Facile N-urethane-protected α-amino/peptide thioacid preparation using EDC and Na2S

Article abstract of DOI:10.1055/s-0031-1290091

We report herein an efficient protocol for the synthesis of N-urethane-protected α-amino/peptide thioacids from their corresponding acids mediated by EDC and Na₂S. The fast reaction under mild conditions enabled the process to be completed in shorter duration with good yield circumventing column purification. The chemistry is compatible with a wide variety of urethane protecting groups, side-chain functionalities, and sterically hindered amino -acids. Georg Thieme Verlag Stuttgart. New York.

Full text of DOI:10.1055/s-0031-1290091

LETTER
89
Facile N-Urethane-Protected a-Amino/Peptide Thioacid Preparation Using
EDC and Na2S
N
-Urethane-Pro
.
tected
a
-A
m
M
ino/Peptide
T
hioacid P
.
reparation Vishwanatha, M. Samarasimhareddy, Vommina V. Sureshbabu*
Peptide Research Laboratory, Department of Studies in Chemistry, Bangalore University,
Dr. B. R. Ambedkar Veedhi, Central College Campus, Bangalore – 560 001, India
E-mail: sureshbabuvommina@rediffmail.com, hariccb@gmail.com, hariccb@hotmail.com
Received 28 June 2011
in-bound peptide are also known.22 With these promising
results and the recent trends in the application of a-amino
thioacids,23,24 there is a pressing need for a rapid and effi-
cient methodology for the preparation of amino and pep-
Abstract: We report herein an efficient protocol for the synthesis of
N-urethane-protected a-amino/peptide thioacids from their corre-
sponding acids mediated by EDC and Na2S. The fast reaction under
mild conditions enabled the process to be completed in shorter du-
ration with good yield circumventing column purification. The tide thioacids.
chemistry is compatible with a wide variety of urethane protecting
groups, side-chain functionalities, and sterically hindered amino
acids.
Owing to its fast reactivity under mild conditions, water-
soluble N-ethyl-N¢-(3-dimethylaminopropyl)carbodiim-
ide (EDC) is often used for the epimerization-free peptide
synthesis.25 Recently, we had the occasion to explore this
reagent for the synthesis of acid azides, ureas, and car-
bamates.26 From these initial studies, we initiated our ex-
amination on the use of EDC in the synthesis of thioacids.
The reaction was fast, and the products were obtained in
good yields.
Key words: N-urethane-protected a-amino thioacids, carbodiim-
ides, sodium sulfide
Protected thioacids masked as thioesters have been em-
ployed as key components in Native Chemical Ligation
(NCL).1 Thioacids [R(C=O)SH] play a pivotal role in or-
ganic synthesis because of their specific reactive profile
and chemical behavior.2,3 They are frequently employed
in peptide and peptidomimetic syntheses as well.4 In par-
ticular, thioacids exhibit varied reactivity towards
amines,5 isonitriles,6 alkyl azides,7 sulfonyl azides,8 isocy-
anates/isothiocyanates,9 aziridines,10 electron-deficient
sulfonamides,11 and alkyl halides12 leading to biologically
desirable mimetics.13,14
In order to optimize the reaction conditions, we studied
the conversion of Fmoc-Ala-OH to its thioacid. Our study
initially used N,N¢-dicyclohexylcarbodiimide (DCC) as
coupling agent and Na2S as hydrosulfide ion source in
various solvents (Table 1, entries 1–6). The first attempt
was performed in MeCN by using three equivalents of
Na2S for three hours which led to thioacid 2b in 54% yield
along with unreacted 1b (Table 1, entry 1). Based on this
preliminary result, the reaction conditions were further
fine-tuned using c different solvents.
Optically active a-amino thioacids are generally prepared
by the activation of the carboxy group followed by pass-
ing H2S gas.15 Alternatively, the Na-protected amino acid
is coupled with 9-fluorenylmethane thiol (FmSH), triphe-
nylmethane thiol (TrtSH), or trimethoxybenzyl thiol
(TmobSH)16 to obtain the corresponding thioester which
is then deprotected to the thioacid. Recently, Danishef-
sky’s group has described the preparation of thioacids by
direct reaction of carboxylic acids with Lawesson’s re-
agent at high temperature.17 To avoid the use of highly
toxic H2S gas, solid NaSH and N2S have been used along
with 1,1¢-carbonyldiimidazole (CDI) as coupling re-
agent.18 However, long reaction times and slow reactivity
can result in low recovery of the product and acyldisul-
fides, unreacted starting material, and other impurities are
often observed as byproducts. Peptide thioacids are gener-
ally synthesized by treatment of Kaiser’s oxime ester with
hexamethyldisilathiane,19 while processes involving acyl-
enzyme thioester intermediates,20 hydrolysis of resin-
bound peptide thioesters,21 and reaction of NaSH with res-
It was observed that the use of THF, dioxane, and DMSO
resulted in poor yields and the formation of unidentified
byproducts (Table 1, entries 2–4), but a yield of 2b of
61% was observed when DMF was used (Table 1, entry
5). Further, we tested the reaction in the presence of HOBt
as an additive in DMF and, although product yield was in-
creased to 69%, 22% of unreacted 1b was still observed
(Table 1, entry 6). To explore the scope and limitations of
carbodiimides, EDC was then chosen. The initial experi-
ments using 1b with EDC/HOBt and Na2S in DMF gave
2b in an acceptable yield of 82% (Table 1, entry 7).
With this combination 11% of 1b was also detected. At
this stage we attempted to isolate pure 2b through column
purification, but this was not satisfactory due to the almost
identical Rf values of both 1b and 2b.15b Subsequently,
EDC in DMF turned out to give satisfactory results in the
model reaction (Scheme 1). To our delight, the desired
product was obtained in 92% yield. Interestingly, no unre-
acted 1b was found which enabled simple recrystalliza-
tion to be used for the isolation of the pure product.
Another test case was conducted with diisopropylcarbodi-
imide (DIC) and DIC/HOBt (Table 1, entries 10 and 11),
SYNLETT 2012, 23, 89–92
x
x
.x
x
.2
0
1
1
Advanced online publication: 07.12.2011
DOI: 10.1055/s-0031-1290091; Art ID: D20211ST
© Georg Thieme Verlag Stuttgart · New York

90
T. M. Vishwanatha et al.
LETTER
Table 1 Optimization of Reaction Conditions for the Synthesis of 2b
Entry
Coupling reagent
DCC–NMM
DCC–NMM
DCC–NMM
DCC–NMM
DCC–NMM
DCC–NMM
EDC
Additive
Solvent
MeCN
THFb
Time (h)
Yield of 2a (%)a
Yield of 1a (%)a
1
2
–
8
8
8
6
5
4
3
3
3
5
4
54
48
57
51
61
69
82
92
89
48
52
25
35
32
31
25
22
11
–
–
3
–
dioxane
DMSO
DMF
4
–
5
–
6
HOBt
DMF
7
HOBt
DMF
8
EDC
–
–
DMF
9
EDC
DMSO
DMF
6
10
11
DIC
51
38
DIC–NMM
HOBt
DMF
a Yields are given according to the mass analysis of the crude reaction mixture measured by LC-MS.
b An aliquot of H2O was added to dissolve N2S.
Table 2 Synthesized N-Urethane-Protected a-Amino Thioacids
and the results were more or less similar to DCC/HOBt.
Thus, optimal conditions for the synthesis of thioacids are
as follows: under argon atmosphere, acid (1 equiv) was
dissolved in DMF (5 mL), EDC (1.1 equiv) was added at
0 °C, after five minutes of stirring a solution of Na2S (3
equiv) in DMF (10 mL) was added, and the reaction mix-
ture was stirred for three hours.
20
Entry Compd Thioacid
[a]D
Yield Mp
2
(c, 1.2, CHCl3) (%)a (°C)
O
O
1
2
2a
2b
89
91
107–109
FmocHN
FmocHN
SH
SH
–11.7
+12.9
78–79
76–78
R
R
EDC, Na2S
DMF, 3 h
SH
OH
PgHN
O
PgHN
3
4
2c
86
90
O
O
FmocHN
SH
Scheme 1 Synthesis of N-urethane-protected a-amino thioacids
O
FmocHN
To illustrate the generality and scope of the optimized
protocol, a range of N-urethane protecting groups and var-
ious side-chain-functionalized amino acids were convert-
ed to the corresponding thioacids, and the results are
summarized in Table 2. In general, good yields are ob-
tained and no significant difference in the reactivity was
observed for all the N-protecting groups. The effect of
side-chain functionalities was also observed by synthesiz-
ing thioacids from the sterically hindered amino acids
Aib, Val, and Ile where a reaction time of up to eight hours
was required to complete the conversion. Our study was
then extended to prepare some peptide thioacids. For this,
a series of di- and tripeptide acids were synthesized using
the EDC method.27 Thus, N-protected a-amino acids were
activated by EDC/HOBt. A fresh solution of O,N-bis/tris-
trimethylsilyl amino acid was added directly in one por-
tion and stirring was continued until completion of the
reaction.
2d
–58.2
101–103
SH
Ot-Bu
O
FmocHN
FmocHN
CbzHN
5
6
7
8
2e
2f
–39.6
–41.0
–49.8
–88.1
88
91
85
86
81–83
87–88
gum
SH
O
SH
O
2g
2h
SH
O
gum
CbzN
SH
Synlett 2012, 23, 89–92
© Thieme Stuttgart · New York

LETTER
N-Urethane-Protected a-Amino/Peptide Thioacid Preparation
91
Table 2 Synthesized N-Urethane-Protected a-Amino Thioacids
Table 3 Synthesized Peptide Thioacids
Entry Compd 3 Peptide thioacid
(continued)
Yield (%)a
78
20
Entry Compd Thioacid
[a]D
Yield Mp
O
2
(c, 1.2, CHCl3) (%)a (°C)
H
N
SH
FmocHN
O
1
3a
O
CbzHN
9
2i
2j
–28.6
–66.7
79
94
121–123
91–93
SH
4
NHBoc
O
O
H
N
2
3
3b
3c
89
78
N
SH
FmocHN
CbzHN
CbzHN
SH
O
10
O
O
H
N
SH
O
O
BocHN
BocHN
SH
11
2k
–55.0
91
65–67
O
O
4
5
3d
3f
91
88
H
N
SH
O
N
H
CbzHN
12
13
2l
86
90
gum
gum
O
O
O
SH
BnO
H
N
O
SH
BocHN
BocHN
2m
–22.7
–10.2
SH
2
BnOOC
a Yields are given after recrystallization of the crude product from
THF–H2O.
O
BocHN
14
2n
90
113–115
SH
OBn
plications. No epimerization in the peptide thioacids has
been detected by chiral HPLC.
a Isolated yield based on 1.
Acknowledgment
Acidification followed by recrystallization of the crude
product led to peptide acids in good yield. The resulting
peptide acids were then converted to their thioacids under
conditions similar to those employed for the preparation
of a-amino thioacids (Table 3).28–30 To determine whether
the reaction conditions lead to any detectable amount of
epimerization in the present protocol, Fmoc-Ala-SH
enantiomers were synthesized, and the optical purity was
measured by chiral HPLC. The D- and L-Fmoc-Ala-SH
were baseline separated with a retention time of 8.4 and
13.5 minutes, respectively.31 In addition, specific rota-
tions of 2b and 2c were recorded as –11.7 and +12.9, re-
spectively. These results indicate that the protocol is free
from epimerization.
We thank the Department of Science and Technology (DST) (Grant
No. SR/S1/OC-26/2008) New Delhi, India for financial support.
References and Notes
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In conclusion, we have demonstrated that N-urethane-pro-
tected a-amino/peptide oxoacids can be readily converted
into their corresponding thioacids in high yields under
mild conditions. The reaction of N-protected a-amino ac-
ids, EDC, and Na2S in DMF led to the desired products in
good yields. Notably, this procedure has been shown to be
fully compatible with all urethane protections as well as a
range of side-chain functionalities. The protocol is opera-
tionally simple, effective, and minimizes side products so
that the crude products can be used directly for further ap-
© Thieme Stuttgart · New York
Synlett 2012, 23, 89–92

92
T. M. Vishwanatha et al.
LETTER
added. The reaction mixture was stirred for 3–4 h (TLC
analysis), and then evaporation of the solvent and
acidification with 1 M HCl furnished pure peptide acid.
(b) For the preparation of O,N-bis-TMS-amino acids, see:
Tantry, S. J.; Vasanthakumar, G.-R.; Sureshbabu, V. V. Lett.
Pept. Sci. 2003, 10, 51.
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2010, 132, 10986.
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(12) Fu, X.; Jiang, S.; Li, C.; Xin, J.; Yang, Y.; Ji, R. Bioorg.
Med. Chem. Lett. 2007, 17, 465.
(28) General Procedure for the Synthesis of Amino/Peptide
Thioacids
To a DMF solution of an acid (1.0 mmol), EDC (1.1 equiv)
was added at 0 °C under a nitrogen atmosphere. After
stirring for 10 min, finely ground Na2S (3 equiv) was added
to the reaction mixture which was allowed for stir for 3–4 h
until the disappearance of the starting material (TLC
analysis). The residue was dissolved in EtOAc (15 mL), and
the solution was then carefully acidified at 0 °C to a pH of 3
by using 1 M KHSO4. The organic layer was then
immediately separated and removed under reduced pressure.
The crude product was triturated with Et2O or recrystallized
with THF–H2O to obtain pure thioacid.
(13) Kolb, J.; Beck, B.; Almstetter, M.; Heck, S.; Herdtweck, E.;
Dömling, A. Mol. Diversity 2003, 6, 297.
(14) Yazmin, T.; Rosa-Bauza Berst, F.; Ellman, J. A.
ChemBioChem. 2007, 8, 981.
(15) (a) Le, H.-T.; Gallard, J.-F.; Mayer, M.; Guittet, E.;
Michelot, R. Bioorg. Med. Chem. 1996, 4, 2201. (b) Hoeg-
Jensen, T.; Jakobsen, H.; Olsen, C. E.; Holm, A.
Tetrahedron Lett. 1991, 32, 7617. (c) Hoeg-Jensen, T.;
Holm, A.; Sorensen, H. Synthesis 1994, 383.
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(b) Crich, D.; Sharma, I. Angew. Chem. Int. Ed. 2009, 48,
2355.
(17) Rao, Y.; Li, X.; Nagorny, P.; Hayashida, J.; Danishefsky,
S. J. Tetrahedron Lett. 2009, 50, 6684.
(18) Monfardini, I.; Huang, J.-W.; Beck, B.; Cellitti, J. F.;
Pellecchai, M.; Dömling, A. J. Med. Chem. 2011, 54, 890.
(19) Schwabacher, A. W.; Maynard, T. L. Tetrahedron Lett.
1993, 34, 1269.
(29) Fmoc-Ile-COSH
Yellow solid; mp 81–83 °C. IR (KBr): nmax = 1689, 1739,
2550, 3342 cm–1. Rf = 0.39 (EtOAc–n-hexane = 60:40). RP-
HPLC: tR = 15.2 (60–100% MeCN, 30 min). ESI-HRMS:
m/z calcd for C21H23NO3S: 392.1296 [M + Na]+; found:
392.1290. 1H NMR (400 MHz, CDCl3): d = 0.88 (t, J = 5.6
Hz, 3 H), 0.98 (d, J = 3.8 Hz, 3 H), 1.12–1.24 (m, 2 H), 2.38–
2.47 (m, 1 H), 4.28 (d, J = 6.7 Hz, 1 H), 4.37 (d, J = 7.1 Hz,
1 H), 4.61 (d, J = 4.4 Hz, 2 H), 5.91 (br s, 1 H), 7.43 (br s, 1
H), 7.26–7.84 (m, 8 H). 13C NMR (100 MHz, CDCl3):
d = 10.8, 14.3, 24.1, 37.0, 46.4, 65.9, 72.8, 125.9, 127.3,
128.6, 128.9, 139.2, 142.6, 155.2, 197.2.
(20) Tan, X.-H.; Yang, R.; Wirjo, A.; Liu, C.-F. Tetrahedron
Lett. 2008, 49, 2891.
(21) Zhang, X.; Lu, X.-W.; Liu, C.-F. Tetrahedron Lett. 2008, 49,
6122.
(30) Fmoc-Ala-Phe-COSH
(22) Shigenaga, A.; Sumikawa, Y.; Tsuda, S.; Sato, S.; Otaka, A.
White solid; mp 126–128 °C. IR (KBr): nmax = 1681, 1748,
1768, 2549, 3328 cm–1. Rf = 0.53 (CHCl3–MeOH = 80:20).
RP-HPLC: tR = 11.4 (60–100% MeCN, 30 min). ESI-
HRMS: m/z calcd for C27H26N2O4S: 497.1511 [M + Na]+;
found: 497.1501. 1H NMR (400 MHz, CDCl3): d = 1.2 (d,
J = 4.8 Hz, 3 H), 2.6 (d, J = 5.6 Hz, 2 H), 2.8 (br s, 1 H), 3.38
(t, J = 7.4 Hz, 1 H), 3.6 (br s, 1 H), 3.9 (t, J = 6.9 Hz, 2 H),
4.1 (m, 1 H), 4.3 (m, 1 H), 6.32 (br s, 1 H), 7.1 (br s, 1 H),
7.2–7.9 (m, 13 H). 13C NMR (100 MHz, CDCl3): d = 17.2,
37.4, 46.8, 51.2, 67.9, 69.7, 125.7, 126.8, 127.2, 127.9,
128.4, 128.9, 131.2, 139.1, 141.4, 143.2, 155.7, 172.1,
197.2.
Tetrahedron 2010, 66, 3290.
(23) (a) Wang, P.; Danishefsky, S. J. J. Am. Chem. Soc. 2010,
132, 17045. (b) Wang, P.; Li, X.; Zhu, J.; Chen, J.; Yuan, Y.;
Wu, X.; Danishefsky, S. J. J. Am. Chem. Soc. 2011, 133,
1597.
(24) Pan, J.; Nelmi, O.; Devarie, B.; Xian, M. Org. Lett. 2011, 13,
1092.
(25) Goyal, N. Synlett 2010, 335; and references cited therein.
(26) Sureshbabu, V. V.; Lalithamba, H. S.; Narandra, N.;
Hemantha, H. P. Org. Biomol. Chem. 2010, 8, 835.
(27) (a) General Procedure for the Preparation of Peptide
Acids
(31) Chiral-HPLC analyses were carried out employing
Chiralpak IA, 250 × 4.6 mm; solvent: hexane–EtOH (7:3);
flow rate: 1.0 mL/min.
A solution of Na-protected amino acid (1 mmol) in dry
CH2Cl2 (5 mL) was cooled to 0 °C, EDC (1 mmol), HOBt
(1.2 mmol), and O,N-bis-TMS-amino acid (1.5 mmol) were
Synlett 2012, 23, 89–92
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