Trifluoroethanethiol: An additive for efficient one-pot peptide ligation - Desulfurization chemistry

Article abstract of DOI:10.1021/ja502806r

Native chemical ligation followed by desulfurization is a powerful strategy for the assembly of proteins. Here we describe the development of a high-yielding, one-pot ligation-desulfurization protocol that uses trifluoroethanethiol (TFET) as a novel thiol additive. The synthetic utility of this TFET-enabled methodology is demonstrated by the efficient multi-step one-pot syntheses of two tick-derived proteins, chimadanin and madanin-1, without the need for any intermediary purification.

Full text of DOI:10.1021/ja502806r

Communication
pubs.acs.org/JACS
Trifluoroethanethiol: An Additive for Efficient One-Pot Peptide
Ligation−Desulfurization Chemistry
Robert E. Thompson,† Xuyu Liu,† Noelia Alonso-García,‡ Pedro Jose Barbosa Pereira,‡ Katrina A. Jolliffe,†
́
and Richard J. Payne*,†
†School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia
‡IBMCInstituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal
S
* Supporting Information
Scheme 1. Native Chemical Ligation−Desulfurization
ABSTRACT: Native chemical ligation followed by
desulfurization is a powerful strategy for the assembly of
proteins. Here we describe the development of a high-
yielding, one-pot ligation−desulfurization protocol that
uses trifluoroethanethiol (TFET) as a novel thiol additive.
The synthetic utility of this TFET-enabled methodology is
demonstrated by the efficient multi-step one-pot syntheses
of two tick-derived proteins, chimadanin and madanin-1,
without the need for any intermediary purification.
hemical synthesis of proteins provides a means by which
residues to Ala following the ligation event.4 This methodology
key structural and functional information on a given target
C
can be elucidated.1 Over the past two decades considerable
advances in this area have been possible owing to the
development of the venerable native chemical ligation method.2
This transformation involves the chemoselective reaction
between a peptide containing a C-terminal thioester and a
peptide bearing an N-terminal Cys residue to afford a native
peptide bond (Scheme 1). Due to the ease of preparation and
stability to long-term storage, alkyl thioesters are often employed
in ligation chemistry. However, this functionality is relatively
inert in the ligation reaction, necessitating the inclusion of a thiol
additive to generate a more reactive peptide thioester as the acyl
donor. A transthioesterification then occurs between the side
chain of the Cys residue and the newly formed thioester moiety,
followed by an irreversible intramolecular S→N acyl transfer to
form a native peptide bond.
Ligation technology has benefited greatly from the introduc-
tion of exogenous thiol additives to improve reaction rates via the
in situ generation of reactive peptide thioesters. In a thorough
study by Johnson and Kent, the relative reactivity of a range of
commercially available thiols was investigated.3 Aryl thiols with
pKa > 6 were shown to afford optimal ligation rates due to two
key reactive properties: (1) the ability to rapidly exchange with
alkyl thioesters to generate aryl thioesters and (2) excellent
leaving group ability upon reaction with the N-terminal Cys
residue. From this study the water-soluble aryl thiol additive
mercaptophenylacetic acid (MPAA, pKa = 6.6) was selected as an
excellent additive that facilitated more rapid ligations than two
other traditionally employed thiol additives, the water-soluble
alkyl thiol mercaptoethanethiolate sodium salt (MESNa, pKa =
9.2) and the sparingly water-soluble thiophenol (pKa = 6.6).
A significant advancement in ligation methodology was the
development of desulfurization chemistry which transforms Cys
sparked interest in the use of the native chemical ligation concept
at a variety of unnatural mercapto- and seleno-amino acids that
can subsequently be converted to native amino acids by
desulfurization or deselenization.5 While desulfurization of Cys
to Ala can be effected through the use of catalytic hydrogen-
ation,4a radical desulfurization4b is the most widely employed
method and has been used in the synthesis of a number of
complex protein targets.6 Given the high-yielding nature of
desulfurization chemistry, the union of this transformation with
efficient ligation chemistry into a one-pot procedure would
represent a powerful addition to the toolbox of methods for use
in chemical protein synthesis. Unfortunately, the necessity of aryl
thiol additives in the ligation reaction prohibits this capability due
to the inherent radical quenching activity of these species.7 As
such, products from ligation reactions require tedious
purification and lyophilization before the purified materials are
submitted to desulfurization. Solutions to this problem have been
sought, including the use of MESNa which, despite significantly
slower ligation rates, does not interfere with desulfurization
chemistry.6g Alternatively, methods to remove aryl thiols from
the reaction mixture have been employed, including extensive
liquid/liquid extraction of aryl thiols such as thiophenol, or solid-
phase extraction procedures.5q Recently, Brik and co-workers
employed a synthetic bifunctional aryl thiol catalyst that could be
captured with an aldehyde-derived solid-supported reagent prior
to the desulfurization reactions.8
In an effort to streamline the two highly efficient reactions into
a straightforward and operationally simple one-pot protocol, we
sought to identify a novel thiol additive capable of facilitating
Received: March 20, 2014
© XXXX American Chemical Society
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dx.doi.org/10.1021/ja502806r | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Communication
Table 1. One-Pot Ligation−Desulfurization Reactions
between Peptide 1 and Thioesters 2−6 Using TFET as a Thiol
Additive
entry
thioester
product
yield (%)
a
b
Figure 1. Kinetics for native chemical ligation reactions between peptide
1 and peptide thioesters Ac-LYRANX-S(CH2)2CO2Et (2−6; for 2, X =
V; 3, X = G; 4, X = F; 5, X = S; 6, X = L). The percent ligated was
calculated at each time point (mean of three independent experiments)
by integrating the areas under the peaks of analytical HPLC
chromatograms at λ = 280 nm. See SI for additional data and analysis.
1
2
3
4
5
Ac-LYRANV-SR (2)
Ac-LYRANG-SR (3)
Ac-LYRANF-SR (4)
Ac-LYRANS-SR (5)
Ac-LYRANL-SR (6)
7
8
84 (62)
a
b
b
b
b
82 (66)
a
9
80 (70)
a
10
11
88 (68)
a
85 (66)
a
Yields over two steps determined by analytical HPLC (see SI for
weighed isolated yields). Isolated yields following HPLC purification
b
rapid ligation without disrupting the subsequent radical
desulfurization. Here we show that 2,2,2-trifluoroethanethiol
(TFET) is an efficient thiol catalyst and, importantly, as an alkyl
thiol permits in situ one-pot desulfurization reactions. To
demonstrate the utility of TFET, we undertook the synthesis
of two small tick-derived proteins, chimadanin and madanin-1,
via one-pot ligation−desulfurization of three peptide fragments
either in the C→N-terminal direction or through kinetically
controlled ligation chemistry9 in the N→C direction, respec-
tively.
TFET (pKa = 7.30) has been shown to be similar to thiophenol
in its propensity to participate in thiol−thioester exchange.10
Owing to the comparatively low pKa of TFET compared with
other alkyl thiols, we envisaged that it would afford exchanged
thioesters with acyl-donor capabilities similar to those of
activated aryl thioesters. Furthermore, the fact that TFET is
relatively volatile (bp 35−37 °C) permits facile removal
following the ligation if necessary (unlike MPAA which can co-
elute with products during HPLC purification).
We began investigating the use of TFET as an additive in
native chemical ligation by comparing it with the commonly
employed aryl thiol additives thiophenol and MPAA and the
alkyl thiol additive MESNa, the most effective alkyl thiol catalyst
currently known. To this end, we studied a challenging model
ligation between model peptide 1 and peptide thioester 2,
bearing a sterically hindered C-terminal Val residue, one of the
slowest sites for native chemical ligation.11 Reactions were
carried out in parallel in ligation buffer comprising 6 M Gn·HCl,
100 mM Na2HPO4 buffer at pH 7.4−7.5 in the presence of 250
mM TFET, PhSH, MPAA, or MESNa as thiol additives.
Gratifyingly, ligation reactions carried out in the presence of
TFET were significantly more rapid than those employing PhSH
and MESNa (see Supporting Information (SI) for data). The
reaction reached completion within 4 h, comparable to the same
transformation employing MPAA, the current gold standard
additive (Figure 1).
determined by optical density at λ = 280 nm; R = (CH2)2CO2Et.
proved to be rapid (Figure 1). Specifically, reaction of 1 with
peptide thioester 3 containing a C-terminal Gly reached
completion in 20 min, while reaction with thioester 6 bearing a
sterically encumbered Leu residue reached completion in 90 min.
An important development in the field of peptide ligation
chemistry was the application of peptide hydrazides12 as masked
peptide thioesters.13 Importantly, we show that TFET is capable
of exchanging with acyl hydrazides bearing both C-terminal Gly
and Val residues, leading to rapid formation of the corresponding
TFET thioesters that were competent in ligation reactions with
model peptide 1 (see SI for details).
With the knowledge that TFET could promote extremely
rapid and high-yielding peptide ligation reactions, we next
investigated its ultimate utility in the context of one-pot ligation−
desulfurization chemistry (Table 1). Ligation reactions between
peptide 1 and peptide thioesters 2−6 in the presence of 2 vol%
(250 mM) TFET and 50 mM TCEP (Table 1) were left to
proceed for 4 h, the time at which the slowest ligation (at Val
thioester 2) was complete (Figure 1). At this stage, the ligation
product was not isolated, but rather the reaction mixture was
thoroughly degassed by sparging with argon in preparation for
the in situ radical desulfurization (see SI for full experimental
details). This also led to the removal of the vast majority of the
dissolved TFET owing to its volatility. At this point additional
TCEP was added to the degassed solution to generate a final
concentration of 200 mM, together with the radical initiator VA-
044 (20 mM) and reduced glutathione5b (40 mM) as a H-atom
source. Reactions were incubated at 37 °C for 16 h to ensure
complete desulfurization. Importantly, all one-pot ligation−
desulfurization reactions proceeded smoothly under these
conditions (80−88% yield as judged by analytical HPLC, see
Figure 2 for crude analytical data for the reaction between
peptide 1 and peptide thioester 4 and the SI for other raw data).
Following HPLC purification, the native peptide products were
isolated in good yields (62−70%, see Table 1) over the two steps,
highlighting the efficiency of the one-pot procedure (Table 1). It
is important to note that while TFET was removed prior to
desulfurization during the degassing step, we also show that
Having demonstrated that TFET was an efficient thiol additive
for ligation at a valyl-thioester, we next investigated the rate of
reaction between 1 and peptide thioesters 3−6 bearing a range of
C-terminal residues, representative of practical ligation junctions
(Figure 1). Gratifyingly, reaction rates between 1 and 3−6 all
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Journal of the American Chemical Society
Communication
Scheme 3. Synthesis of Madanin-1 (17) via a One-Pot
Kinetically Controlled Ligation−Desulfurization with TFET
a
Figure 2. Crude analytical HPLC-MS for one-pot ligation−desulfur-
ization reaction between 1 and 4 (λ = 230 nm). Peak a = 9; peak b = S
P(CH2)2CO2H.
Scheme 2. One-Pot Synthesis of Chimadanin (12) Using
a
TFET
a
Kinetically controlled ligation: 18 (1.2 equiv), 19 (1.0 equiv, 5 mM)
in buffer (6 M Gn·HCl, 100 mM Na2HPO4, 50 mM TCEP), pH 7.4−
7.5, 37 °C, 1 h, then addition of 20 (1.8 equiv), TFET (2 vol%), 37
°C, 12 h. Desulfurization: Ar sparge, adjust to TCEP (200 mM),
reduced glutathione (40 mM), VA-044 (20 mM) in buffer (6 M Gn·
HCl, 100 mM Na2HPO4), 2.5 mM final concn with respect to 21, pH
6.5, 37 °C, 16 h.
synthesis began with the preparation of the requisite fragments
via Fmoc-strategy SPPS, including chimadanin(42-70) (13)
possessing an N-terminal γ-thiol Glu residue, chimadanin(21-
41) (14) bearing an N-terminal thiazolidine and a C-terminal
thioester functionality, and chimadanin(1-20) thioester 15 (see
SI). Peptide 13 (1.2 equiv) bearing an N-terminal γ-thiol Glu
residue was first ligated with peptide thioester 14 (1.0 equiv) in
the presence of TFET. Following completion of the ligation (as
judged by HPLC-MS), the reaction mixture was treated with
methoxyamine at pH 4.2 to unmask an N-terminal Cys residue
and afford intermediate 16. Rather than purifying the
intermediate, the pH of the reaction mixture was adjusted to
6.8 before addition of the N-terminal chimadanin fragment,
peptide thioester 15, and TFET. Ligation of 15 and 16 was again
monitored by HPLC-MS; upon completion, the reaction was
degassed before treatment with additional TCEP, reduced
glutathione, and VA-044 to effect global desulfurization affording
the native protein. Gratifyingly, chimadanin was isolated in 35%
yield over the one-pot, four-step sequence following a single
HPLC purification step (ca. 77% average yield per step).
To further probe the limits of one-pot ligation−desulfurization
reactions employing the TFET additive, we next investigated the
potential of combining kinetically controlled ligation chemistry
with our one-pot methodology to assemble the 60 amino acid
protein madanin-1 (17, Scheme 3), a Cys-free competitive
thrombin inhibitor also produced by the hard tick H.
longicornis.16 The use of a kinetically controlled ligation sequence
would enable the rapid assembly of multiple madanin-1 peptide
segments in the N→C direction without intermediate
purification steps through appropriate reactivity tuning of the
requisite peptide thioesters.9 With a view to future analogue
generation, we were interested in assembling the protein via
three short segments: madanin-1(1-28) (18) as a preformed
a
(i) Ligation: 14 (1.0 equiv) and 13 (1.2 equiv) in buffer (6 M Gn·
HCl, 100 mM Na2HPO4, 25 mM TCEP), pH 6.8, 2.5 mM with
respect to 14, 2 vol% TFET, 30 °C, 2 h. (ii) Thiazolidine
deprotection: 0.2 M methoxyamine (to pH 4.2), 30 °C, 3 h. One-
pot ligation−desulfurization: for ligation, pH adjusted to 7.0, addition
of 15 (1.3 equiv, 3.0 mM) in buffer (6 M Gn·HCl, 100 mM Na2HPO4,
25 mM TCEP), pH 6.8, TFET (2 vol%), 1.0 mM with respect to 16,
30 °C, 18 h; for desulfurization, adjusted to 500 mM TCEP and 40
mM reduced glutathione, Ar sparge, pH adjusted to 6.2, solid VA-044
(20 mM final concn), 37 °C, 5 h.
TFET does not have a detrimental effect on the desulfurization
rate when present in solution (see SI for details).
Having demonstrated the utility of TFET as an additive for
efficient and operationally simple one-pot ligation−desulfur-
ization reactions, we were next interested in extending the scope
of the methodology to the practical synthesis of some small
protein targets. Our first target protein was the 70 amino acid
thrombin inhibitory protein chimadanin (12, Scheme 2)
produced by the hard tick Haemaphysalis longicornis to facilitate
the hematophagous activity of the organism.14 We envisaged the
synthesis of the protein via the assembly of three fragments in the
C→N direction. Specifically, we proposed using a γ-thiol Glu
ligation5q followed by a native chemical ligation−desulfurization
at Cys that would proceed with concomitant desulfurization of
the γ-thiol auxiliary on the Glu residue to generate the native
protein. Importantly, this proposed one-pot strategy would
abolish intermediary purification steps, thus limiting the
exposure of the sensitive γ-thiol moiety to acidic HPLC buffers,
which leads to thiolactamization and peptide cleavage.5q,15 The
C
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Journal of the American Chemical Society
Communication
Commun. 2010, 46, 21. (f) Unverzagt, C.; Kajihara, Y. Chem. Soc. Rev.
2013, 42, 4408. (g) Wang, L.-X.; Amin, M. N. Chem. Biol. 2014, 21, 51.
(2) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science
1994, 266, 776.
TFET-thioester, madanin-1(29-47) (19) bearing an N-terminal
β-thiol Asp residue and an unreactive C-terminal alkyl thioester,
and madanin-1(48-60) (20) possessing an N-terminal Cys
residue (Scheme 3 and SI). Peptide thioester 18, activated as the
preformed TFET-thioester, was first ligated with peptide
thioester 19 bearing an N-terminal β-SH Asp5r moiety and a
C-terminal Thr residue. Following completion of the ligation
after 1 h (as judged by HPLC-MS), peptide 20 was added in
combination with 2 vol% TFET to activate the alkyl thioester and
facilitate a second ligation reaction. Following completion of the
second ligation (12 h), the product 21 was not isolated but rather
subjected to in situ desulfurization of both the Cys and β-thiol
Asp residues to afford the native protein madanin-1 (17) in an
excellent 42% yield over the three steps. To our knowledge, this
represents the first report of a one-pot kinetically controlled
ligation−desulfurization reaction and clearly highlights the utility
of TFET in the context of chemical protein synthesis.
Importantly, the in vitro inhibitory activities of chimadanin (12,
IC50 = 788 nM) and madanin-1 (17, IC50 = 1590 nM) against the
amidolytic activity of thrombin were shown to be similar to that
reported for recombinant madanin-1,16b thus confirming that the
synthetic proteins possessed the expected thrombin-inhibiting
activity (see SI).
In summary, we demonstrate that the alkyl thiol TFET can be
successfully employed as an additive in native chemical ligation
to facilitate ligations with rates comparable to those obtained
with the gold standard additive, MPAA. More importantly,
TFET can be used in ligation−desulfurization chemistry without
the need for intermediate purification or removal/capture from
the reaction mixture. We highlight the utility of TFET as an
additive for one-pot ligation−desulfurization reactions both on
model peptide systems and in the assembly of multiple peptide
fragments to access proteins. Specifically, we used the additive for
the efficient assembly of the tick-derived thrombin inhibitory
proteins chimadanin and madanin-1 through C→N assembly
and kinetically controlled approaches, respectively. Given the
efficiency and simplicity of ligations employing TFET (a
commercially available and affordable reagent), we anticipate
that it will find widespread use in the chemical synthesis of
proteins and post-translationally modified proteins, greatly
improving the efficiency of the processes and reducing handling
and purification of intermediates.
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ASSOCIATED CONTENT
* Supporting Information
Experimental details and analytical data. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
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AUTHOR INFORMATION
Corresponding Author
richard.payne@sydney.edu.au
Notes
■
(13) Zheng, J. S.; Tang, S.; Qi, Y. K.; Wang, Z. P.; Liu, L. Nat. Protoc.
2013, 8, 2483.
The authors declare no competing financial interest.
(14) Nakajima, C.; Imamura, S.; Konnai, S.; Yamada, S.; Nishikado, H.;
Ohashi, K.; Onuma, M. J. Vet. Med. Sci. 2006, 68, 447.
ACKNOWLEDGMENTS
■
(15) Tam, J. P.; Yu, Q. T. Biopolymers 1998, 46, 319.
The authors thank the Australian Research Council for research
funding (Discovery Project 120100194).
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