Metal-free direct amidation of peptidyl thiol esters with α-amino acid esters

Article abstract of DOI:10.1039/c1gc15401j

Metal-free direct amidation of peptidyl thiol esters with α-amino acid esters in the presence of bis(trimethylsilyl) acetamide (BSA) has been developed. This general method provides convenient access to N-protected peptides in good yields under mild condi

Full text of DOI:10.1039/c1gc15401j

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COMMUNICATION
Metal-free direct amidation of peptidyl thiol esters with a-amino acid
esters†
Hao Chen,a Maomao He,a Yaya Wang,a Linhui Zhai,a Yongbo Cui,a Yangyan Li,a Yan Li,b Haibing Zhou,*a
Xuechuan Hong*a and Zixin Denga
Received 12th April 2011, Accepted 29th July 2011
DOI: 10.1039/c1gc15401j
Metal-free direct amidation of peptidyl thiol esters with
a-amino acid esters in the presence of bis(trimethylsilyl)
acetamide (BSA) has been developed. This general method
provides convenient access to N-protected peptides in good
yields under mild conditions and demonstrates a high
tolerance to functionality.
method termed native chemical ligation (NCL) for the coupling
of large peptidic fragments.13 This method is outlined in Scheme
1, which allows for the use of completely unprotected peptide for
the coupling. The coupling process included the intermolecular
transthioesterification and intramolecular S→N acyl transfer.
Nevertheless, this NCL methodology requires a cysteine residue
or a cysteine-mimicking auxiliary of the C-terminal peptide
segment and the rate-limiting step is S→S acyl transfer.12d
To avoid the S→S acyl transfer, Danishefsky and co-workers
reported the NCL reaction using C-terminal ortho-thiophenolic
ester, direct oxo-ester ligation method,14 and direct peptide
synthesis from C-terminal thiol acids and N-terminal peptides
in the presence of HOBT.14i
Amide bond linkage is an ubiquitous and important motif in a
wide range of biological applications (cf. vaccines, antibodies,
enzymes and factors), polymers, chemicals, as well as natural
products.1 Its importance in organic syntheses and medici-
nal chemistry has received enormous attention from organic
chemists and encouraged the development of new synthetic
pathways to construct a C–N bond. The most popular strategy
for amide bond formation relies heavily upon the coupling
reaction of an acid chloride or an activated carboxylic acid
derivative with an amine.2 However, due to some limitations
including the liability of activated carboxylic acid derivatives
and tedious procedures, alternative strategies toward the syn-
thesis of amides have been explored for years. Transition-
metal catalyzed amidation of alkenes,3 aldoximes,4 nitriles,5
haloarenes,6 alkynes,7 alcohols using Ru-, Rh-, and Ag-based
catalytic systems,7a,8 and aldehydes using Cu, Pd, Rh, Ru, and
lanthanide complexes9 with amines, hydrative amide formation
with alkynes,10 and azides in the modified Staudinger reaction11
have been employed for amide synthesis. However, most of these
systems involve toxic solvents and expensive transition-metal
catalysts. Thus, developing new procedures for the synthesis of
amides is highly desirable. Peptide ligation methods have gained
significant attention and have been shown to be particularly im-
portant for the synthesis of large polypeptides or mini-proteins.12
In 1994, Kent and co-workers developed a major breakthrough
Scheme 1 Native chemical ligation.
The earlier research on metal aided direct conversion of thiol
esters to peptides has been reported by Tam15a and Kurosu15b
They have revealed that the thiol esters15c can be sufficiently acti-
vated by silver ion or dimethylaluminum to facilitate the peptide
synthesis. It was also found that amide bonds can be formed by
acyl fluorides with sterically hindered secondary amines in the
presence of BSA in moderate yields.15d,15e Kammermeier and co-
workers reported an example of the peptide synthesis through
a mercaptobenzothiazole thiol ester with a primary amine in
the presence of BSA in N-methylpyrrolidin-2-one (NMP) for
the synthesis of Cefdaloxime.15f However, the information for
metal-free direct conversion of thiol esters to peptides which is
independent of the cysteine is very limited and the reaction has
not been fully investigated. In this communication, we report
the metal-free direct amidation of peptidyl thiol esters with a-
amino acid esters in the presence of BSA in EtOH under mild
conditions without any intermediacy of acyl transfer (Scheme 2).
At the onset of the research, this idea was tested by mixing two
readily available substrates, N-Cbz-L-phenylalanine thiol ethyl
ester 8b and glycine ethyl ester 6a (1.0 equiv.) in the presence of
BSA (1.0 equiv.) under a variety of reaction conditions. When
aKey Laboratory of Combinatorial Biosynthesis and Drug Discovery
(Wuhan University), Ministry of Education, and Wuhan University
School of Pharmaceutical Sciences, Wuhan, 430071, P. R. China.
E-mail: xhy78@whu.edu.cn, zhouhb@whu.edu.cn; Fax: +86
027-6875-9850; Tel: +86 027-6875-2331
bState Key Laboratory of Phytochemistry and Plant Resources in West
China, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming, 650204, P. R. China
† Electronic supplementary information (ESI) available: See DOI:
10.1039/c1gc15401j
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Table 2 Optimization of various silyl compounds for the formation of
peptide 7aa
Scheme 2 Proposed approach for the peptide synthesis.
Entry
1
Silyl compounds
TMSCl
Yield (%)b
90
Table 1 Optimization and thiol esters screening for the formation of
peptide 7aa
2
3
Trace
Trace
Thiol
ester 8
Molar ratio
(8 : 6a)
Equiv. of
BSA
4
Trace
Entry
R1
Yield (%)b
5
6
NEt2TMS
49
73
1
2
3
4
5
6
tBu
Et
p-Tolyl
p-Tolyl
p-Tolyl
p-Tolyl
8a
8b
8c
8c
8c
8c
1 : 1
1 : 1
1 : 1
1 : 2
1 : 2
1 : 2
1
1
1
1
0.5
0
Trace
35
68
90c
7
8
9
80
79
72
37
Trace
a All reactions performed at 0.4–0.6 mmol in EtOH (2 mL) at 40 ◦C for
18 h. b Isolated yield based on the thiol ester. c 22% isolated yield when
the reaction was run in NMP at 40 ◦C for 96 h with (DL)-8c.
the reactions were carried out in acetonitrile, tetrahydrofuran,
toluene, dichloromethane or methanol solvents at ambient
temperature or 40 ◦C, the peptide 7a was not observed at all. A
trace amount of the product 7a was formed in NMP solvent at
40 ◦C for 18 h. However, we were pleased to find that a solution
of these two reactants in ethanol at 40 ◦C for 18 h produced the
desired product 7a in 35% yield (Table 1, entry 2).
a All reactions performed at 0.4–0.6 mmol in EtOH (2 mL) at 40 ◦C for
18 h, 8c : 6a : silyl compound = 1 : 2 : 1 (mol/mol mol-1). b Isolated yield
based on the thiol ester.
extended reaction time (72 h) is usually required for a steric
hindered N-Cbz protected thiol ester (Table 3, entry 3) and
all the reactions can be completely converted to the desired
products over times based on HPLC analysis. However, the
coupling reactions of a-substituted amino acid esters are quite
different and much slower compared to that of Gly-OEt 6a.
Most of them needed an extended reaction time (48–72 h) to
push the reaction further, except when the coupling partner is
a-unsubstituted thiol ester 9b (78% yield, 24 h, Table 3, entry
2) and the yield was decreased due to the steric effect of the
substituted group R4. For example, the reactions of N-Cbz-L-
alanine p-toluene thiol ester 9e with L-leucine methyl ester 6b
and L-tryptophan methyl ester 6c provided peptides 7g and 7h
in 70% and 72% yields respectively (Table 3, entries 6–7) which
are much lower than that of Gly-OEt 6a (97% yield). When the
other N-Cbz protected thiol ester such as 8c, 9f–9g was utilized
as the coupling partner with the a-substituted amino acid ester,
the desired peptide was obtained in a low yield, ranging from
53% to 60% (Table 3, entries 8–9, 12–13). In order to examine
the extent of racemization, an enatiomeric mixture of 7a or 7c
was synthesized in the same manner and HPLC data revealed
that no detectable racemization occurred for the synthesis of L-
7a or L-7c in the BSA-mediated coupling reaction. Significant
racemization was observed (reduced from >99% ee to 54% ee)
for the preparation of N-Ac protected thiol ester 9a according to
the previous DCC procedure. However, no further racemization
occurred in the BSA-mediated coupling reaction in EtOH. The
Having concluded from the above results that the optimal
reaction solvent system was ethanol, the effect of various thiol
esters was further evaluated using similar reaction conditions
(Table 1). Optimization studies revealed that p-toluene thiol ester
provided the best results compared to that of ethyl thiol ester
and t-butyl thiol ester (Table 1, entries 1–3). We found that the
BSA loading is critical determinant of the reaction efficiency
and increasing the BSA equiv (from 0 to 1 equiv.) can boost the
yield significantly, up to 90% yield for p-toluene thiol ester 8c
(Table 1, entries 4–6). More importantly, the yield was improved
as well with increased a-amino ester (Table 1, entry 3 vs. 4).
Various silyl compounds for the coupling were employed to
probe the effect of the additives. The data are listed in Table 2.
Pleasingly, all of the silyl compounds, regardless of TMSOTf,
BSTFA, TMSCl, proceeded smoothly in moderate to good
yields to afford the expected product 7a (49–90% yield). It is
worthy to note that BSA is the most promising coupling additive
for N-Cbz-L-phenylalanine p-toluene thiol ester 8c and glycine
ethyl ester 6a C–N bond formation.
With these optimized conditions in hand, we explored the
scope of the BSA-mediated direct coupling of a variety of
N-protected thiol esters with a-amino acid esters (Table 3).
Generally, the amidation reaction of a-unsubstituted amino
acid esters such as Gly-OEt 6a, proceeds very well to provide
the desired N-Ac or N-Cbz peptides 7a–7b, 7d–7f, 7k and 7n
in 79–97% yields (Table 3, entries 1, 3–5, 10–11 and 14). An
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Table 3 Investigation of BSA-promoted peptide bond formationa
Entry
1
Thiol ester (9)
a-Amino Acid Ester (6)
Peptide (7)
Reaction Time (h)
24
Yield (%)b
91c
6a
7b
7c
7d
2
3
6d
6a
24
72
78c
79
4
6a
6a
7e
72
80
5
6
7
7f
36
48
72
97
70
72
9e
9e
7g
7h
8
8c
8c
8c
6b
7i
48
72
18
36
53
60
90d
85
9
7j
10
11
6a
6a
7a
7k
12
13
9f
6b
6b
7l
48
72
55
57
7m
14
9g
6a
7n
36
92
a All reactions performed at 0.4–0.6 mmol in EtOH (2 mL) at 40 ◦C. b Isolated yield based on the thiol ester. c 9a (54% ee); 7b (54% ee); 7c (>99%
ee); HPLC conditions: Lux 3 mm, Cellulose-2, 4.6 mm ¥ 250 mm, n-hexane/2-propanol = 80 : 20, 206 nm, flow rate = 1.0 mL min-1. d 7a (>99% ee),
Chiral Pak AD-H, 5 mm, 4.6 mm ¥ 250 mm, n-hexane/2-propanol = 70 : 30, 230 nm, flow rate = 0.65 mL min-1.
enantiomeric excess of the corresponding peptide 7b is identical
to that of the thiol ester 9a.
Remarkably, the further screening revealed that our direct
peptide synthesis procedure tolerates a wide range of functional
groups. The amidation was compatible with a variety of
amines containing hydroxyl group or thiol group. The reactions
proceeded smoothly with or without a cysteine –SH residue
and provided the desired products 7o–7q in 60–70% yields
(Scheme 3). Interestingly, when the N-Cbz-L-aspartic bis-p-
toluene thiol ester 11 was applied to the amidation reaction, the
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5 (a) S.-I. Murahashi, T. Naota and E. Saito, J. Am. Chem. Soc., 1986,
108, 7846; (b) C. J. Cobley, M. van den Heuvel, A. Abbadi and J. G.
de Vries, Tetrahedron Lett., 2000, 41, 2467.
6 (a) Y.-S. Lin and H. Alper, Angew. Chem., Int. Ed., 2001, 40, 779;
(b) Y. Uozumi, T. Arii and T. Watanabe, J. Org. Chem., 2001, 66,
5272; (c) P. Nanayakkara and H. Alper, Chem. Commun., 2003, 24,
2384.
7 (a) B. E. Ali and J. Tijani, Appl. Organomet. Chem., 2003, 17, 921;
(b) D. J. Knapton and T. Y. Meyer, Org. Lett., 2004, 6, 687; (c) Y.
Uenoyama, T. Fukuyama, O. Nobuta, H. Matsubara and I. Ryu,
Angew. Chem., Int. Ed., 2005, 44, 1075.
8 (a) T. Naota and S.-I. Murahashi, Synlett., 1991, 10, 693; (b) N. A.
Owston, A. J. Parker and J. M. J. Williams, Org. Lett., 2007, 9, 73;
(c) C. Gunanathan, Y. Ben-David and D. Milstein, Science, 2007,
317, 790; (d) L. U. Nørdstrom, H. Vogt and R. Madsen, J. Am.
Chem. Soc., 2008, 130, 17672; (e) A. J. A. Watson, A. C. Maxwell
and J. M. J. Williams, Org. Lett., 2009, 11, 2667; (f) S. C. Ghosh,
S. Muthaiah, Y. Zhang, X. Xu and S. H. Hong, Adv. Synth. Catal.,
2009, 351, 2643; (g) Y. Zhang, C. Chen, S. C. Ghosh, Y. Li and S. H.
Hong, Organometallics, 2010, 29, 1374; (h) K. Fujita, Y. Takahashi,
M. Owaki, K. Yamamoto and R. Yamaguchi, Org. Lett., 2004, 6,
2785; (i) T. Zweifel, J. V. Naubron and H. Grotzmacher, Angew.
Chem., Int. Ed., 2009, 48, 559; (j) K. Shimizu, K. Ohshima and A.
Satsuma, Chem.–Eur. J., 2009, 15, 9977; (k) G. E. Dobereiner and R.
H. Crabtree, Chem. Rev., 2010, 110, 681.
Scheme 3 Direct peptide synthesis from thiol esters with or without a
cysteine –SH residue.
di-amidation reaction occurred smoothly (11 : 6a : BSA = 1 : 4 : 2
mol : mol : mol) in high yield (Scheme 4). Though the mechanism
of the reaction is clear yet, we speculate that BSA could react
with amines to form N-silylamine intermediates which would
facilitate amide bond formation.15d,15e
9 (a) A. Tillack, I. Rudloff and M. Beller, Eur. J. Org. Chem., 2001, 3,
523; (b) W.-J. Yoo and C.-J. Li, J. Am. Chem. Soc., 2006, 128, 13064;
(c) Y. Suto, N. Yamagiwa and Y. Torisawa, Tetrahedron Lett., 2008,
49, 5732; (d) Y. Tamaru, Y. Yamada and Z. Yoshida, Synthesis, 1983,
6, 474; (e) W.-K. Chan, C.-M. Ho, M.-K. Wong and C.-M. Che, J.
Am. Chem. Soc., 2006, 128, 14796; (f) S. Seo and T. J. Marks, Org.
Lett., 2008, 10, 317; (g) C. Qian, X. Zhang, J. Li, F. Xu, Y. Zhang
and Q. Shen, Organometallics, 2009, 28, 3856; (h) J. M. Li, F. Xu, Y.
Zhang and Q. Shen, J. Org. Chem., 2009, 74, 2575; (i) J. Wang, J. Li,
F. Xu and Q. Shen, Adv. Synth. Catal., 2009, 351, 1363.
Scheme 4 Di-amidation reaction.
In summary, we have developed a new, convenient route
for the synthesis of optically pure N-protected peptides using
a metal-free direct amidation of peptidyl thiol esters with a-
amino acid esters mediated by BSA under mild conditions. This
considerable promising method will be valuable for building a
range of challenging peptide bond which is independent of the
cysteine based NCL strategies. Further investigations regarding
the mechanistic study, reaction scope and their application in
pharmaceutical level discovery research will be reported in due
course.
We are grateful to the National Mega Project on Major
Drug Development (2009ZX09301-014-1), the NSFC (Nos.
20872116, 91017005), and the Fund of State Key Laboratory
of Phytochemistry and Plant Resources in West China (P2010-
KF10).
10 (a) S. Cho, E. Yoo, I. Bae and S. Chang, J. Am. Chem. Soc., 2005,
127, 16046; (b) M. P. Cassidy, J. Raushel and V. V. Fokin, Angew.
Chem., Int. Ed., 2006, 45, 1.
11 (a) E. Saxon and C. R. Bertozzi, Science, 2000, 287, 2007; (b) B. L.
Nilsson, L. L. Kiessling and R. T. Raines, Org. Lett., 2000, 2, 1939;
(c) F. Damkaci and P. DeShong, J. Am. Chem. Soc., 2003, 125, 4408.
12 (a) L. Yang and P. E. Dawson, J. Am. Chem. Soc., 2001, 123, 526;
(b) D. Crich and A. Banerjee, J. Am. Chem. Soc., 2007, 129, 10064;
(c) C. Haase, H. Rohde and O. Seitz, Angew. Chem., Int. Ed., 2008, 47,
6807; (d) C. P. R. Hackenberger and D. Schwarzer, Angew. Chem.,
Int. Ed., 2008, 47, 10030; (e) N. Ollivier, J. Dheur, R. Mhidia, A.
Blanpain and O. Melnyk, Org. Lett., 2010, 12, 5238.
13 (a) P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. H. Kent,
Science, 1994, 266, 776; (b) P. E. Dawson, M. J. Churchill, M. R.
Ghadiri and S. B. H. Kent, J. Am. Chem. Soc., 1997, 119, 4325.
14 (a) Y. Yuan, J. Zhu, X. Li, X. Wu and S. J. Danishefsky, Tetrahedron
Lett., 2009, 50, 2329; (b) Y. Rao, X. Li and S. J. Danishefsky, J. Am.
Chem. Soc., 2009, 131, 12924; (c) J. Chen, P. Wang, J. Zhu, Q. Wan
and S. J. Danishefsky, Tetrahedron, 2010, 66, 2277; (d) J. Chen, Q.
Wan, Y. Yuan, J. Zhu and S. J. Danishefsky, Angew. Chem., Int. Ed.,
2008, 47, 8521; (e) Q. Wan and S. J. Danishefsky, Angew. Chem., Int.
Ed., 2007, 46, 9248; (f) J. D. Warren, J. S. Miller, S. J. Keding and
S. J. Danishefsky, J. Am. Chem. Soc., 2004, 126, 6576; (g) G. Chen,
J. D. Warren, J. Chen, B. Wu, Q. Wan and S. J. Danishefsky, J. Am.
Chem. Soc., 2006, 128, 7460; (h) Q. Wan, J. Chen, Y. Yuan and S. J.
Danishefsky, J. Am. Chem. Soc., 2008, 130, 15814; (i) P. Wang and
S. J. Danishefsky, J. Am. Chem. Soc., 2010, 132, 17045.
Notes and references
1 (a) J. M. Humphrey and A. R. Chamberlin, Chem. Rev., 1997, 97,
2243; (b) T. Cupido, J. Tulla-Puche, J. Spengler and F. Albericio, Curr.
Opin. Drug Discovery Dev., 2007, 10, 768; (c) J. W. Bode, Curr. Opin.
Drug Discovery Dev., 2006, 9, 765; (d) N. Carrillo, E. A. Davalos,
J. A. Russak and J. W. Bode, J. Am. Chem. Soc., 2006, 128, 1452;
(e) B. Shen, D. M. Makley and J. N. Johnston, Nature, 2010, 465,
1027.
2 (a) A. L. J. Beckwith and J. Zabicky, The Chemistry of AmidesWiley,
New York, 1970; (b) R. C. Larock, Comprehensive Organic Transfor-
mationVCH, New York, 1999; (c) K. V. N. S. Srinivas and B. Das, J.
Org. Chem., 2003, 68, 1165.
3 M. Beller, B. Cornils and C. D. Frohning, J. Mol. Catal. A: Chem.,
1995, 104, 17.
4 (a) S. Park, Y. Choi, H. Han, S. H. Yang and S. Chang, Chem.
Commun., 2003, 24, 1936; (b) C. L. Allen, S. Davulcu and J. M. J.
Williams, Org. Lett., 2010, 12, 5096.
15 (a) L. Zhang and J. P. Tam, Tetrahedron Lett., 1997, 38, 4375;
(b) M. Kurosu, Tetrahedron Lett., 2000, 41, 591; (c) H. Tokuyama, S.
Yokoshima, T. Yamashita, S.-C. Lin, L. Li and T. Fukuyama, J. Braz.
Chem. Soc., 1998, 9, 381–387; (d) H. Wenschuh, M. Beyenmann, R.
Winter and M. Bienert, Tetrahedron Lett., 1996, 37, 5483; (e) H. Kim,
B. Gardner and M. Kahn, Tetrahedron Lett., 1995, 36, 6013; (f) B.
Kammermeier, G. Beck, J. Reitz, C. Sommer, T. Wollmann and H.
Jendralla, Org. Process Res. Dev., 1997, 1, 121.
2726 | Green Chem., 2011, 13, 2723–2726
This journal is
The Royal Society of Chemistry 2011
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