An improved method for the preparation of protected (R)-2-methylcysteine: Solution-phase synthesis of a glutathione analogue

Article abstract of DOI:10.1055/s-0030-1259021

A synthetic method for the preparation of (R)-2-methylcysteine that dramatically improves the overall yield of this important unnatural amino acid has been refined. The key steps in the preparation of (R)-2-methylcysteine were improved such that necessary

Full text of DOI:10.1055/s-0030-1259021

LETTER
2941
An Improved Method for the Preparation of Protected (R)-2-Methylcysteine:
Solution-Phase Synthesis of a Glutathione Analogue
I
mprove
d
Prepara
t
o
ion of
(
R
)-2-
u
Methylcyste
g
a
Department of Chemistry and Biochemistry, The University of Southern Mississippi,
118 College Drive #5043, Hattiesburg, MS 39406, USA
b
Department of Chemistry, University of Wisconsin-Oshkosh, 800 Algoma Blvd., Oshkosh, WI 54901, USA
Fax +1(601)2666075; E-mail: Douglas.Masterson@usm.edu
Received 22 August 2010
butyl mercaptan and paraformaldehyde in dichlo-
romethane. The reaction could be monitored visually as
the solution became homogenous upon addition of HCl.
Once the reaction had reached completion, the solvent and
Abstract: A synthetic method for the preparation of (R)-2-methyl-
cysteine that dramatically improves the overall yield of this impor-
tant unnatural amino acid has been refined. The key steps in the
preparation of (R)-2-methylcysteine were improved such that nec-
essary intermediates were prepared in high yields and of sufficient volatiles were removed under reduced pressure at room
purity to avoid the need for distillation or column chromatography.
The (R)-2-methylcysteine was prepared (>90% ee) in appropriately
protected form and used in a novel solution phase synthesis of a glu-
The resulting liquid was found to be essentially pure 1
tathione analogue.
temperature and the resulting suspension was vacuum fil-
tered to remove the white insoluble material that formed.
(87% corrected yield) contaminated with residual dichlo-
romethane. The purity of 1 was found to be sufficient for
use in the alkylation of dimethyl methylmalonate.
Key words: amino acids, asymmetric synthesis, peptides, glu-
tathione, bioorganic chemistry
O
O
SCH2Cl
Unnatural amino acids have found numerous uses in re-
cent years, and several reports have demonstrated the ben-
efits of using unnatural amino acids in foldamers,1
peptidomimetics,2 enzyme inhibitors,3 and as ligands.4
One class of amino acids that has received considerable
attention is the a-methyl amino acids, which have demon-
strated a remarkable ability to retard the degradation of
peptides by hydrolytic enzymes as well as improve the
stability of secondary structure.5–6 Various synthetic strat-
egies have been devised for the preparation of a-methyl
amino acids.7–16 One of the most versatile methods of
preparation is the conversion of chiral acid ester interme-
diates, as illustrated in Scheme 1, into amino acids.17–18
The acid ester methodology allows for the preparation of
various amino acid classes as well as both enantiomers of
the amino acid by straightforward synthetic manipulation.
MeO
OH
1
1. NaH, THF
2. pig liver
esterase
S
O
O
4
MeO
OMe
2
steps
OMe
O
H
N
O
HO
O
S
6
The main drawback to the preparation of 4 is the synthesis
of alkylating agent 1. Compound 1 has been prepared us- Scheme 1
ing tert-butyl mercaptan, paraformaldehyde, and either
TMSCl or HCl.11 The resulting mixture is then distilled
Diester 3 was prepared in a manner similar to that reported
in the literature. Scheme 2 illustrates the preparation of 3
using our modified reaction conditions. The crude diester
3 was worked up in the normal manner and the resulting
oil was placed under vacuum overnight to provide 3 in
sufficient purity to proceed to the pig liver esterase (PLE)-
mediated hydrolysis step without the need for column
chromatography or lengthy distillation procedures. The
acid ester 4 was prepared as previously reported and con-
verted into the protected amino acid (6) as illustrated in
Scheme 2 in good overall yield and enantiomeric purity.17
under reduced pressure to provide 1 in 20–60% yield. We
report here an improved procedure for the preparation of
1 that avoids the need for distillation. Compound 1 is then
converted into protected (R)-2-methyl cysteine (6) in
good yield and enantioselectivity. Compound 6 was used
to prepare a novel glutathione analogue to illustrate the
utility of 6 in solution phase peptide synthesis.19
Compound 1 was prepared as outlined in Scheme 2. An-
hydrous HCl was passed through a suspension of tert-
SYNLETT 2010, No. 19, pp 2941–2943
Advanced online publication: 03.11.2010
DOI: 10.1055/s-0030-1259021; Art ID: S05310ST
x
x
.x
x
.2
0
1
0
To illustrate the utility of 6 in solution phase synthesis of
peptides, we chose to prepare an analogue of reduced and
© Georg Thieme Verlag Stuttgart · New York

2942
D. S. Masterson et al.
LETTER
O
O
moved using standard procedures, providing sulfide 11 as
the trifluoroacetate salt. Interestingly, 11 was isolated as a
single diastereoisomer, as evidenced by HPLC and NMR
analyses.
a
b
MeO
OMe
SCH2Cl
SH
St-Bu
1 (87%)
3 (quantitative)
Sulfide 11 was transformed into the reduced glutathione
analogue 12 by removal of the tert-butyl protecting group
by using mercuric acetate, in quantitative yield.20 Peptide
12 was then transformed into the disulfide 13 by passage
of oxygen through a basic aqueous solution of 12 in good
yield as the trifluoroacetate salt (Scheme 4). The overall
yield of 13 starting from tert-butyl mercaptan was 21%
over 12 steps.
c
Moz
O
O
O
H
N
OPMB
d (80%)
e (98%)
HO
MeO
OH
O
St-Bu
St-Bu
4 (73%, 91% ee)
6 (78% two steps)
O
NH3
O
Scheme 2 Reagents and conditions: (a) CH2Cl2, paraformaldehyde,
HCl(g); (b) dimethyl methylmalonate (2), NaH, THF; (c) PLE, pH 7.2
phosphate buffer; (d) (i) diphenylphosphoryl azide, Et3N, (CH2Cl)2;
(ii) p-methoxybenzyl alcohol; (e) LiOH, THF, H2O, Et3NBnBr.
H
N
a
OH
11
HO2C
N
H
O
SH
12 (100%)
O
b
NHMoz
St-Bu
a
EtO2C
N
H
+
NH3Cl
6
EtO2C
O
NH3
O
H
7 (84%)
N
OH
HO2C
N
H
b
O
O
S
S
O
O
HN
Ot-Bu
Ot-Bu
O
O
H
c
NH2
N
H
EtO2C
N
EtO2C
N
N
CO2H
H
H
HO
N
O
H
O
NH3
O
St-Bu
St-Bu
8 (76%)
9 (88%)
13 (88%)
Scheme 4 Reagents and conditions: (a) (i) Hg(OAc)2, TFA; (ii)
H2S/H2O; (b) NH4OH, H2O (pH 8.0), O2.
d (97%)
e (89%)
O
NH3
O
In conclusion, we have prepared the alkylating agent 1 in
excellent yield by using an improved synthetic method
that avoids the need to purify the product by distillation.
Compound 1 was found to be of sufficient purity to use in
the alkylation of dimethyl methylmalonate giving product
3 in high yield and purity. Compound 3 was ultimately
transformed into the Moz-protected amino acid 6, which
was shown to be a suitable reagent for solution phase syn-
thesis of glutathione analogues 12 and 13. We are in the
process of examining 12 and 13 in biological assays and
will report on these efforts in due course.
H
N
OH
HO2C
N
H
O
St-Bu
11 (86% two steps)
Scheme 3 Reagents and conditions: (a) DIPEA, PyBroP, CH2Cl2;
(b) 10% TFA, CH2Cl2, thioanisole; (c) Boc-L-glutamic acid 1-tert-
butyl ester, DIPEA, PyBroP, CH2Cl2; (d) LiOH, THF, H2O; (e) 50%
TFA, CH2Cl2.
oxidized glutathione. The first coupling step involved the
formation of the peptide bond between 6 and glycine ethyl
ester, as shown in Scheme 3, to provide fragment 7 in
good yield. We found that using bromotripyrrolidino-
phosphonium hexafluorophosphate (PyBroP) as the cou-
pling agent provided superior results compared to using
N,N-dicyclohexylcarbodiimide (yield with DCC = 52%).
The Moz carbamate protecting group in 7 was removed
using 10% trifluoroacetic acid (TFA) and thioanisole as
cation scavenger, giving 8 in good yield. Fragment 8 was
then coupled with Boc-L-glutamic acid 1-tert-butyl ester,
giving 9 in good yield. Again, the use of PyBroP was
found to be important for obtaining good yields in the cou-
pling. Next, the ester and Boc protecting groups were re-
Acknowledgment
We would like to thank the National Science Foundation for support
of this work (NSF-CAREER, MCB-0844478) and for funds used to
acquire an NMR spectrometer (CHE-0840390). We would also like
to thank the University of Southern Mississippi Department of Che-
mistry and Biochemistry.
References and Notes
(1) Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore,
J. S. Chem. Rev. 2001, 101, 3893.
(2) Steer, D. L.; Lew, R. A.; Perlmutter, P.; Smith, A. I.;
Aguilar, M.-I. Curr. Med. Chem. 2002, 9, 811.
Synlett 2010, No. 19, 2941–2943 © Thieme Stuttgart · New York

LETTER
Improved Preparation of Protected (R)-2-Methylcysteine
2943
(3) Ghorai, M. K.; Das, K.; Kumar, A. Tetrahedron Lett. 2007,
48, 2471.
(13) Pattenden, G.; Thom, S. M.; Jones, M. F. Tetrahedron 1993,
49, 2131.
(4) Vicario, J. L.; Badía, D.; Carrillo, L.; Reyes, E.; Etxebarria,
J. Curr. Org. Chem. 2005, 9, 219.
(5) Shao, H.; Zhu, Q.; Goodman, M. J. Org. Chem. 1995, 60,
790.
(14) Mulqueen, G. C.; Pattenden, G.; Whiting, D. A. Tetrahedron
1993, 49, 5359.
(15) Boyce, R. J.; Mulqueen, G. C.; Pattenden, G. Tetrahedron
Lett. 1994, 35, 5705.
(6) Long, Y.-Q.; Xue, T.; Song, Y.-L.; Liu, Z.-L.; Huang, S.-X.;
Yu, Q. J. Med. Chem. 2008, 51, 6371.
(7) Smith, N. D.; Goodman, M. Org. Lett. 2003, 5, 1035.
(8) Smith, N. D.; Wohlrab, A. M.; Goodman, M. Org. Lett.
2005, 7, 255.
(9) Seebach, D.; Sting, A. R.; Hoffmann, M. Angew. Chem., Int.
Ed. Engl. 1996, 35, 2708.
(16) Boyce, R. J.; Pattenden, G. Synlett 1994, 587.
(17) Kedrowski, B. L. J. Org. Chem. 2003, 68, 5403.
(18) Masterson, D. S.; Roy, K.; Rosado, D. A.; Fouche, M.
J. Pept. Sci. 2008, 14, 1151.
(19) For an example of a racemic synthesis, see: Zhdanko, A. G.;
Gulevich, A. V.; Nenajdenko, V. G. Tetrahedron 2009, 65,
4692.
(10) Seebach, D.; Weber, T. Tetrahedron Lett. 1983, 24, 3315.
(11) Seebach, D.; Weber, T. Helv. Chim. Acta 1984, 67, 1650.
(12) Pattenden, G.; Thom, S. M. J. Chem. Soc., Perkin Trans. 1
1993, 1629.
(20) Nishimura, O.; Kitada, C.; Fujino, M. Chem. Pharm. Bull.
1978, 26, 1576.
Synlett 2010, No. 19, 2941–2943 © Thieme Stuttgart · New York