromides that have a large amount of steric
hindrance due to their highly branched nature (e.g.
1-bromo-2,2-dimethylpropane) are still inherently
inefficient.
Two other thiols (Table 2, entries 4 and 10) were also
studied to ascertain the effects of the amino group of
cysteine (the amino group is absent in 3-mercaptopropi-
onic acid, entry 4; cysteine methyl ester, entry 10,
contains a free amino group) on the utility of the
method involving sodium ethoxide. For the reaction of
3-mercaptopropionic acid with 1-bromododecane, an
enormous improvement in yield was obtained using the
new method (the yields using the method involving
sodium ethoxide and the literature method of Brown et
al. are 68 and 21%, respectively; Table 2). In contrast,
the yield obtained for the reaction involving the cys-
teine methyl ester was reduced when using sodium
ethoxide. These results suggest that while the method
utilizing sodium ethoxide is clearly superior to its
ammonia/methanol counterpart in some instances, in
particular for reactions involving branched alkyl bro-
mides, the reactions involving either a cysteine deriva-
tive bearing a free amino group or a less-hindered alkyl
bromide (such as a straight-chain alkyl bromide or an
isoprenyl bromide) benefit little from using this method.
Given the above results, the general utility of the
method involving a sodium alkoxide was explored.
Sodium ethoxide was selected for these studies rather
than sodium methoxide since the formation of a precip-
itate when using the former provides a convenient
visual indicator that the reaction is progressing. The
reactions between N-acetyl cysteine and a variety of
alkyl bromides were examined under these conditions
(Table 2). Good yields were obtained for many of the
bromides used, in several cases marking a substantial
improvement over the results obtained using the litera-
ture method. In fact, for some of the alkyl bromides
(e.g. 1-bromo-2-ethylbutane, Table 2, entry 2) the
desired product was generated in excellent yield,
whereas no product was obtained following the litera-
ture method. For the isoprenyl bromides (Table 2,
entries 8 and 9), comparable yields were obtained using
the new method and the literature procedure. However,
there was little or no reaction between N-acetyl cysteine
One concern with using such relatively harsh basic
conditions is the danger of racemization. Comparison
of N-acetyl-S-dodecyl-L-cysteine samples made via the
ammonia/methanol and sodium ethoxide methods

D. A. Perrey, F. M. Uckun / Tetrahedron Letters 42 (2001) 1859–1861
1861
found identical optical rotations, indicating that no
racemization had occurred. However, in the cysteine
methyl ester case, this concern was well founded as the
optical rotations obtained were widely different, which
incidentally may account for the lower yield using this
substrate.
Abe, K.; Fukui, S. Experimentia 1979, 35, 1275–1276.
3. Yang, C.-C.; Marlowe, C. K.; Kania, R. J. Am. Chem.
Soc. 1991, 113, 3177–3178.
4. Brown, M. J.; Milano, P. D.; Lever, D. C.; Epstein, W.
W.; Poulter, C. D. J. Am. Chem. Soc. 1991, 113, 3176–
3177.
5. Or, Y. S.; Clark, R. F.; Luly, J. R. J. Org. Chem. 1991, 56,
3146–3149.
6. Xue, C.-B.; Becker, J. M.; Naider, F. Tetrahedron. Lett.
1992, 33, 1435–1438.
7. (a) Perrey, D. A.; Narla, R. K.; Uckun, F. M. Bioorg.
Med. Chem. Lett. 2000, 10, 547–549; (b) Perrey, D. A.;
Scannell, M. P.; Narla, R. K.; Uckun, F. M. Bioorg. Med.
Chem. Lett. 2000, 10, 551–552.
8. A typical experiment was performed as follows: Freshly
cut sodium metal (0.51 g, 22 mmol) was dissolved in
anhydrous ethanol (50 mL) under an atmosphere of nitro-
An efficient reaction for the S-alkylation of cysteine
provides a useful tool for exploring structure-activity
relationships in cysteine-containing inhibitors and/or
substrates. Elucidation of a variety of different experi-
mental conditions under which this reaction can be
performed is extremely useful for the synthesis of a
wide variety of different compounds destined for bio-
logical testing. The method presented here provides a
valuable complement to the methods already in the
literature, giving access in particular to branched cys-
teine derivatives that are otherwise difficult to synthe-
size.
gen. To the solution was added N-acetyl-L-cysteine (1.63
g, 10 mmol) followed by 1-bromo-2-methylpropane (1.51
g, 1.20 mL, 11 mmol) and the reaction mixture heated at
reflux for 4 h, during which time a white precipitate was
formed. Upon cooling, the reaction was quenched with a
small amount of water, the solvent was removed under
reduced pressure and the residue redissolved in ethyl ace-
tate. The solution was washed with 1 M HCl and satu-
rated brine, dried over anhydrous MgSO4 and the solvent
removed under reduced pressure to give the product, N-
References
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