Triple Function of 4-Mercaptophenylacetic Acid Promotes One-Pot Multiple Peptide Ligation

Article abstract of DOI:10.1002/anie.201809765

One-pot multiple peptide ligation is a key technology to improve the efficiency of chemical protein synthesis. One-pot repetitive peptide ligation requires a cycle of three steps: peptide ligation, removal of a protecting group, and inactivation of the deprotection reagent. However, previous strategies are not sufficient because of harsh deprotection conditions, slow deprotection rates, and difficulty in quenching the deprotection reagent. To address these issues, we developed a rapid, efficient deprotection and subsequent quenching strategy using an allyloxycarbonyl group to protect the N-terminal cysteine residue. 4-Mercaptophenylacetic acid (MPAA), a thiol additive for native chemical ligation, functioned not only as a scavenger for π-allyl palladium complexes, but also as a quencher of palladium(0) complexes. By utilizing the multifunctionality of MPAA, we carried out a one-pot five-segment ligation to afford histone H2AX (142 amino acids), which was isolated in 59 % yield.

Full text of DOI:10.1002/anie.201809765

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Title: Triple Function of 4-Mercaptophenylacetic Acid Promotes One-
Pot Multiple Peptide Ligation
Authors: Naoki Kamo, Gosuke Hayashi, and Akimitsu Okamoto
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Triple Function of 4-Mercaptophenylacetic Acid Promotes One-
Pot Multiple Peptide Ligation
Naoki Kamo, Gosuke Hayashi and Akimitsu Okamoto[a,b]*
[
a]
[a]
Abstract: One-pot multiple peptide ligation is a key technology to
improve the efficiency of chemical protein synthesis. One-pot repeti-
tive peptide ligation requires a cycle of three steps: peptide ligation,
removal of a protecting group, and inactivation of the deprotection
reagent. However, previous strategies are not sufficient because of
harsh deprotection conditions, slow deprotection rates, and difficulty
in quenching the deprotection reagent. To address these issues, we
developed a rapid, efficient deprotection and subsequent quenching
strategy using an allyloxycarbonyl group to protect the N-terminal
cysteine. 4-Mercaptophenylacetic acid (MPAA), which is a thiol
additive of native chemical ligation, functioned not only as a scaven-
ger for a π-allyl palladium complex, but also as a quencher of palla-
dium(0) complex. Utilizing the multifunctionality of MPAA, we
achieved the first one-pot five-segment ligation to afford histone
H2AX (142 amino acids) in 59% isolated yield.
plexes based on the Tsuji–Trost reaction. It was envisaged that
the proposed catalytic mechanism would reduce the amount of
the deprotection reagents. We expected that 4-
mercaptophenylacetic acid (MPAA), a thiol additive to accelerate
[7]
NCL reaction, would function as the nucleophile for the π-allyl
[8]
palladium complex to facilitate the Alloc deprotection. Moreo-
ver, it is known that sulfur-containing compounds decrease the
reactivity of metal complexes because of the poisoning effects. [
We expected that MPAA would be utilized for quenching of the
palladium complexes after deprotection. Using these three func-
tions of MPAA, we anticipated that efficient one-pot multiple
peptide ligation would be accomplished through the repetitive
cycles of three steps: NCL, removal of the Alloc groups, and
quenching of the palladium complexes (Scheme 1A).
9]
Chemical protein synthesis has become one of the most
powerful methods to obtain a variety of tailor-made proteins.
This chemical method has enabled us to incorporate various
modifications into proteins at specific sites.[1] To achieve the
chemical synthesis of large proteins, native chemical ligation
NCL)[ is the most widely employed method to condense two
2]
(
side-chain unprotected peptide segments between an N-terminal
cysteine (Cys) and a C-terminal thioester. To ligate multiple
peptide segments, the N-terminal Cys of middle peptide seg-
ments should be protected to avoid polymerization and/or cy-
clization. For the one-pot multiple peptide ligation to improve
synthesis efficiency, there are three key steps: peptide ligation,
removal of protecting groups, and quenching of deprotection
reagents. However, it has been difficult to repeat these three
steps efficiently. For example, thiazolidine (Thz), removed with
methoxyamine, is a widely used protecting group of N-terminal
Cys for the ligation of several peptide segments.[3] However, the
removal of a Thz group requires a long reaction (>3h). Moreover,
during the subsequent NCL reaction, methoxyamine would react
with thioesters to generate a (N-methoxy)carboxamide.[4] There-
fore, rapid deprotection under mild conditions and complete
quenching of deprotection reagents without affecting the subse-
quent NCL reaction are required for efficient one-pot multiple
peptide ligation.
Scheme 1. Summary of the present research. (A) General scheme of multiple
peptide ligation in a one-pot manner. (B) Triple functions of MPAA in this study.
Herein, we report the first one-pot multiple peptide ligation
utilizing the triple functions of MPAA as (I) a thiol additive for
NCL, (II) a scavenger for the π-allyl complex, and (III) a quench-
er of palladium complexes (Scheme 1B). Finally, we accom-
plished the chemical synthesis of histone H2AX, a variant of
histone H2A, in high yield through one-pot five-segment ligation.
We began to investigate the function of MPAA as a scaven-
ger for the π-allyl palladium complex and a quencher for palladi-
um complexes. We prepared N-allyloxycarbonyl-S-tritylcysteine
(
Alloc-Cys(Trt)-OH), which would be applied to Fmoc solid-
Recently, Brik and coworkers reported the removal of pro-
tecting groups for N-terminal Cys, such as Thz or acetamidome-
thyl groups, with palladium complexes under NCL conditions.[5]
However, a substantial amount of palladium (15–40 equiv.) is
required to complete the deprotection, which would not be ade-
quate for the repetitive cycles of ligation in a one-pot approach.
Therefore, we decided to employ allyloxycarbonyl (Alloc) pro-
tecting groups,[6] which are removed with palladium (0) com-
phase peptide synthesis (SPPS) (Figure S1). This amino acid
was condensed with the peptide extended on the resin to afford
peptide 1 (Figure S2). Then, we tried to remove the Alloc group
of peptide 1 with palladium complexes employing 3,3',3''-
phosphanetriyltris(benzenesulfonic acid) trisodium salt (TPPTS)
as a ligand for palladium (Figure S3). In the absence of MPAA,
the reaction did not reach completion, which indicated that the
generated π-allyl palladium complex was trapped without the
attack by external nucleophiles (Figure S3B). By contrast, the
deprotection reaction with 2 equiv. of Pd/TPPTS complex
reached completion within 10 min in the presence of MPAA, and
allyl-MPAA was detected in the HPLC chart (Figure S3C). This
suggested that MPAA functioned as a scavenger for the π-allyl
palladium complex, and facilitated the removal of the Alloc group.
We attempted to achieve complete inactivation of the
Pd/TPPTS complex after removal of the Alloc group. We hy-
pothesized that ligand exchange with MPAA on palladium would
inactivate the Pd/TPPTS complex. The ligand exchange of the
[
[
a]
b]
N. Kamo, Dr. G. Hayashi, Prof. Dr. A. Okamoto
Department of Chemistry and Biotechnology, Graduate School of
Engineering, The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
Prof. Dr. A. Okamoto
Research Center for Advanced Science and Technology
The University of Tokyo
4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan
E-mail: okamoto@chembio.t.u-tokyo.ac.jp
Supporting information for this article is given via a link at the end of
the document.
31
Pd/TPPTS complex with MPAA was confirmed by P NMR
analysis (Figure 1). In the absence of MPAA, the dissociation of
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TPPTS from palladium was not observed (Figure 1C). However,
in the presence of MPAA (10 equiv. versus the Pd/TPPTS com-
plex), a signal derived from free TPPTS was observed after 10
min reaction (Figure 1D), suggesting that MPAA coordinated to
palladium and the dissociation of TPPTSs occurred.
saturated after the coordination of MPAA, and the formation of
16- or 14-electron complexes, which are important intermediates
for deprotecting the Alloc group, was prevented.[ Furthermore,
the replacement of MPAA with TPPTS changed the electron
density of palladium complexes, which could hamper the oxida-
tive addition of Alloc groups. It is notable that during removal of
the Alloc group by the Pd/TPPTS complex, inactivation of a
palladium complex by ligand exchange proceeded simultane-
ously and these two reactions reached completion within 10 min
10]
(
Scheme 2). When we compared MPAA with other thiophenol
derivatives as for scavengers and quenchers, MPAA and 4-
hydroxythiophenol functioned similarly (Figure S6). We decided
to employ MPAA, which is the most water-soluble among the
thiophenol derivative, for further experiments.
Figure 1. Analysis of ligand exchange of Pd/TPPTS complex with MPAA and
31
TCEP by P NMR measurement. NMR spectra of (A) mixture of TPPTS and
TPPTS oxide, (B) Pd/TPPTS complex. NMR spectra of Pd/TPPTS complex
after 10 min stirring (C) without MPAA, (D) with 10 equiv. of MPAA.
Table 1. Deprotection of the Alloc group of peptide 1 employing the Pd/TPPTS
complex after ligand exchange.
Scheme 2. Proposed mechanism of removal of Alloc groups and simultane-
ous inactivation of the Pd/TPPTS complex by ligand exchange with MPAA.
Conversion yield (%)[a]
Entry
3 min
94
5 min
82
10 min
94
Inactivation of the Pd/TPPTS complex by ligand exchange
enabled deprotection of the Alloc group and subsequent NCL
reaction in one-pot manner (Figure S7). The Alloc group of
peptide 1 was removed with 2 equiv. of Pd/TPPTS complex
within 10 min, and the Pd/TPPTS complex was inactivated by
ligand exchange with MPAA for a further 10 min. Then, peptide
1
2
3
Only water
100 mM MPAA
NCL buffer
69
40
>5
50
23
5
[
(
a] The conversion yields were calculated from peak areas analyzed by HPLC
shown in Figure S4).
3, bearing an Alloc-protected Cys at its N-terminus and a 2-
We examined the effect of the ligand exchange on the activi-
mercaptoethane sulfonate sodium salt (MESNa) thioester at its
C-terminus, was added to ligate with 2 for 2 h (Figure S7). The
HPLC profile showed a major peak corresponding to the desired
ligated product 4, which was isolated in 90% yield. As a result, a
cycle of removal of the Alloc groups, quenching of Pd/TPPTS
complex, and subsequent NCL reaction was accomplished
utilizing the triple function of MPAA. When the inactivation time
was less than 10 min, the self-cyclization and dimerization of
peptide 3 were observed as by-products (Figure S8). Therefore,
the inactivation of the Pd/TPPTS complex by ligand exchange
with MPAA was an important step to suppress the generation of
these by-products.
Using these interesting features of MPAA, we tried the first
one-pot five-segment ligation to afford a model polypeptide (41
amino acids), which was a modified peptide sequence of C-
terminal histone H4. The polypeptide was divided into five pep-
tide segments at the Cys sites (peptides 2, 3, 5, 7, 9), and each
segment was synthesized through Fmoc SPPS (Figure S9).
ty of the Pd/TPPTS complex. Pd/TPPTS solutions containing
only water (Table 1, entry 1), 100 mM MPAA (entry 2), or NCL
buffer (entry 3) were considered. At each time point (3 min, 5
min, or 10 min), peptide 1 bearing Alloc-protected Cys at its N-
terminus was added, then the removal of the Alloc group with
the Pd/TPPTS complex was analyzed by HPLC (Figure S4). In
the presence of only water, the activity of the Pd/TPPTS com-
plex was retained even after 10 min stirring. By contrast, in the
presence of 100 mM MPAA or in the NCL buffer, the activity of
the Pd/TPPTS complex was gradually reduced as the stirring
time increased. Under these conditions, the palladium complex
was completely inactivated after 10 min stirring. These results
indicated that the Pd/TPPTS complex could be inactivated by
ligand exchange with MPAA in the NCL buffer. We also found
that 40 mM tris(2-carboxyethyl)phosphine (TCEP) could inacti-
vate the Pd/TPPTS complex (Figure S4). Moreover, the removal
of the Alloc group did not proceed with the palladium complex
inactivated by ligand exchange, even after 20 h reaction at 37 C
(
Figure S5). We concluded that palladium complexes might be
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COMMUNICATION
Figure 2. Synthesis of a model peptide using one-pot five-segment ligation. (A) Synthetic route. (B) One-pot ligation monitored by analytical HPLC (gradient: 10–
5% for 35 min) monitored at 220 nm. 4, 6 and 8 = Alloc-protected peptides 4, 6, and 8. # = a ligation product between peptides 7 and 9. * = a ligation product
between peptides 5, 7, and 9. (C) HPLC profile (gradient: 10–45% for 35 min) and MALDI-TOF mass spectrum of purified peptide 10.
4
One-pot ligation of these five peptide segments was con-
ducted according to the synthetic procedure shown in Figure 3A.
All the NCL reactions and the removal of Alloc groups were
to initiate the fourth NCL reaction. After 2 h, full-length H2AX
with four Cys mutations (19) was observed as the major product
(Figure 3A, S13). The reaction product was purified by HPLC,
and the desired peptide 19 was obtained in 59% isolated yield
from 11, suggesting that each reaction step proceeded in 93%
yield on average. The purity of peptide 19 was confirmed by
HPLC and MALDI-TOF MS (Figure 3A). Such a high isolated
yield of the synthetic H2AX after one-pot five-segment ligation
suggests that our ligation method is more efficient than previous
attempts, which achieved one-pot four-segment ligation (tri-
2 4
conducted in NCL buffer (6 M Gn·HCl, 200 mM NaH PO , 100
mM MPAA, 40 mM TCEP) at 37 °C. Peptides 2 and 3 (1.1 equiv
versus peptide 2) were dissolved in NCL buffer and the NCL
reaction reached completion within 1 h to afford peptide 4 (Fig-
ure 2B). Pd/TPPTS complex (2 equiv.) was added to the reac-
tion solution, and the mixture was stirred for 20 min. Then, pow-
dered peptide 5 was added to initiate the second NCL reaction.
These operations were repeated for the second deprotection
and the third NCL and deprotection (Figure 2B). Finally, pow-
dered peptide 9 was added to initiate the fourth NCL reaction.
HPLC analysis after 2 h showed a major peak corresponding to
the desired ligated peptide 10 (Figure S10). We identified the
other HPLC peaks as deriving from the hydrolysis of thioesters
or cyclization of added peptide segments. The reaction product
was purified by HPLC, the desired peptide 10 was obtained in
fluoroacetamidomethyl group, 45%; seleno- and thioesters,
29%; N-sulfanylethylanilide, 20%).[
13-15]
A total of seven steps
were completed within 15 h, and furthermore, the concentration
of the peptide elongated from the first peptide did not change
greatly (initial, 2.1 mM; final, 1.7 mM) because only small
amounts of additives are used for the entire synthetic procedure.
66% isolated yield from 2, suggesting that each reaction step
(
four ligations and three deprotection steps) proceeded in 94%
yield on average. The purity of peptide 10 was confirmed by
HPLC and MALDI-TOF MS (Figure 2C).
Based on the successful one-pot five-segment peptide liga-
tion, we next attempted the chemical synthesis of full-length
human histone H2AX, a variant of histone H2A. This protein is
exchanged with canonical H2A in chromatin fibers mainly in S
phase. When a double-strand DNA break occurs during DNA
replication, the S139 at the C-terminus of H2AX is phosphory-
lated by some kinases to form gamma-H2AX (γ-H2AX).[11] This
modification contributes to the recruitment of DNA-repairing
proteins. Recently, relationships between PTMs on H2AX have
been energetically investigated.[12]
H2AX bearing 142 amino acids does not contain Cys resi-
dues, which are necessary for peptide ligation by the NCL reac-
tion. Therefore, we introduced four Cys mutations at four Ala
2
1
53
86
122
sites (A C, A C, A C, A C), and full-length H2AX was divid-
Figure 3. Synthesis of histone H2AX. (A) a) HPLC chart of the fourth NCL
reaction between peptides 17 and 18 for 2 h (gradient: 10–66% for 35 min). #
ed into five peptide segments (Scheme S1, peptides 11, 12, 14,
6, and 18), which were synthesized by Fmoc SPPS (Figure
=
allyl-MPAA. * = mixture of a self-cyclized formation of peptide 14 and a
1
ligation product of peptides 14, 16, and 18. b) HPLC chart, and c) MALDI-TOF
mass spectrum of purified peptide 19. (B) Size-exclusion chromatography
analysis of reconstituted H2AX–H2B dimer (Protein Data Bank entry 3AFA of
H2A–H2B dimer) containing either recombinant H2AX or synthetic H2AX.
S11).
The one-pot five-segment ligation using the Pd/TPPTS complex
was conducted according to the synthetic procedure shown in
Scheme S1. Each NCL reaction and the deprotection of the
Alloc groups by the Pd/TPPTS complex were monitored by
HPLC (Figure S12). Finally, 1.2 equiv of peptide 18 was added
Subsequent desulfurization, which converted mutated Cys
residues into the original Ala residues, afforded peptide 20 (full-
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COMMUNICATION
length H2AX) in 79% isolated yield (Figure S14). Analysis by
atomic absorption spectrophotometer showed that palladium
content attached on chemically synthesized H2AX was
Keywords: chemical protein synthesis • peptides • palladium •
native chemical ligation
0
.00096% (Figure S15).
The heterodimer of synthetic H2AX (20) and H2B was recon-
[
1]
a) L. Zhou, M. T. Holt, N. Ohashi, A. Zhao, M. M. Müller, B. Wang, T. W.
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stituted to examine whether synthetic H2AX would be folded
properly. Recombinant H2AX or synthetic H2AX were dissolved
in denaturing conditions, and recombinant H2B was added in
separate experiments. These solutions were dialyzed for one
day, and analyzed by size-exclusion chromatography. The
chromatography profile showed a major peak corresponding to
the H2AX–H2B heterodimer (Figure 3B). Each heterodimer was
collected and analyzed by HPLC. The ratios of the peak areas of
synthetic H2AX to H2B were almost the same as those of re-
combinant H2AX to H2B (Figure S16). These results indicated
that synthesized H2AX was properly folded to form a H2AX–
H2B heterodimer, just as recombinant H2AX was.
In conclusion, we have devised an efficient one-pot multiple
peptide ligation employing the triple function of MPAA. We fo-
cused on the scavenging and poisoning effect of MPAA on
palladium complexes, and utilized these features to accomplish
the inactivation of the Pd/TPPTS complex after the removal of
Alloc groups. From clear HPLC data and the high isolated yield
of H2AX, we envisage that six or more peptide segments could
be also assembled in a one-pot manner using our method.
Therefore, our strategy is adequate for the chemical synthesis of
large multifunctionalized proteins. We expect that this technique
will lead to the elucidation of biological functions of proteins and
the development of new medicines.
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The authors declare no conflict of interest.
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Entry for the Table of Contents (Please choose one layout)
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Naoki Kamo, Gosuke Hayashi and
Akimitsu Okamoto*
Page No. – Page No.
Triple Function of 4-
Mercaptophenylacetic Acid Promotes
One-Pot Multiple Peptide Ligation
Efficient one-pot multiple peptide ligation was accomplished through repetitive
native chemical ligation (NCL) and the removals of allyloxycarbonyl (Alloc) groups
with palladium complexes. In this reaction system, we utilized triple functions of 4-
mercaprophenylacetic acid (MPAA).
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