Peptide thioester formation by an N to S acyl shift reaction at the cysteinyl prolyl cysteine position

Article abstract of DOI:10.1246/bcsj.20100005

In protein synthesis, peptide thioesters are frequently used as key building blocks for the ligation strategy. We report herein on the synthesis of a peptide thioester using an N to S acyl shift reaction. A peptide containing a CysProCys (CPC) sequence was transformed into a peptide diketopiperazine (DKP) thioester under acidic conditions. In this reaction, the CPC moiety undergoes a tandem N to S acyl shift, followed by DKP formation to give the desired thioester. Since proteins containing a CPC sequence can be prepared by recombinant DNA techniques, the CPC peptide represents a new method for preparing peptide thioesters as recombinant proteins for protein synthesis.

Full text of DOI:10.1246/bcsj.20100005

570
Bull. Chem. Soc. Jpn. Vol. 83, No. 5, 570–574 (2010)
© 2010 The Chemical Society of Japan
Peptide Thioester Formation by an N to S Acyl Shift Reaction
at the Cysteinyl Prolyl Cysteine Position
Toru Kawakami,* Sakiko Shimizu, and Saburo Aimoto*
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871
Received January 6, 2010; E-mail: kawa@protein.osaka-u.ac.jp, aimoto@protein.osaka-u.ac.jp
In protein synthesis, peptide thioesters are frequently used as key building blocks for the ligation strategy. We report
herein on the synthesis of a peptide thioester using an N to S acyl shift reaction. A peptide containing a Cys-Pro-Cys
(CPC) sequence was transformed into a peptide diketopiperazine (DKP) thioester under acidic conditions. In this reaction,
the CPC moiety undergoes a tandem N to S acyl shift, followed by DKP formation to give the desired thioester. Since
proteins containing a CPC sequence can be prepared by recombinant DNA techniques, the CPC peptide represents a new
method for preparing peptide thioesters as recombinant proteins for protein synthesis.
The peptide thioester is one of key building blocks in protein
to design a novel structure without an ester group, which would
make it possible to use genetically engineered proteins as
peptide thioester precursors, without the need for a special
translation system. We report herein on the synthesis of a
peptide DKP thioester using a peptide containing a Cys-Pro-
Cys sequence (CPC-peptide 1b).
synthesis using the ligation strategy.1 The focus of our research
has been on the use of the N to S acyl shift reaction for
preparing peptide thioesters.2-4 A thiol-containing auxiliary,2,5
such as a 4,5-dimethoxy-2-mercaptobenzyl group, and a
cysteine residue3,4,6 mediate the N-S acyl shift reaction, and
can be used to produce peptide thioesters. We previously
reported that a peptide containing a cysteinyl prolyl ester
(CPE) moiety (CPE peptide 1a) is spontaneously transformed
into a peptide diketopiperazine (DKP) thioester 2 via an N-S
acyl shift, followed by the formation of a DKP derivative
(Scheme 1).4 The CPE peptide 1a can be directly utilized in a
ligation reaction with a peptide containing a cysteine residue at
the N-terminus (Cys-peptide 3) in a one pot reaction to give
the ligated peptide 4. In this reaction, the ester group plays a
crucial role as a leaving group during DKP formation. The CPE
peptide can be readily prepared by 9-fluorenylmethoxycarbonyl
(Fmoc) solid phase peptide synthesis (SPPS) and, quite
recently, a reconstituted cell free protein expression system
enabled us to genetically produce the CPE peptide, which can
be further transformed into a cyclic peptide.7 The next step was
Results and Discussion
We initially prepared a peptide library containing a H-Ala-
Lys-Leu-Arg-Phe-Gly-Cys-Pro-Xaa-Xbb-Xcc-Arg-NH2 (5)
structure, in which the Cys-Pro sequence (bold) was fixed and
in which Xaa-Xbb-Xcc represents random sequences of 3
amino acid residues consisting of Asn, Asp, Cys, Gln, Gly, His,
Leu, or Ser (Scheme 2). The library was treated with an excess
amount of H-Cys-Tyr-NH2 (6) in a carbonate buffer at pH 8.2.
A ligated product, H-Ala-Lys-Leu-Arg-Phe-Gly-Cys-Tyr-
NH2 (7), would be expected to be produced, if the active
sequence is contained in the library. A library, in which the
Xaa residue was fixed with 8 different amino acid residues
consisting of Asn, Asp, Cys, Gln, Gly, His, Leu, or Ser,
respectively, and Xbb and Xcc contained random residues, was
HS
O
O
O
peptide 1
S
N
N
X
CONHR2
peptide 1
N
HN
H
O
O
R1
O
1a: X = O, R1 = H
2
1b: X = NH, R1 = CH2SH
HS
peptide 2
HS
H2N
O
3
O
peptide 2
peptide 1
N
H
O
4
Scheme 1. Peptide DKP thioester formation from CPE and CPC peptides and ligation with a Cys-peptide.
Published on the web April 29, 2010; doi:10.1246/bcsj.20100005

T. Kawakami et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 5 (2010)
571
H-Ala-Lys-Leu-Arg-Phe-Gly-Cys-Pro-Xaa-Xbb-Xcc-Arg-NH2 (5)
Xxx: Asn, Asp, Cys, Gln, Gly, His, Leu, or Ser
H-Cys-Tyr-NH2 (6)
H-Ala-Lys-Leu-Arg-Phe-Gly-Cys-Tyr-NH2 (7)
Scheme 2. Search for ligation activity in the CP library.
O
S
peptide
O
H
N
N
H2N
NHR
HS
b
O
O
O
O
H
a
HX
9
N
N
peptide
N
NHR
or
b
H
O
O
O
HX
8
HS
H2N
NHR
O
a
N
X = O or S
peptide
N
X
H
O
O
10
O
O
O
O
O
S
H2N
peptide
NHR
peptide
S
N
N
HN
H2N
X
+
O
O
O
2
c
11
H2N
HX
NHR
12
Scheme 3. Proposed mechanism for the formation of DKP thioesters from CPC or CPS peptides.
reacted with the Cys-peptide 6. The ligated product 7 was
observed as a small but distinct peak by RP-HPLC and mass
spectroscopy (MS), when a Ser or Cys residue was located at
the Xaa position (data not shown). When a Ser residue was
located at the Xaa position and the Xbb or Xcc position was
fixed with the 8 different amino acid residues, respectively,
the results were similar; a signal corresponding to peptide 7
was observed in the majority of cases, although the ligation
efficiency was low. The results indicate that only a Ser or Cys
residue at the Xaa position is required for activation of the
peptide for ligation, whereas the reaction appears to proceed in
low yield under neutral conditions.
We hypothesized that this ligation reaction proceeds via
the formation of the peptide DKP thioester 2 from a
peptide 8 containing Cys-Pro-Ser or Cys-Pro-Cys sequences
(Scheme 3). In this scenario, an N-S/O acyl shift reaction at
the second Cys or Ser residue (path b) would result in the
production of a (thio)ester structure 10, which is structurally
similar to the CPE peptide. Once the CPE structure is
constructed, the ligation reaction would be expected to proceed
via an N-S acyl shift reaction at the first Cys residue (path a)
followed by DKP formation (path c), liberating the C-terminal
Ser/Cys-peptide 12. The order of reactions “a” and “b” would
not be critical for the success of the overall reaction.
Since the equilibrium between a thioester and an amide
forms at a Cys site shifts in favor of the thioester under the
acidic conditions,3 in a subsequent experiment, some of the
selected peptides, H-Ala-Lys-Leu-Arg-Phe-Gly-Xaa-Xbb-
Xcc-Y (13), shown in Table 1, were treated with a 0.1 M
hydrochloric acid solution at 110 °C in an evacuated, sealed
tube for 2 h. In the reaction of H-Ala-Lys-Leu-Arg-Phe-Gly-
Cys-Pro-Cys-NH2 (13a), the mass number corresponding to
the DKP derivative of thioester 14 was observed. The other
major peaks were the CPC peptide 13a, the C-terminally
hydrolyzed CPC peptide, H-Ala-Lys-Leu-Arg-Phe-Gly-
Cys-Pro-Cys-OH (13a¤), and the hydrolysis product, H-Ala-
Lys-Leu-Arg-Phe-Gly-OH (15), respectively (Figure 1A).
The proportions of these compounds, based on peak areas,
are shown in Table 1. The proportion of DKP thioester 14
(17%) was in good agreement with its isolated yield (12%).
When a Gly residue was added after the CPC sequence (peptide
13c), the reaction proceeded quite similarly to the reaction
without the Gly residue, indicating that additional residues can
be attached at the C-terminus of the CPC sequence (Entry 3).
The CPC peptides with a C-terminal carboxylic acid 13b and
13d, which are analogs of 13a and 13c, respectively, gave
almost the same results (Entries 2 and 4). Furthermore, the CPC
peptide 13i, which contains 5 additional amino acid residues

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Bull. Chem. Soc. Jpn. Vol. 83, No. 5 (2010)
Peptide Thioester Formation by N-S Acyl Shift
Table 1. Formation of DKP Thioester 14 from Peptide 13
H-Ala-Lys-Leu-Arg-Phe-Gly-Xaa-Xbb-Xcc-Y (13)
0.1 M HCl, 110 °C, 2 h
(A)
13a
14
15
13a'
O
O
25
20
15
10
H-Ala-Lys-Leu-Arg-Phe-Gly
S
N
HN
14
O
Proportion of peak area
on RP-HPLC/%a)
Entry 13 Xaa Xbb Xcc
Y
0
10
20
30
13 (13¤)b) 14
15f)
Retention time / min
1
2
3
4
5
6
7
8
13a Cys Pro Cys NH2
13b Cys Pro Cys OH
13c Cys Pro Cys Gly-NH2 2 (34)
13d Cys Pro Cys Gly-OH 36 (®) 16 (2)c) 23
13e Cys Pro Ser NH2
13f Cys Pro Gly NH2
13g Cys Ala Cys NH2
13h Ser Pro Cys NH2
21 (17) 17 (2)c) 26
14'
(B)
32 (®) 19 (1)c) 25
17 (2)c) 23
14
33 (39)
2 (61)
1
4
16
20
24
16
25
20
15
10
15
23 (28) 4d)
29 (30) 7e)
a) The proportions of 13, 13¤, 14, and 15 were determined by
absorbance at 220 nm. b) In bracket the proportion of the
hydrolysis product at the C-terminal amide, H-Ala-Lys-Leu-
Arg-Phe-Gly-Xaa-Xbb-Xcc-OH (13¤), is shown. c) In bracket
the proportion of DKP thioester with an epimerized DKP
moiety. d) DKP [cyclo(-Cys-Ala-)] thioester 17. e) DKP
[cyclo(-Ser-Pro-)] ester 18. f) H-Ala-Lys-Leu-Arg-Phe-
Gly-OH (15).
0
10
20
30
Retention time / min
H-Cys-Tyr-NH2 (6)
(C)
7
after the CPC moiety, was also transformed into the DKP
thioester 14 in an isolated yield of 11% with the CPC peptide
recovered in 23%, and the cleaved C-terminal peptide, H-Cys-
Ala-Pro-Ala-Arg-Gly-OH (16) was isolated in a yield of 30%
(Scheme 4). This result supports the proposed reaction mech-
anism shown in Scheme 3. Furthermore, the production of a
peptide thioester via recombinant DNA techniques is quite
promising, because the C-terminal structure after the CPC
sequence is flexible and proteins containing the CPC sequence
can easily be prepared as recombinant proteins.
25
20
15
10
On the other hand, when the Xbb or Xcc position of the Cys-
Pro-Cys sequence was substituted with other residues, the
yields of DKP thioester were decreased substantially, although
similar levels of the hydrolysis product 15 was observed
(Entries 5-7). This indicates that the S-peptide, corresponding
to structure 9, is directly hydrolyzed prior to the formation of
the DKP derivative. When the Cys residue at the Xaa position
was substituted with Ser, the corresponding DKP ester 18 was
obtained (Entry 8). In comparison with the substitution at the
Xcc position with Ser (Entry 5), a thioester might be superior to
an ester (structure 11 in Scheme 3), in terms of producing a
DKP structure under the acidic conditions used in this reaction.
In the reaction of CPC peptide 13a in 0.1 M hydrochloric
acid, extensive hydrolysis was observed while the CPC peptide
continued to be present. Mass numbers corresponding to
the DKP, cyclo(-Cys-Pro-), and H-Cys-Pro-Cys-OH were
observed in the reaction mixture, which indicated that the
hydrolysis occurred before and after the formation of the
0
10
20
30
Retention time / min
Figure 1. RP-HPLC elution profiles of reaction mixtures.
DKP thioester formation from CPC peptide 13a (A) in
0.1 M HCl at 110 °C for 2 h and (B) in HFBA at 110 °C for
4 h. (C) Ligation of the crude DKP thioester, prepared in
HFBA, and Cys-peptide 6. Column: YMC-Pack ProC18
(4.6 © 150 mm). Eluent: 0.1% TFA in aq acetonitrile,
¹1
1.0 mL min
.
DKP thioester. To reduce the extent of hydrolysis, the reaction
was carried out in heptafluorobutyric acid (HFBA) vapor at
110 °C in an evacuated, sealed tube. After a 4-h reaction, the
CPC peptide 13a had disappeared and the extent of hydrolysis
was reduced (Figure 1B). A peak corresponding to DKP
thioester 14 was observed and a new peak, indicated as 14¤ in
the figure, appeared, whose mass number was identical to that

T. Kawakami et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 5 (2010)
573
H-Ala-Lys-Leu-Arg-Phe-Gly-Cys-Pro-Cys-Ala-Pro-Ala-Arg-Gly-OH (13i)
14 + H-Cys-Ala-Pro-Ala-Arg-Gly-OH (16)
Scheme 4. DKP thioester formation and cleavage of the C-terminal portion from the CPC peptide.
for DKP thioester 14. DKP thioester 14¤ was determined to be
an epimer of 14 in the DKP moiety, cyclo(-Cys(R)-Pro-),
which was confirmed by RP-HPLC, in which product 14¤
co-eluted with the epimers in the DKP moiety, although
cyclo(-D-Cys(R)-Pro-) and cyclo(-Cys(R)-D-Pro-) were not
separated on a C18 column. Although DKPs are known to be
epimerized easily in the presence of a base,8 it is not clear at
this stage that the DKP was epimerized in the formation in
HFBA. The crude mixture of DKP peptides 14 and 14¤ was
reacted with an excess amount of Cys-peptide 6 to give a single
ligated isomer 7 in a yield of 17% based on CPC peptide 13a
(Figure 1C).
separately introduced. After combining all of the resins again,
the peptide chain was elongated by the usual SPPS to give a H-
Ala-Lys(Boc)-Leu-Arg(Pbf)-Phe-Gly-Cys(Trt)-Pro-Ser(tBu)-
Xbb-Xcc-Arg(Pbf)-NH-resin, in which the Xaa position was
fixed with a Ser residue, and both Xbb and Xcc positions
contained 8 different amino acid residues. The peptides were
cleaved from the resin, and partially purified by RP-HPLC, in
which the peptide-fractions were combined together and
lyophilized to give a library of H-Ala-Lys-Leu-Arg-Phe-
Gly-Cys-Pro-Ser-Xbb-Xcc-Arg-NH2.
Reaction of Peptide Library 5 with Cys-peptide 6.
A
typical reaction procedure is as follows: A peptide library 5
(0.5 mg) was treated with Cys-peptide 6 (0.5 mg) in a carbonate
buffer solution (pH 8.2) (60 ¯L) containing 20 mM tris(2-
carboxyethyl)phosphine (TCEP) at room temperature for
24 h. After adding a solution of 0.2 M dithiothreitol (DTT)
(0.12 mL), followed by stirring for 1 h, the reaction mixture
was analyzed by RP-HPLC with detection by absorbance at
220 and 280 nm, and the fraction containing a ligated product 7
was analyzed by MS. In the reactions in which the Xaa residue
was fixed with a Cys or Ser residue, a ligated peptide was
observed both by RP-HPLC and MS. 7: MALDI-TOF: found
m/z 956.7, calcd for (M + H)+ 956.5.
Conclusion
In conclusion, a peptide containing a Cys-Pro-Cys (CPC)
sequence can be transformed into a peptide DKP thioester and
a C-terminal peptide with the N-terminal Cys residue. The
reaction required acidic conditions and a high temperature,
and current efforts are directed toward optimizing the reaction.
It is noteworthy that recombinant proteins containing the
CPC sequence would be transformed into the thioester, since
proteins containing a CPC sequence can be prepared by
recombinant DNA techniques. So far, the peptide thioesters
are prepared, via recombinant DNA techniques, by the use of
intein domains, which catalyze protein splicing reactions.9 The
CPC peptide will provide an alternate approach for preparing
recombinant peptide thioesters.
Formation of Peptide DKP Thioester 14 under Acidic
Conditions. Typical Procedures: 0.1 M HCl: A solution of
peptide 13 (0.20 ¯mol) containing 10 mM TCEP and 0.1 M
HCl (0.20 mL) in an evacuated, sealed tube was heated at
110 °C for 2 h. The reaction mixture (30 ¯L) was directly
analyzed by RP-HPLC, with detection by absorbance at
220 nm, and the fractions were applied to ESI-MS. Isolated
yield was determined by quantitative amino acid analysis.
HFBA vapor:12 A small glass tube (6 mm © 30 mm), con-
taining lyophilized peptide 13 (0.20 ¯mol), was placed in a
large glass tube (12 mm © 105 mm), that contained HFBA
(0.10 mL), evacuated and then flame sealed. It was incubated at
110 °C for 4 h. A 50% aqueous acetonitrile solution was added
to the reaction mixture, which was then lyophilized. The crude
mixture was dissolved in 5% acetonitrile (0.10 mL), an aliquot
of the solution (30 ¯L) was analyzed by RP-HPLC and the
fractions were analyzed by ESI-MS.
Experimental
Peptide Synthesis. Peptides were prepared by the standard
Fmoc solid phase peptide synthesis (SPPS) by using an
automated peptide synthesizer, ACT440³ (aapptec, Louisville,
KY), or a manual procedure by employing N,N-diisopropyl-
carbodiimide and 1-hydroxybenzotriazole as activating re-
agents. Peptides were cleaved from the solid support by
treatment with reagent B,10 purified by RP-HPLC on a C18
column using a linear increasing gradient of acetonitrile in
water/0.1% TFA, and confirmed by MALDI-TOF MS on a
Bruker AutoFLEX spectrometer or ESI-MS on a Thermo
Finnigan LCQ· DECA XP spectrometer.
Preparation of Peptide Library 5. A library was prepared
by the split-and-mix synthesis method.11 A typical procedure
for the synthesis of a peptide containing a Ser residue at
the Xaa site is as follows: After Fmoc-Arg(Pbf)-OH was
introduced to a Rink amide resin, the resin was divided into
8 aliquots. To each resin, 8 different amino acid derivatives,
Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-
OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH,
Fmoc-Leu-OH, or Fmoc-Ser(tBu)-OH, were separately intro-
duced. All resins were combined, and divided into 8 aliquots
again. To each resin, 8 different amino acid derivatives were
13a: MS (ESI): found m/z 993.7, calcd for (M + H)+ 993.5;
amino acid analysis: ProndGly1Ala0.91CysndLeu0.90Phe0.87
Lys0.98Arg0.93
13b: MS (ESI): found m/z 994.6, calcd for (M + H)+ 994.5;
amino acid analysis: ProndGly1Ala0.97CysndLeu0.97Phe0.99
Lys0.97Arg0.97
13c: MS (ESI): found m/1050.5, calcd for (M + H)+
1050.5; amino acid analysis: ProndGly2Ala0.97CysndLeu0.98
Phe0.99Lys1.0Arg0.98
13d: MS (ESI): found m/z 1051.5, calcd for (M + H)+
1051.5; amino acid analysis: ProndGly2Ala0.99CysndLeu0.98
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Bull. Chem. Soc. Jpn. Vol. 83, No. 5 (2010)
Peptide Thioester Formation by N-S Acyl Shift
Phe0.99Lys0.98Arg0.96
13e: MS (ESI): found m/z 977.7, calcd for (M + H)+ 977.5;
amino acid analysis: Ser0.86ProndGly1Ala0.98CysndLeu0.98
Phe0.93Lys0.98Arg0.97
13f: MS (ESI): found m/z 947.5, calcd for (M + H)+ 947.5;
amino acid analysis: ProndGly2Ala1.0CysndLeu0.99Phe0.91
Lys1.0Arg1.0
13g: MS (ESI): found m/z 967.7, calcd for (M + H)+ 967.5;
amino acid analysis: Gly1Ala2.0CysndLeu0.96Phe0.92Lys0.98
Arg0.98
13h: MS (ESI): found m/z 977.7, calcd for (M + H)+ 977.5;
amino acid analysis: Ser0.83ProndGly1Ala0.99CysndLeu0.97
Phe0.95Lys0.99Arg0.97
13i: MS (ESI): found m/z 1446.7, calcd for (M + H)+
1446.7; amino acid analysis: ProndGly2Ala2.9CysndLeu0.98
Phe1.0Lys0.99Arg2.0
14: MS (ESI): found m/z 873.5, calcd for (M + H)+ 873.5;
amino acid analysis: ProndGly1Ala1.0CysndLeu0.96Phe0.99
Lys0.97Arg1.0
14¤: MS (ESI): found m/z 873.5, calcd for (M + H)+ 873.5;
amino acid analysis: ProndGly1Ala1.2CysndLeu0.96Phe1.0Lys0.96
Arg0.99
15: MS (ESI): found m/z 691.6, calcd for (M + H)+ 691.4;
amino acid analysis: Gly1Ala1.0Leu0.96Phe0.99Lys0.98Arg1.0
16: MS (ESI): found m/z 574.3, calcd for (M + H)+ 574.3;
amino acid analysis: ProndGly1Ala2.2CysndArg1.1
17: MS (ESI): found m/z 847.5, calcd for (M + H)+ 847.5.
18: MS (ESI): found m/z 857.7, calcd for (M + H)+ 857.5.
Ligation of Peptide DKP Thioester 14 with Cys-peptide
6. Peptide thioester 14 was prepared by the reaction of CPC
peptide 13a (0.2 mg, 0.20 ¯mol) in HFBA vapor in an
evacuated, sealed tube at 110 °C for 4 h. After the addition of
aqueous acetonitrile, followed by lyophilization, the crude
material was reacted with Cys-peptide 6 (0.5 mg, 1.8 ¯mol) in a
tricine buffer solution (pH 8.2, 0.10 mL) containing 10 mM
TCEP at 37 °C for 1 h. Ligated peptide 7 was isolated by RP-
HPLC and the yield was determined by quantitative amino acid
analysis to be 17% based on CPC peptide 13a. 7: ESI-MS:
found m/z 956.6, calcd for (M + H)+ 956.5; amino acid
.
This research was supported, in part, by The Naito
Foundation and Grants-in-Aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science and
Technology, Japan.
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analysis: Gly1Ala1.0CysndLeu0.98Tyr0.82Phe0.90Lys1.2Arg1.0
.