Development of a Chemical Methodology for the Preparation of Peptide Thioesters Applicable to Naturally Occurring Peptides Using a Sequential Quadruple Acyl Transfer System

Article abstract of DOI:10.1002/open.201500086

Peptide thioesters are very useful in protein chemistry, and chemistry- and biochemistry-based protocols are used for the preparation of thioesters. Among such protocols, only a few biochemistry-based approaches have been use for naturally occurring peptide sequences. The development of chemistry-based protocols applicable to natural sequences remains a challenge, and the development of such methods would be a major contribution to protein science. Here, we describe the preparation of peptide thioesters using innovative methodology that features nickel(II)-mediated alcoholysis of a naturally occurring peptide sequence, followed by O-N and N-S acyl transfers. This protocol involves sequential quadruple acyl transfer, termed SQAT. Notably, the SQAT system consists of sequential chemical reactions that allow naturally occurring peptide sequences to be converted to thioesters without requiring an artificial chemical unit. SQATs: Peptide thioesters are an important compound class in peptide chemistry, but chemistry-based protocols to synthesize natural-peptide-based thioesters are challenging. Here, a sequential quadruple acyl transfer (SQAT) protocol is described for the preparation of thioesters. Importantly, the SQAT system can be applied to naturally occurring peptides, converting them to their corresponding thioesters without the need for an artificial chemical unit.

Full text of DOI:10.1002/open.201500086

DOI: 10.1002/open.201500086
Development of a Chemical Methodology for the
Preparation of Peptide Thioesters Applicable to Naturally
Occurring Peptides Using a Sequential Quadruple Acyl
Transfer System
Yusuke Tsuda, Akira Shigenaga, Kohei Tsuji, Masaya Denda, Kohei Sato, Keisuke Kitakaze, Takahiro Nakamura,
Tsubasa Inokuma, Kohji Itoh, and Akira Otaka*[a]
Peptide thioesters are very useful in protein chemistry, and
chemistry- and biochemistry-based protocols are used for the
preparation of thioesters. Among such protocols, only a few
biochemistry-based approaches have been use for naturally oc-
curring peptide sequences. The development of chemistry-
based protocols applicable to natural sequences remains a chal-
lenge, and the development of such methods would be
a major contribution to protein science. Here, we describe the
preparation of peptide thioesters using innovative methodolo-
gy that features nickel(II)-mediated alcoholysis of a naturally
occurring peptide sequence, followed by OÀN and NÀS acyl
transfers. This protocol involves sequential quadruple acyl
transfer, termed SQAT. Notably, the SQAT system consists of se-
quential chemical reactions that allow naturally occurring pep-
tide sequences to be converted to thioesters without requiring
an artificial chemical unit.
tocols applicable to expressed proteins remains necessary. Re-
ported chemical methods include the NÀS acyl-transfer-medi-
ated conversion of specific cysteinyl sequences to thio-
esters[9,10] and the S-thiocarbonylation of a cysteinyl residue fol-
lowed by acyl-guanidine-mediated cleavage;[11] however, these
pioneering attempts still require further investigation to
enable their practical use. During the progress of the present
work, Macmillan and co-workers developed a chemical method
based on NÀS acyl-transfer chemistry.[12] However, an innova-
tive protocol for thioester preparation applicable to a naturally
occurring sequence is still required because the present proto-
cols do not always afford satisfactory results. Thus, we have ap-
proached the subject from a different perspective and report
herein a chemical approach to peptide thioesters production
that can be applied to naturally occurring peptide sequences.
We began our research by evaluating the applicability of the
nickel(II)-assisted peptide bond hydrolysis developed by Bal
and co-workers[13] to thioester synthesis. In nickel(II)-assisted
hydrolysis, the peptide bond preceding the serine or threonine
in the sequence S/T-X-H-Z (where X and Z can be any amino
acid residues except proline) is hydrolyzed. Mechanistic investi-
gations of the hydrolysis indicated that two crucial steps are
involved in the reaction: 1) formation of a square-planer nick-
el(II)-bound active complex consisting of the imidazole nitro-
gen and the three preceding amide nitrogens; 2) formation of
O-peptidyl intermediates resulting from NÀO acyl transfer of
the amide bond preceding the serine or threonine residue and
subsequent hydrolysis of the O-acyl intermediate (Scheme 1). A
similar NÀO acyl transfer is also seen in intein-mediated pro-
tein splicing, and this prompted us to investigate the nickel(II)-
mediated hydrolysis in the preparation of thioesters.
There is increasing interest in the challenge of preparing pep-
tide/protein thioesters, through chemical or biochemical
means, because of their great utility in protein synthesis using
native chemical ligation (NCL).[1] Protocols using chemical devi-
ces for the synthesis of peptide thioesters have been exten-
sively reported in the literature.[2–4] However, one major short-
coming of the use of chemical devices is incompatibility with
expressed proteins. Engineered intein[5,6] or sortase-mediat-
ed[7,8] methodologies have enabled the preparation of thioest-
ers from expressed proteins, in which thioester formation relies
on the enzyme or enzyme-like activity of sortase or intein, re-
spectively. Although such biochemical processes have gradual-
ly become popular, the development of chemistry-based pro-
Initially, we examined the applicability of the nickel(II)-
mediated hydrolysis to the peptide sequence: H-
[a] Y. Tsuda, Dr. A. Shigenaga, K. Tsuji, M. Denda, K. Sato, K. Kitakaze,
T. Nakamura, Dr. T. Inokuma, Prof. K. Itoh, Prof. A. Otaka
Institute of Health Bioscience and Graduate School of Pharmaceutical-
Sciences, Tokushima University, Shomachi, Tokushima 770-8505 (Japan)
E-mail: aotaka@tokushima-u.ac.jp
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/open.201500086.
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
made.
Scheme 1. Formation of O-acyl peptide intermediates during nickel(II)-medi-
ated hydrolysis of peptide bond preceding Ser/Thr-X-His-Z sequence.
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AKLRFGCPSRHWKFL-NH2 (1),[14] with the anticipated one-step
formation of a thioester: H-AKLRFG-SR (2). The reasons for se-
lecting peptide 1 were based on Aimoto’s experimental results,
in which a cysteinyl prolyl ester afforded peptide thioesters.[9]
However, subjecting peptide 1 to the nickel(II)-mediated reac-
tion failed to afford thioester 2.[15]
Table 1. Nickel(II)-mediated conversion of 5a to oxyesters.
We speculated that one possible reason for the failure was
related to the presence of the cysteinyl residue close to the
nickel(II)-bound active complex. We therefore examined the
hydrolysis of a peptide, H-AKLRFGAPSRHWKFL-NH2 (3), with
alanine substituted for cysteine. Although the nickel(II)-mediat-
ed hydrolysis of 3 (100 mm NiCl2, 508C, pH 8.2) did not go to
completion even after 48 h, we found that the reaction in the
presence of 0.2m tris(hydroxymethyl)aminomethane (Tris) af-
forded a small amount of a Tris adduct, H-AKLRFGAP-Tris (4),
on the processed N-peptide, the N-terminal half of the cleaved
peptide (see Figure S1 in the Supporting Information). It is
worth noting that an appropriate oxygen nucleophile has
a good chance of being involved in the nucleophilic conver-
sion of the serine-isopeptide intermediate to the correspond-
ing oxyester via OÀO intermolecular acyl transfer. This suggest-
ed the feasibility of a stepwise conversion of an SRHW-contain-
ing peptide to a thioester through an oxyester. We therefore
next used Ac-LYRAASRHWKFL-NH2 (5a), which has a more
scissile alanyl–serine linkage, to examine the conversion to an
oxyester.
Entry
Conditions[a]
NiCl2 [mm]
Nucleophile
FC[b]
pH
[d]
1
2
3
4
5
6
7
8
10
10
10
10
10
1
20
10
10
10
1
8.2
8.2
8.2
8.2
8.2
8.2
8.2
7.8
8.6
8.2
8.2
8.2
50% (v/v) TFE
–
–
[d]
50% (v/v) iPrOH
10% (v/v) MeOH
30% (v/v) MeOH
50% (v/v) MeOH
30% (v/v) MeOH
30% (v/v) MeOH
30% (v/v) MeOH
30% (v/v) MeOH
30% (v/v) MeOH
30% (v/v) MeOH
30% (v/v) MeOH
0.44
0.70
0.72
0.69
0.68
0.61
0.53
0.43
0.59
0.65
9
10[c]
11
12
10
[a] Reactions were performed in 0.2m HEPES buffer at 378C for 12 h in
the presence of 1 mm of 5a. [b] The fraction converted (FC) was deter-
mined by HPLC separation and integration (integ.) of 8a (or 9) as a frac-
tion of the sum of the integration of unreacted 5a+hydrolyzed 6a+8a
(or 9). [c] In the presence of 6m Gn·HCl. [d] Oxyesters were not obtained.
Standard hydrolysis reaction of 5a in 0.2m 2-[4-(2-hy-
droxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer
(pH 8.2) in the presence of 10 mm nickel(II) chloride at 378C
went to completion within 12 hours to give the corresponding
hydrolyzed peptides, Ac-LYRAA-OH (6a) and H-SRHWKFL-NH2
(7). Based on these hydrolysis conditions, the nickel(II)-mediat-
ed conversion of 5a to the corresponding oxyester in the pres-
ence of alcohols was examined (Table 1). The use of trifluoroe-
thanol (TFE) or propan-2-ol as a nucleophile failed to yield the
corresponding oxyester. However, methanol participated in the
nucleophilic attack to yield the methyl ester peptide,
Ac-LYRAA-OMe (8a). The fraction converted increased with in-
creasing methanol concentration (entries 3–5 Table 1). Across
the nickel(II) concentration range (1–20 mm), no significant dif-
ferences were observed (entries 4, 6 and 7 in Table 1). Metha-
nolysis at pH 8.2 gave the best result (entries 4, 8 and 9 in
Table 1).[16] The nickel(II)-mediated reaction tolerated the pres-
ence of guanidine hydrochloride (Gn·HCl), although the frac-
tion converted decreased (entry 10 in Table 1). Conversion to
OÀS acyl-transfer step under acidic conditions using an alterna-
tively synthesized l-alanine-containing peptide.[20]
To develop an alternative methodology to the OÀS acyl-
transfer step, we focused on an innovative protocol involving
a peptide hydrazide/azide, reported by Liu and co-workers.[21]
As such, we next examined thioester synthesis from methyl
ester 8a using hydrazide/azide. The nickel(II)-mediated alcohol-
ysis of 5a (0.2m HEPES, 10 mm NiCl2, 50% (v/v) MeOH, pH 8.2,
378C, for 12 h), followed by addition of hydrazine monohy-
drate (NH2NH2·H2O) to the reaction mixture (final concentra-
tion: 5% (v/v) NH2NH2·H2O) with additional reaction for 1 hour
at 258C, gave the peptide hydrazide, Ac-LYRAA-NHNH2 (11 a),
in 80% isolated yield.[22] Furthermore, the resulting 11 a was
converted to the corresponding sodium mercaptoethanesulfo-
nate (MESNa) thioester, Ac-LYRAA-SCH2CH2SO3Na (13a), using
Liu’s conditions via the peptide azide, Ac-LYRAA-N3 (12a), and
no epimerization was observed in the sequence of reactions.[23]
We named this thioesterification system SQAT, because the
thioesters were produced by sequential quadruple acyl transfer
(NÀO, OÀO, OÀN, and NÀS acyl transfers) as shown in
Scheme 2.
a
dithiodiethyl (DTDE; HOCH2CH2S-SCH2CH2OH) oxyester,
Ac-LYRAA-OCH2CH2S-SCH2CH2OH (9), also proceeded (en-
tries 11 and 12 in Table 1). Here, DTDE was selected due to the
anticipation of the OÀS acyl-transfer-mediated conversion of
the DTDE oxyester to a thioester.[4] Among several attempts to
achieve conversion to the thioesters,[17] the use of 0.1% (v/v)
trifluoromethanesulfonic acid/5% (v/v) para-thiocresol in tri-
fluoroacetic acid successfully converted 9 to the corresponding
methylphenyl thioester, Ac-LYRAA-SPh(4-Me) (10), via
a tandem thiol switch.[18,19] However, this procedure is accom-
panied by the formation of a considerable amount of alanine-
epimerized peptide. The origin of this was shown to be an
These results encouraged us to examine the applicability of
the protocol to the 20 other naturally occurring amino acids
(X)–serine junctions in Ac-LYRAXSRHWKFL-NH2 (5) (Table 2).
Several X-SRHW sequences were proven to be potential sites
for thioester synthesis.
The feasibility of preparing thioesters using the SQAT system
was confirmed by NCL-mediated syntheses of C-type and A-
type natriuretic peptides (CNP and ANP, 53- and 28- residue
naturally occurring peptides, respectively). For the preparation
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of CNP (14) and ANP (15), 43-residue nickel-sensitive SRHW-
fused peptide 16 (CNP 53 1–36+SRHWKFL-NH2) and 29-resi-
due peptide 17 (ANP 28 1–22+SRHWKFL-NH2) were synthe-
sized respectively. Treatment of 43-residue CNP peptide 16
with 10 mm nickel(II) chloride/50% (v/v) methanol in 0.2m
HEPES, pH 8.2, at 378C for 6 hours, followed by hydrazine mon-
ohydrate treatment at 258C for 1 hour, yielded a 36-residue
peptide hydrazide 18 as the main processed N-peptide via the
peptide methyl ester 19 in 69% isolated yield. Treatment of
the resulting hydrazide 18 with 20 mm sodium nitrite in 6m
Gn·HCl–0.2m sodium phosphate, pH 3.0, at 08C for 1 hour
gave the corresponding peptide azide. Without purification,
the peptide azide was treated with 200 mm (4-carboxyme-
thyl)thiophenol (MPAA) in 6m Gn·HCl–0.2m sodium phosphate,
pH 7.0, at room temperature for 1 hour, affording the MPAA
thioester, which was then subjected to NCL with the N-termi-
nal cysteinyl CNP (37–53) fragment 20 to yield the reduced
form CNP 53 21 in 47% isolated yield after HPLC purification.
Folding of the reduced material 21 in 6m Gn·HCl–0.1m sodium
phosphate, pH 7.3/DMSO (9:1) afforded CNP 14.
Scheme 2. Sequential quadruple acyl transfer (SQAT) system for thioester
synthesis.
Table 2. Conversion of peptides 5 to peptide hydrazides 11.[a]
Next, applicability of the SQAT system to cysteine-containing
peptides was verified by conversion of the cysteine-containing
ANP precursor 17 to the corresponding peptide hydrazide 22
(Scheme 3 and Figure 1). Experimental manipulation similar to
that employed for the conversion of 16 to 18 was conducted
for the preparation of 22, except for trapping of nickel(II) by
the addition of ethylenediaminetetraacetic acid (EDTA) to the
Entry
Peptide 5
X
FC to 11[b]
1
2
3
4
5
6
7
8
5a
5b
5c
5d
5e
5 f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
A
G
D
E
N
Q
S
0.61 (80%[c])
0.66
0.40
0.69
[d]
–
–
[e]
0.42
0.48
T
[f]
9
C
P
V
M
L
I
Y
F
H
K
R
W
–
10
11
12
13
14
15
16
17
18
19
20
0.09
0.12[g]
0.58
0.66[h]
[g]
–
0.40
0.41
0.57
0.65
0.59
0.62
5s
5t
[a] Peptide 5 (1 mm) in 0.2m HEPES was treated in the presence of
10 mm NiCl2 and 50% (v/v) MeOH, pH 8.2, at 378C for 24 h, followed by
addition of NH2NH2·H2O (final concentration: 5% (v/v) NH2NH2) and left to
react at 258C for a further 3 h. [b] The fraction converted (FC) was deter-
mined by HPLC separation and integration (integ.) of 11 as a fraction of
the sum of the integration of unreacted 5+hydrolyzed 6+8+11.
[c] Under optimized conditions (see main text), 11 a was obtained in 80%
isolated yield. [d] a,b-Dihydrazide peptide was obtained. [e] A mixture of
a and g-hydrazide peptides was obtained. [f] No N-processed peptides
(6i, 8i, and 11 i) were observed. [g] Although the initial NÀO acyl shift
proceeded, subsequent reactions did not proceed to completion. [h] For
satisfactory HPLC purification, N-terminally extended peptide Ac-KLYR-
ALSRHWKFL-NH2 (5m) was used.
Figure 1. HPLC monitoring of reactions for the synthesis of ANP: a) nickel(II)-
mediated methanolysis (t=0 h); b) nickel(II)-mediated methanolysis (t=3 h);
c) hydrazinolysis (t=3 h); d) azidation (t=0 h); e) azidation (t=1 h); f) Thioly-
sis (t=1 h); g) native chemical ligation (NCL) (t=0 h); h) NCL (t=4 h).
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terization of the generated pro-
tein thioesters is underway, and
the results of this study will be
presented in due course. Finally,
we believe that the SQAT system
described here will become
a useful chemical procedure for
thioester preparation as a com-
plement to other available pro-
tocols including the intein-medi-
ated procedures.
Acknowledgements
This research was supported in
part by a Grant-in-Aid for Scientif-
ic Research (KAKENHI) from the
Japan Society for the Promotion
of Science (JSPS) and research
grants from the Takeda Science
Foundation (Osaka, Japan) and
the Uehara Memorial Foundation
(Tokyo, Japan). K.S. is grateful for
a scholarship from the Yoshida
Scholarship Foundation (Tokyo,
Japan). Y.T. and M.D. thank the
Japan Society for the Promotion
of Science (JSPS) for fellowships.
Scheme 3. Chemical synthesis of ANP using the sequential quadruple acyl transfer (SQAT) system. a) Peptide 17
(1 mm) was treated in 0.2m HEPES in the presence of 10 mm NiCl2 and 50% (v/v) MeOH, pH 8.2, at 378C for 3 h,
followed by addition of EDTA into the reaction mixture. b) NH2NH2·H2O was added into the reaction mixture (final
concentration: 5% (v/v) NH2NH2) and left to react at 258C for a further 3 h. c) Peptide 22 was treated in 0.2m
sodium phosphate in the presence of 6m Gn·HCl and 20 mm NaNO2, pH 3.0, at À108C for 1 h. d) MESNa-contain-
ing buffer (6m Gn·HCl, 0.2m sodium phosphate, 200 mm MESNa) was added into the reaction mixture, and the
pH of the solution was adjusted to pH 7.0 by using 2 m NaOH (aq). Then, the reaction mixture was stored at
room temperature for 1 h. e) Peptide 24 and thiophenol were added into the reaction mixture (final concentra-
tion: peptide 23 (1.5 mm), 24 (2.0 mm), 5% (v/v) thiophenol) and left to react at 378C for a further 4 h.
reaction mixture before hydrazinolysis. Without treatment with
EDTA, desired peptide disappeared upon HPLC analysis of reac-
tion mixture after hydrazinolysis. This is probably attributable
Keywords: acyl transfer · native chemical ligation · nickel(II)-
mediated alcoholysis · peptide hydrazides · peptide thioesters
to formation of insoluble metal nickel species by reduction of
nickel(II) with hydrazine, on which the cysteinyl peptides were
adsorbed. Application of the modified SQAT system with the
additional EDTA treatment successfully generated thioester 23
for ANP synthesis.[24] NCL of 23 with N-terminal cysteinyl pep-
tide (ANP 23–28 24), followed by folding gave ANP 15.
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´
´
[13] a) A. Kre¸z˙el, E. Kopera, A. M. Protas, J. Poznanski, A. Wysłouch-Cieszyn-
[22] Nickel(II)-mediated hydrolysis and hydrazinolysis did not proceed in the
presence of hydrazine.
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´
Kre¸z˙el, A. M. Protas, A. Belczyk, A. Bonna, A. Wysłouch-Cieszynska, J.
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the SQAT-mediated conversion. See, Figure S10 and S11 in the Support-
ing Information.
´
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[14] SRHW is the best nickel(II)-sensitive sequence identified to date by pep-
tide library screening. See Ref. [13a].
[24] During azidation, S-nitrosyl peptide 26 was formed; however, subse-
quent thiolysis regenerates the thiol group.
[15] Exposure of peptides possessing a cysteine residue close to the SRHW
sequence afforded processed C-peptide, whereas no cysteine-contain-
ing N-peptides were obtained (Entry 9, Table 2).
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Saito, H. Sakuraba, K. Itoh, Mol. Ther. 2011, 19, 1017–1024.
[16] Because initial methanolysis was carried out in a mixed solvent (MeOH/
H2O), the best conversion yields were around 70%. Attempts to in-
crease the conversion yield have been carried out. The use of MeOH as
a solvent sometimes causes solubility problems for proteins.
Received: March 31, 2015
Published online on April 28, 2015
ChemistryOpen 2015, 4, 448 – 452
www.chemistryopen.org
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ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim