Construction of diverse peptide structural architectures: Via chemoselective peptide ligation

Article abstract of DOI:10.1039/d1sc01174j

Herein, we report the development of a facile synthetic strategy for constructing diverse peptide structural architectures via chemoselective peptide ligation. The key advancement involved is to utilize the benzofuran moiety as the peptide salicylaldehyde ester surrogate, and Dap-Ser/Lys-Ser dipeptide as the hydroxyl amino functionality, which could be successfully introduced at the side chain of peptides enabling peptide ligation. With this method, the side chain-to-side chain cyclic peptide, branched/bridged peptides, tailed cyclic peptides and multi-cyclic peptides have been designed and successfully synthesized with native peptidic linkages at the ligation sites. This strategy has provided an alternative strategic opportunity for synthetic peptide development. It also serves as an inspiration for the structural design of PPI inhibitors with new modalities. This journal is

Full text of DOI:10.1039/d1sc01174j

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Construction of diverse peptide structural
architectures via chemoselective peptide ligation†
Cite this: DOI: 10.1039/d1sc01174j
a
Carina Hey Pui Cheung,‡a Jianchao Xu,‡a Chi Lung Lee,
Yanfeng Zhang,a
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
c
a
Ruohan Wei,a Donald Bierer,b Xuhui Huang
and Xuechen Li
*
Herein, we report the development of a facile synthetic strategy for constructing diverse peptide structural
architectures via chemoselective peptide ligation. The key advancement involved is to utilize the
benzofuran moiety as the peptide salicylaldehyde ester surrogate, and Dap–Ser/Lys–Ser dipeptide as the
hydroxyl amino functionality, which could be successfully introduced at the side chain of peptides
enabling peptide ligation. With this method, the side chain-to-side chain cyclic peptide, branched/
bridged peptides, tailed cyclic peptides and multi-cyclic peptides have been designed and successfully
synthesized with native peptidic linkages at the ligation sites. This strategy has provided an alternative
strategic opportunity for synthetic peptide development. It also serves as an inspiration for the structural
design of PPI inhibitors with new modalities.
Received 1st March 2021
Accepted 13th April 2021
DOI: 10.1039/d1sc01174j
rsc.li/chemical-science
based drugs, peptide drugs being able to bind to the PPI
targets more specically implies less off-target effects.12 More-
Introduction
over, peptide drugs have also been reported to have less
cytotoxicity.13
Protein–protein interactions (PPIs) are involved in many bio-
logical processes, such as antigen–antibody, ligand–GPCRs, and
substrate–enzymes.1–3 Abnormal PPIs driving signaling changes
can be pathogenic, hence PPIs are considered as the therapeutic
targets for various diseases.4–7 Interests in targeting disease-
associated PPIs have been growing in both academia and
industry. The rationale behind the inhibitor design is to block
the PPIs through mimicry of the topologically dened regions.
However, molecular targeting in PPIs is extremely challenging
due to the at and shallow binding pocket in proteins.4,8 Hence,
PPIs have long been considered to be undruggable using
traditional small molecules with molecular weight less than 500
as the disrupters/inhibitors.9 To address this issue, middle-
sized modalities, such as peptides and peptidomimetics have
been explored.10,11 Synthetic peptides have been reported to
have higher potency and specicity in targeting PPIs, not only
because they possess the capability in binding to the large
grooves on the interacting face, but their residues can also be
modied to mimic the conformational features of the protein
domain at the binding interface and disrupt the PPIs.8 More
importantly, compared with the traditional small molecules-
Although the peptides represent a promising class of thera-
peutic drugs in various therapeutic areas, they suffer from some
limitations.6 For example, linear peptides are not stable in vivo
and are prone to protease degradation.4,14,15 They also have poor
permeability to access desirable intracellular targets.4,8
Furthermore, the binding of peptides to the PPI interface may
not be always thermodynamically favorable as they will need to
overcome the entropic penalty to reorganize themselves into its
constrained bioactive state.4 To overcome the intrinsic
aDepartment of Chemistry, State Key Lab of Synthetic Chemistry, The University of
Hong Kong, Hong Kong. E-mail: xuechenl@hku.hk
bDepartment of Medicinal Chemistry, Bayer AG, Aprather Weg 18A, 42096 Wuppertal,
Scheme 1 (a) Synthesis of Dap–Ser dipeptide. (i) isobutyl chlor-
oformate, DIEA, dry CH2Cl2, 0 ꢀC, 2 h; (ii) Fmoc-Dap-OH, 1 h, 66%. (b)
Synthesis of the benzofuran building block. (iii) NaBH4, EtOH, 0 ꢀC, 1 h,
92%; (iv) PPh3$HBr, dry ACN, reflux, overnight, 87%; (v) DIC, dry
CH2Cl2, 0 ꢀC, overnight; (vi) Et3N, dry toluene, reflux, overnight, 57%
over two steps; (vii) LiOH, THF/H2O ¼ 3 : 1, rt, 6 h; (viii) 4 N HCl in
dioxane, rt, 2 h; (ix) Fmoc-Cl, dioxane/H2O, NaHCO3, overnight, 68%
over three steps.
Germany
cDepartment of Biological and Chemical Engineering, The Hong Kong University of
Science and Technology, Clear Water Bay, Kowloon, Hong Kong
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d1sc01174j
‡ Equal contribution.
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limitations of the natural peptides, structural modications biological potency and binding affinity.17 Successful examples
with unnatural elements are being explored. One of the most of stapled peptides offering therapeutic modality include all-
notable strategies is to develop side chain-to-side chain cyclic hydrocarbon-linked stapled peptide ALRN-6924 which is
peptides from the mimicry of interfacial a-helical domains.16–18 under clinical development as an anti-cancer drug targeting
Previous studies have shown that via varying the structural HDM2/p53.19,20 Other examples include peptides targeting HIV
design including the stapled positions, structures, lengths and integrase, BCl-2 and b-catenin.21–23 Apart from the stapling
peptide sequence, one can change the dynamics of the peptide– chemistry, some other motifs have been explored, including the
protein interaction8 thus optimize the engagement of inhibitors b-strands mimetics24 and loops motif25 that display more
at the PPIs interface. Peptide drugs with optimized structural complex topologies. Examples of tertiary mimetics as PPI
designs were found having higher protease resistance, inhibitors has also been reported, including a- and a/b-peptides
derived from the “Z-domain” scaffold.26 As PPIs have pivotal
roles in the regulation of biological systems, novel and practical
tools for the generation of new peptide architectures and
structural complexities will be worth being explored.
Here, we report the development of chemical ligation
chemistry for constructing diverse peptide structural motifs,
including side chain-to-side chain cyclic peptides, branched
and bridged peptides, tailed cyclic peptides and multi-cyclic
peptides. We expect these peptides will represent new struc-
tural motifs and offer new modalities for developing inhibitors
of PPIs with enhanced stability and binding affinity.
Results and discussion
Our design involves using chemoselective peptide ligation to
link the side chain unprotected (cyclic)peptide segments for
architecture construction. To this end, the reacting groups
necessary for executing ligation need be installed at the side
chain of the peptide. This is a challenging task and has not been
well explored in the literature. The notable native chemical
Fig. 1 Planned peptide architectures via peptide ligation: side chain-
to-side chain cyclic peptide and three classes of structural motifs.
ligation (NCL) requires C-terminal thioesters and N-terminal
cysteinyl peptide fragments as the requisite reacting
Class I: bridged peptides and branched peptides; class II: tailed cyclic
peptides; class III: bridged cyclic and embedded bicyclic peptides.
Fig. 2 (A) Schematic diagram of side chain-to-side chain cyclic peptides obtained via Ser/Thr ligation. (B) Chemoselective peptide stapling via
side chain-to-side chain Ser/Thr ligation with different peptide sequences. D0 ¼ Asp(benzofuran), E0 ¼ Glu(benzofuran), X: Dap–Ser dipeptide. (a)
Percentage conversion to the product based on UPLC-MS analysis of the crude reaction mixture. (b) Isolated yield based on ligation. (C) UV
traces of linear peptide D, crude reactions of ozonolysis, Ser/Thr ligation and acidolysis respectively.
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counterparts.27 A side chain NCL was reported between the N- by acidolysis, the native peptidic linkage is generated at the
linked glycosyl auxiliary to the thioester side chain of an N- ligation junction. Apart from linear peptide/protein synthesis,
terminal aspartate oligopeptide.28 A drawback is that extra intramolecular Ser/Thr ligation has been developed for peptide
orthogonal protecting groups for Asp/Glu are needed for selec- cyclization to produce head-to-tail cyclic peptides of various
tive deprotection of the aspartic acid side chain protecting sizes.33–39 In this paper, we ought to further expand the scope of
group prior to thioester formation.28 Another limitation of this Ser/Thr ligation by solving the problem of introducing the
technique is that the thioester generated will be easily decom- peptide salicylaldehyde ester on the side chain, with which to
posed upon piperidine treatment in Fmoc-solid phase peptide construct diverse peptide scaffolds.
synthesis (SPPS).29
To install these two reactive groups onto the side chain of the
Peptide C-terminal salicylaldehyde esters have been devel- unprotected peptides for executing side-chain STL, we rst
oped to ligate side chain unprotected peptides with N-terminal identied the Dap–Ser or Lys–Ser dipeptide as the side chain
Ser/Thr/Cys/Pen for Ser/Thr ligation (STL)30,31 and Cys/Pen hydroxyl amino functionality. The Dap–Ser or Lys–Ser dipeptide
ligation (CPL)32 used in chemical protein synthesis. The che- was successfully synthesized via mixed anhydride derivatives
moselective reaction between the two counterparts affords an from isobutyl chloroformate (Scheme 1a). Boc-Ser(tBu)-OH was
acid-cleavable N,O/S-benzylidene acetal intermediate. Followed rst dissolved in dry CH2Cl2, isobutyl chloroformate and DIEA
were then added at 0 ꢀC to afford the mixed anhydride. The
amine nucleophile, Fmoc-Dap-OH or Fmoc-Lys-OH, was added
aer an hour to generate the dipeptide ready for SPPS.
The introduction of the salicylaldehyde ester to the side
chain of Asp/Glu of the peptide is very challenging. As priorly
mentioned, there is no simple and direct method reported yet
for synthesizing peptides with Asp/Glu side chain active esters.
The complication occurs as the coupling of Asp (O-salicylalde-
hyde ester) or any Asp active ester building block will lead to
aspartimide formation during SPPS or the condensation step.
Moreover, the Asp/Glu active esters cannot survive piperidine
treatment during Fmoc-SPPS. All previous strategies for
synthesizing peptide C-terminal salicylaldehyde esters are not
applicable for introducing salicylaldehyde esters at the peptide
side chain.31 To solve this problem, we have come to identify
a novel synthetic strategy utilizing the benzofuran moiety as the
peptide crypto-salicylaldehyde ester. Upon ozonolysis, the
benzofuran moiety could readily become a salicylaldehyde ester
for subsequent chemoselective ligation.
Previous synthetic routes to benzofuranylalanine via transi-
tion metal-catalyzed or chemoenzymatic synthesis were re-
ported.40,41 In our design, preparation of the benzofuran moiety
started from salicylaldehyde. Salicylaldehyde was rst treated
with reducing agent NaBH4, followed by reux with
Fig. 3 Class I: branched peptides and bridged peptides. Entry 3(a) UV
trace of purified ester A. (b and c) UV traces of crude reactions of Ser/
Thr ligation and acidolysis of compound 3. Entry 5(a) UV trace of
purified ester A (b and c). UV traces of crude reactions of Ser/Thr
ligation and acidolysis of compound 5.
Table 1 Sequences of model peptides synthesizeda
Entry
A
B
%
1
2
3
4
5
6
7
8
Ac-DTTAD0A-NH2
H-SNVKAQFL-NH2
H-SKAKL-NH2
H-SKAKL-NH2
H-TLHAPTD-OH
Ac-(K–S)SKAKL-NH2
Ac-(K–S)SKAKL-NH2
Ac-(K–S)SARKYFAGNLPE-NH2
Ac-(K–S)SKAKL-NH2
H-NIGTYLP(K–S)NVK-NH2
Ac-ARE(K–S)TPEP-NH2
Ac-ARE(K–S)TPEP-NH2
78.2b, 36.6c
80.0b, 39.4c
95.0b, 39.1c
89.6b, 32.8c
90.5b, 48.5c
91.9b, 53.0c
95.6b, 37.4c
88.9b, 33.3c
93.4b, 31.1c
>98b, 38.4c
91.7b, 34.7c
Ac-SRQQGESNQERGARD0RL-NH2
Ac-D0SERVELRKLQDV-NH2
H-VIGGVGD0N-NH2
Ac-DTTAD0A-NH2
Ac-SRQQGESNQERGARD0RL-NH2
Ac-SRQQGESNQERGARD0RL-NH2
Ac-D0SERVELRKLQDV-NH2
Ac-D0SERVELRKLQDV-NH2
Ac-D0SERVELRKLQDV-NH2
Ac-LSQD0RG-NH2
9
10
11
D0 ¼ Asp(benzofuran), (K–S) ¼ Lys–Ser dipeptide. b Percentage conversion to product based on UPLC-MS analysis of the crude reaction mixture.
a
c
Isolated yield based on ligation.
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With these building blocks in hand, we continued to incor-
porate them into the peptide to execute our construction plan.
As a proof of concept, the linear peptide with Dap–Ser and
Asp(benzofuran) could be readily synthesized under standard
Fmoc-SPPS conditions.
Aer global deprotection, the resultant side chain unpro-
tected peptides were treated with ozonolysis in H2O/ACN (v : v ¼
1 : 1) with 0.7% TFA under 0 ꢀC for 1 minute, affording the
peptide salicylaldehyde ester smoothly. It is noting that
oxidation-prone residues are not compatible with the ozone
treatment, i.e. Cys and Met react with ozone at the sulydryl to
give sequential O-atom addition products,42 and Trp becomes
kynurenine aer ozonolysis.43 Therefore, these amino acid
residues are excluded from the chemical space available for this
chemistry. Subsequently, side chain-to-side chain Ser/Thr liga-
tion with unprotected peptides was performed. The peptide was
dissolved in pyridine acetate buffer at 0.5 mM concentration,
which spontaneously proceeded to cyclize, followed by acid-
olysis to cleave the N,O-benzylidene acetal intermediate to
afford the cyclic peptides with an overall yield of ꢁ35% aer one
HPLC purication. With this strategy, we were able to generate
Fig. 4 Class II: tailed cyclic peptides. Entry 18(a) UV trace of purified
ester A. (b and c) UV traces of crude reactions of Ser/Thr ligation and
acidolysis of compound A. (d) UV trace of crude reaction of ozonolysis
of compound A0. (e and f) UV traces of crude reactions of Ser/Thr Asp–Dap/Lys lactam linkage with a hydrophilic hydroxyl group
ligation and acidolysis of compound A0 + B.
on different peptide sequences (Fig. 2). Notably, the chemo-
selective cyclization can tolerate most of the side chain func-
tionalities presenting in the peptide except oxidation-prone
residues.
These results were very encouraging and opened up new
triphenylphosphine hydrobromide. The generated 2-hydrox-
ybenzyltriphenylphosphonium bromide was then added to Boc-
Asp-OMe and transformed into an activated ester intermediate
upon DIC treatment (Scheme 1b). Subsequently, the resulted
intermediate was reuxed with triethylamine to offer the
benzofuran moiety. Finally, the desired benzofuran building
block was obtained by C-terminal methyl ester group depro-
tection, followed by N-terminal Boc group deprotection and
Fmoc group installation. As the Fmoc-Asp(benzofuran)-OH
moiety is stable under both acidic and basic conditions during
peptide synthesis, it could be directly used in Fmoc-SPPS. We also
obtained Fmoc-Glu(benzofuran)-OH in similar route (see ESI†).
opportunities to construct novel peptide structures via chemical
ligation. We envisaged that our strategy could be exibly
applied to afford innovative peptide modalities via ligating
peptides with side chain salicylaldehyde ester with other
peptides carrying Dap/Lys–Ser dipeptide or N-terminal Ser/Thr/
Cys/Pen. Thus, in addition to the side chain-to-side chain cyclic
peptides, we continued to apply this new chemistry to synthe-
size three classes of structural motifs, as shown in Fig. 1.
The rst class includes branched peptide and bridged
peptide (Fig. 3 and Table 1). The peptide with the side chain
Table 2 Sequences of model peptides synthesizeda
Entry
Sequence
%
12
13
H-SDKITED0A-NH2
>98b, 42.3c
H-SNKQARD0KIYQSQT-NH2
95.2b, 46.1c
A0 + B
%
A0
Sequence
A
B
Entry
Sequence
%
Sequence
14
15
16
17
18
19
H-SKAKL-NH2
H-TLHAPTD-NH2
Ac-(K–S)SKAKL-NH2
Ac-ARE(K–S)TPEP-NH2
Ac-(K–S)SARKYFAGNLPE-NH2
H-NIGTYLP(K–S)NVK-NH2
92.9b, 39.5c
93.2b, 41.2c
>98b, 53.3c
91.5b, 52.5c
81.2b, 49.0c
>98b, 52.3c
H-SD0LSQRG-SAL ester
>98b, 48.0c
D0 ¼ Asp(benzofuran), (K–S) ¼ Lys–Ser dipeptide, SAL ¼ salicylaldehyde. b Percentage conversion to product based on UPLC-MS analysis of the
a
crude reaction mixture. c Isolated yield based on ligation.
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ligate with another peptide carrying side chain Dap–Ser or Lys–
Ser under the Ser/Thr ligation conditions to produce bridged
peptides (Table 1, entries 5–11). Such structural motifs will
allow combining two functional peptides to generate a hybrid
peptide with dual activities.
The second class includes tailed cyclic peptides (Fig. 4 and
Table 2). Due to the compact structures of cyclic peptides and
their ultra-stability against enzymatic degradation and physical
denaturation, cyclic peptides have attracted a lot of attention in
the pharmaceutical industry. In this regard, the development of
effective methods for peptide cyclization has been an inten-
sively researched subject. In our design, a peptide with the side
chain salicylaldehyde ester and N-terminal Ser/Thr could cyclize
under the Ser/Thr ligation condition to generate head-to-side
chain cyclic peptides (Table 2, entries 12 and 13). Further-
more, a cyclic peptide with the side chain salicylaldehyde ester
could react with another peptide with side chain Lys–Ser to
generate cyclic peptide with a branched tail (Table 2, entries 14–
19). Our strategy not only provides an effective synthetic method
but also generates new structural motifs.
The third class of peptide structural architectures includes
the bridged cyclic and embedded bicyclic peptides (Fig. 5 and
Table 3). One side chain-to-tail cyclic peptide with free N-
terminal Cys could react with another head-to-tail cyclic
peptide with side chain salicylaldehyde ester under Cys ligation
conditions to generate a bridged cyclic peptide, which will
provide a way to conjugate cell-penetrating peptide to a peptide
ligand. Alternatively, the embedded bicyclic peptides could be
synthesized by performing rst the side chain-to-tail Ser/Thr
ligation involving the side chain Lys–Ser and C-terminal
Fig. 5 Class III: bridged cyclic and embedded bicyclic peptides. Entry
20(a and b) UV traces of crude reactions of Ser/Thr ligation and
acidolysis of compound A. (c) UV trace of crude reaction of ozonolysis
of compound A0. (d and e) UV traces of crude reactions of Ser/Thr
ligation and acidolysis of compound B. (f) UV trace of crude reaction of
Thz deprotection of compound B0. (g and h) UV traces of crude
reactions of Cys/Pen ligation and acidolysis of compound A0 + B0.
salicylaldehyde reacted with another peptide carrying N-
terminal Ser/Thr under the Ser/Thr ligation conditions to
produce the branched peptide (Table 1, entries 1–4). Alterna-
tively, the peptide with side chain salicylaldehyde ester can
Table 3 Sequences of model peptides synthesizeda
A0
B0
A
B
A0 + B0
Entry Sequence
Sequence
%
Sequence
Sequence
%
%
20
Boc-(Thz)(K–S)LSQRG-SAL ester
88.9b, 37.2d 84.3b, 50.4c
90.1b, 35.4d 89.9b, 45.8c
H-SD0LSQRG-SAL ester
>98a, 48.0b
21
Boc-(Thz)NFS(K–S)QSNKRFLSKTQG-SAL ester
A0
A00
A
Entry
Sequence
Sequence
%
Sequence
%
22
Fmoc-SKFT(K–S)SEND0ILSQRG-SAL ester
70.4b, 32.6e
80.2b, 29.5c
a
b
D0 ¼ Asp(benzofuran), (K–S) ¼ Lys–Ser dipeptide, SAL ¼ salicylaldehyde. Percentage conversion to the ligation product based on UPLC-MS
c
d
analysis of the crude reaction mixture. Isolated yield based on ligation. Isolated yield over 2 steps (intramolecular STL and one-pot Thz
deprotection). Isolated yield over 2 steps (intramolecular STL and one-pot Fmoc deprotection).
e
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salicylaldehyde ester, followed by the head-to-side chain Ser/Thr Zhang and R. Wei performed the chemical synthesis of building
ligation involving the N-terminal Ser/Thr and the side chain blocks and side chain-to-side chain cyclic peptides. C. H. P.
salicylaldehyde ester. Based on this synthetic route, we envi- Cheung performed the chemical synthesis of the peptide
sioned that cyclic peptides of a higher order would be achiev- structural motifs.
able through the rational design of combining the above
bridged cyclization and embedded bicyclization approaches.
Conflicts of interest
Conclusions
There are no conicts to declare.
In summary, we have developed a facile synthetic strategy for
constructing diverse peptide structural motifs via chemo- Acknowledgements
selective peptide ligation. The key advancement of our synthetic
This work was supported by the Hong Kong Research Grant
strategy is to utilize the benzofuran moiety as the peptide sali-
cylaldehyde ester surrogate, and Dap–Ser/Lys–Ser dipeptide as
the hydroxyl amino functionality. These two building blocks
could be incorporated into the peptide side chain under the
standard Fmoc-SPPS conditions. Aer global deprotection, the
desired peptide salicylaldehyde ester could be efficiently
generated upon ozonolysis. Subsequently, Ser/Thr ligation or
Cys/Pen ligation is performed to covalently link up the two
reacting counterparts to form different peptide architectures. As
a proof of concept, different models of side chain-to-side chain
cyclic peptides were successfully synthesized. We then proposed
that the application scope of this strategy could be further
expanded to afford innovative peptide modalities. In addition to
the side chain-to-side chain cyclic peptides, three classes of
structural motifs have been designed and successfully synthe-
sized by ligation. Peptides with side chain salicylaldehyde ester
have reacted smoothly with different peptide counterparts
carrying Dap/Lys–Ser dipeptide or N-terminal Ser/Thr/Cys/Pen
to generate the native peptidic linkages at the ligation sites.
These results have demonstrated the exibility of the chemical
ligation approach to construct diverse peptide structural
architectures. As ozone is a strong oxidant, the oxidizable amino
acid acids (i.e. Trp, Met, and Cys) are not compatible with this
Council of Hong Kong (HKUST C6009-15G, 17309616, AoE/M-
09/12 and AoE/P-705/16).
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