Tandem thiol switch synthesis of peptide thioesters via N-S acyl shift on thiazolidine

Article abstract of DOI:10.1021/ol202047q

An efficient "thiol switch" approach for the synthesis of peptide thioesters via an acid-catalyzed N-S acyl shift and a thioester exchange reaction in tandem with concurrent removal of protecting groups is described. This method employs novel 2-(thiomethyl)thiazolidine (TMT)-anchored resins and is fully compatible with Fmoc chemistry.

Full text of DOI:10.1021/ol202047q

ORGANIC
LETTERS
2011
Vol. 13, No. 19
5176–5179
Tandem Thiol Switch Synthesis of Peptide
Thioesters via NꢀS Acyl Shift on
Thiazolidine
Rohit K. Sharma and James P. Tam*
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive,
Singapore 637551
jptam@ntu.edu.sg
Received July 28, 2011
ABSTRACT
An efficient “thiol switch” approach for the synthesis of peptide thioesters via an acid-catalyzed NꢀS acyl shift and a thioester exchange reaction
in tandem with concurrent removal of protecting groups is described. This method employs novel 2-(thiomethyl)thiazolidine (TMT)-anchored
resins and is fully compatible with Fmoc chemistry.
Peptide thioesters are useful building blocks for protein
modification and immobilization strategies in peptide and
protein arrays.1ꢀ3 Moreover, theyare required for segment
coupling reactions such as chemical ligation,3 Staudinger
ligation,4 and Agþ-mediated thioester ligation.5 While
peptide thioesters are compatible with the Boc/Bzl
chemistry during solid-phase peptide synthesis (SPPS),2
they are incompatible with the Fmoc-based methods
because of their susceptibility to piperidine deprotection1
steps. This has led to early attempts at developing Fmoc-
compatible methods for preparing peptide thioesters based
on optimized Fmoc deprotection cocktails,6 activation of
protected peptides in solution,7 and thiol-labile safety-
catch linkers.8
Recently, there has been increasing interest in methods
involving acyl-transfer reactions for the Fmoc-compatible
synthesis of peptide thioesters.9ꢀ17 In principle, preparing
an ester or a thioester from an amide is the reverse process
of a ligation scheme. In practice, such a reversal is generally
thermodynamically disfavored and would require activation,
(1) (a) Wieland, T.; Bokelmann, E.; Bauer, L.; Lang, H. U.; Lau, H.
Liebigs Ann. Chem. 1953, 583, 129–149. (b) Liu, C. F.; Tam, J. P. J. Am.
Chem. Soc. 1994, 116, 4149–4153. (c) Dawson, P. E.; Muir, T. W.; Clark-
Lewis, I.; Kent, S. B. Science 1994, 266, 776–779. (d) Tam, J. P.; Lu,
Y.-A.; Liu, C.-F. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 12485–12489.
(e) Tam, J. P.; Lu, Y.-A.; Yang, J.-L.; Chiu, K.-W. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 8913–8918. (f) Tam, J. P.; Yu, Q.; Miao, Z. Biopolymers
1999, 51, 311–332. (g) Mende, F.; Seitz, O. Angew. Chem., Int. Ed. 2011,
50, 1232–1240.
(2) Hojo, H.; Aimoto, S. Bull. Chem. Soc. Jpn. 1991, 64, 111–117.
(3) (a) Haase, C.; Seitz, O. Angew. Chem., Int. Ed. 2008, 47, 1553–
1556. (b) Rohde, H.; Seitz, O. Biopolymers 2010, 94, 551–559. (c) Blanco-
Canosa, J. B.; Dawson, P. E. Angew. Chem., Int. Ed. 2008, 47, 6851–
6855. (d) Ficht, S.; Payne, R. J.; Guy, R. T.; Wong, C. Chem.;Eur. J.
2008, 14, 3620–3629.
(6) Li, X. Q.; Kawakmi, T.; Aimoto, S. Tetrahedron Lett. 1998, 39,
8669–8672.
(7) Futaki, S.; Sogawa, K.; Maruyama, J.; Asahara, T.; Niwa, M.
Tetrahedron Lett. 1997, 38, 6237–6240.
(8) Backes, B. J.; Virgilio, A. A.; Ellman, J. A. J. Am. Chem. Soc.
1996, 118, 3055–3056.
(9) (a) Hojo, H.; Onuma, Y.; Akimoto, Y.; Nakahara, Y.; Nakahara,
Y. Tetrahedron Lett. 2007, 48, 25–28. (b) Hojo, H.; Murasawa, Y.;
Katayama, H.; Ohira, T.; Nakahara, Y.; Nakahara, Y. Org. Biomol.
Chem. 2008, 6, 1808–1813.
(10) Nagaike, F.; Onuma, Y.; Kanazawa, C.; Hojo, H.; Ueki, A.;
Nakahara, Y. Org. Lett. 2006, 8, 4465–4468.
(11) Ohta, Y.; Itoh, S.; Shigenaga, A.; Shintaku, S.; Fujii, N.; Otaka,
A. Org. Lett. 2006, 8, 467–470.
(12) Tsuda, S.; Shigenaga, A.; Bando, K.; Otaka, A. Org. Lett. 2009,
11, 823–826.
(4) (a) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2000,
2, 1939–1941. (b) Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. Org. Lett.
2000, 2, 2141–2143.
(5) (a) Blake, J. Int. J. Pept. Protein Res. 1981, 17, 273–274. (b)
Aimoto, S. Biopolymers 1999, 51, 247–265. (c) Chen, G.; Wan, Q.; Tan,
Z.; Kan, C.; Hua, Z.; Ranganathan, K.; Danishefsky, S. J. Angew.
Chem., Int. Ed. 2007, 46, 7383–7387.
(13) Kawakami, T.; Sumida, M.; Nakamura, K.; Vorherr, T.;
Aimoto, S. Tetrahedron Lett. 2005, 46, 8805–8807.
(14) (a) Ollivier, N.; Behr, J.-B.; El-Mahdi, O.; Blanpain, A.; Melnyk,
O. Org. Lett. 2005, 7, 2647–2650. (b) Ollivier, N.; Dheur, J.; Mhidia, R.;
Blanpain, A.; Melnyk, O. Org. Lett. 2010, 12, 5238–5241.
r
10.1021/ol202047q
Published on Web 09/08/2011
2011 American Chemical Society

conformational assistance, or both to enable an internal
hydroxyl or thiol nucleophile to undergo an intramolecular
NꢀO or NꢀS acyl shift reaction to form an ester or
thioester from an amide.9ꢀ14
Scheme 1. Preparation of starting Materials for Synthesis of
Peptide Thioesters
The NꢀO or NꢀS acyl shift is known to occur in the
early stage of protein splicing to afford an ester or
thioester.1b The acyl shift is proximity driven and confor-
mationally assisted with the scissile peptide bond in a cis
conformation. Among the naturally occurring peptide
bonds, only the tertiary Xaa-Pro bonds can favorably exist
in a cis conformation without enzymatic assistance. To
mimic the NꢀS acyl shift in protein splicing, many sought
to develop tertiary amide or proline-like surrogates with a
thiol handle to enable a ciscoid NꢀS acyl shift of a tertiary
amide bond to form a thioester. Tertiary amide surrogates
include structures such as N-alkylated Cys and its
mimetics,9 thiol-substituted proline,10 oxazolidinone,11
anilide,12 or modified benzyl13 groups. However, the re-
ported conformationally assisted NꢀS acyl methods are
often complicated by synthetic complexity in their pre-
paration, by side reactions at either the activation or the
conversion step, and in some cases, by the need of an
additional step for their removal.
Based on our previous ligation chemistry using thiazo-
lidoine and oxazolidoine as a proline surrogate,18 we
envisioned that a thiazolidine with a 2-thiomethyl group
could undergo a proximity-driven NꢀS acyl shift reaction
in a cis conformation, mimicking the NꢀS acyl shift of
protein splicing. Herein, we describe the use of 2-thio-
methylthiazolidine as an Fmoc-compatible proline surro-
gate to enable a tandem acid-catalyzed thiol switch to
afford a thioester, first through an NꢀS acyl-transfer
reaction and then a SꢀS (thiol thioester) exchange. To
obtain the desired 2-thiomethylthiazolidine, our scheme
began with2-mercaptoethanol 1, which was protected with
a trityl group upon treatment withtriphenylmethyl alcohol
in the presence of trifluoroacetic acid (TFA) in CHCl3
(Scheme 1a)16 to afford tritylthioethyl alcohol 2 in 85%
yield within 2 h. The purified alcohol 2 was then subjected to
oxidation by reacting it with pyridinium chlorochromate2
(PCC) under anhydrous conditions for 6 h to obtain the
corresponding tritylthioethyl aldehyde 3 in 70% yield.
The desired thiazolidine can be synthesized from alde-
hyde 3 by allowing it to undergo intermolecular cyclization
with a moiety containing a 1,2-aminothiol functionality as
previously reported by our group.18 The unprotected
cysteine anchored on an Fmoc-compatible resin support
would provide such a functionality. Keeping this in mind,
the synthesis of unprotected cysteine-anchored resins was
carried out, first by coupling Fmoc-Cys(S-t-Bu)-OH 4 to
the Wang or Rink-amide resin 5 using benzotriazole-1-yl-
oxytris(dimethylamino)phosphonium hexafluorophosphate
(BOP) and N,N-diisopropylethylamine (DIEA), followed
by the removal of Fmoc group (Scheme 1b). Treatment
with 2-mercaptoethanol (10%, v/v) in DMF for 12 h re-
sulted in removal of tert-butylsulfenyl protecting group,
thereby rendering the unprotected cysteine 6 with desired
1,2-aminothiolfunctionality. The presenceof freethiol was
confirmed by treating 6 with Ellman’s reagent [5,50-dithiobis-
(2-nitrobenzoic acid)], leading to the appearance of a red
solution.
To synthesize the thiazolidine ring with a highly acid-
sensitive trityl moiety both on the thiol side chain and resin
supports (e.g., Rink resin), the tritylthioethyl aldehyde 3
was reacted with resin-coupled unprotected cysteine 6
under neutral conditions for 24 h in DCM (Scheme 2).
Treatment of resin beads with Ellman’s reagent did not
produce red color, confirming the absence of free thiol
group. To further characterize the cyclized product, the
reaction was reproduced in the solution phase, confirming
the quantitativeformation of thiazolidine ring(Supporting
Information Figure 3). The intermolecular cyclization was
efficient and proceeded via an imine capture between the
free amino group of 6 and the aldehyde carbonyl of 3,
followed by ringꢀchain tautomerization by the free thiol
group leading to the formation of tritylthiomethylthiazo-
lidineꢀWang (TMTꢀW) or ꢀRink amide (TMTꢀR)
resin 7. Collectively termed as TMT resins, their main
advantage is that the suitably placed trityl protected thiol
(15) (a) Swinnen, D.; Hilvert, D. Org. Lett. 2000, 2, 2439–2442. (b)
Sewing, A.; Hilvert, D. Angew. Chem., Int. Ed. 2001, 40, 3395–3396. (c)
Sun, Y.; Lu, G.; Tam, J. P. Org. Lett. 2001, 3, 1681–1684. (d) Raz, R.;
Rademann, J. Org. Lett. 2011, 13, 1606–1609. (e) Kang, J.; Macmillan,
D. Org. Biomol. Chem. 2010, 8, 1993–2002. (f) Hogenauer, T. J.; Wang,
Q.; Sanki, A. K.; Gammon, A. J.; Chu, C. H. L.; Kaneshiro, C. M.;
Kajihara, Y.; Michael, K. Org. Biomol. Chem. 2007, 5, 759–762. (g)
Zheng, J.; Chang, H.; Wang, F.; Liu, L. J. Am. Chem. Soc. 2011, 133,
11080–11083. (h) Dheur, J.; Ollivier, N.; Vallin, A.; Melnyk, O. J. Org.
Chem. 2011, 76, 3194–3202. (i) Dheur, J.; Ollivier, N.; Melnyk, O. Org.
Lett. 2011, 13, 1560–1563. (j) Kang, J.; Richardson, J. P.; Mcmillan, D.
Chem. Commun. 2009, 4, 407–409.
(16) K. Nakamura, K.; Mori, H.; Kawakami, T.; Hojo, H.; Nakahara,
Y.; Aimoto, S. Int. J. Pept. Protein Res. 2007, 13, 191–202.
(17) Eom, K. D.; Tam, J. P. Org. Lett. 2011, 13, 2610–2613.
(18) (a) Botti, P.; David Pallin, T.; Tam, J. P. J. Am. Chem. Soc. 1996,
118, 10018–10024. (b) Tam, J. P.; Yu, Q.; Yang, J. J. Am. Chem. Soc.
2001, 123, 2487–2494.
Org. Lett., Vol. 13, No. 19, 2011
5177

moiety can be released at the completion of peptide synth-
esis as a latent thiolate for the NꢀS acyl transfer reaction.
varying concentrations of TFA, trifluoromethanesulfonic
acid (TfOH) and TC. Since the NꢀS acyl shift is dependent
on the acidity function of the TfOH/TFA deprotecting
solution, we tested a suitable acidic condition by varying
the concentration of TfOH in three increments, 0.1, 0.25,
and 1%, while keeping the concentrations of TFA and TC
constant. Initially, TR6-TMT-W resin 9a was treated
with 0.1% TfOH and 5% TC (v/v) in TFA for 2 h. It led
to the formation of the desired thioester TR6-TC 12a (m/z
722) as the major product in MS profile with an isolated
yield of 55% (Figure 1a), which was confirmed by MS/MS
sequencing (Supporting Information Figure 8). Apart
from the desired thioester product, the MS profile also
revealed minor peaks at m/z 616 (<5%) and m/z 781
(<8%). The former peak corresponded to TR6-OH sug-
gesting the incomplete attachment of the Fmoc-Cys(S-t-
Bu)-OH to the resin, and the latter indicated an impurity
peak which seems to be generated during peptide synthesis.
A side reaction in preventing the NꢀS acyl shift is the
trifluoroacetylation of the thiol group, with an observed
peak at m/z 838.
Scheme 2. Preparation of Peptide Thioesters via Acid-Catalyzed
NꢀS and then SꢀS Acyl Shifts
The secondary amine of the synthesized thiazolidine ring
offers a suitable site for carrying out the solid-phase
peptide synthesis. Fmoc-protected amino acids were se-
quentially coupled to the TMT-resins in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
hexafluorophosphate (HATU) and DIEA, resulting in the
formation of TMT-resin-coupled product 8 (Scheme 2).
Subsequently, the Fmoc group was removed and other
amino acids were further coupled via regular Fmoc-SPPS
steps to afford an orthogonally protected peptide-TMT 9.
Recently, our group has reported an efficient Fmoc-
compatible method for preparing peptide thioesters via an
acid-catalyzed tandem “thiol switch” of esters by an
intramolecular OꢀS acyl shift followed by trapping the
rearranged thioester with a large excess of thiocresol (TC)
to form a stable thioester.17 We envisioned that this
approach might also work for the NꢀS acyl transfer
reaction via the 2-thiomethylthiazolidine surrogate. In this
context, the protected peptide-TMT 9 was treated with
Figure 1. RP-HPLC profiles of the preparation of TR6 12a from
TMTW-TR6 9 using of 5% TC, TFA, and (A) 0.1% TfOH; (B)
0.25% TfOH; (C) 1% TfOH.
The yield of 12a increased to 69% as the concentration
of TfOH was increased to 0.25% (v/v) in TFA (Figure 1b).
However, when the TfOH concentration was increased to
1% (v/v) of TFA, there was also an increase in competing
reactions leading to a slight decrease in the thioester yield
to 65% (Figure 1c). Considering this almost unchanged
(19) (a) Lu, Y.-A.; Tam, J. P. Org. Lett. 2005, 7, 5003–5006. (b)
Payne, R. J.; Ficht, S.; Greenberg, W. A.; Wong, C. Angew. Chem., Int.
Ed. 2008, 47, 4411–4415.
(20) Eom, K. D.; Miao, Z.; Yang, J.-L.; Tam, J. P. J. Am. Chem. Soc.
2003, 125, 73–82.
(21) (a) Tam, J. P.; Heath, W. F.; Merrifield, R. B. J. Am. Chem. Soc.
1983, 105, 6442–6455. (b) Tam, J. P.; Heath, W. F.; Merrifield, R. B.
J. Am. Chem. Soc. 1986, 108, 5242–5251. (c) Chen, J.; Warren, D.; Wu,
B.; Chen, G.; Wan, Q.; Danishefsky, S. J. 2006, 47, 1969-1972.
5178
Org. Lett., Vol. 13, No. 19, 2011

yield of thioester for a 4-fold increase in the TfOH con-
centration, the use of 0.25% TfOH (v/v) of TFA appeared
to be a practical compromise so as to permit sufficient acid
strength to protonate the amide carbonyl to effect the NꢀS
acyl shift but not too strong to increase competing side
reactions. To investigate suitable conditions for the second
thiol switch of the thioester intermediates 10 and 11 by a
thioester exchange, the concentration of TC was varied
from 2.5% to 5% to 10%. These chagnes effectively
resulted in the formation of TR6-TC in respective yields
of 45%, 69%, and 73%. As the thioester yield did not
increase much after 5%TC, we postulated that the suitable
concentration of TC and TfOH for the acid-catalyzed
tandem NꢀS-acyl transfer reaction and subsequent thioe-
ster exchange using TMT handle appears to be 5% TC (or
lower %) and 0.25% TfOH (v/v) of TFA, respectively.
Previously, we showed the L- and D-form of TIGGIR-TC
and TIGGIr-TC, respectively, are well separated by
HPLC.17 We did not detect TIGGIr-TC by HPLC, suggest-
ing racemization is minimal under the proposed conditions.
It should noted that TC also serves as a scavenger under the
proposed conditions.
To determine the applicability of the current method for
the Fmoc-compatible Rink amide resins, the Rink amide
resin-coupled TR6-TMT was subjected to deprotection,
NꢀS acyl transfer, and thioester exchange using the identi-
fied conditions. It resulted in the formation of thioester
TR6-TC 12a in 84% yield (Figure 2), suggesting that Rink
amide resin is even more appropriate for thioester prepara-
tion than the Wang resin based on the proposed thiazoli-
dine surrogate. The increase in yield is likely attributed to
the ease of attachment of the TMT-handle to the Rink
amide resins. This result also suggests that the proposed
TMT-handle can be used for any Fmoc-compatible resin to
afford a peptide thioester. Another important advantage of
the tandem thiol switch reaction is that it provides a
solution to circumvent the issues for a separate step of
activation and auxiliary removal by allowing the deprotec-
tion of side chains under a one-pot reaction. Compared to
OꢀS acyl shift, the thiol switch reaction seems to be cleaner
for the NꢀS acyl shift under similar acidic conditions
because of the absence of the reversible alkylation of the
guandino side chain of Arg by hydroxyalkylthiol handle.17
In summary, the synthesis of peptide thioester via acid-
catalyzed NꢀS-acyl shift on a 2-thiomethylthiazolidine3
framework is a simple, novel, and practical Fmoc-based
method for solid-phase peptide synthesis. It employs sim-
ple starting materials and is compatible with the Wang or
Rink-amide resins. The proposed TMT resin 7 is relatively
easy to synthesize and provides with a thioester surrogate
where routine Fmoc-base peptide synthesis can be pursued
to obtain the desired peptide thioester in relatively good
yield after an acid-catalyzed tandem thiol switch reaction.
The observed NꢀS acyl shift was dependent on acidity,
and the protonation state of the amide carbonyl as the
formation of thioester 12 was accelerated by the addition
of a catalytic amount of TfOH. The proposed NꢀS acyl
shift scheme bears similarity to the “low TFMSA” and
“low HF” conditions with an acidity function of ꢀ5.2 and
could be potentially used for ligation, convergent synth-
esis, cysteine-free ligation,19 and tandem ligation20 as well
as for the synthesis of glyco- and phosphopeptides.5c,21
Figure 2. RP-HPLC profile of the preparation of TR6 12a from
TMTR-TR6 9 using 0.25% TfOH and 5% TC (v/v) of TFA.
The versatility of the acid-catalyzed NꢀS acyl shift
reaction using the TMT handle was further verified with
two additional peptide thioesters, RG9-TC 12b and KV6-
TC 12c, which were synthesized using TMT-Wang resin 9
under similar conditions in 65% and 57% yields, respec-
tively. Mechanistically, the acid-catalyzed thioester forma-
tion from peptide-TMT 9 resulted from a combination of
two “thiol switch” reactions. The first thiol switch takes
place after the release of the TMT handle 9 during the acid
deprotection step to permit an acid-catalyzed intramole-
cular NꢀS acyl shift of 10 to 11 followed by the second
thiol switch via a thioester exchange from 11 to 12 by
thiocresol under a timely activation by TfOH/TFA proto-
nating the amide carbonyl 10 at the C-terminus (Scheme 2).
Acknowledgment. This research was supported in part
by Biomedical Research Council (BMRC 09/1/22/19/612)
of A*STAR and Academic Research Fund (ARC21/08) of
Ministry of Education in Singapore.
Supporting Information Available. General procedures
and additional HPLC, NMR, and MS data. This material
is available free of charge via the Internet at http://pubs.
acs.org.
Org. Lett., Vol. 13, No. 19, 2011
5179