Biomimetic synthesis of cyclic peptides using novel thioester surrogates

Article abstract of DOI:10.1002/bip.22308

Acyl shifts involving N-S and S-S rearrangements are reactions central to the breaking of a peptide bond and forming of thioester intermediates in an intein-catalyzed protein splicing that ultimately leads to the formation of a new peptide bond by an uncatalyzed S-N acyl shift reaction. To mimic these three acyl shift reactions in forming thioesters and the subsequent peptide ligation, here we describe the development of two 9-fluorenylmethoxycarbonyl (Fmoc)-compatible thioester surrogates that can undergo uncatalyzed N-S, S-S, and S-N acyl shifts for preparing thioesters and cyclic peptides. These surrogates were incorporated as a C-terminal amido moiety of a target peptide using Fmoc chemistry by solid-phase synthesis, and then transformed into a thioester or thiolactones via two acyl shift reactions with or without the presence of an external thiol under acidic conditions. The proposed intein-mimetic thioester surrogates were prepared using readily available starting materials including N-methyl cysteine or 2- thioethylbutylamide. A key functional moiety shared in their design is the thioethylamido (TEA) moiety, which is essential to effect a proximity-driven N-S acyl shift under a favorable five-member ring transition in the breaking of a peptide bond. Thus, the tandem series of acyl shifts effected by a TEA moiety in a thioester surrogate together with a thioethylamino moiety of an N-terminal Cys residue in a linear peptide precursor are chemical mimics of an intein, as they mediate both excision and ligation reactions in forming cyclic peptides including cyclic conotoxin and sunflower trypsin inhibitor described herein. VC 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci).

Full text of DOI:10.1002/bip.22308

Biopolymers: Peptide Science
Biomimetic Synthesis of Cyclic Peptides using Novel
Thioester Surrogates
Xinya Hemu, Misako Taichi, Yibo Qiu, DingꢀXiang Liu, James P. Tam
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551
Correspondence to: James P. Tam; eꢀmail: jptam@ntu.edu.sg
Contract grant sponsors: Agency for Science Technology and Research (A*STAR) and National Research Foundation (NRF) of
Singapore
ABSTRACT:
Acyl shifts involving N-S and S-S rearrangements are reactions central to the breaking of a peptide bond
and forming of thioester intermediates in an intein-catalyzed protein splicing that ultimately leads to the
formation of a new peptide bond by an uncatalyzed S-N acyl shift reaction. To mimic these three acyl shift
reactions in forming thioesters and the subsequent peptide ligation, here we describe the development of
two 9-fluorenylmethoxycarbonyl (Fmoc)-compatible thioester surrogates that can undergo uncatalyzed
N-S, S-S and S-N acyl shifts for preparing thioesters and cyclic peptides. These surrogates were
incorporated as a C-terminal amido moiety of a target peptide using Fmoc chemistry by solid-phase
synthesis, and then transformed into a thioester or thiolactones via two acyl shift reactions with or
without the presence of an external thiol under acidic conditions. The proposed intein-mimetic thioester
surrogates were prepared using readily available starting materials including N-methyl cysteine or 2-
thioethylbutylamide. A key functional moiety shared in their design is the thioethylamido moiety, which is
essential to effect a proximity-driven N-S acyl shift under a favorable five-member ring transition in the
breaking of a peptide bond. Thus, the tandem series of acyl shifts effected by a thioethylamido moiety in a
thioester surrogate together with a thioethylamino moiety of an N-terminal Cys residue in a linear
peptide precursor are chemical mimics of an intein, as they mediate both excision and ligation reactions
in forming cyclic peptides including cyclic conotoxin and sunflower trypsin inhibitor described herein.
Keywords: thioester, thioethylamido, thioester surrogate, N-S acyl shift, intein mimetic, cyclic peptides,
conotoxin, sunflower trypsin inhibitor
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an
‘Accepted Article’, doi: 10.1002/bip.22308
© 2013 Wiley Periodicals, Inc.

Biopolymers: Peptide Science
Page 2 of 17
INTRODUCTION
Cyclic peptides, naturally occurring or synthetic, are generally referred to peptides with an endꢀtoꢀend or
headꢀtoꢀtail linkage of the peptide backbone through an amide bond. Consequently, cyclic peptides
without ends are resistant to degradation by exopeptidases and are more conformationally restricted than
their linear counterparts. These advantages have long been recognized in drug design to increase the
metabolic stability of a bioactive linear peptide.1 For a synthetic cyclic peptide derived from a linear
peptide, there is an additional advantage as a neoꢀepitope can be created by joining the amino acids of the
Nꢀ and Cꢀtermini. Such an epitope can become a new bioactive fragment and can be exploited in the drug
design of a peptide biologic.
For preparing cyclic peptides, a common approach is to use unprotected peptides containing a
thioester.2 Thioesters are key functional groups in various synthetic strategies for peptide cyclization and
ligation.3ꢀ8 Over the years, different methods for preparing thioesters or their precursors compatible with
tꢀbutoxycarbonyl (Boc) and fluorenylmethoxycarbony (Fmoc) chemistries have been developed for solidꢀ
phase synthesis of peptides and proteins. Thioesters are susceptible to base and intolerant to repetitive
piperidine deprotection steps in Fmoc chemistry. Boc chemistry, which utilizes repetitive acid
deprotection steps, is an effective approach for preparing peptide thioesters by assembling a peptide chain
directly on a thioester linker functionalized on resin supports. However, the repetitive use of
trifluoroacetic acid in deprotection steps may pose problems in preparing glycoꢀ or phosphopeptides. In
addition, the use of HF in the final cleavage steps requires a specialized apparatus and safety regulations,
which limit the extensive application of Boc chemistry for preparing peptide thioesters. At present, Fmoc
chemistry is the method of choice for solidꢀphase syntheses of many peptides. These needs have
prompted strong interests in the development of Fmocꢀcompatible schemes for preparing thioesters.
Fmocꢀcompatible methods of preparing peptide thioesters include mild basic deprotection cocktails,9
baseꢀtolerant thioester linkers,10 “safetyꢀcatch” auxiliaries,11ꢀ15 use of a partially protected peptide,16 and
amideꢀ or esterꢀbased surrogates that can be transformed into thioesters via an Nꢀ or OꢀS acyl shift
reaction, respectively. In recent years, the approach using amidoꢀthioester surrogates has gained
popularity and examples include Nꢀ4,5ꢀdimethoxyꢀ2ꢀmercaptoꢀbenzyl (Dmmb),17 Nꢀalkylated Cys,18 bisꢀ
19
20
(sulfanylehtylamino) (SEA)
or bisꢀ(mercaptoethyl) amide (BMEA),
thioꢀproline,21 and thioꢀ
thiazolidine.22
A particularly appealing aspect of the amideꢀbased thioester surrogates is that they are generally
stable to both Bocꢀ and Fmocꢀchemistries in solidꢀphase synthesis. A functional group essential in the
amido thioester surrogate approach is the thioethylamido moiety (TEA) to effect a proximityꢀdriven NꢀS
acyl shift reaction via a fiveꢀmember ring transition state to form the desired thioester. A similar
functional group, the thioethyl amino moiety of an Nꢀterminal cysteine is the key moiety to drive the
reverse reaction on the cysteineꢀthioester ligation via an SꢀN acyl shift. Thus, the interconversion of the
TEA and TEꢀamino moieties represents the reversible reactions of a XꢀCys bond to form a thioester or an
amide bond, a principle which has been exploited in the general design of amido thioester surrogate
methods. However, current methods in preparing these TEAꢀbased thioester surrogates are fairly
complicated, and a simple and practical method or a new design of TEAꢀbased thioester surrogates would
be desirable.
As previously discussed, the amidoꢀthioester formation via an NꢀS acyl shift is a reverse reaction of
peptide ligation. It is thermodynamically unfavorable and requires a cis conformation of a tertiary XꢀCys
amide bond to undergo an NꢀS acyl shift, often under acidic conditions. Furthermore, the general scheme
of a tertiary amideꢀbased thioester surrogate to form a cyclic peptide bears a certain similarity to protein
splicing, which undergoes a series of acyl shift reaction including an intramolecular NꢀS acyl shift to form
a thioester (Figure 1). In particular, a Cꢀterminal amidoꢀthioester surrogate mimics the overall effect of an
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Biopolymers: Peptide Science
inteinꢀmediated splicing process to give an endꢀtoꢀend cyclic peptide via a series of intramolecular acyl
shift reactions, ending with the excision of the thioester surrogate.
Inteinꢀmediated splicing involves four acyl shifts to excise an intein from its flanking exteins and
rejoin the Nꢀ and Cꢀexteins by a peptide bond (Figure 2a).23ꢀ25 The first reaction is an NꢀS acyl shift to
break the peptide bond at the Nꢀsplicing site of the Nꢀextein/intein junction, forming a branched
intermediate in which the Nꢀextein is linked to the side chain of the Nꢀterminal Cys of intein by a
thioester bond. This is followed by an SꢀS acyl shift, a thioester exchange reaction, between the Nꢀ
terminal Cys of Cꢀextein and the newly formed thioester bond at the Nꢀsplicing site, resulting in the
formation of the second branched intermediate. To excise the intein from this branched intermediate, an
NꢀN acyl shift occurs at the Cꢀterminal Asn residue of an intein to yield the third branched intermediate of
Nꢀ and Cꢀexteins linked by a thioester bond. The final SꢀN acyl between the Nꢀterminal Cys of the Cꢀ
extein and the newly formed thioester is uncatalyzed and results in a new peptide bond formation between
the Nꢀ and Cꢀexteins to complete the splicing process. Thus, the Nꢀterminal Cys of both intein and Cꢀ
extein, play a key role in the breaking and making of peptide bonds through the NꢀS, SꢀS and SꢀN acyl
shift reactions, whereas the thioethylamido (TEA) moieties at the splicing sites play the essential role in
these acyl shift reactions.
The presence of a thioethylamino moiety as an Nꢀterminal Cys together with a Cꢀterminal TEAꢀ
containing thioester surrogate in a cysteineꢀrich peptide makes it an ideal candidate for macrocyclization
through a series of NꢀS, SꢀS and SꢀN acyl shift reactions (Figure 2). In addition, a cysteineꢀrich peptide
precursor would provide a choice of multiple available cyclization sites. Moreover, the sideꢀchain thiols
of cysteineꢀrich peptides can replace the role of external thiols in the SꢀS acyl shift reaction to give a
series of thiolactone intermediates instead of a Cꢀterminal thioester. The Nꢀterminal cysteine would
eventually join the Cꢀterminus of the peptide precursor by expansion of thiolactone rings through
reversible SꢀS exchange reactions, leading to a SꢀN acyl shift reaction to afford a new NꢀtoꢀC peptide
bond to form an endꢀtoꢀend macrocycle. This approach of entropyꢀdriven thiolactone ring expansion
accelerates peptide cyclization and has been known as the thia zip reaction in preparing cyclic
peptides.8,26ꢀ28 Thus we envisioned that, in the synthesis of endꢀtoꢀend cyclic peptides, a Cꢀterminal TEA
thioester surrogate can mimic the role of intein in protein splicing to mediate NꢀS, SꢀS and SꢀN acyl shifts,
leading to both thioester formation and ligation of the Nꢀ and Cꢀtermini as an endꢀtoꢀend peptide
macrocycle as well as their excision from the precursor sequence (Figure 1 and 2b).
Here we report the development of two TEA thioester surrogates that are compatible with Fmoc
chemistry (Figure 3). Both surrogates were prepared using readily available starting materials from
commercial sources, FmocꢀMeCys(Trt) and 2ꢀthioethylbutylamine. They were readily attached to a
highly acidꢀlabile resin support functionalized by amine (Rink amide resin) or chlorotrityl chloride, to
form MeCys and thioethylbutylamido (TEBA) resins, respectively. The MeCys or TEBA moieties on
these resins served as the starting point for a solidꢀphase synthesis using Fmoc chemistry. They also
served as the Cꢀterminal residue of a peptide precursor that can be transformed into a peptide thioester
(Figure 2b). Nꢀalkyl cysteine derivatives have been previously reported by Hojo et al.18 However, MeCys
as a thioester surrogate has not been studied in detail or used for synthesis of cyclic peptides. Similarly,
we reported the conformationallyꢀconstrained thiomethylthiazolidine (TMT) carboxylic acid as an amidoꢀ
thioester surrogate.22 Both MeCys and TMT resins have limitations, which led us to the development of
the simplified TEBA thioester surrogates for preparing cyclic peptides.
RESULTS AND DISCUSSION
Two bioactive peptides were used to illustrate the efficacy of the proposed TEAꢀbased thioester
surrrogates. The family of ωꢀconotoxins is a potent N-type calcium channel inhibitor secreted by marine
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Biopolymers: Peptide Science
Page 4 of 17
cone snails.29 They contain three conserved disulfide bonds in a cystineꢀknot motif. Synthetic ωꢀ
conotoxin MVIIA is 1000ꢀfold more potent than morphine and a FDAꢀapproved analgesic drug for the
management of chronic and neuropathic pain.30,31 However, ωꢀconotoxin MVIIA has low metabolic
stability, which might be enhanced by backbone cyclization as an endꢀtoꢀend macrocycle. Thus we
designed a prototype of cyclic ωꢀconotoxin cCG29 based on the 25ꢀresidue ωꢀconotoxin MVIIA to
improve
its
metabolic
stability
(Figure
4).
The
cyclic
analog
cCG29
(CKGKGAKCSRLMYDCCTGSCRSGKCꢀGGPG) was designed by adding a flexible fourꢀresidue linker
GGPG at the Cꢀterminus as a spacer to facilitate its endꢀtoꢀend cyclization. The choice of a Glyꢀrich
linker also allowed it to be used as a cyclization site. The commercially available FmocꢀMeCys(Trt) was
used as a thioester surrogate in the solidꢀphase synthesis of cCG29 as a model peptide mimicking an
intein for preparing cyclic peptides.
The second bioactive peptide selected in this study was sunflower trypsin inhibitor (SFTIꢀ1). SFTIꢀ1
is a naturallyꢀoccuring cyclic peptide and is one of the smallest and most potent trypsin inhibitor known.
It contains 14 amino acids (GRCTKSIPPICFPD) that form two βꢀsheets stabilized by an intramolecular
disulfide bond and an endꢀtoꢀend cyclic backbone.32 In its synthesis and endꢀtoꢀend cyclization, we used
our novel TEAꢀthioester surrogate, the TEBA linker. The Arg2ꢀCys3 junction was selected as the
cyclization point. A linear precursor of SFTI with Cꢀterminal TEBA moiety was prepared for thia zip
cyclization to afford the reduced SFTIꢀ1.
MeCys as a TEA Thioester Surrogate in the Synthesis of Cyclic ωꢀConotoxin
The unprotected linear CG29 precursor with a Cꢀterminal MeCys thioester surrogate was prepared using
Fmoc chemistry (Figure 5). FmocꢀGly was coupled to Rink amide resins to give FmocꢀGlyꢀNHꢀresin 1.
The commercially available FmocꢀMeCys(Trt) was then coupled to the Fmocꢀdeprotected glycine using
DIC/HOBt to give FmocꢀMeCys(Trt)ꢀGlyꢀRink amide resin 2 as a functionalized resin. The Cꢀterminal
Gly of CG29 sequence was coupled to the hindered secondary amine of MeCys(Trt) on resin 2 using a
more reactive coupling reagent HATU/DIEA with repeated couplings to afford FmocꢀGlyꢀMeCys(Trt)ꢀ
GlyꢀRink amide resin 3. This coupling reaction was monitored by acetaldehyde/chloranil that reacted with
a secondary amine to give a greenꢀblue color. The remaining sequence of CG29 was coupled to resin 3 by
a stepwise Fmoc solidꢀphase synthesis using PyBOP/DIEA in a microwaveꢀassisted peptide synthesizer
to give CG29ꢀMeCys(Trt)ꢀGlyꢀRink amide resin 4. The unprotected peptide CG29ꢀMeCysꢀGlyꢀNH2 5
was removed from resin 4 by treating with TFA/TIS/H2O (95/2.5/2.5, v/v) for 2 h to give a 21% isolated
yield after HPLC purification and lyophilization.
The acidꢀcatalyzed NꢀS acyl shift of CG29ꢀMeCysꢀGlyꢀNH2 5 to give its thioester intermediate 6 was
performed in an aqueous solution at pH 2. In the absence of an external thiol, the internal thiols of the
cyclized CG29 spontaneously underwent a series of SꢀS acyl shift reactions to form CG29 thiolactones
8aꢀf. For example, after treating the cyclized peptide CG29ꢀMeCysꢀGlyꢀNH2 5 in a pH 2 solution at 40°C
for 36 h, we isolated five out of six expected thiolactone isomers and labeled these peaks as 8aꢀd and 8f
in the RPꢀHPLC traces (Figure 6c). The peaks 8aꢀd and 8f were characterized by MALDIꢀTOF mass
spectrometry, and all gave the same m/z 2896.4 as the cyclic CG29 9, which suggested that they were
thiolactone isomers. These thiolactones could be distinguished from the cyclic peptide 9 by monitoring at
wavelength 260 nm in HPLC traces because thioesters and thiolactones are strongly absorbed at 260 nm
whereas the absorbance of amide bonds is considerably weaker at this UV wavelength. Observation of
thiolactone formation as a result of SꢀS acyl shift reactions indicated that sideꢀchain thiols actively
participated in transesterification. Thus we expected that cysteineꢀrich peptides, such as CG29 containing
six cysteines, were able to cyclize without addition of external thiols. The series of thiolactones with
different ring sizes were exchangable and collectively acted as the starting materials for the subsequent
endꢀtoꢀend backbone cyclization reaction. This cyclization reaction was performed at pH 7.5 buffered by
0.1 M phosphate buffer for 2 h at room temperature in 86% yield (Figure 6e). TCEP (10 mM) was added
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Biopolymers: Peptide Science
in this cyclization buffer to prevent disulfide formation. The SꢀS exchange reactions were favored under
pH 7.5 to permit the expansion of thiolactone rings as an endꢀtoꢀend thiolactone formed by the Nꢀ and Cꢀ
termini, and which led to a proximityꢀdriven SꢀN acyl shift to form a backbone amide of the desired
macrocycle. This thiolactoneꢀassisted zipping mechanism was earlier descibed by our laboratory as the
thia zip cyclization.8,27 From a synthetic peptide precursor with thioester surrogate to endꢀtoꢀend cyclized
peptide product, the MeCysꢀmediated cyclization of cysteineꢀrich peptide ressembled the mechanism of
inteinꢀmediated protein splicing. Only a single purification step by RPꢀHPLC was required after
completion of cyclization. Thus this method could afford a good yield in the preparation of a circular
protein such as CG29.
In the presence of an external thiol, a stable thioester was obtained from the peptide precursor CG29ꢀ
MeCysꢀGlyꢀNH2 5. The reaction was performed in a pH 2 solution at 40°C using an excessive amount of
an external thiol (50 eq. MESNa). After a 24ꢀh treatment, two thioesters and one thiolactone were isolated
by RPꢀHPLC (Figure 6d) as CG29ꢀMES 7 (76%), MeCys thioester 6 (5%) and a thiolactone CG29 8f
(4%), respectively. Compared to the NꢀS/SꢀS acyl shift reactions performed without an external thiol, the
HPLC traces of thioesterification reaction were simplified by the addition of an excessive amount of
external thiol, and CG29ꢀMES 7 was the most abundent thioester product. The thiolactone 8f with the
retention time at 43.6 min was closest to the retention time of cyclic CG29 9 at 43.9 min and we
speculated that it could be the endꢀtoꢀend thiolactone, based on the similarity in conformation with the
endꢀtoꢀend cyclized CG29 9. The thia zip cyclization was performed at pH 7.5 for 2 h at room
temperature. The cyclic CG29 9 was transformed from 6, 7 and 8f via SꢀS and SꢀN tandem acyl shift
reactions to the desired cyclic CG29 (Figure 6e). The oxidative folding of cyclic CG29 was conducted
using a highly diluted solution (10 ꢁM) in 0.1 M ammonium phosphate buffer at pH 8 containing 2 M
ammonium sulfonate and oxidized/reduced glutathionine (peptide:GSSG:GSH= 1:10:100 eq) at 4°C for
72 h to give ~15% folded cyclic conotoxin CG29. The native disulfide connectivity was characterized by
partial reduction/Sꢀalkylation and chymotrypsin digestion followed by MS/MS analysis.
Side Reactions and Rationale to Develop Simplified TEXA Types of TEA thioester surrogates
The use of MeCys(R) (R, the Sꢀprotecting group) thioester surrogate has several limitations. First is the
slow coupling of a Cꢀterminal amino acid derivative of a target peptide with the secondary amine of
MeCys(R) on resin. The reactivity of this secondary amino group is low thus needs multiple couplings
for completion. Hojo et al. reported a method to circumvent the difficulty of the coupling problem of the
MeCys(R) on resin by a solutionꢀphase preparation of different dipeptide units, which can be easily
introduced on resin to give MeCys(R)ꢀcontaining peptides.33,34
The second drawback of the MeCys(R) surrogate group was the formation of a MeAla(Pip) side
product (+51 Da) during Fmocꢀsynthesis of a peptide containing a Cꢀterminal MeCys(Trt) on resin. This
35
side reaction was previously reported by Lukszo et al.
as a baseꢀcatalyzed βꢀelimination on a Cꢀ
terminal Cys(Trt) or Cys(Acm) followed by a Michael addition of piperidine to form 3ꢀ(1ꢀ
piperidinyl)alanine. MeCys(Trt) was also susceptible to this side reaction and the side product was
accumulated during each piperidine deprotection step in Fmoc solidꢀphase synthesis. This synthetic
problem was not ressolved by addition of a spacer residue Gly between MeCys(Trt) and the resin support.
In the microwaveꢀassisted peptide synthesis of CG29ꢀMeCys(Trt)ꢀGlyꢀNH2 5, the side product CG29ꢀ
MeAla(Pip)ꢀGlyꢀNH2 accumulated to 10% at the completion of the synthesis, suggesting that an average
of 0.3% side reaction per deprotection cycle.
The third drawback of using MeCys(Trt) was that it was an expensive reagent, costing about 1,000
USD per gram.
These limitations led us to develop other amido types thioester surrogates, such as the 2ꢀ
thiomethylthiazolidine (TMT)ꢀcarboxylicꢀ acid thioester surrogate (Figure 3b), which we have reported
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Biopolymers: Peptide Science
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previously.22 The thiaꢀproline moiety of the TMT thioester surrogate facilitates an NꢀS acyl shift by
enhancing a cis conformation and suppressing the βꢀelimination side reaction.
To completely eliminate the βꢀelimination side reaction, it would be desirable to remove the electron
withdrawing αꢀcarbonyl moiety of a MeCys(R) and the αꢀCH susceptible to abstraction by piperidine
during the deprotection cycles. To achieve this objective, we have developed a novel family of TEAꢀ
thioester surrogates, which we designated as the TEXA thioester surrogates.
The TEXA thioester surrogates (where X=methyl, ethyl, propyl or butyl) were designed by removing
the Cꢀterminal reactive carbonyl functionality. Without the carboxylic group, the TEXA design could
decrease the steric hindrance during the coupling reaction and eliminate the side reaction of βꢀelimination
and the piperidine addition product. TEXA has an advantage in that it uses the resin support as a Sꢀ
protecting group for the thioethyl moiety. Such a strategy further simplifies the synthesis of
functionalization of the TEA thioester. An example of a TEXA thioester surrogate, TEBA (where
X=butyl) is described below (Figure 3c).
Thioethylbutylamide (TEBA) as a TEA Thioester Surrogate in the Syntheis of SFTI
The preparation of SFTI using the TEBA linker commenced with the attachment of the commercially
available (2ꢀbutylamino)ꢀethanethiol 10 to ClꢀTrt(2ꢀCl) resin to give TEBAꢀTrt(2ꢀCl) resin 11 (Figure 7).
The Cꢀterminal FmocꢀArg(Pbf) (4 eq) was coupled to the secondary amine 11 using HATU/DIEA for 1 h
to give FmocꢀArg(Pbf)ꢀTEBA resin 12. The amino group on 11 was found to be more reactive than that
on MeCys due to the absence of the carbonyl group. Acetaldehyde/chloranil method for detection of a
secondary amino group confirmed that the condensation reaction was complete in a single coupling
reaction. Peptide elongation of the SFTI linear precursor was carried out by a manually stepwise coupling
protocol using Fmoc amino acid derivative/HBTU/HOBt/DIEA in DMF for 30 min for each coupling
reaction. As for Fmoc deprotection, 20% morpholine/DMF 36 (5 min x 4) was used, rather than piperidine,
for prevention of aspartimide formation at Asp14ꢀGly1. The protected SFTIꢀTEBA resin 13 was treated
with TFA/TIS/H2O (95/2.5/2.5) to remove all protecting groups to give SFTIꢀTEBA 14 without any
aspartimide formation in 17% yield from ClꢀTrt(2ꢀCl) resin after purification by RPꢀHPLC.
After investigating a suitable pH as an optimal condition for a tandem NꢀS and SꢀS acyl shift reaction
(data not shown), we selected pH 3 with MESNa as an external thiol incubated at 40oC. When 14 was
treated with MESNa in an aqueous solution buffered at pH 3 and 40oC for 15 h, the thioester 15 derived
from the NꢀS acyl shift of 14 was observed first and then converted to the MES thioester 16, and
thiolactone 17 (24%) was also observed on HPLC traces. Both forms underwent a thia zip cyclization
smoothly to obtain 18 in 52% yield after adjusting the pH to 7 in the presence of TCEP. The disulfide
formation using DMSO as the oxidant was successfully carried out under a neutral condition to afford
SFTIꢀ1 in 82% yield (Figure 8).
CONCLUSIONS
The TEA thioester surrogates are chemical mimics of inteins as they fulfill their roles, just as inteins do in
protein splicing, to enable both excision and ligation reactions in forming endꢀtoꢀend cysteineꢀrich cyclic
peptides, examples of which include cyclized conotoxin and SFTI. Under acid conditions the thioester
surrogate, such as MeCys in a cysteineꢀrich peptide, undergoes an NꢀS acyl shift to form a thioester, after
which it undergoes a series of SꢀS acyl shifts to form a thiolactone with the excision of the MeCys moiety.
These thiolactones eventually undergo an SꢀN acyl shift via a thia zip cyclization at basic pH to form the
peptide backbone of an endꢀtoꢀend macrocycle. A simplified version of MeCys is the TEBA thioester
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Biopolymers: Peptide Science
surrogate, which eliminates the side reactions associated with the MeCys thioester surrogate in peptide
synthesis.
TEA thioester surrogates may have additional applications in sequential peptide ligation to prepare
large proteins as the difference in suitable pH range for NꢀS, SꢀS and SꢀN acyl shift reaction may allow
selective activation of TEA thioester surrogates attached on Cꢀtermini of peptide segments.
MATERIALS AND METHODS
General
Fmoc amino acid derivatives were purchased from ImpexꢀChem including FmocꢀAlaꢀOH, Fmocꢀ
Arg(Pbf)ꢀOH, FmocꢀGlyꢀOH, FmocꢀIleꢀOH, , FmocꢀCys(Trt)ꢀOH, FmocꢀLys(Boc)ꢀOH, FmocꢀTyr(tBu)ꢀ
OH, FmocꢀPheꢀOH, FmocꢀThr(tBu)ꢀOH, FmocꢀValꢀOH and FmocꢀTrp(Boc)ꢀOH. FmocꢀMeCys(Trt)ꢀOH
was purchased from Advamac. Wang resin (0.9 mmol/g), Chlorotrityl chloride resin (1.2 mmol/g), Rink
amide resin (0.4 mmol/g), HOBt and BOP were purchased from Novabiochem. DIEA, 2ꢀmercaptoethanol,
triphenylmethyl alcohol, pyridinium chlorochromate, thiocresol (TC), DIC, and LꢀCysteine hydrochloride
were purchased from Aldrich. TFA was purchased from Alfa Aesar. PyBOP, HOAt, HATU and HBTU
were purchased from GL Biochem. Solvents DMF, DCM, piperidine, morpholine and diethyl ether were
purchased from Merck.
The Cꢀterminal FmocꢀXaa was coupled manually in all peptides. The rest of peptide sequences were
synthesized by automated microwave peptide synthesizer (CEM, Liberty1) using 5 eq. of PyBOP and 10
eq. of DIEA for coupling and 20% piperidine (0.1 M HOBt) in DMF for deprotection.
Synthesis of CG29ꢀMeCysꢀGlyꢀNH2 on Rink amide resin
The Rink amide resin (1 g, 0. 34 mmol) was swollen for 10 min in DCM (10 mL) and the resin was
filtered and washed with DCM (1 min x 2). The spacer amino acid FmocꢀGly (1.36 mmol, 404 mg) was
manually coupled with 4 eq. of DIC (1.36 mmol, 143 ꢂl) and 4 eq. of HOBt (1.36 mmol, 184 mg) in
DMF (10 ml) (shaking for 1 h at room temperature) to give FmocꢀGlyꢀNH resin 1. The resin was washed
by DMF and DCM respectively followed by deprotection using 20% piperidine solution in DMF (5 min x
2). A mixture of 2 eq. FmocꢀMeCys(Trt)ꢀOH (0.68 mmol, 408 mg), 4 eq. DIC (1.36 mmol, 143 ꢂl) and 4
eq. HOAt (1.36 mmol, 185 mg) in DMF (10 ml) was added into resinꢀcontaining reaction vessel. The
reaction was performed by shaking for 2 h at room temperature. Fmoc was deprotected by 20% piperidine
in DMF (5 min x 2 at room temperature) and resin was washed (DCM x 2, DMF x 2) to give MeCys(Trt)ꢀ
Glyꢀamide resin 2. FmocꢀGly (1.36 mmol, 404 mg) was coupled manually to the secondary amine on
MeCys(Trt) of resin 2 by 4 eq. HATU (1.36 mmol, 517 mg) and 6 eq. DIEA (2.04 mmol, 341 ꢂl) to
obtain resin 3 after triple coupling. The completion of reaction was examed by acetaldehyde /chloranil
test (2% acetaldehyde, 2% chloranil in DMF, 5 min at room temperature). The substitution of FmocꢀGlyꢀ
MeCys(Trt)ꢀGlyꢀamide resin 3 (1.2 g) was ~0.28 mmol/g. 350 mg resin 3 (0.1 mmol) was used for
synthesis of the fullꢀlength CG29 using microwaveꢀassisted peptide synthesizer using PyBOP/DIEA
(5/10 eq) to give CG29ꢀMeCysꢀGlyꢀamide resin 4 (783 mg). A mixture of 5 ml TFA/TIS/H2O (95/2.5/2.5,
v/v) was added to 200 mg resin 4 to remove side chain deprotection group and cleave peptide from solid
support. The reaction mixture was shaken for 2 h at room temperature and then added to chilled diethyl
ether for peptide precipitation. The precipitate was dried in vaccuo and purified by RPꢀHPLC to give
CG29ꢀ MeCysꢀGlyꢀNH2 5 (17.6 mg, 21% isolated yield).
Synthesis of SFTI on thiolethylbutylamido (TEBA) resin using ClꢀTrt(2ꢀCl) resin
ClꢀTrt(2ꢀCl) resin (0.5 g, 0.6 mmol) was swollen for 30 min in DCM (10 mL) and the resin was filtered
and washed with DCM (1 min x 2). (2ꢀbutylamino)ethanethiol 10 (0.3 mmol, 44 ꢂl) in DCM (20 ml) was
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added, and the suspension was shaken for 1 h at room temperature. 3.6 mmol DIEA (3.6 mmol, 627 ꢂl) in
MeOH was added, and the reaction mixture shaken for 10 min to quench the unreacted resin. The resin
was washed with DCM and DMF, respectively, to give TEA resin 11. A mixture of FmocꢀArg(Pbf) (1.2
mmol, 779 mg), HATU (1.2 mmol, 456 mg) in DMF and DIEA (1.8 mmol, 314 ꢂl) was added to resin 11.
The suspension was shaken for 1 h and coupling procedure was duplicated to give FmocꢀArg(Pbf)ꢀTEBA
resin 12. The rest of peptide sequence was synthesized manually to give SFTIꢀTEBA resin 13. A mixture
of TFA/TIS/H2O (95/2.5/2.5, v/v) was added to peptide resin 13 and the reaction mixture was shaken for
2 h. After dropwise addition of the reaction mixture to chilled diethyl ether for precipitation, the
precipitate was dried in vaccuo and purified by RPꢀHPLC to give SFTIꢀTEBA 14 (27.4 mg, 17% yield).
Cyclization of cysteineꢀrich peptide conotoxin CG29
CG29ꢀMeCysꢀGlyꢀNH2 5 (1 ꢂmol, 3.1 mg) was dissolved in 5 mM H2SO4 solution (pH 2, 1 mL) at a
concentration of 1 mM with 50 eq. MESNa (50 ꢂmol, 8.2 mg) and incubated at 40°C for 24 h. The
formation of thioesterꢀform 6, MES thioester 7 and thiolactones 8aꢀf from 5 was monitored by RPꢀHPLC.
TCEP (10 ꢂmol, 2.8 mg) were added to the reaction mixture to avoid disulfide formation. The pH of
reaction mixture remained about pH 2 after addition of TCEP. After adjusting the pH to 7.5, thia zip
cyclization was performed at room temperature with gentle stirring for 2 h to give cyclic reduced CG29 9
(comparative yield 73%). In the second cyclization reaction, CG29ꢀMeCysꢀGlyꢀNH2 5 (1 ꢂmol, 3.1 mg)
was dissolved in 5 mM H2SO4 solution (pH 2, 1 mL) at a concentration of 1 mM without addition of
MESNa. The reaction was conducted at 40°C for 36 h when 5 was not observed in HPLC monitoring.
Thioesterꢀform 6 and five out of six thiolactone 8aꢀf was isolated from HPLC and identified by MALDIꢀ
TOP mass spectrometry. After adjusting the pH to 7.5, thia zip cyclization was performed at room
temperature with gentle stirring for 2 h to give cyclic reduced CG29 9 (comparative yield 86%). To a
solution of cyclic CG29 9 (1 ꢂmol, 2.9 mg) in 0.1 M ammonium phosphate buffer (pH 8, 100 ml) that
contained 2 M ammonium sulfonate, a redox reagent including oxidized/reduced glutathionine (molar
ratio of peptide:GSSG:GSH= 1:10:100) was added to catalyze disulfide formation at 4°C for 72 h to give
14% folded cyclic conotoxin CG29 with native disulfide connectivity. The disulfide connectivity was
analyzed by partial reduction/alkylation method. 300 ꢂg folded cCG29 was reduced by TCEP (10 mM)
for 30 min and Sꢀalkylated by 50 mM Nꢀethylmaleimide (NEM) for another 30 min to obtain
intermediates with one or two pairs of intact disulfide bonds. The 1SS and 2SS species were isolated by
RPꢀHPLC and reduced completely by 20 mM DTT followed by 50 mM iodoacetamide (IAM) alkylation.
Chymotrypsin treatment opened the cyclic backbone at Tyr13ꢀAsp14 site to give linear alkylated products
for MS/MS analysis.
Cyclization of cysteineꢀrich peptide trypsin inhibitor SFTI
SFTIꢀTEBA 14 (5.85 ꢂmol, 9.64 mg) was dissolved in 0.1 M sodium phosphate buffer (pH 3, 1.95 mL) at
a concentration of 5 mM with MESNa (292 ꢂmol, 48.0 mg) and incubated at 40°C for 18 h. The
formation of S form 15, MES thioester 16 and thiolactones 17a and 17b from 16 was monitored by RPꢀ
HPLC. TCEP (58.5 ꢂmol, 16.8 mg) and 0.1% sodium phosphate (pH 3, 3.9 mL) were added to the
reaction mixture to avoid disulfide formation and dilute the reaction mixture, respectively. After adjusting
the pH to 7 by addition of 2 N NaOH, thia zip cyclization was performed at room temperature with gentle
stirring for 4 h to give cyclic reduced SFTI 18 (4.6 mg, 52%). To a solution of 18 (16.2 ꢂmol, 24.5 mg) in
0.1 M sodium phosphate (pH 7.5, 15.2 mL), DMSO (800 L, 5%, v/v) were added and the solution was
allowed to stand for 15 h at room temperature. The reaction mixture was quenched with 1N HCl for a
final pH of 2 and purified by using RPꢀHPLC to give SFTIꢀ1 (20.2 mg, 82%).
RPꢀHPLC and mass spectrometry
Peptides with different Cꢀterminal thioester surrogates and their intermediates were monitored by
analytical RPꢀHPLC (highꢀpressure analytical column Aeris Peptide 3.6u XBꢀC18, 250 x 4.6 mm, HPLC
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Biopolymers: Peptide Science
gradient 5ꢀ35% acetonitrile in 0.1% TFA for 60 min) and MALDIꢀTOF mass spectrometry (reflectronꢀ
mode) or ESI. The sequence analysis was performed by MS/MS.
REFERENCES
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2. Tam, J. P.; Lu, Y. A.; Eom, K. D. Tetrahedron Lett 1997, 38, 5599ꢀ5602.
3. Tam, J. P.; Wong, C. T. T. J Biol Chem 2012, 287, 27020ꢀ27025.
4. Tam, J.; Xu, J. Pept Sci 2001, 60, 194ꢀ205.
5. Mende, F.; Seitz, O. Angew Chem Int Ed 2011, 50, 1232ꢀ1240.
6. Dawson, P. E.; Muir, T. W.; ClarkꢀLewis, I.; Kent, S. B. Science 1994, 266, 776ꢀ779.
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8. Tam, J. P.; Lu, Y. A. Prot Sci 1998, 7, 1583ꢀ1592.
9. Li, X. Q.; Kawakami, T.; Aimoto, S. Tetrahedron Lett 1998, 39, 8669ꢀ8672.
10. Raz, R.; Rademann, J. Org Lett 2011, 13, 1606ꢀ1609.
11. Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi, A. J Am Chem Soc 1999, 121, 11369ꢀ11374.
12. Camarero, J. A.; Hackel, B. J.; de Yoreo, J. J.; Mitchell, A. R. J Org Chem 2004, 69, 4145ꢀ4151.
13. BlancoꢀCanosa, J. B.; Dawson, P. E. Angew Chem Int Ed 2008, 47, 6851ꢀ6855.
14. Tofteng, A. P.; Sørensen, K. K.; CondeꢀFrieboes, K. W.; HoegꢀJensen, T.; Jensen, K. J. Angew Chem Int Ed 2009, 48,
7411ꢀ7414.
15. Fang, G.ꢀM.; Li, Y.ꢀM.; Shen, F.; Huang, Y.ꢀC.; Li, J.ꢀB.; Lin, Y.; Cui, H.ꢀK.; Liu, L. Angew Chem Int Ed 2011, 50, 7645ꢀ
7649.
16. Aimoto, S. Biopolymers 1999, 51, 247ꢀ265.
17. Kawakami, T.; Akaji, K.; Aimoto, S. Org Lett 2001, 3, 1403ꢀ1405.
18. Hojo, H.; Onuma, Y.; Akimoto, Y.; Nakahara, Y.; Nakahara, Y. Tetrahedron Lett 2007, 48, 25ꢀ28.
19. Ollivier, N.; Dheur, J.; Mhidia, R.; Blanpain, A.; Melnyk, O. Org Lett 2010, 12, 5238ꢀ5241.
20. Hou, W.; Zhang, X.; Li, F.; Liu, C.ꢀF. Org Lett 2010, 13, 386ꢀ389.
21. Nagaike, F.; Onuma, Y.; Kanazawa, C.; Hojo, H.; Ueki, A.; Nakahara, Y.; Nakahara, Y. Org Lett 2006, 8, 4465ꢀ4468.
22. Sharma, R. K.; Tam, J. P. Org Lett 2011, 13, 5176ꢀ5179.
23. Xu, M.; Southworth, M.; Mersha, F.; Hornstra, L. Cell 1993, 75, 1371ꢀ1377.
24. Xu, M. Q.; Comb, D. G.; Paulus, H.; Noren, C. J.; Shao, Y.; Perler, F. B. EMBO J 1994, 13, 5517ꢀ5522.
25. Muir, T.; Sondhi, D.; Cole, P. Proc Natl Acad Sci USA 1998, 95, 6705ꢀ6710.
26. Tam, J.; Lu, Y.; Yang, J.; Chiu, K. Proc Natl Acad Sci USA 1999, 96, 8913.
27. Tam, J.; Lu, Y.; Yu, Q. J Am Chem Soc 1999, 121, 4316ꢀ4324.
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29. Olivera, B. M.; Gray, W. R.; Zeikus, R.; Mcintosh, J. M.; Varga, J.; Rivier, J.; Desantos, V.; Cruz, L. J. Science 1985, 230,
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FIGURE 1 Comparison of (a) intein-catalyzed protein splicing (I, intein; N, N-extein; C, C-extein) and (b)
thioethylamide (TEA)-mediated peptide cyclization using thioester surrogate (TS).
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Biopolymers: Peptide Science
FIGURE 2 Acyl shift reactions common to intein-meidated protein splicing and TEA-mediated peptide
cyclization using thioester surrogates in chemical synthesis. (a) Four acyl shift reactions found in intein-
mediated protein splicing. I, intein; N, N-extein; C, C-extein. (b) Three acyl shift reactions found in peptide
cyclization mediated by TEA thioester-surrogate. N, N-terminus; C, C-terminus.
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FIGRUE 3 The design of thioethylamide (TEA) thioester surrogates including (a) N-methyl Cys (MeCys), (b)
thiomethyl-thiazolidine (TMT) carboxylic acid and (c) thioethylalkylamide (TEXA) (where X = M for methyl, E
for ethyl, P for propyl, B for butyl).
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Biopolymers: Peptide Science
FIGURE 4 Strategy for the cyclization of ωꢀconotoxin MVIIA by a short linker sequence.
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FIGURE 5 Preparation of peptide-MeCys-Gly-NH2 on Rink amide resins using Fmoc synthesis. i = Fmoc-Gly,
DIC, HOBt, ii = 20% piperidine deprotection, iii = Fmoc-MeCys(Trt), DIC, HOBt,iv = Fmoc-Arg(Pbf), HATU,
DIEA, DMF, v = TFA/TIS/H2O (95/2.5/2.5, v/v), 2 h.
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Biopolymers: Peptide Science
FIGURE 6 Schema and HPLC analysis of CG29-MeCys-NH2 5 cyclization via tandem thiol switch reactions
mediated by intramolecular thiols or external thiols. The first N-S acyl shift of CG29-MeCys-Gly-NH2 5 in pH
2 solution resulted in the formation of the peptide thioester 6 that subsequently was trapped by internal
thiols to give intramolecular thiolactones 8a-d and 8f (c), or transformed into stable thioester CG29-MES 7
in the presence of external thiol MESNa (d). Thia-zip cyclization was performed at pH 7.5 at room
temperature for 2 h to give cyclic CG29 9 (e).
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FIGURE 7 The synthesis scheme of SFTI-TEAB on Cl-Trt(2-Cl) resin. i = compound 10 (2-
butylamino)ethanethiol, CH2Cl2, 30 min, ii = Fmoc-Arg(Pbf), HATU, DIEA, 1 h, iii = TFA/TIS/H2O
(95/2.5/2.5, v/v), 2 h.
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FIGURE 8 One-pot tandem thiol switch and cyclization of SFTI-TEAB. i = MESNa, 0.1 M sodium phosphate
(pH 3), 40°C, 17 h, ii = TCEP, pH 7 (addition of 2 N NaOH), rt, 3 h, iii = DMSO, 0.1 M sodium phosphate
(pH 7.5), 15 h.
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