The use of a cysteinyl prolyl ester (CPE) autoactivating unit in peptide ligation reactions

Article abstract of DOI:10.1016/j.tet.2009.03.008

A peptide containing a cysteinyl prolyl ester (CPE) moiety at the C-terminus (CPE peptide) is spontaneously transformed into a diketopiperazine thioester via an intramolecular N-S acyl shift reaction, followed by diketopiperazine formation. The CPE peptide can be ligated with a Cys-peptide in a one-pot procedure. The peptide diketopiperazine thioester can also be transformed into a peptide thioester by intermolecular thiol-thioester exchange with external thiol compounds such as sodium mercaptoethanesulfonate. Since CPE peptides can be prepared by standard Fmoc solid-phase synthesis, it is a versatile alternative to the peptide thioester, providing a flexible ligation strategy that promises to be useful in polypeptide synthesis.

Full text of DOI:10.1016/j.tet.2009.03.008

Tetrahedron 65 (2009) 3871–3877
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
The use of a cysteinyl prolyl ester (CPE) autoactivating
unit in peptide ligation reactions
*
*
Toru Kawakami , Saburo Aimoto
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A peptide containing a cysteinyl prolyl ester (CPE) moiety at the C-terminus (CPE peptide) is sponta-
neously transformed into a diketopiperazine thioester via an intramolecular N–S acyl shift reaction,
followed by diketopiperazine formation. The CPE peptide can be ligated with a Cys-peptide in a one-pot
procedure. The peptide diketopiperazine thioester can also be transformed into a peptide thioester by
intermolecular thiol–thioester exchange with external thiol compounds such as sodium mercaptoetha-
nesulfonate. Since CPE peptides can be prepared by standard Fmoc solid-phase synthesis, it is a versatile
alternative to the peptide thioester, providing a flexible ligation strategy that promises to be useful in
polypeptide synthesis.
Received 6 February 2009
Received in revised form 3 March 2009
Accepted 3 March 2009
Available online 9 March 2009
Keywords:
CPE autoactivating unit
Peptide ligation
Peptide thioester
N–S acyl shift reaction
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
and cleaved by acidolysis by treatment with an acid, such as an-
hydrous hydrogen fluoride to give the peptide thioester.2,6 While
Chemical ligation methodology is a powerful and useful strategy
for protein synthesis. The method involves the use of a peptide
thioester as a building block, which can be prepared by either
chemical or biological methods.1 In the thioester method, a par-
tially protected peptide thioester is used as a building block and
condensed in the presence of silver ions as an activating reagent for
the thioester.2 Native chemical ligation permits chemoselective
ligation, in which an unprotected peptide thioester can be ligated
with a cysteinyl peptide in an aqueous buffer solution.3 In the
extended chemical ligation strategy, a thiol auxiliary, attached to
the N-terminal amino group, is used instead of a cysteine residue
and an unprotected peptide thioester is used as a building block,
thus retaining the advantageous features of the native chemical
ligation reaction.4,5
a peptide thioester can be readily prepared by the Boc SPPS
method, the preparation of glycosylated7 or phosphorylated8
thioesters remains a formidable task. In addition, the preparation
of
a peptide thioester using the 9-fluorenylmethoxycarbonyl
(Fmoc) SPPS method continues to be a challenge, because the
thioester decomposes in the presence of piperidine, a reagent that
is commonly used to remove the Fmoc group. Weak nucleophilic
bases can be used in a direct synthesis: a mixture of 1-methyl-
pyrrolidine, hexamethylenimine, and 1-hydroxybenzotriazole
(HOBt) can be used to remove the Fmoc group, thus permitting the
isolation of the intact thioester. Using this approach, a peptide
thioester can be prepared in a straightforward manner,9 but the
chiral amino acid residue at the thioester position is racemized to
some extent during the reaction.10 Indirect methods have also been
developed. For example, a peptide chain can be elongated on the
sulfonamide linker, and the linker can then be activated and
replaced by an external thiol.11 This procedure is in widespread use,
but has some drawbacks. For example, the sulfonamide moiety can
undergo acetylation during the capping cycle12 and methionine
Although these ligation methods were originally developed for
use in conjunction with a peptide thioester, improved methods for
preparing such thioesters would be highly desirable. Peptide
thioesters can be prepared in a straightforward manner using
tert-butoxycarbonyl (Boc) solid-phase peptide synthesis (SPPS)
methodology. Starting from
a
thioester resin, such as Boc-
residues are frequently alkylated during the activation step.13
A
Gly-SCH2CH2CO- -Ala-MBHA resin, the peptide chain is elongated
b
number of synthetic methods for preparing peptide thioesters
based on the Fmoc SPPS procedure have been reported. These
methods include the use of a Wang linker, which is replaced by
a thiol,14 an aryl hydrazine linker,15 an in situ O to S acyl shift re-
action,16 trithioortho esters,17 and an O-aminoanilide moiety,
which is activated to give the N-acylurea, and thiolysis to give
* Corresponding authors. Tel.: þ81 6 6879 8602; fax: þ81 6 6879 8603.
E-mail addresses: kawa@protein.osaka-u.ac.jp (T. Kawakami), aimoto@protein.
osaka-u.ac.jp (S. Aimoto).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.03.008

3872
T. Kawakami, S. Aimoto / Tetrahedron 65 (2009) 3871–3877
a thioester.18 Each method may overcome some of the difficulties
associated with peptide thioester synthesis by the Fmoc SPPS
procedure.
HS
O
O
O
O
peptide 1
S
N
N
O
peptide 1
N
H
HN
In our strategy, the basis for preparing a peptide thioester in-
volves an N to S acyl shift reaction. We previously reported on
a method for preparing peptide thioesters, based on an N–S acyl
shift reaction mediated by a 4,5-dimethoxy-2-mercaptobenzyl
(Dmmb) group,19 which was originally reported as a removable
auxiliary for extended chemical ligation.4c The Dmmb group, when
attached to an amide, can be removed by treatment with a strong
acid, such as trifluoromethanesulfonic acid, whereas an S-peptide
(thioester intermediate) is formed under weakly acidic conditions,
such as in the presence of trifluoroacetic acid (TFA), as the result of
an intramolecular N–S acyl shift reaction.20 The S-peptide can be
transformed into the corresponding stable peptide thioester by an
intermolecular thiol–thioester exchange reaction.19–21 The N–S acyl
shift reaction was also used to prepare peptide thioesters by other
groups.22
The Cys-containing peptide 1 can also be converted into S-
peptide 2 by an intramolecular N–S acyl shift reaction under
acidic conditions such as in the presence of TFA (Scheme 1).20
This S-peptide can easily be transformed into the original peptide
even under weakly acidic conditions such as a 0.1% TFA solution,
which is frequently used in RP-HPLC eluents.20 These findings
suggest that the amide and thioester forms of the Cys-containing
peptide are present in equilibrium and that the amide form is the
dominant species. If the equilibrium could be controlled by
blocking the amino group, which forms as the result of the N–S
acyl shift reaction, it would be possible to produce a peptide
thioester based on an N–S acyl shift reaction at the Cys residue.
R1
O
O
5
6
HS
H2N
peptide 2
HS
O
7
O
peptide 2
peptide 1
N
H
O
8
Scheme 3. Thioester formation and ligation of CPE peptide.
contains no thioester moiety itself. Moreover, the autoactivating
function of the CPE unit can be quenched by introducing a thiol
blocking group in the Cys residue, which would prevent inter- and
intramolecular ligation itself, and the direction of ligation at the N
or C terminus could be controlled,25 thus providing a flexible
ligation strategy for polypeptide synthesis using multi-component
peptide building blocks. In this report, we provide a detailed
description of such thioester formation and ligation using the CPE
autoactivating unit.
2. Results and discussion
2.1. Preparation of a peptide containing CPE unit
A peptide containing the CPE unit can be readily prepared by
standard Fmoc SPPS because no thioester bond is present during
the chain elongation cycles. A typical procedure is shown in the
model peptide synthesis (Scheme 4). Glycolic acid was first
attached to the Rink amide resin 9, and Fmoc-Pro-OH was then
introduced. It was necessary to introduce the dipeptide, Fmoc-Xaa-
Cys(Trt)-OH (11), in order to avoid DKP formation from Cys-Pro
ester residues. Dipeptide 11 was conveniently prepared by one-pot
reaction of Fmoc-Xaa-OSu, which was formed by mixing Fmoc-Xaa-
OH, N-hydroxysuccinimide (HOSu), and N-(3-dimethylamino-
propyl)-N0-ethylcarbodiimide hydrochloride, with H-Cys(Trt)-OH
in the presence of N,N-diisopropylethylamine (DIEA) in a mixed
solvent of dichloromethane and N,N-dimethylformamide (DMF)
(1:1). When dipeptide 11 was introduced using 1-hydroxybenzo-
triazole (HOBt) in combination with diisopropylcarbodiimide
O
HS
S
O
peptide
peptide
peptide
N
H
peptide
H2N
O
1
O
2
Scheme 1. Equilibrium between amide and thioester at the cysteine residue.
In 1985, Zanotti et al. reported on a reaction in which the
phenylacetyl cysteinyl proline p-nitrophenyl active ester 3 is con-
verted into the diketopiperazine (DKP) thioester 4 under reductive
conditions (Scheme 2).23 The reaction would be predicted to pro-
ceed via the reduction of the disulfide, a subsequent N–S acyl shift
reaction, followed by diketopiperazine formation with the proline
active ester. As a result, a peptide thioester is spontaneously pro-
duced by an intramolecular reaction. This reaction would be ideal
for blocking the amino group, since it would stabilize the thioester
and prevent the S–N acyl shift reaction from occurring.
(DIPCI), the model peptide contained ca. 40% D-Cys residues. The
racemization was reduced to less than 10%, when 3-hydroxy-1,2,3-
bezotriazin-4(3H)-one (HOObt)26 was used in place of HOBt. The
peptide chain was then elongated by standard Fmoc chemistry to
give the protected peptide resin, Fmoc-His(Trt)-Pro-Ile-Arg(Pmc)-
Fmoc-HN
1) piperidine
9
t
BuS
O
S
O
O
O
Ph
2) HOCH2CO2H, DIPCI, HOBt
3) Fmoc-Pro-OH, HBTU, DIEA
S
N
Ph
N
O
NO2
N
HN
H
Fmoc-Pro-OCH2CO-NH
O
10
O
3
4
1) piperidine
Scheme 2. Formation of the thioester of DKP.23
2) Fmoc-Xaa-Cys(Trt)-OH (11),
DIPCI, HOObt
Fmoc-Xaa-Cys(Trt)-Pro-OCH2CO-NH
1) piperidine
12
To test this hypothesis, we designed a cysteinyl prolyl ester
(CPE) unit, without thioester and active ester moieties in the pep-
tide (Scheme 3).24 We expected that this CPE peptide 5 would be
spontaneously transformed into the Ca-DKP thioester 6 by an
intramolecular reaction, and would then react with a cysteinyl
peptide 7 in the same manner as occurs in the case of native
chemical ligation. The CPE peptide is an alternative to the peptide
thioester, and can be prepared by standard Fmoc (SPPS) because it
2) Fmoc-amino acid, DIPCI, HOBt
Fmoc-His(Trt)-Pro-Ile-Arg(Pmc)-Xaa-Cys(Trt)-Pro-OCH2CO-NH
13
1) TFA
2) RP-HPLC
14
Fmoc-His-Pro-Ile-Arg-Xaa-Cys-Pro-OCH2CO-NH2
Scheme 4. Preparation of CPE peptide.

T. Kawakami, S. Aimoto / Tetrahedron 65 (2009) 3871–3877
3873
HS
O
HS
H
N
HS
O
N
O
H
N
Fmoc-HPIR
N
H
DILLG-NH2
+
DILLG-NH2
Fmoc-HPIR
R1
O
14
H2N
N
H
R1
O
16
O
CONH2
15
O
Scheme 5. CPE ligation.
20% (14c), 26% (14d), respectively, based on the Fmoc content of the
Fmoc-Pro-OCH2CO-NH-resin (10).
100
90
80
70
60
50
40
30
20
10
0
2.2. Direct ligation of CPE peptide with Cys-peptide
We assumed that the peptide containing the CPE unit would be
spontaneously transformed into the DKP thioester in neutral buffer
solutions, and when mixed with a Cys-peptide, the ligation reaction
would occur in one pot. The CPE peptide, Fmoc-His-Pro-Ile-Arg-
Gly-Cys-Pro-OCH2CONH2 (14a), was reacted with a Cys-peptide,
Cys-Asp-Ile-Leu-Leu-Gly-NH2 (15) in sodium phosphate buffer so-
lutions in the range of pH 7.3–8.3 at 37 ꢀC (Scheme 5). The ligated
product, Fmoc-His-Pro-Ile-Arg-Gly-Cys-Asp-Ile-Leu-Leu-Gly-NH2
(16a) was produced. The time course for product yield is shown in
Figure 1. The rate of the reaction increased at higher pH. At pH 8.3,
the yield of ligated peptide reached 90% after 24 h (Fig. 2). A small
peak (5%), corresponding to the hydrolysis product, Fmoc-His-Pro-
Ile-Arg-Gly-OH (17a) was observed on the RP-HPLC elution profile.
Ligations of different amino acid residues, Gly, Ala, Val, and Ser,
were carried out under similar reaction conditions. Table 1 shows
data on the yields of products after a 24-h reaction at pH 8.4.
Ligated products 16b–d were obtained in good yields similar to that
for 16a. In the case of ligation at chiral amino acid residues, small
amounts of epimerized product were observed. The following
: pH 8.3
: pH 7.8
: pH 7.3
0
5
10
reaction time / h
15
20
25
Figure 1. Time course for the ligation of Fmoc-His-Pro-Ile-Arg-Gly-Cys-Pro-OCH2
-
CONH2 (14a) with Cys-Asp-Ile-Leu-Leu-Gly-NH2 (15) in the range of pH 7.3–8.3 at
37 ꢀC to yield Fmoc-His-Pro-Ile-Arg-Gly-Cys-Asp-Ile-Leu-Leu-Gly-NH2 (16a). pH: 8.3
(C), 7.8 (:), 7.3 (-).
D-amino acid-containing ligated peptides were observed: 1%
(
D
-Ala), 2% ( -Val), or 5% ( -Ser), respectively. Epimerization of the
D
D
Xaa-Cys(Trt)-Pro-OCH2CONH-resin (13). Finally, the CPE peptide,
Fmoc-His-Pro-Ile-Arg-Xaa-Cys-Pro-OCH2CONH2 (14) (Xaa¼a, Gly;
b, Ala; c, Val; d, Ser) was obtained by treatment with TFA, followed
by RP-HPLC purification. Isolated yields were 30% (14a), 22% (14b),
Ser residue during the ligation was slightly higher than for amino
acids containing aliphatic side chains. It has also been reported that
the highly reactive His-thioester undergoes slight epimerization
during ligation,5f,27 but that this can be suppressed by conducting
the reaction at a lower pH (vide infra).
16a
2.3. Transformation of CPE peptide into peptide thioester
In order to investigate the reaction of the CPE peptide in more
detail, the CPE peptide 14a was treated without a Cys-peptide in
a sodium phosphate buffer (pH 8.0), and subjected to RP-HPLC
and mass analysis. On the RP-HPLC, a small peak, corresponding
to the DKP thioester 18a (R1¼H) was observed, which further
reacted with peptide 14a to give a branched dimeric thioester 19a
(R1¼H) (Scheme 6, Fig. 3). The hydrolysis product 17a was pro-
duced in the prolonged reaction and, after a 24-h reaction, 17a
was produced as the main product. When an excess of sodium
50
40
*
17a
30
20
Table 1
CPE ligation yields for different amino acid residuesa
Entry
Peptide (Xaa)
Yieldb,c/%
Epimerization D
-Xaab/%
1
2
3
4
16a (Gly)
16b (Ala)
16c (Val)
16d (Ser)
82
72
74
75
d
1
2
0
10
retention time / min
20
30
5
a
Figure 2. RP-HPLC elution profile of a reaction mixture of Fmoc-His-Pro-Ile-Arg-Gly-
Cys-Pro-OCH2CONH2 (14a) and Cys-Asp-Ile-Leu-Leu-Gly-NH2 (15) at pH 8.3 and 37 ꢀ
Fmoc-His-Pro-Ile-Arg-Xaa-Cys-Pro-OCH2CONH2 (14) was ligated with Cys-Asp-
Ile-Leu-Leu-Gly-NH2 (15) at pH 8.4 and 37 ꢀC for 24 h to give Fmoc-His-Pro-Ile-Arg-
Xaa-Cys-Asp-Ile-Leu-Leu-NH2 (16).
C
for 24 h giving Fmoc-His-Pro-Ile-Arg-Gly-Cys-Asp-Ile-Leu-Leu-Gly-NH2 (16a). * non-
peptidic compound. Column: YMC-Pack ProC18 (4.6ꢁ150 mm), eluent: 0.1% TFA in
aqueous acetonitrile, 1.0 mL/min.
b
Determined by RP-HPLC.
c
Excluding epimerized products.

3874
T. Kawakami, S. Aimoto / Tetrahedron 65 (2009) 3871–3877
HS
O
O
O
O
H
N
H
N
N
O
Fmoc-HPIR
S
N
Fmoc-HPIR
N
H
R1
HN
R1
O
14
O
CONH2
18
H2O
O
HSCH2CH2SO3Na
O
H
N
Fmoc-HPIR
Fmoc-HPIR
O
H
S
H
R1
N
N
Fmoc-HPIR
Fmoc-HPIR
OH
17
SCH2CH2SO3H
O
H
R1
R1
N
N
N
H
20
R1
O
O
O
CONH2
19
Scheme 6. Thioester formation from the CPE peptide.
of thioesters. The extent of epimerization was as follows: 5% (20b,
Ala), 1% (20c, Val), and 26% (20d, Ser), respectively, after a 6-h
reaction. The extent of epimerization of the Ser residue was
exceptionally high and, when examined more closely, it was found
to increase with reaction time. The epimerization of Ser-thioester
20d increased from 12% (2 h) to 26% (6 h). The isolated peptide
14a
19a
thioester of
8.2. These findings clearly indicate that epimerization occurs after
thioester formation. The -proton of the thioester is acidic, is
L-Ser 20d underwent epimerization when treated at pH
a
18a
deprotonated in the presence of a base and, as a result, the amino
acid undergoes racemization.28 The order of racemization was in
the order of Ser>Ala>Val, of the amino acid residues tested in this
study, which is in good agreement with results observed for the
alkaline hydrolysis of peptides.29 Amino acid residues with ali-
phatic side chains, such as Val residue, would be expected to be
more resistant to deprotonation, and, in the case of an electron-
withdrawing group such as a Ser residue, deprotonation would be
accelerated. When the CPE peptide 14d was reacted with
HSCH2CH2SO3Na at pH 7.3, Ser-thioester 20d was formed in 65%
yield after 10 h and 80% after 24 h, and the degree of epimerization
was 9% and 16%, respectively (Fig. 6). Under lower pH conditions,
the thioester was relatively stable to hydrolysis, and epimerization
50
40
30
20
17a
0
10
retention time / min
20
Figure 3. RP-HPLC elution profile for the reaction of Fmoc-His-Pro-Ile-Arg-Gly-Cys-
Pro-OCH2CONH2 (14a) in sodium phosphate buffer (pH 8.0) for 8 h. The DKP thioester
18a was produced along with the branched dimeric thioester 19a, a hydrolysis product
17a, and DKP, cyclo(-Cys-Pro-). Column: YMC-Pack ProC18 (4.6ꢁ150 mm), eluent: aq
acetonitrile containing 0.1% TFA, flow rate: 1.0 mL/min.
20a
2-mercaptoethanesufonate was added to the reaction mixture,
the peptide thioester, Fmoc-His-Pro-Ile-Arg-Gly-SCH2CH2SO3H
(20a) was formed in 72% yield, after a 6-h reaction at pH 8.8 and
37 ꢀC (Fig. 4). When the reaction was continued, the thioester was
gradually hydrolyzed. The time course for the reaction is shown
in Figure 5(A). When the pH of the reaction buffer was reduced to
below 8, thioester formation was slower, but the thioester that
was formed, was relatively stable to hydrolysis (Fig. 5(B)).
The CPE peptides containing four different amino acid residues,
Gly, Ala, Val, and Ser at the thioesterification sites were applied to
the formation of the peptide thioesters 20 by reaction with sodium
2-mercaptoethanesufonate (Fig. 6). At pH 8.2 and 37 ꢀC, the rates of
thioester formation were very similar for all of the different amino
acid residues used, and the yield of thioester reached about 70%
after 6 h. The thioesters of Gly, Ala, and Ser underwent gradual
hydrolysis with increasing reaction time, but the Val-thioester was
stable after a 24-h reaction. A thioester with a bulky side chain
group would likely be more resistant to hydrolysis. Epimerization
was observed to some extent in cases of chiral amino acid residues
50
17a
40
14a
30
20
0
10
retention time / min
20
Figure 4. RP-HPLC elution profile for the reaction of a mixture of Fmoc-His-Pro-Ile-
Arg-Gly-Cys-Pro-OCH2CONH2 (14a) and HSCH2CH2SO3Na at pH 8.8 and 37 ꢀC for 6 h to
give Fmoc-His-Pro-Ile-Arg-Gly-SCH2CH2SO3H (20a). Column: YMC-Pack ProC18
(4.6ꢁ150 mm), eluent: 0.1% TFA in aqueous acetonitrile, 1.0 mL/min.

T. Kawakami, S. Aimoto / Tetrahedron 65 (2009) 3871–3877
3875
epimerization is minimal. The CPE peptide is one of the alternatives
to the peptide thioester, for use as a building block in polypeptide
synthesis by ligation, and can be readily prepared by Fmoc SPPS.
The CPE peptide can be also transformed into the corresponding
peptide thioester via an intermolecular thiol–thioester exchange
reaction after Ca-DKP thioester formation. Suppression of hydro-
lysis and epimerization of the thioester in the case of sensitive
amino acid residues, such as Ser, need to be investigated further,
although these are suppressed to some extent, when lower pH
reaction conditions are employed. At this stage, the CPE peptide is
the reagent of choice for use as a building block in one-pot thiol-
mediated ligation reactions, since undesirable reactions, such as
epimerization, can be avoided and yields are generally better than
stepwise procedures through isolation of the peptide thioester.
A
100
80
60
40
20
0
: 20a
: 17a
: 14a
0
5
10
reaction time / h
15
20
25
3. Experimental
3.1. General
100
80
60
40
20
0
B
The amino acid derivatives used were of the
unless otherwise noted. Fmoc-amino acid derivatives were
L-configuration,
purchased from the Peptide Institute, Inc (Minoh, Japan). 4-(N-a-9-
: pH 8.8
: pH 8.3
: pH 7.8
: pH 7.3
: pH 6.8
Fluorenylmethoxycarbonyl-amino-20,40-dimethoxybenzyl)phenoxy
resin (Fmoc-Rink amide resin) were purchased from Watanabe
Chemical Ind., Ltd (Hiroshima, Japan).
HPLC was carried out on a reversed phase column, YMC-Pack
ProC18 (4.6ꢁ150 mm), Hydrosphere C18 (4.6ꢁ150 mm), YMC Chi-
ral NEA(S) (4.6ꢁ150 mm), or YMC-Pack ProC18 (10ꢁ250 mm) (YMC
Co., Ltd., Kyoto, Japan) using a linear increasing gradient of aceto-
nitrile in water/0.1% TFA, and detection was by an absorbance
measurement at 220 nm. ESI-MS spectra were recorded on
a Thermo Finnigan LCQÔ DECA XP spectrometer. MALDI-TOF-MS
spectra were recorded on a Bruker AutoFLEX spectrometer. Peptide
yields were determined by amino acid analyses, unless otherwise
noted, which were performed on a Hitachi L-2000 amino acid an-
alyzer after hydrolysis with constant boiling point HCl (Nacalai
Tesque) at 110 ꢀC for 24 h in an evacuated sealed tube.
0
10
20 30
reaction time / h
40
50
Figure 5. Time course for the reaction of the CPE peptide, Fmoc-His-Pro-Ile-Arg-Gly-
Cys-Pro-OCH2CONH2 (14a) with HSCH2CH2SO3Na at 37 ꢀC to yield Fmoc-His-Pro-Ile-
Arg-Gly-SCH2CH2SO3Na (20a). (A) Reaction at pH 8.8: 20a (C); 17a (:); 14a (-). (B)
Yields of 20a in the reaction at the pH of 8.8 (C), 8.3 (B), 7.8 (:), 7.3 (6), and 6.8 (-).
was suppressed somewhat, even in the case of the sensitive Ser-
thioester. Furthermore, when the peptide thioester is immediately
reacted with a Cys-peptide, these undesirable reactions are sup-
pressed to minimum levels, and the desired ligated product is
produced in good yield (vide supra).
3.2. Preparation of Fmoc-His-Pro-Ile-Arg-Xaa-Cys-Pro-
In conclusion, a peptide containing the CPE unit at the C-ter-
minus can be spontaneously transformed into a peptide thioester
through an N–S acyl shift reaction, followed by DKP formation.
When the CPE peptide is mixed with a Cys-peptide, the ligation
reaction proceeds well through thioester formation, followed by
native chemical ligation, in a one-pot process. The ligation can be
carried out using a variety of amino acid residues and the extent of
OCH2CONH2 (14)
3.2.1. A typical procedure for preparation of Fmoc-His-Pro-Ile-
Arg-Gly-Cys-Pro-OCH2CONH2 (14a)
Fmoc-Rink amide resin (Fmoc-NH-resin) (9) (1.00 g, 0.65 mmol/g)
was treated with 1-methyl-2-pyrrolidinone (NMP) (8 mLꢁ3), fol-
lowed by 20% piperidine (8 mL) for 5, 10, and 10 min. After washing
with NMP (8 mLꢁ5), the resin was treated with a solution containing
glycolic acid (99 mg, 1.3 mmol), HOBt$H2O (0.23 g, 1.5 mmol), and
DIPCI (0.23 mL, 1.5 mmol) in NMP (5 mL) for 16 h. The resin was
washed with NMP (8 mLꢁ5), then treated for 8 h with a solution
prepared by mixing Fmoc-Pro-OH (0.68 g, 2.0 mmol), 1-[bis(dime-
thylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophos-
phate (HBTU) (0.76 g, 2.0 mmol), and DIEA (0.52 mL, 3.0 mmol) in
DMF 5 mL for 2 min. The resin was washed with NMP (8 mLꢁ3), fol-
lowed by MeOH (8 mLꢁ3), and dried in vacuo. Fmoc-Pro-OCH2CONH-
resin (10) was obtained in 1.14 g (Fmoc: 0.46 mmol/g).
Resin 10 (0.520 g, 0.24 mmol) was treated with NMP (3 mLꢁ3),
followed by an NMP solution (5 mL) containing 10% Ac2O and 5%
DIEA for 10 min. After washing with NMP (3 mLꢁ3), the resin was
treated with 20% piperidine (3 mL) for 5, 5, and 10 min, and washed
with NMP (3 mLꢁ5). The resin was treated with a solution con-
taining Fmoc-Gly-Cys(Trt)-OH (0.32 g, 0.50 mmol), HOObt (90 mg,
0.55 mmol), and DIPCI (0.092 mL, 0.60 mmol) in NMP (4 mL) for
15 h. The resin was washed with NMP (3 mLꢁ3), followed by MeOH
(5 mLꢁ3), and dried in vacuo. Fmoc-Gly-Cys(Trt)-Pro-OCH2CONH-
resin (12a) was obtained in 0.608 g (Fmoc: 0.31 mmol/g).
100
90
80
: 20a (Gly)
: 20b (Ala)
: 20c (Val)
: 20d (Ser)
70
60
50
40
30
20
10
0
: 20d (Ser, pH 7.2)
0
5
10
reaction time / h
15
20
25
Figure 6. Time course for the reaction of the CPE peptide, Fmoc-His-Pro-Ile-Arg-Xaa-
Cys-Pro-OCH2CONH2 (14) with HSCH2CH2SO3Na at pH 8.2 and 37 ꢀC to yield Fmoc-His-
Pro-Ile-Arg-Xaa-SCH2CH2SO3H (20). Peptide thioester (Xaa): 20a (Gly, C), 20b (Ala,
:), 20c (Val, -), 20 (Ser, A), and 20d (Ser, at pH 7.2, >).

3876
T. Kawakami, S. Aimoto / Tetrahedron 65 (2009) 3871–3877
Resin 12a (0.249 g) was applied to an automated peptide syn-
thesizer, ACT 440 (aapptec, Louisville, KY). The peptide chain was
Phe-OH (pH 8.0). The solution was stirred at room temperature, and
an aliquot (5 L) was removed to quench the reaction with acetic
acid (8 L). The reaction was analyzed by RP-HPLC on YMC-Pack
U
m
elongated by the following steps: NMP wash (1 minꢁ3); 20% pi-
peridine in the NMP treatment (5, 5, 10 min); NMP wash
(1 minꢁ6); coupling with the Fmoc-amino acid derivative
(0.50 mmol) using DIPCI and HOBt$H2O for 1.5 h; NMP wash
(1 minꢁ3); capping with a solution containing 10% Ac2O, 5% DIEA in
NMP for 10 min. After drying, 0.335 g of the Fmoc-His(Trt)-Pro-Ile-
Arg(Pmc)-Gly-Cys(Trt)-Pro-OCH2CONH-resin (13a) was obtained.
Resin 13a (0.335 g) was treated with a solution containing 2%
triisopropylsilane, 5% phenol, and 5% water in TFA (4.0 mL) for
2 h. The peptide was precipitated by adding cold ether and the
precipitate was washed with ether (20 mLꢁ3). The crude mate-
rial was passed through a disposable cartridge, TOYO pack ODS-
M (Tosoh, Tokyo, Japan), with 50% acetonitrile, and freeze-dried
to give the crude peptide (0.112 g). After purification by RP-HPLC
on YMC-pack ProC18 (1ꢁ25 cm), 45 mg of Fmoc-His-Pro-Ile-Arg-
m
ProC18 (4.6ꢁ150 mm).
cyclo(-Cys(Fmoc-His-Pro-Ile-Arg-Gly-)-Pro-) (18a): MS (MALDI-
TOF): found m/z 983.4, calcd for (MþH)þ 983.5; amino acid anal-
ysis: Pro1.1Gly1.3Cys0.86Ile0.85His0.81Arg1.
Fmoc-His-Pro-Ile-Arg-Gly-Cys(Fmoc-His-Pro-Ile-Arg-Gly-)-Pro-OCH2
-
CONH2 (19a): MS (MALDI-TOF) found m/z 1840.3, calcd for (MþH)þ
1840.9; amino acid analysis: Pro2.2Gly2.2Cys0.50Ile1.8His1.7Arg2.
Fmoc-His-Pro-Ile-Arg-Gly-OH(17a):MS(MALDI-TOF):foundm/z801.3,
calcd for (MþH)þ 801.4; amino acid analysis: Pro0.59Gly1.1Ile0.95His0.88Arg1.
cyclo(-Cys-Pro-): MS (ESI): found m/z 201.2, calcd for (MþH)þ
201.1.
3.5. Analysis of formation of peptide thioester 20 from CPE
peptide 14
Gly-Cys-Pro-OCH2CONH2 (14a) was obtained (30 mmol, 30%
Fmoc-His-Pro-Ile-Arg-Xaa-Cys-Pro-OCH2CONH2 (14) (3–5 mM)
based on the Fmoc content in 10). Compound 14a: MS (MALDI-
TOF): found m/z 1058.6, calcd for (MþH)þ 1058.5; amino acid
was dissolved in a mixture of acetonitrile (30
sodium phosphate buffer (70 L) containing
HSCH2CH2SO3Na, 0.01 M TCEP, and 0.10 M Boc-Phe-OH at appro-
priate pH. The solution was stirred at 37 ꢀC, and an aliquot (5
L)
was removed to quench the reaction with acetic acid (8 L). The
mL) and a 0.2 M
m
0.4 M
analysis: Pro1.8Gly1CysndIle1.0His0.84Arg0.92
.
Fmoc-His-Pro-Ile-Arg-Ala-Cys-Pro-OCH2CONH2 (14b): 22%; MS
(MALDI-TOF): found m/z 1072.8, calcd for (MþH)þ 1072.5; amino
acid analysis: Pro1.8Ala1.1CysndIle1.2His0.93Arg1.
m
m
reaction was analyzed by RP-HPLC on YMC-Pack ProC18
(4.6ꢁ150 mm) for 20a–c or YMC Hydrosphere C18 (4.6ꢁ150 mm)
for 20d. The yield of peptides was calculated based on the peak
area compared with that of Boc-Phe-OH as an internal standard.
The epimerized products were confirmed by MS and RP-HPLC
Fmoc-His-Pro-Ile-Arg-Val-Cys-Pro-OCH2CONH2 (14c): 20%; MS
(MALDI-TOF): found m/z 1100.9, calcd for (MþH)þ 1100.5; amino
acid analysis: Pro2.0CysndVal1Ile0.94His0.84Arg0.94
.
Fmoc-His-Pro-Ile-Arg-Ser-Cys-Pro-OCH2CONH2 (14c): 26%; MS
(MALDI-TOF): found m/z 1088.3, calcd for (MþH)þ 1088.5; amino
acid analysis: Ser0.57Pro2.4CysndIle0.97His0.87Arg1.
analyses in comparison with the peptides containing
residues, which were prepared separately.
D-amino acid
Fmoc-His-Pro-Ile-Arg-Gly-SCH2CH2SO3H (20a): MS (MALDI-TOF):
found m/z 925.3, calcd for (MþH)þ 925.4; amino acid analysis:
Pro0.60Gly1.1Ile0.92His0.86Arg1.
3.3. Analysis of ligation of CPE peptide 14 with Cys-peptide 15
Peptides, Fmoc-His-Pro-Ile-Arg-Xaa-Cys-Pro-OCH2CONH2 (14)
(3–5 mM) and Cys-Asp-Ile-Leu-Leu-Gly-NH2 (15) (6–9 mM) were
Fmoc-His-Pro-Ile-Arg-Ala-SCH2CH2SO3H (20b): MS (MALDI-TOF):
found m/z 939.2, calcd for (MþH)þ 939.4; amino acid analysis:
dissolved in a mixture of acetonitrile (25
phosphate buffer (75 L) containing 0.01 M tris(2-carboxy-
ethyl)phosphine (TCEP), and 50 mM Boc-Lys(Cl-Z)-OH at appro-
priate pH. The solution was stirred at 37 ꢀC, and an aliquot (5
L)
was removed to quench the reaction with acetic acid (8 L) and
0.5 M DTT (8 L). The reaction was analyzed by RP-HPLC on YMC-
mL) and a 0.2 M sodium
ProndAla1.1Ile1His0.84Arg0.92
.
m
Fmoc-His-Pro-Ile-Arg-Val-SCH2CH2SO3H (20c): MS (MALDI-TOF):
found m/z 967.3, calcd for (MþH)þ 967.4; amino acid analysis:
Pro0.83Val1.0Ile0.99His0.79Arg1.
Fmoc-His-Pro-Ile-Arg-Ser-SCH2CH2SO3H (20d): MS (MALDI-TOF):
found m/z 955.3, calcd for (MþH)þ 955.4; amino acid analysis:
Ser0.97Pro1.2Ile0.96His0.88Arg1.
m
m
m
Pack ProC18 (4.6ꢁ150 mm) for 16a–c or YMC CHIRAL NEA(S)
(4.6ꢁ150 mm) for 16d. The yield of peptides was calculated based
on the peak area compared with that of Boc-Lys(Cl-Z)-OH as an
internal standard. Epimerized products were confirmed by MS and
RP-HPLC analyses in comparison with the peptides containing
Acknowledgements
This research was supported, in part, by Grants-in-Aid for Sci-
entific Research from the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
D
-amino acid residues, which were prepared separately.
Fmoc-His-Pro-Ile-Arg-Gly-Cys-Asp-Ile-Leu-Leu-Gly-NH2
(16a):
MS (MALDI-TOF): found m/z 1414.9, calcd for (MþH)þ 1414.7;
amino acid analysis: Asp0.99Pro1.1Gly2CysndIle1.9Leu2.0His0.84Arg0.92
.
Fmoc-His-Pro-Ile-Arg-Ala-Cys-Asp-Ile-Leu-Leu-Gly-NH2 (16b): MS
References and notes
(MALDI-TOF): found m/z 1428.8, calcd for (MþH)þ 1428.7; amino acid
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