A facile, inexpensive and scalable route to thiol-protected α-methyl cysteine

Article abstract of DOI:10.1055/s-0032-1318316

A facile, scalable synthesis of α-methyl cysteine with three alternate thiol protecting groups (trityl, allyl and tert-butyl) is described. The thiol-protected amino acids are obtained in six steps from l-cysteine ethyl ester and are ideally suited for a range of natural product and solid-phase peptide synthesis applications. Georg Thieme Verlag Stuttgart - New York.

Full text of DOI:10.1055/s-0032-1318316

LETTER
▌591
lAetteFr acile, Inexpensive and Scalable Route to Thiol-Protected α-Methyl
Cysteine
Thiol-Protected α-Methyl Cysteine
Heather J. Johnston, Alison N. Hulme*
School of Chemistry, University of Edinburgh, Joseph Black Building, West Mains Road, Edinburgh, EH9 3JJ, UK
Fax +44(131)6506453; E-mail: Alison.Hulme@ed.ac.uk
Received: 22.01.2013; Accepted: 06.02.2013
Abstract: A facile, scalable synthesis of α-methyl cysteine with
three alternate thiol protecting groups (trityl, allyl and tert-butyl) is
described. The thiol-protected amino acids are obtained in six steps
from L-cysteine ethyl ester and are ideally suited for a range of nat-
ural product and solid-phase peptide synthesis applications.
O
N
O
O
N
N
O
H
O
Me
N
N
S
N
Me
N
H
O
Key words: natural products, α-methyl cysteine, solid-phase pep-
tide synthesis, camphorsultam auxiliary, P2-Et
O
OH
Br
Both the R- and S-enantiomers of α-methyl cysteine occur
in Nature and they are often found incorporated within a
thiazoline ring.1 Many α-methyl cysteine containing natu-
ral products exhibit interesting pharmaceutical properties,
for example bisebromoamide (1; Figure 1) has anticancer
activity at nanomolar levels against a wide range of cancer
lines;2 thiangazole (2) has anti-HIV activity and is highly
selective for HIV-1 over HIV-2;3 desferrithiocin (3) is an
iron chelator with antineoplastic activity4 and didehy-
dromirabazole A (4) is an anticancer agent which exhibits
selectivity for solid tumours.5 α-Methyl cysteine is also
frequently used in the field of peptide mimetics where
quaternisation of the α-centre: (i) prevents racemisation,
which otherwise is comparatively facile under both acidic
and basic conditions;6 (ii) restricts rotation about the N–Cα
(Φ) and Cα–C(O) (Ψ) bonds, which can stabilise a pre-
ferred peptide conformation;7 and (iii) enhances stability
towards enzymatic degradation, thus increasing biologi-
cal half-life.8
bisebromoamide (1)
S
S
NHMe
O
N
Ph
N
N
S
O
N
S
thiangazole (2)
N
OH
S
S
N
N
CO2H
N
N
S
S
desferrithiocin (3)
didehydromirabazole A (4)
Figure 1 α-Methyl cysteine containing natural products
Three thiol protecting groups [S-trityl (Tr), S-allyl (Allyl)
and S-tert-butyl (t-Bu)] were targeted to provide appropri-
ate oxidation-resistant, bench-stable thiol-protected deriv-
atives.
A comprehensive review of approaches to the asymmetric
synthesis of α-methyl cysteine was published by Singh in
2004 and identifies five common strategies: thiolation of
a bromomethyl bislactam ether; regioselective ring open-
ing of a chiral aziridine or β-lactone; utilisation of See-
bach’s ‘self-regeneration of chirality’ approach;
enzymatic resolution; and use of a camphorsultam chiral
auxiliary to direct methylation of a thiazoline.6 Alternate
routes have since been published, but these often require
either expensive, non-commercial phase-transfer cata-
lysts,9 utilise time-consuming enzymatic resolution,7,10 or
employ microwave technologies.8 Our goal was to opti-
mise a reliable, inexpensive, scalable route to optically
pure α-methyl cysteine for solid-phase peptide synthesis
(SPPS), peptoid mimetic and natural product synthesis ap-
plications.
Whilst the asymmetric allylation, or benzylation, of ap-
propriately functionalised thiazolines may be achieved
using both cinchona and tertiary ammonium salt based
PTCs;9 the yields and ee values obtained for aliphatic al-
kylation are generally poor. Thus in selecting a scalable
synthetic route to α-methyl cysteine we were attracted to
the precedented use of camphorsultam to direct the alkyl-
ation of thiazolines; not least due to the excellent handling
properties which this auxiliary conveys (including the
generation of crystalline intermediates, and its ease of re-
moval and recycling) which we believed would outweigh
the disadvantages of an auxiliary-based approach when
working on a gram scale.11 The required thiazoline precur-
sor 6 (Scheme 1), was readily formed by reacting (R)-cys-
teine ethyl ester hydrochloride 5 and ethyl benzimidate
hydrochloride in the presence of a tertiary amine base.
SYNLETT 2013, 24, 0591–0594
Advanced online publication: 25.02.2013
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6
DOI: 10.1055/s-0032-1318316; Art ID: ST-2013-D0069-L
© Georg Thieme Verlag Stuttgart · New York

592
H. J. Johnston, A. N. Hulme
LETTER
was purified by column chromatography to give a colour-
less solid, methylated thiazoline 9, as a single diastereo-
mer. While the mass spectrometric and [α]D values of this
compound matched those of the literature, there was a sig-
nificant shift in the δ value which we observed in the 1H
NMR for the protons of the newly introduced methyl
group (δ = 1.69 vs. δ = 2.5011). However, comparison of
our NMR data to that of the oxazoline reported by S. Jew
et al.,15 and other similar thiazolines,1,16 gave us full con-
fidence in the outcome of this methylation reaction.
SH
S
b
a
Ph
EtO2C
EtO2C
NH2•HCl
N
6
5
S
X
c
Ph
N
Ph
N
N
N
S
S
O
O
O
O
O
O
9
8a: X = S
8b: X = O
A two-step procedure for the removal of the auxiliary and
hydrolysis of the thiazoline ring was found to provide op-
timum yields. The auxiliary was efficiently removed with
LiOH (1 M aq); the (1S)-(–)-2,10-camphorsultam 7 was
recovered in 89% yield and could be recycled following
recrystallisation from ethanol. The thiazoline ring was
then cleaved using HCl (6 M aq) to liberate α-methyl cys-
teine. Purification of the α-methyl cysteine from the ben-
zoic acid by-product was attempted using DOWEX
50WX8-200 resin with an initial eluant of pH 3 citrate
buffer followed by 5% ammonium hydroxide solution to
liberate the amino acid. Whilst removal of the by-product
was successful, NMR analysis indicated the presence of
citric acid (which acts as a stabiliser for the reduced cys-
teine). Any attempts to further purify 10 using this process
resulted in dimerisation to the corresponding cystine; a
process which could be readily followed by NMR (cyste-
ine CH2 – CHAHB δ 2.91, CHAHB δ 3.18 vs. cystine CH2
– CHAHB δ 3.20 CHAHB δ 3.58). Fortunately, an alterna-
tive strategy employing azeotropic removal of residual
water from the crude product mixture using toluene17
caused the hydrochloride salt of α-methyl cysteine 10 to
precipitate allowing its facile separation from the reaction
by-product. Using this method, the hydrochloride salt 10
was isolated in excellent yield (89% over two steps).
d,e
SH
N
N
N
P
N
N
P
N
N
P2-Et
HO2C
NH2•HCl
10
Scheme 1 Synthesis of α-methyl cysteine. Reagents and conditions:
(a) PhC(=NH)OEt·HCl, MeOH, Et3N, r.t., 24 h (95%); (b) (1S)-(–)-
2,10-camphorsultam (7), Me3Al, toluene, 55 °C, 24 h (76%); (c) P2-
Et, MeI, TBAB, CH2Cl2, –78 °C, 1 h (99%); (d) LiOH (1 M aq), THF,
r.t., 30 min; (e) HCl (6 M aq), reflux, 24 h (89% over 2 steps).
Subsequent coupling to (1S)-(–)-2,10-camphorsultam (7)
was achieved using AlMe3; but while the reaction itself
was not particularly problematic, purification proved
troublesome. The desired product 8a is described in the
literature as colourless and crystalline,11 however purifi-
cation by column chromatography using a range of sol-
vent systems rarely provided pure product and
recrystallisation also proved unsuccessful. The optimised
purification method developed involved gentle heating of
the crude product in diethyl ether followed by filtration.12
Using this method, a colourless crystalline product 8a was
obtained in good yield (as an ca. 1:1 epimeric mixture in
the thiazoline ring) which could be used directly in the
subsequent step.
To provide derivatives of α-methyl cysteine with extend-
ed bench-stability, three different sulfur protecting groups
were targeted: the S-trityl (Tr), S-allyl (Allyl) and S-tert-
butyl (t-Bu) groups (11a–c, Scheme 2). Whilst all three
protecting groups are widely employed in SPPS strategies
only the Tr- and t-Bu-protected derivatives of (R)-10 have
previously been reported.
Removal of the enolisable α proton in 8a gives a planar
intermediate,11 the faces of which may be readily discrim-
inated in the presence of the bulky camphorsultam, allow-
ing for highly diastereoselective methylation. Pennington
et al. have used BuLi in the presence of HMPA to effect
this transformation,11 but the yields obtained were modest
and the use of HMPA on scale is not desirable.13 In con-
trast, phosphazene bases provide an attractive alternative
as extremely strong, non-ionic, non-charged nitrogen bas-
es which generate highly reactive ‘naked’ enolates.14
Phosphazene bases also exhibit very low nucleophilicity
and hence are comparatively inert towards the electrophil-
ic component of a reaction. We opted to use the phospha-
zene base P2-Et,15 as methylation of the oxazoline 8b has
been reported to proceed in high yield with excellent dia-
stereoselectivity in the presence of this base.15 The reac-
tion of 8a was found to proceed in high yield with one
equivalent of P2-Et in one hour in the presence of the
phase-transfer catalyst TBAB. Attempts to reduce the
loading of P2-Et to substoichiometric levels (e.g. 10
mol%) were unsuccessful, resulting in sluggish reaction
times and poor yields of product (ca. 50%). The product
SH
S
PG
a, b or c
HO2C
NH2•HCl
HO2C
NH2
10
11a: PG = Tr
11b: PG = Allyl
11c: PG = t-Bu
Scheme 2 Thiol-protection of α-methyl cysteine. Reagents and con-
ditions: (a) H3PO4 (85% aq), Ph3COH, toluene, reflux, 1 h (92%); (b)
H2C=CHCH2Br, NH4OH (2 M aq), r.t., 18 h (78%); (c) t-BuOH, HCl
(37% aq), 50 °C, 18 h (81%).
Trityl protection was achieved using a method developed
by M. Yus et al.18 Phosphoric acid (85%, aq) was added to
a solution of 10 and triphenyl methanol in toluene. Reflux
for one hour gave the salt of 11a in high yield as a colour-
Synlett 2013, 24, 591–594
© Georg Thieme Verlag Stuttgart · New York

LETTER
Thiol-Protected α-Methyl Cysteine
593
(15) Lee, J.; Lee, Y.-I.; Kang, M. J.; Lee, Y.-J.; Jeong, B.-S.; Lee,
J.-H.; Kim, M.-J.; Choi, J.-Y.; Ku, J.-M.; Park, H.-G.; Jew,
S.-S. J. Org. Chem. 2005, 70, 4158.
(16) Liu, Y.; Liu, J.; Qi, X.; Du, Y. J. Org. Chem. 2012, 77, 7108.
(17) (a) Bhansali, P.; Hanigan, C. L.; Casero, R. A.; Tillekeratne,
L. M. V. J. Med. Chem. 2011, 54, 7453. (b) Matsumoto, S.;
Murao, H.; Yamaguchi, T.; Izumida, M. U.S. Patent Appl.
US 20070010689 A1, 2007.
less crystalline solid. Introduction of the allyl group to
give 11b was achieved using allyl bromide and NH4OH (2
M aq) following a procedure reported by Y. Tsantrizos et
al.19 Finally, the tert-butyl group was introduced follow-
ing a slightly modified version of the procedure described
by Chimiak et al.;20 thus 10 was dissolved in HCl (37%
aq) and heated at 50 °C in the presence of tert-butyl alco-
hol to produce 11c.
(18) Almena, J.; Foubelo, F.; Yus, M. J. Org. Chem. 1996, 61,
1859.
In conclusion, we report a simple, inexpensive, scalable
and repeatable synthesis of α-methyl cysteine (five steps
from commercial cysteine ethyl ester hydrochloride, 64%
overall yield). This route can be easily adapted to incorpo-
rate alternate sulfur protecting groups, which has been il-
lustrated by the synthesis of three different species.21 It is
anticipated that these products will find application in
SPPS as their Fmoc derivatives, incorporation into the
synthesis of a range of natural products and, in the case of
11b, might provide an elimination-resistant modified cys-
teine with potential for RCM peptide stapling,22,23 and
bioorthogonal protein modification24 through cross-
metathesis25 and the thiol-ene click (TEC) reaction.26
(19) Goudreau, N.; Brochu, C.; Cameron, D. R.; Duceppe, J.-S.;
Faucher, A.-M.; Ferland, J.-M.; Grand-Maître, C.; Poirier,
M.; Simoneau, B.; Tsantrizos, Y. S. J. Org. Chem. 2004, 69,
6185.
(20) Pastuszak, J. J.; Chimiak, A. J. Org. Chem. 1981, 46, 1868.
(21) (2R)-2-Amino-2-methyl-3-[(triphenylmethyl)sulfan-
yl]propanoic Acid Phosphate Salt11
H3PO4 (0.2 mL; 85% aq) was added to a stirred solution of
α-methyl cysteine hydrochloride 10 (100 mg, 0.58 mmol)
and trityl alcohol (0.15 g, 0.58 mmol) in toluene (5 mL) at
r.t. The reaction mixture was heated at reflux for 1 h, cooled
to r.t. and then the reaction mixture was concentrated in
vacuo. H2O (5 mL) was added and the crude product was
stirred for a further 30 min, then filtered and recrystallised
from MeOH to give the desired product 11a as a colourless
solid (202 mg, 92%); Rf 0.21 (MeOH–CH2Cl2, 1:9); [α]D
+30.0 (c 1.00, MeOH); mp 178–180 °C [lit.11 mp 179–180
ºC]. IR (neat): 3471 (NH), 2580–3600 (OH), 1730 (C=O),
1630 (Ar), 1593 (Ar), 1512 (Ar) cm–1. 1H NMR (500 MHz,
DMSO): δ = 7.24–7.38 (m, 15 H, ArH), 3.50 (br s, 2 H,
NH2), 2.45 (d, J = 11.7 Hz, 1 H, CHACHB), 2.39 (d, J = 11.7
Hz, 1 H, CHACHB), 1.22 (s, 3 H, CCH3). 13C NMR (126
MHz, DMSO): δ = 171.12 (C), 143.96 (3 × C), 129.08 (6 ×
CH), 128.11 (6 × CH), 126.88 (3 × CH), 65.99 (C), 58.67
(CH2), 21.96 (CH3), trityl C absent. MS (ESI+, MeOH): m/z
(%) = 378 (24) [M + H]+, 243 (100), 179 (10). 1H NMR and
13C NMR spectroscopic data were in good agreement with
the literature.
Acknowledgment
We thank the EPSRC (DTA) and Cancer Research UK for funding.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett. Included are
the preparation of α-methyl cysteine 10, and 1H NMR and 13C NMR
spectra for all compounds.SnuIpgfoip
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References and Notes
(2R)-2-Amino-2-methyl-3-[(prop-2-en-1-yl)sulfanyl]-
(1) Han, F. S.; Osajima, H.; Cheung, M.; Tokuyama, H.;
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(3) Boyce, R. J.; Mulqueen, G. C.; Pattenden, G. Tetrahedron
1995, 51, 7321.
propanoic Acid27
Allyl bromide (0.08 mL, 0.87 mmol) was added to a stirred
solution of α-methyl cysteine hydrochloride 10 (100 mg,
0.58 mmol) in NH4OH (2 mL, 2 M aq) at r.t. The reaction
mixture was stirred at r.t. for 18 h then the product was
concentrated in vacuo. The crude product was then
recrystallised from EtOH to give the desired product 11b as
a colourless solid (79.6 mg, 78%); Rf 0.45 (MeOH–CH2Cl2,
1:9); [α]D +25.0 (c 0.40, H2O); mp 257–259 °C [lit.23 mp 260
°C]. IR (neat): 3454 (NH), 3419 (NH), 2700–3230 (OH),
1738 (C=O), 1605 (C=C), 1597 (COO–) cm–1. 1H NMR (400
MHz, D2O): δ = 5.69–5.83 (m, 1 H, CH=CH2), 5.08–5.20
(m, 2 H, CH=CH2), 3.08–3.22 (m, 2 H, CH2), 3.05 (d, J =
14.5 Hz, 1 H, CHACHB), 2.71 (d, J = 14.5 Hz, 1 H,
CHACHB), 1.45 (s, 3 H, CCH3). 13C NMR (126 MHz, D2O):
δ = 175.30 (C), 133.72 (CH), 118.33 (CH2), 61.14 (C), 36.85
(CH2), 34.94 (CH2), 22.20 (CH3). MS (ESI+, MeOH–
CH2Cl2): m/z (%) = 176 (29) [M + H]+, 159 (25), 144 (24),
136 (30), 114 (27), 110 (40). IR data were in good agreement
with literature.
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(2R)-2-Amino-3-(tert-butylsulfanyl)-2-methylpropanoic
Acid Hydrochloride Salt17b
HCl (2.5 mL; 37% aq) was added to a stirred solution of α-
methyl cysteine hydrochloride 10 (100 mg, 0.58 mmol) in
t-BuOH (0.56 g, 5.8 mmol) at r.t. The reaction mixture was
heated to 50 °C and stirred for ca. 18 h at the same
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© Georg Thieme Verlag Stuttgart · New York
Synlett 2013, 24, 591–594

594
H. J. Johnston, A. N. Hulme
LETTER
the solvent had been removed. On standing overnight at r.t.
colourless crystals formed. The crystals were collected by
filtration to give the desired product 11c as a colourless solid
(90.3 mg, 81%); Rf 0.10 (MeOH–CH2Cl2, 1:9); [α]D –50.0 (c
0.40, H2O); mp 272–274 °C [lit.17b mp 272–275 °C]. IR
(neat): 3389 (NH), 3333 (NH), 2750–3100 (OH), 1734
(C=O) cm–1. 1H NMR (500 MHz, D2O): δ = 3.13 (d, J = 13.4
Hz, 1 H, CHAHBS), 2.93 (d, J = 13.4 Hz, 1 H, CHAHBS), 1.56
(s, 3 H, CCH3), 1.26 [s, 9 H, C(CH3)3]. 13C NMR (126 MHz,
D2O): δ = 173.31 (C), 60.07 (C), 43.51 (C), 34.09 (CH2),
29.74 (3 × CH3), 21.79 (CH3). MS (ESI+, MeOH–CH2Cl2):
m/z (%) = 192 (58) [M + H]+, 137 (72), 136 (100), 119 (88),
101 (81), 90 (72). 1H NMR and 13C NMR spectroscopic data
were in good agreement with the literature.
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Synlett 2013, 24, 591–594
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