Fmoc-SPPS chemistry compatible approach for the generation of (glyco)peptide aryl thioesters

Article abstract of DOI:10.1016/j.tetlet.2011.03.064

A new approach is described for the general Fmoc-based solid-phase synthesis of (glyco)peptide aryl thioesters. A peptide alkyl oxoester obtained by standard Fmoc-based chain elongation undergoes an O-to-S acyl shift, and is followed by alkyl thioester exchanges with a large excess of aryl thiol, affording the corresponding peptide aryl thioester. The newly developed methodology is useful for the chemical synthesis of post-translationally modified proteins because of its compatibility with standard Fmoc-SPPS conditions. In addition, the peptide aryl thioesters are essential intermediates for chemical synthesis of proteins by kinetically controlled convergent strategy.

Full text of DOI:10.1016/j.tetlet.2011.03.064

Tetrahedron Letters 52 (2011) 2655–2660
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Fmoc-SPPS chemistry compatible approach for the generation
of (glyco)peptide aryl thioesters
Ji-Shen Zheng a,b, Wei-Xian Xi b, Feng-Liang Wang a, Juan Li b, , Qing-Xiang Guo a,
⇑
⇑
a Department of Chemistry, University of Science and Technology of China, Hefei 230026, China
b Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new approach is described for the general Fmoc-based solid-phase synthesis of (glyco)peptide aryl thi-
oesters. A peptide alkyl oxoester obtained by standard Fmoc-based chain elongation undergoes an O-to-S
acyl shift, and is followed by alkyl thioester exchanges with a large excess of aryl thiol, affording the cor-
responding peptide aryl thioester. The newly developed methodology is useful for the chemical synthesis
of post-translationally modified proteins because of its compatibility with standard Fmoc-SPPS condi-
tions. In addition, the peptide aryl thioesters are essential intermediates for chemical synthesis of pro-
teins by kinetically controlled convergent strategy.
Received 28 January 2011
Revised 2 March 2011
Accepted 14 March 2011
Available online 21 March 2011
Ó 2011 Elsevier Ltd. All rights reserved.
C-Terminal peptide thioesters have been shown to be crucial
intermediates for the synthesis and modification of proteins
through native chemical ligation (NCL)1 or the thioester method.2
These methods have led to the practical chemical syntheses of a
large variety of small proteins (ꢀ100 amino acids).3,4 Considering
that a typical protein domain in nature consists of 200–300 amino
acids, the synthesis of average sized proteins is still one significant
challenge.5 Recently, an efficient strategy for the synthesis of large
protein molecules has been developed as the kinetically controlled
convergent ligation of multiple peptide fragments. This method
utilized the large reactivity difference between the peptide aryl
thioesters and alkyl thioesters. The peptide aryl thioesters would
react with a bifunctional cysteinyl peptide alkyl thioesters and
the alkyl thioesters functional group should remain unreacted
(Scheme 1).6 In addition, the peptide aryl thioesters can be used
in auxiliary mediated ligation7 and Ag+-free thioester methods.8
Therefore, the synthesis of peptide aryl thioesters has attracted
much attention.
methodology for peptide aryl thioesters synthesis is rarely
reported.8,16 This may be attributed to the instablity of the thioes-
ters’ functionality to the nucleophile reagents such as piperidine re-
quired for repeated Fmoc removal. Recently, the synthesis of
peptide thioesters through an intramolecular O-to-S17 or N-to-S18
acyl shift has been reported. These approaches allowed the success-
ful synthesis of cyclic peptide thioesters, phosphorylated and
acetylated histone H2B, as well as crambin.19 Herein, we report a
novel method for the generation of a peptide aryl thioester. This
method is based on peptide alkyl oxoesters which is compatible
with the mild Fmoc-chemistry for the peptide assembly on resin.
The peptide aryl thioester is obtained by in situ O-to-S acyl shift,
transthioesterfication with a large excess of aryl thiol, and the final
deprotection by TFA (Scheme 2b).
Inspired by Danishefsky’s aryl O-to-S acyl shift strategy to gen-
erate peptide aryl thioesters (Scheme 2a),17 we thought that this
kind of structure would be employed for the synthesis of peptide
aryl thioesters by Fmoc-based SPPS, and then we tested the stabil-
ity of phenolic ester 1 in Fmoc removal condition (piperidine/
DMF = 1:4). However, the compound is rapidly decomposed within
5 min. Subsequently, we focused on Botti’s peptide alkyl oxoester
218,19c which would be a better candidate than aryl oxoesters
because of their pKa’s difference.
Currently, aryl thioesters are prepared usually by tert-butoxy-
carbonyl solid-phase peptide synthesis (Boc-SPPS).6,9,10 Unfortu-
nately, the Boc protocol requires the use of harsh acidic reagents
(such as HF which needs special precaution and apparatus for its
handling) that are not compatible with synthesis of phospho-11,12
and glyco-peptides.13–15
The mild Fmoc-SPPS (Fmoc = 9-fluorenylmethoxycarbonyl), on
the other hand, is a preferable strategy for the synthesis of phos-
pho- and glyco-peptide thioesters. However, the Fmoc-based
StBu
S
H
N
O
Bz-HN
O
O
Bz-HN
O
S
SEt
O
⇑
Corresponding authors. Tel.: +86 10 62796753; fax: +86 10 62771149 (J.L.).
E-mail address: ljuan@mail.tsinghua.edu.cn (J. Li).
1
2
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.03.064

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J.-S. Zheng et al. / Tetrahedron Letters 52 (2011) 2655–2660
C-to-N
O
N-to-C
O
O
S
O
O
Thz Pep 4
R
Pep 5
Pep 6
Pep 1
Pep 2
Pep 3
Thz
Cys
Cys
R
Cys
SAr
SAr
SAr
S
KCL
1) KCL
2) Thz to Cys
1) NCL
2) Thz to Cys
SAr:
S
KCL
NCL
TTCCPSIVARSNFNACRLPGTPEALCATYTGCIIIPGATCPGDYAN
folding & disulfide formation
Crambin[V15A]
Scheme 1. Fully convergent synthesis of the model protein crambin via kinetically controlled ligation (KCL).6
a. Phenolic ester mediated NCL
b. Peptide alkyl oxoester mediated
synthesis of peptide aryl thioesters
by Danishefsky et.al in 2006
SEt
StBu
S
S
O
O
NHBoc
H
N
Peptide 1
O
O
Peptide
OtBu
PH 7-8
O
excess R-SH
Reduce Disulfide
HS
O
SH
H
N
Peptide 1
O
O
NHBoc
Peptide
OtBu
O-to-S
Acyl shift
HO
O
O
O
O-to-S Acyl shift
O
Peptide 1
NCL
S
HO
O
O
NH
NHBoc
Peptide
OtBu
Peptide 2
H2N
HS
NH
S
1) exchange with ArSH
2) Deprotection by TFA
O
H
Peptide 2
Peptide 1
N
O
NH
SAr
O
Peptide
HS
Scheme 2. The strategy for the synthesis of peptide aryl thioesters.
(entry 8). Besides, using aprotic solvent such as methanol
instead of water, exchange also gave a high yield (80%, Table 2,
entry 1).
We tested the model alkyl oxoester 2 for the aryl thioester. It
was reported that the hydrolysis of the oxoester structure 2
was a major completing reaction during the native chemical liga-
tion through in situ O-to-S acyl shift. To reduce the amount of
hydrolyzed material in aryl thiol exchange reaction, we exploited
a series of conditions (Table 1). We thought that under anhydrous
reaction conditions, the hydrolysis should be suppressed. Unfor-
tunately, the yield for peptide aryl thioesters is low (entry 1). It
is found that different organic solvent ratios resulted in the
various final yields of this exchange reaction. 40–60% NMP
(N-methyl-2-pyrrolidone)/H2O are the optimal solvent condition
(entries 5–7) for aryl thioesters. If NMP concentration is less than
30%, the ester hydrolysis becomes a serious competing reaction
We next examined the synthesis of alkyl oxoesters with Gly,
Ala, Phe, Leu and Val as the C-terminal amino acids to test the gen-
eral applicability of this method. The result for the preparation of
aryl thioesters is shown in Table 2. The data indicate that the trans-
thioesterification yield is highly sensitive to the side chain steric
hindrance and the new method is applicable to the synthesis of
peptide aryl thioesters with acceptable yields (entries 1, 3 and 5).
However, the yield is low for more sterically hindered amino acids
(entries 6 and 7). In addition, when H2O is replaced with tertiary
butyl alcohol (tBuOH) the hydrolysis of alkyl oxyesters could be

J.-S. Zheng et al. / Tetrahedron Letters 52 (2011) 2655–2660
2657
Table 1
Preparation of aryl thioesters using alkyl oxoesters in different NMP aqueous solution
StBu
S
50mM TCEP•HCl,
100mM p-toluenethiol
O
O
Bz-HN
H
S
1% DIPEA in NMP/H2O
Bz-HN
N
O
O
2.0mM
SH
Thiol exchange
Reduction
SH
Bz-HN
S
O
O
H
O
H
N
Bz-HN
N
O-to-S
HO
Acylshift
O
O
Entry
NMP aq (%)
Yielda (%)
1
2
3
4
5
6
7
8
100
99
90
70
60
50
40
30
33
42
54
54
83
74
79
36
a
All the yields were measured by HPLC.
Table 2
Preparation of peptide aryl thioesters at different amino acid residues of alkyl oxoesters
StBu
50mM TCEP•HCl,
S
O
S
O
100mM p-toluenethiol
1% DIPEA
H
Bz-Gly-HN
Bz-Gly-HN
N
O
R
R
O
Entry
R
Solvent
Yieldsa (%)
1
2
3
4
5
6
7
H
CH3
CH3
CH3
CH2Ph
i-Bu
i-Pr
NMP/MeOH/H2O (50:45:5)
80
26
40
70
36
NMP/MeOH/H2O (50:45:5)
NMP/tBuOH/H2O (50:45:5)
tBuOH/H2O (95:5)
NMP/tBuOH/H2O (50:45:5)
NMP/tBuOH/H2O (50:45:5)
NMP/tBuOH/H2O (50:45:5)
16
<10
a
All the yields were measured by HPLC.
reduced, and the aryl thioester obtained in 70% yield for Ala oxyes-
ter (entry 4).
xyl group to a thiol group is unfavorable in those conditions. When
the pH is increased, the thioester intermediate decreases. Also the
ester 3 will be hydrolyzed within 1 h at high pH (pH 8, Fig. 1C).
Therefore, we are in favor of the mechanism that the alkyl thioester
intermediates are obtained through in situ O-to-S acyl shift. Then
they proceed through the final transthioesterification step under
a large excess of aryl thiol to generate the target peptide aryl
thioesters.
Using this method, we synthesized two model glycopeptide thi-
oesters (Scheme 3). The glycopeptide alkyl oxoester was prepared
according to the literature:19c couplings of amino acids with
HBTU/HOBt in DMF and deprotections using 20% piperidine/DMF.
After the peptide assembly, the final peptide resin was treated with
50 mM TCEP.HCl, 100 mM p-toluenethiol, and 1% N,N-diisopropyl-
ethylamine (DIPEA) in tBuOH/H2O (95:5, v/v) for 10 h at rt for
completion of the transthioesterification with p-toluenethiol. After
removal of solvents by lyophilization, the crude peptide was
treated with Reagent K (TFA/phenol/water/thioanosole/EDT = 82.5:
To study the mechanism of the ‘O-to-S’ acyl shift reaction and
the major intermediate of this equilibrium reaction, we used the
NMR method. Because of the different chemical shift of carbonyl
carbon of the thioester (ꢀ200 ppm) and oxoester (ꢀ172 ppm)
intermediates, the ‘O-to-S’ acyl shift reaction could be monitored
by 13C NMR analysis with a 13C-labeled substrate. Thus, 13C-labeled
carbonyl carbon of ester bond was incorporated into oxoester sub-
strate 3.
Upon the addition of tris(2-carboxyethyl)-phosphine hydro-
chloride (TCEP.HCl, 20 equiv) to 3 in NMP/water (40:60, v/v), disul-
fide reduction was completed rapidly, and the resulting 13C NMR
spectrum was obtained at room temperature at different pH
(Fig. 1). According to the 13C NMR spectra of 3, it is clear that the
oxoester intermediate is thermodynamically predominant and
the thioester is the minor intermediate under acidic conditions
(pH <7, Fig. 1A, B). In other words, acyl shift process from a hydro-

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J.-S. Zheng et al. / Tetrahedron Letters 52 (2011) 2655–2660
Figure 1. 13C NMR spectra of ‘O-to-S’ shift (300 MHz).
5:5:5:2.5 (v/w/v/v/v)) for 2–3 h at rt to obtain the unprotected
crude glycopeptide aryl thioesters. The result of the synthesis is
shown in Table 3 and the MALDI–TOF of the two crude peptides
is shown in Figure 2. The mass data show a good purity of the
desired glycopeptide aryl thioester. It is easy to purify the peptide
by RP-HPLC.
In conclusion, in this Letter we present a novel method
for the preparation of (glyco)peptide aryl thioesters. The strat-
egy relies on the stability of the ester bond toward nucleo-
philes, the in situ O-to-S acyl transfer for peptide alkyl
thioesters, and the final transthioesterification for the target
peptide aryl thioesters. The mechanism is demonstrated using
13C NMR tool. This new method is useful for the chemical
synthesis of post-translationally modified proteins because of
its compatibility with standard Fmoc-SPPS conditions. The
preparation of peptide aryl thioester for chemical synthesis of
protein by kinetically controlled convergent strategy is in
progress.

J.-S. Zheng et al. / Tetrahedron Letters 52 (2011) 2655–2660
2659
StBu
S
StBu
StBu
S
S
HBTU, HOBt
TBAF
+ H2N
OH
NH
NH
O
TBSO
TBSO
ArSH:
HO
O
O
Fmoc-AA-OH
HBTU, HOBt
Rink Amide Resin
SH
StBu
S
StBu
S
Glc
O
O
Fmoc-SPPS
NH
O
NH
peptide
BocHN
Fmoc-AA
O
O
O
OtBu
TCEP•HCl, ArSH, DIPEA
Glc
Glc
peptide
Aryl thioesters
O
O
TFA
peptide
BocHN
SAr
SAr
OtBu
Scheme 3. Fmoc-based synthesis of glycopeptide aryl thioesters.
[M+H]
H-IIe-Ser-Thr[(Glc(4Ac))]-Gly-
Leu-Tyr-Lys-Gly-Ala-SAr
H-Leu-Ala-Thr[Glc(4Ac)]-Gly-
[M+H]
Ser-Lys-Ala-Thr-IIe-Gly-SAr 1354.7
1345.63
Peptide aryl thioester
Peptide aryl thioester
H-IIe-Ser-Thr[(Glc(4Ac))]-Gly-
Leu-Tyr-Lys-Gly-Ala-OH
Hydrolysis product
[M+Na]
1376.7
[M+H]
[M+K]
1239.61
1392.7
980
1100
1220
1340
1460
1580
900
1050
1200
1350
1500
1650
m/z
m/z
(a)
(b)
Figure 2. MALDI–TOF of two crude glycopeptide aryl thioesters (a, b) exchanged from the resin without any purification.
3. (a) Dawson, P. E.; Kent, S. B. H. Annu. Rev. Biochem. 2000, 69, 923–960; (b) Kent,
S. B. H. Chem. Soc. Rev. 2009, 38, 338–351.
Table 3
Preparation of glycopeptide aryl thioesters
4. (a) Aimoto, S. Curr. Org. Chem. 2001, 5, 45–87; (b) Casi, G.; Hilvert, D. Curr. Opin.
Struct. Biol. 2003, 13, 589–594; (c) Nilsson, B. L.; Soellner, M. B.; Raines, R. T.
Annu. Rev. Biophys. Biomol. Struct. 2005, 34, 91–118; (d) Li, J.; Zheng, J. S.; Shen,
F.; Fang, G. M.; Guo, Q. X.; Liu, L. Prog. Chem. 2007, 19, 1866–1882; (e) Dirksen,
A.; Dawson, P. E. Curr. Opin. Chem. Biol. 2008, 12, 760–766; (f) Haase, C.; Seitz, O.
Angew. Chem., Int. Ed. 2008, 47, 1553–1556; (g) Hackenberger, C. P. R.;
Schwarzer, D. Angew. Chem., Int. Ed. 2008, 47, 10030–10074.
5. Lee, J. Y.; Bang, D. Biopolymers 2010, 94, 441–447.
Sequence
m/z
Yieldsa
(%)
H-Leu-Ala-Thr-[b-
Gly-SArb (aryl thioester a)
H-IIe-Ser-Thr[b- -Glc(OAc)4]-Gly-Leu-Tyr-Lys-Gly-Ala-
SArb (aryl thioester b)
D-Glc(OAc)4]-Gly-Ser-Lys-Ala-Thr-IIe-
1354.7 25
1345.6 19
D
6. Bang, D.; Pentelute, B. L.; Kent, S. B. H. Angew. Chem., Int. Ed. 2006, 45, 3985–
3988.
a
Isolated yield based on the amino groups in the initial resin.
SAr denotes SPhCH3.
b
7. Li, J.; Cui, H. K.; Liu, L. Tetrahedron Lett. 2010, 51, 1793–1796.
8. (a) Chen, G.; Wan, Q.; Tan, Z.; Kan, C.; Hua, Z.; Ranganathan, K.; Danishefsky, S.
J. Angew. Chem., Int. Ed. 2007, 46, 7383–7387; (b) Hojo, H.; Murasawa, Y.;
Katayama, H.; Ohira, T.; Nakaharaa, Y.; Nakahara, Y. Org. Biomol. Chem. 2008, 6,
1808–1813.
9. Dawson, P. E.; Churchill, M. J.; Ghadiri, M. R.; Kent, S. B. H. J. Am. Chem. Soc.
1997, 119, 4325–4329.
Acknowledgment
10. (a) Bang, D.; Pentelute, B. L.; Gates, Z. P.; Kent, S. B. Org. Lett. 2006, 8, 1049–
1052; (b) Fang, G. M.; Cui, H. K.; Zheng, J. S.; Liu, L. Chem. Biol. Chem. 2010, 11,
1061–1065.
11. Muir, T. W.; Sondhi, D.; Cole, P. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6705–
6710.
The study was supported by the NSFC grants (Nos. 21002056
and 20932006).
References and notes
12. Huse, M.; Holford, M. N.; Kuriyan, J.; Muir, T. W. J. Am. Chem. Soc. 2000, 122,
8337–8338.
13. Shin, Y.; Winans, K. A.; Backes, B. J.; Kent, S. B. H.; Ellman, J. A.; Bertozzi, C. R. J.
Am. Chem. Soc. 1999, 121, 11684–11689.
14. Tolbert, T. J.; Wong, C. H. J. Am. Chem. Soc. 2000, 122, 5421–5428.
1. Dawson, P. E.; Muir, T. W.; Clarklewis, I.; Kent, S. B. H. Science 1994, 266, 776–
779.
2. Hojo, H.; Aimoto, S. Bull. Chem. Soc. Jpn. 1991, 64, 111–117.

2660
J.-S. Zheng et al. / Tetrahedron Letters 52 (2011) 2655–2660
15. Miller, J. S.; Dudkin, V. Y.; Lyon, G. J.; Muir, T. W.; Danishefsky, S. J. Angew.
Chem., Int. Ed. 2003, 42, 431–434.
(e) Nakamura, K.; Mori, H.; Kawakami, T.; Hojo, H.; Nakahara, Y.; Aimoto, S.
Int. J. Pept. Res. Ther. 2007, 13, 191–202; (f) Ozawa, C.; Hojo, H.; Nakahara, Y.;
Katayama, H.; Nabeshima, K.; Akahane, T.; Nakahara, Y. Tetrahedron 2007, 63,
9685–9690; (g) Ozawa, C.; Katayama, H.; Hojo, H.; Nakahara, Y.; Nakahara, Y.
Org. Lett. 2008, 10, 3531–3533; (h) Tsuda, S.; Shigenaga, A.; Bando, K.; Otaka, A.
Org. Lett. 2009, 11, 823–826; (i) Kawakami, T.; Aimoto, S. Tetrahedron 2009, 65,
3871–3877.
16. Blanco-Canosa, J. B.; Dawson, P. E. Angew. Chem., Int. Ed. 2008, 47, 6851–6855.
17. (a) Warren, J. D.; Miller, J. S.; Keding, S. J.; Danishefsky, S. J. J. Am. Chem. Soc.
2004, 126, 6576–6578; (b) Botti, P.; Manganiello, S.; Gaertner, H. Org. Lett.
2004, 6, 4861–4864; (c) Chen, G.; Warren, J. D.; Chen, J. H.; Wu, B.; Wan, Q.;
Danishefsky, S. J. J. Am. Chem. Soc. 2006, 128, 7460–7462.
18. (a) Ohta, Y.; Itoh, S.; Shigenaga, A.; Shintaku, S.; Fujii, N.; Otaka, A. Org. Lett.
2006, 8, 467–470; (b) Nakamura, K.; Sumida, M.; Kawakami, T.; Vorherr, T.;
Aimoto, S. Bull. Chem. Soc. Jpn. 2006, 79, 1773–1780; (c) Nagaike, F.; Onuma, Y.;
Kanazawa, C.; Hojo, H.; Ueki, A.; Nakahara, Y. Org. Lett. 2006, 8, 4465–4468;
(d) Kawakami, T.; Aimoto, S. Tetrahedron Lett. 2007, 48, 1903–1905;
19. (a) George, E. A.; Novick, R. P.; Muir, T. W. J. Am. Chem. Soc. 2008, 130, 4914–
4924; (b) Chiang, K. P.; Jensen, M. S.; McGinty, R. K.; Muir, T. W. Chem. Biol.
Chem. 2009, 10, 2182–2187; (c) Zheng, J. S.; Cui, H. K.; Fang, G. M.; Xi, W. X.;
Liu, L. Chem. Biol. Chem. 2010, 11, 511–515.