Chemical and Ribosomal Synthesis of Topologically Controlled Bicyclic and Tricyclic Peptide Scaffolds Primed by Selenoether Formation

Article abstract of DOI:10.1002/anie.201813827

Bicyclic and tricyclic peptides have emerged as promising candidates for the development of protein binders and new therapeutics. However, convenient and efficient strategies that can generate topologically controlled bicyclic and tricyclic peptide scaffolds from fully-unprotected peptides are still much in demand, particularly for those amenable to the design of biosynthetic libraries. In this work, we report a reliable chemical and ribosomal synthesis of topologically controlled bicyclic and tricyclic peptide scaffolds. Our strategy involves the combination of selenoether cyclization followed by disulfide or thioether cyclization, yielding desirable bicyclic and tricyclic peptides. This work thus lays the foundation for developing peptide libraries with controlled topology of multicyclic scaffolds for in vitro display techniques.

Full text of DOI:10.1002/anie.201813827

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Accepted Article
Title: Chemical and Ribosomal Synthesis of Topologically Controlled
Bicyclic and Tricyclic Peptide Scaffolds Primed by Selenoether
Formation
Authors: Yizhen Yin, Qianran Fei, Weidong Liu, Zhuoru Li, Hiroaki
Suga, and Chuanliu Wu
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201813827
Angew. Chem. 10.1002/ange.201813827
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COMMUNICATION
Chemical and Ribosomal Synthesis of Topologically Controlled
Bicyclic and Tricyclic Peptide Scaffolds Primed by Selenoether
Formation
[a]
[b]
[b]
[b]
[a]
[b]
Yizhen Yin+, Qianran Fei+, Weidong Liu, Zhuoru Li, Hiroaki Suga,* and Chuanliu Wu*
Dedication ((optional))
Abstract: Bicyclic and tricyclic peptides have emerged as promising
candidates for the development of protein binders and new
therapeutics. However, convenient and efficient strategies that can
generate topologically controlled bicyclic and tricyclic peptide
scaffolds from fully-unprotected peptides are still much in demand,
particularly for those amenable to the design of biosynthetic libraries.
In this work, we report a reliable chemical and ribosomal synthesis of
topologically controlled bicyclic and tricyclic peptide scaffolds. Our
strategy involves the combination of selenoether cyclization followed
by disulfide or thioether cyclization, yielding desirable bicyclic and
tricyclic peptides. This work thus lays the foundation for developing
peptide libraries with controlled topology of multicyclic scaffolds for in
vitro display techniques.
These reagents can react with cysteine residues in peptides
under mild conditions to afford multicyclic scaffolds. However,
cyclizing a peptide containing four cysteines through two identical
crosslinks yields three different bicyclic scaffolds, while cyclizing
through two different crosslinks or a crosslink containing four thiol-
reactive groups potentially yields six different bicyclic or tricyclic
scaffolds. Importantly, the simultaneous presence of different
isomers might lead to bias against structurally less favored but
more constrained isomers. Cyclization strategies relying on the
regioselective thioether formation to control the topology have the
potential to overcome these problems,[7] but such strategies are
rarely amenable to biosynthesis of multicyclic peptides. In nature,
regioselective thioether formation can be achieved for the
biosynthesis of ribosomally synthesized and post-translationally
modified peptides (e.g., lanthipeptides), which involves
Multicyclic peptides occupy a unique chemical space between
larger biologics and small molecules, which have emerged as
promising candidates for the development of new protein binders
and potent therapeutics.[1] In nature, the structural diversity of
disulfide-constrained peptides can be evolutionarily acquired
through synergistic manipulations of primary sequences, cysteine
[
8]
biosynthetic enzymes and leader sequences. Therefore, there
is still need for more convenient and efficient strategies that can
generate topologically controlled multicyclic peptide scaffolds
without significant increase in synthetic complexity.
Here we report a reliable regioselective cyclization strategy for
generating topologically controlled bicyclic and tricyclic peptide
scaffolds that is amenable to the in vitro translation system. Owing
to the higher nucleophilicity of selenocysteine (Sec) over cysteine
(Cys), Sec can preferentially react with halogenated residues to
achieve regioselective cyclization of peptides containing both Sec
and Cys residues (Figure 1). By varying the pattern of the
crosslinking residues (i.e., Sec, Cys and halogenated residues)
and the secondary crosslinks (i.e., alkylation or arylation
reagents), multicyclic peptide scaffolds can be designed and
synthesized in controlled manners (Figure 1). Moreover, by taking
advantage of genetic coding reprogramming using flexizymes
(
Cys) patterns and disulfide connectivities.[2] However, disulfide-
constrained peptide scaffolds cannot be well controlled with the
exception of the naturally occurring primary sequences which
generally govern the tertiary folding via intramolecular interactions
between residues and their backbones. Moreover, artificially or
randomly made peptides containing multiple Cys residues may
suffer from the formation of isomers and undergo disulfide
isomerizations.[3]
By utilizing multi-step bioorthogonal crosslinking, protecting
groups, or C2/C3-symmetric small molecules as crosslinks,
topologically controlled multicyclic peptide scaffolds have been
obtained.[1e, 4, 5] Among such strategies for peptide cyclization or
crosslinking, those that rely on reagents containing two or more
thiol-reactive groups such as bis-(bromomethyl)benzene and tris-
integrated with a customized reconstituted translation system (FIT
[9, 10]
system),
both Sec and the halogenated residues can be
incorporated into peptide chains, thus enabling the regioselective
cyclization of biosynthetic peptides into the desirable bicyclic or
tricyclic peptide scaffolds described above. This work will benefit
the development of ribosomally expressed topologically
controlled multicyclic peptide libraries.
(
bromomethyl)benzene (TBMB) have proven to be useful.[1c, 6]
[
[
a]
Dr. Y. Yin and Prof. H. Suga, Department of Chemistry, School of
Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo,
Our study was inspired by recent studies showing that
selenoether-bridged peptides can be synthesized by Se-
alkylation of Sec with halogenated residues under milder
conditions at lower pH as compared with thioether counterpart via
113-0033 (Japan). E-mail: hsuga@chem.s.u-tokyo.ac.jp.
b]
Q. Fei, W. Liu, Z. Li and Prof. C. Wu, Department of Chemistry,
College of Chemistry and Chemical Engineering, The MOE Key
Laboratory of Spectrochemical Analysis and Instrumentation,
Xiamen University, Xiamen, 361005 (P.R. China). E-mail:
chlwu@xmu.edu.cn.
S-alkylation of Cys.[11] This selectivity owes to the fact that the pK
a
value for the selenol in Sec is ~3‒5 units lower than that for the
[+
]
These authors contributed equally to this work.
[12]
thiol in Cys.
However, it remains unexplored if the
intramolecular Se-alkylation of Sec can take place more efficiently
than the competing S-alkylation of Cys to allow for selective
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cyclization of peptides into designer structures. Thus, we first
designed and synthesized a model peptide (1) composed of
primarily glycine (G) and lysine (K) residues containing a single
Sec (U) residue with the N-chloracetyl-tryptophan (ClAc-W) at the
N-terminus and two Cys (C) by employing Fmoc-based solid-
phase peptide chemistry (Figure 2a). The side chain of the Sec
residue was protected with the standard p-methoxybenzyl (Mob)
group, which can be facilely removed during the peptide cleavage
from the resin by adding 2,2’-dithiobis(5-nitropyridine) (DTNP) to
the standard trifluoroacetic acid (TFA) cocktail.[13] After the
peptide cleavage and deprotection, the crude peptide was
precipitated into ice-cold diethyl ether, which was then dissolved
into 0.1% TFA aqueous solution and purified by high performance
liquid chromatography (HPLC). Dissolving the isolated peptide
The topology of the bicyclic peptide was further confirmed by
characterizing the crosslink connectivity by tryptic digestion and
mass spectral analysis of the peptide fragments (Figure S1),
indicating the feasibility of generating the expected ring-bridged
peptide bicycles crosslinked via a selenoether and a disulfide
bond from peptides with a ClAc-W―C―U―C framework (―
represents a 3-mer peptide segment). By varying the pattern of
the crosslinking residues, two other peptide bicycles could be
generated, that was the ring-fused and ring-tandem peptide
bicycle from model peptides (2 and 3) with a ClAc-W―C―C―U
and ClAc-W―U―C―C framework, respectively (Figure 2,
Figures S2 and S3). Furthermore, we demonstrated that varying
the composition and length of the peptide (4) does not affect the
efficiency of the regioselective cyclization (Figure S4).
into
an
aqueous
solution
containing
tris(2-
carboxyethyl)phosphine (TCEP, 5 e.q., as a reducing reagent)
and L-ascorbic acid sodium salt (10 e.q., as a protecting reagent
for deselenization[14]) led to a quantitative conversion into a sole
dehalogenated product (1d) as monitored by HPLC and mass
spectrometry (MS) (Figure 2b and Figure S1). We observed the
formation of a sole product (1o; i.e., HPLC peak-to-peak
conversion) when the dehalogenated product was dissolved into
a phosphate buffer containing 0.5 mM oxidized glutathione
(
GSSG) at pH 7.4 (Figure 2b), implying that both the
dehalogenated and the oxidative crosslinking reactions occurred
in the selective manner.
Figure 2. a) Synthetic route for the peptides containing Sec and N-terminal
chloracetyl; b-e) HPLC chromatograms showing regioselective cyclization of
peptide 1, 2, and 3 in aqueous buffers. 1d, 2d and 3d are dehalogenated
monocycles; 1o, 2o and 3o are oxidized bicycles. All peptides are C-terminally
aminated.
As disulfide bond can be reduced by dithiothreitol (DTT) or tris(2-
carboxyethyl)phosphine (TCEP), selectively crosslinking the
reduced Cys residues with diverse crosslinkers containing two
thiol-reactive groups could generate a range of structurally
controlled bicyclic peptide scaffolds (Figure 1). Thus, we chose
three of the many reported crosslinkers (i.e., diiodomethane, 1,3-
bis(bromomethyl)benzene, and, hexafluorobenzene) to test the
Figure 1. Schematic illustration of the regioselective cyclization of peptides
containing one halogenated residue (X; chloroacetyl amino acid), one
selenocysteine (U) and two or three cysteines (C) into structurally controlled
bicyclic and tricyclic peptides. Note that, not all possible patterns of X/U/C are
listed. Structures of the crosslinkers with two or three thiol-reactive groups used
for the secondary crosslinking were given in the boxes.
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COMMUNICATION
concept by diversifying bicyclic peptide scaffolds.[6b,
15]
As
challenging due to near-random crosslinking of Cys residues.[1f, 7]
To overcome this limitation, we have utilized our genetic code
reprogramming approach using flexizymes integrated with a
expected, these crosslinking reactions all led to rapid and
quantitative cyclization of the model peptide (1) into the expected
structures (Figures 3a and Figure S5) using TCEP as the reducing
agent, and the presence of the selenoether bond and amino
groups in lysine residues seemed not affect the reactions. In
addition, the selenoether crosslinks are stable to the treatment of
TCEP and high concentrations of thiol compounds such as DTT
and glutathione (GSH), which not only enables for the one-pot
production of bicyclic peptides in high yields without unwanted
side reactions, such as deselenization reaction and thiol-
selenoether exchanges,[13, 16] but also affords diverse stable
bicyclic scaffolds tolerant to intracellular reducing conditions.
custom-made in vitro translation apparatus (referred to the FIT
system),[
9, 10]
and demonstrated the ribosomal synthesis of bi- and
tricyclic peptides. This approach relies on preferential thioether-
macrocyclization where the second Cys residue (underlined) in
ClAc-X-C―C―C selectively reacts with N-terminal ClAc group to
form the smallest cyclic peptide via a thioether bond. The
remaining two Cys residues oxidatively form a disulfide bond to
yield a bicyclic scaffold.[18] Alternatively, ClAc-X-C―C―C―C
selectively forms a tricyclic scaffold by the combination with
selective self-thioether formation of the second Cys residue
(
underlined) with the ClAc group and the post-translational
To extend the above strategy to designing structurally controlled
tricyclic peptide scaffolds, two model peptides (5 and 6) exhibiting
crosslinking of the remaining three Cys resides using TBMB.[
19]
However, this approach does not allow for insertion of a peptide
segment between ClAc-X and C , since this would result in loss
1
of selectivity where any downstream Cys sidechain could react
with the ClAc group. Therefore, we applied the method of the
thioether/selenoether chemistry devised in this work to the
ribosomal synthesis of topologically controlled bicyclic peptides.
respectively
W―C―C―C―U framework were designed and synthesized
Figure 3b, Figures S6 and S7). After the formation of the
selenoether bond, the peptide could subsequently react with
,3,5-tris-(bromomethyl)benzene (TBMB) to yield the expected
the
ClAc-W―C―U―C―C
and
ClAc-
(
1
tricyclic peptide scaffolds as a sole product (Figure 3b and Figure
S7). The above strategy using the combination of selenoether and
disulfide/thioether bonds in designer peptides enables us to
synthesize bicyclic or tricyclic peptides in the topologically con-
trolled manner.
Figure 4. Overview of the expression of regio-selectively cyclized peptides in
Figure 3. a) HPLC chromatograms showing the cyclization of 1d (100 μM) by
reactions with diiodomethane (5 mM in aqueous THF solution), 1,3-
bis(bromomethyl)benzene (0.5 mM in phosphate buffer), hexafluoro-benzene
the FIT system. All peptides are C-terminally carboxylic acids.
(
25 mM in DMF solution), respectively; b) HPLC chromatograms showing the
We first synthesized seleno-L-cysteine-3,5-dinitrobenzyl ester
cyclization of peptide 5 (100 μM) by reaction with TBMB (0.5 mM) in phosphate
buffer at pH 7.4. TCEP was added as the reducing reagent to prevent disulfide
formation. All peptides are C-terminally aminated.
L
( Sec-DBE) , which was charged onto tRNAs by dFx (Figure 4
2
and Figure S8). Then, we demonstrated that 2-chloracetyl-D-
D
tyrosine (ClAc- Tyr) and Sec could be simultaneously
incorporated into model peptides (7 and 8) by assigning them to
the initiator and elongator AUG codons respectively and the
Ribosomal synthesis of macrocyclic peptides will generate
unique peptide libraries with the complexity of sequence space as
D
fMet
L
simultaneous addition of ClAc- Tyr-tRNA CAU and Sec-
1
3
high as 10 . When such libraries are appropriately displayed on
mRNA in combination with the genetic code reprogramming, e.g.
RaPID (Random nonstandard Peptides Integrated Discovery)
system, extremely potent ligand macrocycles can be readily
identified.[17] On the other hand, the expression of topologically
controlled multicyclic peptides via disulfide bonds have been
tRNAAsn-E2CAU (Figure 4).[9] The cyclization reaction between the
incorporated Sec and ClAc- Tyr can take place immediately after
D
the expression, as indicated by the MALDI-TOF mass spectra
(
Figure 5a). To demonstrate if the Se-alkylation of Sec by ClAc-
D
Tyr in peptides can proceed over the competing S-alkylation of
Cys under the biological expression condition, we then
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Angewandte Chemie International Edition
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multicyclic scaffold diversities in
future for screening bioactive
molecules or drug discovery.
In
summary,
we
have
developed a reliable strategy for
the design and synthesis of
topologically controlled bicyclic
and tricyclic peptide scaffolds.
We demonstrated that the
regioselective cyclization of Sec
with halogenated residues in
peptides can proceed with a
high specificity over the
competing S-alkylation reaction
of the cysteine thiol, which
leaves the free cysteine
residues for further crosslinking
to
generate
topologically
attractive peptide scaffolds. As
both
cyclization
the
regioselective
secondary
and
crosslinking can be performed
under physiological conditions
with no obvious side reactions,
our strategy is amenable to the
in vitro translation system,
which allows the generation of
bicyclic and tricyclic peptides in
the
FIT
system
without
Figure 5. MALDI-TOF MS and MS/MS characterization of ribosomally-expressed peptides; a) MALDI-TOF MS of implication of isomer formations.
selenoether-bridged peptide 7 and 8; b) MALDI-TOF MS/MS analysis of selenoether-bridged peptide 9 and 10; the asterisk
Further work will focus on
in the figures indicates the parent ion; the parent ion [M+H] of peptide 9 in the figure is 3093.57; the parent ion [M+H]+ of
+
peptide 10 in the figure is 3092.46. c) MALDI-TOF MS of 9 regioselectively cyclized through a selenoether bond and a developing mRNA displayed
disulfide bond (9o; GSSG oxidation) or a thioether crosslink (Pm9; modification with 2,6-bis (bromomethyl) pyridine).
libraries based on these bicyclic
or tricyclic peptide scaffolds.
incorporated both Cys and Sec residues into model peptides (9
and 10), and characterized the peptides expressed in the FIT
system using MALDI-TOF MS/MS (Figure 5b). The result clearly
shows that the ClAc group only reacted with the selenol of the Sec
residue during and after the peptide expression, revealed by the
identical MS/MS spectra/fragmentation between ribosomally
expressed and chemically synthesized peptides (Figures S11 and
S12). This result is also in a good agreement with our previous
result of chemical synthesis. These findings suggest the feasibility
of extending our regioselective cyclization strategy to
synthesizing topologically controlled bicyclic and tricyclic peptide
scaffolds in the FIT system.
The selenoether and thioether crosslinks are both stable under
reducing conditions such as in the cytosol, making these
multicyclic peptides interesting scaffolds for the further design or
screening of potent bioactive molecules.
Acknowledgements
We thank Drs. Yichao Huang, Christos Tsiamantas for their
suggestions and assistance during experiments and paper
preparation. This work was supported by Japan Agency for
Medical Research and Development (AMED) the Basic Science
and Platform Technology Program for Innovative Biological
Medicine and Platform for Drug Discovery, Informatics, and
Structural Life Science to H.S., a Japan Society for the Promotion
of Science Postdoctoral Fellowship for Foreign Researchers
(P16412) to Y.Y, and the National Natural Science Foundation of
China (91853112, 21822404, and 21521004) to C.W..
A set of model peptides containing three Cys residues and one
D
Sec and ClAc- Tyr were also designed and expressed in the FIT
system (peptide 11 and 12). MALDI-TOF MS/MS confirmed the
D
regioselective cyclization of the incorporated Sec with ClAc- Tyr
(
Figures S10 and S11). After the expression, disulfide bond
formation or thioether crosslinking can be achieved by
subsequently removing the excess reducing reagent DTT in the
expression system by means of acetone precipitation. The
efficient conversion of these reduced peptides into their oxidized
or thioether-crosslinked forms was indicated by the complete
disappearance of the reduction peaks and/or the appearance of
the corresponding peaks of the bicyclic products in MALDI-TOF
mass spectra (Figure 5c). These results lay the foundation to
developing mRNA displayed peptide libraries with large
Keywords: chemical • ribosomal • selenocysteine• FIT system •
topology• multicyclic peptides
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[
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5
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COMMUNICATION
Yizhen Yin, Qianran Fei, Weidong Liu,
Zhuoru Li, Hiroaki Suga,* and Chuanliu
Wu*
Page No. – Page No.
Chemical and Ribosomal Synthesis of
Topologically Controlled Bicyclic and
Tricyclic Peptide Scaffolds Primed by
Selenoether Formation
Topologically controlled
multicyclic peptides
Flexible in-vitro
translation system
We report a reliable chemical and ribosomal synthesis of topologically controlled bicyclic
and tricyclic peptide scaffolds. Our strategy involves the combination of selenoether
cyclization followed by disulfide or thioether cyclization, yielding desirable bicyclic and
tricyclic peptides. This work thus lays the foundation for developing peptide libraries with
controlled topology of multicyclic scaffolds for in vitro display techniques.
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