Synthesis of thioester peptides for the incorporation of thioamides into proteins by native chemical ligation

Article abstract of DOI:10.1002/psc.2589

Thioamides can be used as photoswitches, as reporters of local environment, as inhibitors of enzymes, and as fluorescence quenchers. We have recently demonstrated the incorporation of thioamides into polypeptides and proteins using native chemical ligation (NCL). In this protocol, we describe procedures for the synthesis of a thioamide precursor and an NCL-ready thioamide-containing peptide using Dawson's N-acyl-benzimidazolinone (Nbz) process. We include a description of the synthesis by NCL of a thioamide-labeled fragment of the neuronal protein α-synuclein. Copyright 2014 European Peptide Society and John Wiley & Sons, Ltd. Thioamides can be used as photoswitches, as reporters of local environment, as inhibitors of enzymes, and as fluorescence quenchers. We have recently demonstrated the incorporation of thioamides into polypeptides and proteins using native chemical ligation (NCL). In this protocol, we describe procedures for the synthesis of a thioamide precursor and an NCL-ready thioamide-containing peptide using Dawson's N-acyl-benzimidazolinone (Nbz) process. We include a description of the synthesis by NCL of a thioamide-labeled fragment of the neuronal protein α-synuclein. Copyright

Full text of DOI:10.1002/psc.2589

Protocol
Received: 31 August 2013
Revised: 21 October 2013
Accepted: 24 October 2013
Published online in Wiley Online Library: 9 January 2014
(wileyonlinelibrary.com) DOI 10.1002/psc.2589
Synthesis of thioester peptides for the
incorporation of thioamides into
proteins by native chemical ligation‡
Solongo Batjargal, Yun Huang, Yanxin J. Wang and E. James Petersson*
Thioamides can be used as photoswitches, as reporters of local environment, as inhibitors of enzymes, and as fluorescence quenchers.
We have recently demonstrated the incorporation of thioamides into polypeptides and proteins using native chemical ligation (NCL).
In this protocol, we describe procedures for the synthesis of a thioamide precursor and an NCL-ready thioamide-containing peptide
using Dawson’s N-acyl-benzimidazolinone (Nbz) process. We include a description of the synthesis by NCL of a thioamide-labeled
fragment of the neuronal protein α-synuclein. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: Thioamide; Native Chemical Ligation; Thioester; Fmoc SPPS
Scope and Comments
*
Correspondence to: E. James Petersson, Department of Chemistry, University of
Pennsylvania, Philadelphia, PA, USA. E-mail: ejpetersson@sas.upenn.edu
The thioamide is a nearly isosteric substitution of the carbonyl
oxygen in the peptide bond with a sulfur atom [1]. Conservative
backbone modification with a thioamide has been used to ana-
lyze the importance of specific hydrogen bonds to the folding
of secondary structure motifs and to coupled protein folding
and binding processes [2,3]. Because of its enhanced proteolytic
‡
This article is published in Journal of Peptide Science as part of the Special Issue
devoted to contributions presented at the Chemical Protein Synthesis Meeting,
April 3–6, 2013, Vienna, edited by Christian Becker (University of Vienna, Austria).
Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA
J. Pept. Sci. 2014; 20: 87–91
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

BATJARGAL ET AL.
stability and modulated activity, thioamide substitution has also
been applied in several biologically active oligopeptides [4–6].
For example, Fischer and coworkers showed that a thioamide-
containing peptide substrate resisted hydrolysis by prolyl
oligopeptidase [5]. The red-shifted absorption band of the
thioamide peptide bond can be used in several different ways
to study structural changes in the backbone with high spatial
and temporal control. The π-to-π* transition at 270 nm and the
n-to-π* transition at 340 nm give rise to thioamide-specific CD
signatures that can be identified against a background of
oxoamide signal at shorter wavelengths [7,8]. Wildemann et al.
assembled ribonuclease S (RNAse S) through noncovalent associ-
ation of a proteolytic fragment of RNAse S and a synthetic
thiopeptide and turned its enzymatic activity on and off through
cis/trans photoisomerization of the thioamide [9]. Our laboratory
expanded the utility of thioamides by demonstrating that
a thioamide can act as a quencher of various fluorophores
such as endogenous Tyr or Trp, and the unnatural amino
acids p-cyanophenylalanine, 7-methoxycoumarin-4-ylalanine, and
acridon-2-ylalanine [10–12]. Quenching can occur through either
Förster resonance energy transfer or photo-induced electron
transfer mechanisms, depending on the identity of the chromo-
phore. The placement of thioamides is expected to be tolerated
in almost any position in a protein because of its small size,
although the sulfur substitution may prove disruptive to hydro-
gen bonding in some cases [7,13–15]. Despite these attractive
applications, the incorporation of thioamides has traditionally
been limited to peptides made through solid phase peptide
synthesis (SPPS).
Recently, we have shown that a thioamide can be incorporated
into a full-length protein by exploiting the native chemical liga-
tion (NCL) reaction [16,17]. NCL requires two fragments: one
bearing an N-terminal Cys and the other bearing a C-terminal
thioester [18]. The synthesis of the N-terminal Cys fragment is
straightforward, but the development of an efficient strategy
for synthesizing a thioamide-containing C-terminal thioester
using Fmoc-based SPPS has required considerable study. Boc-
based SPPS, commonly used in the NCL community, is not viable
as the acidic cleavage conditions degrade the thioamide peptide.
We have previously published syntheses using solution-phase
thioesterification with PyBOP activation as well as on-resin
thioesterification using an Aimoto-type Cys-Pro-Gly linker where
the Cys is protected as a disulfide and Gly is replaced by glycolic
acid (CbPGo) (Figure 1) [16,19]. However, the former method could
lead to epimerization at the α-carbon of the C-terminal residue and
often suffers from poor solubility of the protected peptides [20].
Although the latter method alleviates the epimerization problem,
it requires one to synthesize a dipeptide with the C-terminal residue
of the thioester fragment coupled to Cys, which tends to couple to
the Pro on the resin in a very low yield. In unpublished work, we
have used an α-hydroxycysteine (Cya) strategy developed by Muir
and Botti to generate thiopeptide thioesters (Figure 1) [21,22].
While the latent thioester of Cya did ligate rapidly, the synthesis
of Cya is non-trivial (11% yield over five steps) and may be a barrier
for some users. In contrast to these three methods, Dawson’s
N-acyl-benzimidazolinone (Nbz) strategy could be used to
generate thioamide-containing thioester peptides in relatively
high yields and involves a simple synthesis using commercially
available components [23]. After elongation of the peptide
chain on 3,4-diaminobenzoyl (Dbz) resin, the introduction of
4-nitrophenylchloroformate closes the ring to form the N-acylurea
moiety, Nbz.
In this protocol, we describe our exploration of the compatibil-
ity of thioamides with the acylating conditions and report an
optimized procedure for the synthesis of Nbz-thiopeptides. In
addition, we describe the synthesis of the Fmoc-based SPPS
thioamide precursor, thiocarboxybenzotriazole 4a, using 4-nitro-
1,2-phenylendiamine and P4S10 (Scheme 1) [24]. The use of
4-nitro-1,2-phenylenediamine presents several advantages over
N-Boc-phenylenediamine, which was used in our previous reports:
(i) 4-nitro-1,2-phenylenediamine is generally more affordable
than N-Boc-phenylenediamine, (ii) the removal of the Boc
protecting group with TFA is omitted, simplifying the procedure
slightly, and (iii) the nitro route is compatible with amino acids
bearing acid-labile protecting groups such Glu or Asp. There are
some advantages to the Boc route; Boc-protection suppresses
the formation of a benzimidazole (BzIm) side product during the
thionation step (conversion of 2b to 3b), and the lack of the nitro
group in the benzotriazole products (e.g. 4b) makes them more
stable. Nonetheless, the nitro route (i.e. 2a–4a) is generally prefer-
able. The most common thionation reagents are Lawesson’s
reagent (LR) and P4S10 [25,26]. Although LR is reported to have
higher thionation yields, this was not observed for certain amino acids
in our hands. For Ala, thionation with LR was higher yielding (89% vs
10%). However, the yield for the rest of the amino acids tested was
limited to 10–35%, and recycling of the unreacted oxoanilide (2a)
Figure 1. Other Fmoc-based thioesterification methods tested. Top: off-resin activation of protected peptides. Middle: on-resin synthesis of protected
Cys-Pro-glycolic acid (CbPGo) linker, with off-resin activation by reduction of t-Bu disulfide. Bottom: on-resin incorporation of α-hydroxycysteine (Cya),
with off-resin activation by reduction of t-Bu disulfide.
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SYNTHESIS OF THIOAMIDE-CONTAINING THIOESTER PEPTIDES
Scheme 1. Synthesis of Fmoc-thiovaline-benzotriazole derivatives 4a. Reagents and conditions: (i) NMM, isobutylchloroformate, 4-nitro-1,2-
phenylenediamine, THF, overnight, r.t. (90%), (ii), P4S10, Na2CO3, THF, r.t. (86%), and (iii) NaNO2, AcOH, H2O, r.t. (90%). Compounds 2b, 3b, and 4b are
shown for discussion purposes.
was necessary to obtain a reasonable thionation yield. In contrast,
thionation with P4S10 proved to be more efficient and higher yielding,
particularly for β-branched amino acids such as valine.
generated in situ reacts with the primary amine of compound
3a, forming a benzotriazole through intramolecular diazonium
cyclization. This reaction completed in 30 min, and compound
4a precipitated upon the addition of cold water to the reaction
solution. Purification via silica chromatography afforded very
pure 4a as an orange solid in a 90% yield. However, Ala and other
amino acids with protected side chains such as Asp degraded in the
process of chromatographic purification. Thus, we recommend
minimal handling of the benzotriazoles to prevent hydrolysis and
degradation through cyclization. As long as the aminothioacyl-
anilide (e.g. 3a) is pure going into the cyclization reaction, precipi-
tation provides sufficiently pure material for peptide coupling. After
precipitation followed by filtration, the nitrobenzotriazole 4a was
dried in the presence of P2O5 under vacuum at r.t. overnight and
was used for peptide synthesis. The detailed procedures and 1H
and 13C NMR spectra for compound 2a, 3a, and 4a are reported
in the Supporting Information.
Here, we describe the preferred synthetic strategy for the
thioamide precursor, an efficient method to obtain thiopeptide
thioesters using Dawson’s resin, and the use of a thiopeptide
thioester in an NCL reaction to synthesize a fragment of the
Parkinson’s disease protein α-synuclein, Ac-αS1-19 V′3 (the prime
symbol denotes a backbone thiocarbonyl at the indicated resi-
due) [27,28]. Recently, Lee and coworkers have characterized
the structure and binding affinity of this fragment to the calcium
signaling protein calmodulin (CaM) [29]. Our laboratory is cur-
rently exploring the αS/CaM interaction using thioamide fluores-
cence quenching.
Experimental Procedure
Optimized Thiovaline Precursor Synthesis
Thiopeptide–Nbz Synthesis
The synthesis of thiovaline precursor 4a is presented as a general
method. We chose to compare methods using Fmoc-Val-OH (1)
because the thionation yield of this amino acid was particularly
low using previously published methods. As a first step, the
carboxyl group of Fmoc-Val-OH was amidated with 4-nitro-1,
2-phenylenediamine, a latent benzotriazole. To a solution of Fmoc-
Val-OH in THF stirred in an NaCl/ice bath at À10 °C, NMM and
isobutylchloroformate were added to generate Fmoc-Val-anhydride
in situ, and then, 4-nitro-1,2-phenylenediamine was added. After
stirring overnight at room temperature (r.t.), the reaction solvent
THF was removed by rotary evaporation. The crude mixture
was dissolved in DMF, and a saturated KCl solution was added,
resulting in the precipitation of a yellow solid. This precipitation
removes the NMM salts effectively, although organic side prod-
ucts precipitate too. Because of the poor solubility of compound
2a in DCM, complete purification via silica chromatography was
difficult. Thus, compound 2a was isolated in 90% yield with 73%
purity after KCl precipitation.
The thiopeptide Ac-MDV′FMKGL-Nbz (7) was synthesized on
commercially available Dawson Dbz AM resin (Novabiochem®, San
Diego, CA, USA). After removal of the Fmoc protecting group, the first
amino acid was loaded by HATU/DIPEA activation. The peptide was
elongated by standard SPPS procedures with HBTU/DIPEA activation.
Thiovaline was introduced by adding the preactivated derivative
Fmoc-thioval-nitrobenzotriazole (4a) with DIPEA but without HBTU.
The last amino acid was loaded as Ac-Met to avoid undesired acety-
lation on the Dbz group when an acetylating reagent such as Ac2O is
used. After assembly of peptide 5, the resin was treated with p-
nitrophenyl chloroformate to form the peptidyl carbamate 6, which
was then converted to peptide–Nbz resin by a subsequent treatment
of DIPEA. The Nbz peptide 7 was cleaved from resin by TFA treat-
ment and purified by preparative HPLC. The detailed procedures
for peptide synthesis are provided in the Supporting Information.
Native Chemical Ligation
Thionation of Fmoc-Val-nitroanilide 2a with LR resulted in
either low conversion at r.t. or formation of significant amounts of
a benzimidazole side product at 70°C. On the other hand,
thionation with P4S10 was high yielding with minimal side product
formation. To a solution of P4S10 and anhydrous Na2CO3 in THF,
compound 2a was added and stirred at r.t under slow argon flow.
The reaction was monitored by TLC to track the consumption of
2a. Upon completion, the reaction solution was filtered through
Celite, washed with 5% NaHCO3, and purified on a silica column
to afford pure compound 3a as yellow solid in 86% yield. Purifica-
tion of P4S10 by Soxhlet extraction is often recommended. We find
this to be unnecessary and obtain high yields with commercial
P4S10 stored in a dessicator at r.t. and used directly.
To a solution of 6 M guanidinium hydrochloride (Gdn•HCl) and
200 mM sodium phosphate, triscarboxyethyl phosphine and
thiophenol were added such that their final concentrations were
20 mM and 1% (v/v), respectively. It is crucial to adjust the pH of
the solution to 7.0 and to degas it with Ar before use in ligation.
Ac-αS1-8 V′3-Nbz (7, 0.35 μmol) and αS9-19C9 (8, 0.51 μmol) were
dissolved in NCL buffer, and the reaction was initiated upon
combining the two peptides. After purging with Ar briefly, the
reaction solution was placed in an incubator and shaken at
1000 rpm at 37 °C overnight. An aliquot (25 μl) was taken out
periodically, quenched by the addition of 975 μl of 0.1% TFA in water,
and stored at À20 °C until analysis by analytical reversed phase HPLC.
Conversion to the product, Ac-αS1-19 V′3 (9), was greater than 95% by
peak area (relative to unreacted 7 and hydrolyzed 7) after 30min
To form the benzotriazole 4a, NaNO2 was added to compound
3a in glacial acetic acid diluted with 5% water. Nitrous acid
J. Pept. Sci. 2014; 20: 87–91 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

BATJARGAL ET AL.
Scheme 2. Synthesis of Ac-αS1-8 V´3-Nbz (7). Reagents and conditions: (i) SPPS on Dbz AM resin, r.t., (ii) p-nitrophenyl chloroformate, r.t., (iii) DIPEA/DMF,
r.t., and (iv) TFA/TIPS/thioanisole/DCM (80 : 5 : 2.5 : 12.5), r.t.
Figure 2. Native chemical ligation to form Ac-αS1-19 V´3 (9). Left: reaction scheme. Right: reaction monitored by HPLC (monitored at 215 nm) and MALDI
MS of product (9) peak (m/z Calcd: 2026.0, Found: 2026.3). Asterisk indicates the expected mass of Ac-αS1-19 (oxoamide at position 3).
(Figure 2). The thiopeptide reactant and products are sufficiently sta-
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J. Pept. Sci. 2014; 20: 87–91 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci