Coupling of sterically demanding peptides by β-thiolactone-mediated native chemical ligation

Article abstract of DOI:10.1039/c7sc04744d

The ligation of sterically demanding peptidyl sites such as those involving Val-Val and Val-Pro linkages has proven to be extremely challenging with conventional NCL methods that rely on exogenous thiol additives. Herein, we report an efficient β-thiolactone-mediated additive-free NCL protocol that enables the establishment of these connections in good yield. The rapid NCL was followed by in situ desulfurization. Reaction rates between β-thiolactones and conventional thioesters towards NCL were also investigated, and direct aminolysis was ruled out as a possible pathway. Finally, the potent cytotoxic cyclic-peptide axinastatin 1 has been prepared using the developed methodology.

Full text of DOI:10.1039/c7sc04744d

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Coupling of sterically demanding peptides by b-
thiolactone-mediated native chemical ligation†
Cite this: DOI: 10.1039/c7sc04744d
Huan Chen,a Yunxian Xiao,a Ning Yuan,c Jiaping Weng,a Pengcheng Gao,a
a
Leonard Breindel,b Alexander Shekhtmanb and Qiang Zhang
*
The ligation of sterically demanding peptidyl sites such as those involving Val–Val and Val–Pro linkages has
proven to be extremely challenging with conventional NCL methods that rely on exogenous thiol additives.
Herein, we report an efficient b-thiolactone-mediated additive-free NCL protocol that enables the
establishment of these connections in good yield. The rapid NCL was followed by in situ desulfurization.
Reaction rates between b-thiolactones and conventional thioesters towards NCL were also investigated,
and direct aminolysis was ruled out as a possible pathway. Finally, the potent cytotoxic cyclic-peptide
axinastatin 1 has been prepared using the developed methodology.
Received 2nd November 2017
Accepted 2nd January 2018
DOI: 10.1039/c7sc04744d
rsc.li/chemical-science
additive-free NCL followed by desulfurization, and provides
a signicant advance in protein synthesis.
Introduction
NCL is typically initiated by adding an exogenous thiol
additive such as 4-mercaptophenylacetic acid11 (MPAA 1)
(Fig. 2a); a reversible transthioesterication between an acti-
vated thioester and cysteine provides a tethered peptidyl
adduct, which then undergoes a facile N–S shi to form a new
amide bond. There are two critical drawbacks associated with
the use of 1. When the coupling partners involve bulky side-
chains, the MPAA thiol exchange outcompetes the forward N–S
shi, resulting in the disintegration of the tethered peptide and
low yields. In addition, it necessitates the inclusion of a step to
remove the thiol additive prior to the desulfurization. Although
there are multiple procedures for simplied MPAA removal,7,11
Despite the major advances in the synthetic coupling of
peptides and proteins such as the use of Native Chemical
Ligation (NCL) procedures1 and metal free desulfurization,2 the
accomplishment of ligations3 involving amino acids with
sterically demanding side chains continues to be problematic
(Fig. 1a). In the previous reports by Bertozzi,4 Danishefsky4 and
others,4 the ligation sites involving valine or proline were fur-
nished in low yield and required an extended reaction time.
This challenge is particularly relevant for the synthesis of
membrane proteins in which amino acids such as Val, Ile, Leu,
and Ala constitute more than one-third of the transmembrane
domains.5 Thus, the need to develop methodologies for the
ligation of these residues is tremendous. Although considerable
efforts have been directed towards improving the ligation effi-
ciency at sterically hindered sites,6,7 such as the elegant report
by Dong8 on the internal activation of thioesters promoting the
occurrence of ligation at proline sites, the problem remains and
continues to place severe limitations on the ability to access
membrane proteins by NCL.9,10 Herein we describe a protocol
that utilizes readily available b-thiolactones to facilitate the
challenging peptide ligation at sterically demanding Val–Ala,
Val–Leu, Val–Val, and Val–Pro sites (Fig. 1b). This allows the
connection of two bulky peptidyl residues, through a one-pot
aDepartment of Chemistry, University at Albany, State University of New York, 1400
Washington Avenue, Albany, NY 12222, USA. E-mail: qzhang5@albany.edu
bDepartment of Chemistry, University at Albany, State University of New York, 1400
Washington Avenue, Albany, NY 12222, USA
cState Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical
Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7sc04744d
Fig. 1 The coupling of sterically hindered peptides with b-thiolactone
by NCL.
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thiolactones 2 and 3 (ref. 18) in high yield (Table 1). Under the
standard NCL conditions with the presence of MPAA, the
mixture of thietan 2 and cysteine ester 4 produced the dipeptide
5 in 82% yield. The ligation between 2 and 4 in the absence of 1
was completed within 30 min, and the dipeptide 5 was ob-
tained. Subsequent in situ desulfurization cleanly provided Ala–
Ala dipeptide 6. Although b-thiolactones are known to undergo
reactions at their carbon sites,19 we did not observe any by-
products. Aer screening, the use of Na2HPO4 buffer provided
the best result, furnishing the dipeptide 6 in 84% yield over two
steps. The addition of Gn$HCl lowered the reaction yield, and
the presence of TCEP did not change the ligation outcome.
Under the optimized reaction conditions, the scope of the
reaction was investigated. It was found that L-cysteine boosted
the reaction yield to 90% when coupled to thiolactone 2 (Table
1, entry 1). The reaction between 2 and L-Pen20 9c smoothly
yielded the Ala–Val dipeptide 10c. The coupling of thiolactone 2
and selenocystine 9d generated the desired product 10b in two
steps.21 Interestingly, the mass spectrum of the initial ligated
dipeptide was messy, and no meaningful mass peaks could be
identied; however, aer the desulfurization, the mass spec-
trum shows that dipeptide 10b can be detected as the major
product. We believed that our observation was caused by the
poor solubility of the intermediate dipeptide. The
penicillamine-derived thiolactone 3 was next investigated. It
was reacted with cysteine to provide the corresponding dipep-
tide 10d in 81% yield. The thiolactone-bearing dipeptide 7,
prepared using a literature reported procedure,15 was mixed
with dipeptide 9g in the NCL buffer to produce tetrapeptide 10l
in excellent yield (80%). Finally, the Pen dipeptide 8 was
examined. The tripeptide 10m and tetrapeptides 10n and 10o
could be prepared in good yield. To further explore the reaction
scope and limitations, we embarked on studies of more
extended peptides (Table 2). The thiolactone bearing peptides
were prepared using an EDC/HOOBt coupling method23 fol-
Fig. 2 Complication of the NCL when an external thiol activator is
added.
reverse phase preparative HPLC purication remains the most
effective technique. The additional purication and subsequent
requisite lyophilization further lower the reaction yield and
efficiency.
Results and discussion
We envisioned that the constraints mentioned above could be lowed by global sidechain deprotection. The peptides 11–15
overcome if b-thiolactones12 were used as thioester surrogates13 were generated without epimerization.24 Ligation between
(Fig. 2b). Considering that the concept of using macro- peptidyl fragments 11 and 16a provided polypeptide 17a in good
thiolactones as thioesters has been elegantly illustrated by the yield. The reaction between 12 and 16a afforded 17b in 86%
Nishiuchi group,14 the relief of the b-thiolactone ring strain yield aer two steps. The reaction between thiolactone 11 and
might promote ligation without pre-activation. Furthermore,
a
3-mercapto-leucine-bearing peptide25 16b provided the
difficulty of regenerating the thietane could circumvent the desired product 17c in high yield. Interestingly, considering the
need for the thiol additive, which might not only enable in situ notorious difficulty of amide bond generation between a valine
desulfurization, but it might also prevent the reversible trans- and valine or proline residues,4 we attempted to construct Val–
thioesterication and improve the reactivity at the sterically Val and Val–Pro bonds using our protocol. The coupling
hindered sites. This accessible methodology could provide between 12 and 16c occurred smoothly and the overall Val–Val
a signicant advance in protein synthesis and eliminate the linked polypeptide 17e was produced in good yield over two
additional steps typically required for peptide ligation, while steps (76%). Finally, the possibility of ligating valine and proline
achieving the synthesis of sterically challenging proteins and constructs was examined. The linkage of 12 and 16d was
peptides with high efficiency. Pioneering work,15 elegantly successfully achieved in 57% yield in a one-pot fashion. A few
illustrated by Crich and co-workers, has demonstrated the more examples of coupling between thiolactones and 4-
connections of single amino acids in the presence of exogenous mercapto-prolines were probed, and the yields varied based on
thiol activators, however the investigation of b-thiolactones in the sequences (42% for 17g and 32% for 17h). The b-thiolactone
the context of polypeptides as well as at sterically hindered sites mediated ligation has a wide functional group tolerance;
has been elusive.16 Our approach commenced with the cycliza- a variety of amino acid residues have been investigated, and the
tion of commercially available Boc-L-cysteine and Boc-L-peni- side-chains of Asp, Lys, Arg, His, Thr, Glu, Gln, and Tyr do not
cillamine (Boc-Cys and Boc-Pen respectively)15,17 to furnish interfere with the NCL (Table 2 entries 9 and 10). It is worth
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Table 1 Scope of the b-thiolactone mediated one-pot NCL and desulfurization methoda
Entry
Thiolactones
Ligating amino acids/peptides
Products
Yield (%)
90%
1
53%
80%
2
2
2
3
4
81%
77%
5
6
2
3
67%
69%
9f
7
8
3
71%
4
79%
80%
9
7
7
9f
10
9g
62%
11
4
72%
77%
12
13
8
8
9f
9g
a
Reagents and conditions: (a) buffer (0.1 M Na2HPO4, 0.01 M TCEP, pH ¼ 7.8), rt, 1–8 h; (b) desulfurization buffer (0.5 M TCEP pH ¼ 7.2, 0.1 M VA-
044, t-BuSH), 37 ꢀC, 2 h.
noting that among all the ligations described in Table 2, the HPLC purication and the known obstacle of thiol removal
initial NCL conversions were quantitative or near-quantitative;26 required for hydrophobic residues,27 this method still provides
it was the subsequent desulfurization that signicantly reduced a fundamental improvement over current protocols.
the overall reaction yield. More specically, regarding the
Intrigued by the facile b-thiolactone reactivity, we next
sequence 17h in entry 8 which gave the lowest yield, the investigated the reaction mechanism and rate. Reaction rates of
precursor product (bis-thiol) was isolated in 54% yield prior to the thiolactone and other known activated thioesters10,28 were
desulfurization. Considering the yield reduction from prep- compared (Fig. 3a). By monitoring with LC-MS, the ligation of
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a
Table 2 Studies of the b-thiolactone mediated one-pot NCL method with polypeptides22
Entry
Thiolactones
Ligating peptides
Products
Yield (%)
84%
1
2
86%
73%
81%
76%
3
4
5
57%
42%
6
7
32% (54%)
82%
8
9
83%
10
a
Reagents and conditions: (a) EDC (2.5 equiv.), HOOBt (2.5 equiv.), CH2Cl2, ꢂ20 ꢀC, (b) TFA/H2O/TIPS ¼ 95/2.5/2.5; NCL conditions: 0.1 M
Na2HPO4, 0.01 M TCEP, pH ¼ 7.8, rt, 1–8 h; desulfurization: buffer (0.5 M TCEP pH ¼ 7.2, 0.1 M VA-044, t-BuSH), 37 ꢀC, 2–3 h. * ¼ isolated
yield aer the initial NCL shown in parentheses.
the L-cysteine and thiolactone-bearing peptide 13 was deter- 16a were consumed, furnishing 17i exclusively. The direct
mined to be completed within 5 min. Analogously, peptides that aminolysis product 17j was not observed, which suggested that
possessed phenyl selenoesters 13a/13c and phenyl thioesters thietan-mediated peptide ligation did indeed proceed by the
13b/13d were subjected to identical reaction conditions. Aer NCL pathway.29
5 min, alanine phenyl selenoester 13a was consumed by 55%
With our enhanced understanding of the strain facilitated
and alanine phenyl thioester 13b was converted by 4%. More peptide ligation, we explored an expansion of the reaction scope
intriguingly, the analogous valine phenyl selenoester 13c and and application by pursuing the synthesis of a cyclic peptide
thioester 13d were merely consumed by 4% and 1% respectively axinastatin 1 (20) with our methodology (Scheme 1). Axinastatin
during the same period. This experiment illustrated that b-thi- 1 is a naturally occurring cyclic heptapeptide which was isolated
olactone was a superior thioester compared to existing activated in minute quantities from a marine sponge (4.5 ꢁ 10ꢂ5
%
thioesters regarding the reaction rate. Using a cross-over yield).30 It shows potent cytotoxicity against a leukemia cancer
experiment, we probed whether the reaction proceeded cell line (ED50 ¼ 0.21 mg mLꢂ1). Structurally, 20 contains three
through an NCL process or an undesired aminolysis pathway valines and two prolines in the seven amino acid backbone.
(Fig. 3b). A combination of peptides 13, 16a and 16f were mixed Importantly, Pro2 and Pro6 of the naturally occurring axinas-
in the NCL buffer. Aer 60 min, two major peaks appeared in tatin 1 are in trans and cis congurations, respectively. The
the LC trace. One peak corresponded to peptide 17i, which is synthesis herein commenced with the preparation of 18 using
the desired NCL product, and another was identied as the an Fmoc solid phase peptide synthesis with a TGT resin, and the
unreacted peptide 16f. At 120 min, the trace amounts of 13 and thiolactone 19 was produced through the coupling of 3a and the
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Scheme 1 Synthesis of cytotoxin axinastatin 1.
Conclusions
In summary, we have successfully developed a novel one-pot
protocol for peptide ligations at sterically demanding sites
utilizing the strained b-thiolactone. The strain not only greatly
accelerates the reaction and enables in situ thiol removal, but
also provides a practical method for the otherwise challenging
ligation linking sterically hindered peptidyl residues. The
reaction scopes and limitations have been evaluated and dis-
cussed, and the b-thiolactones proved to be a much more rapid
thioester surrogate compared to conventional thioesters.
Furthermore, the mode of the reaction has been veried to be
an NCL pathway. The versatility of the method was demon-
strated in part by the synthesis of axinastatin 1. Overall, this
strain-driven thietan-mediated ligation provides a powerful tool
for the synthesis of peptides and proteins. In particular, our
method could make the hydrophobic regions of membrane
proteins33 more synthetically accessible. Studies towards the
synthesis of complex membrane proteins using our approach
will be reported in due course.
Conflicts of interest
There are no conicts to declare.
Fig. 3 Reaction rate and mechanistic investigation. Reagents and
conditions: NCL condition for (a) and (b): 0.1 M Na2HPO4, 0.01 M
TCEP, pH ¼ 7.8, rt.
Acknowledgements
Support for this work was provided by: the National Science
Foundation (CHE 1710174), the University at Albany-SUNY to Q.
Zhang, and a China Research Council Scholarship to P. Gao.
Thanks are extended to Prof. Rabi Musah (SUNY Albany) and
Prof. Paramjit Arora (NYU) for helpful suggestions. The early
work performed by Kyle McHugh is acknowledged.
peptide 18 without noticeable epimerization.23 The thiolactone
19 was subjected to a one-pot NCL followed by desulfurization
to successfully produce the cyclic peptide 20 intramolecularly in
1
good yield. The high resolution MS, H, and 13C NMR data of
synthesized axinastatin 1 are identical to the reported ones.31
The proper proline conformations were conrmed by 2D NMR
experiments.32
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