Generation of an S-peptide via an N-S acyl shift reaction in a TFA solution

Article abstract of DOI:10.1246/bcsj.79.1773

An N-S acyl shift reaction of thiol-containing peptides in a trifluoroacetic acid (TFA) solution was confirmed by a combination of ¹³CNMR spectroscopy, reversed-phase (RP) HPLC, and MS analyses. A model peptide containing a cysteine residue was

Full text of DOI:10.1246/bcsj.79.1773

ꢀ 2006 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 79, No. 11, 1773–1780 (2006) 1773
Generation of an S-Peptide via an N–S Acyl Shift
Reaction in a TFA Solution
Ken’ichiroh Nakamura,1 Megumi Sumida,1 Toru Kawakami,1
ꢀ1
Thomas Vorherr,2 and Saburo Aimoto
1Institute for Protein Research, Osaka University, Osaka 565-0871
2Bachem Holding AG, CH-4416 Bubendorf, Switzerland
Received April 24, 2006; E-mail: aimoto@protein.osaka-u.ac.jp
An N–S acyl shift reaction of thiol-containing peptides in a trifluoroacetic acid (TFA) solution was confirmed by a
combination of 13C NMR spectroscopy, reversed-phase (RP) HPLC, and MS analyses. A model peptide containing a
cysteine residue was transformed into an S-peptide in a TFA solution. The S-peptide was quickly transformed to the
original peptide during RP-HPLC analysis even when an eluent that contained 0.1% TFA was used. A peptide that
had an N-2-mercapto-4,5-dimethoxybenzyl (Dmmb) group was also transformed into an S-peptide, forming a thioester
bond with the thiol of the Dmmb group by TFA treatment. The generated S-peptide was isolated by directly injecting the
reaction mixture into a RP-HPLC and was readily converted to the corresponding 2-sulfoethyl thioester via an intermo-
lecular thiol exchange reaction with sodium 2-mercaptoethanesulfonate. The 2-sulfoethyl peptide thioester is widely
used as a building block in polypeptide synthesis. The N–S acyl shift reaction observed in peptides with or without
an auxiliary group provides a new route for the preparation of peptide thioesters.
N–S acyl shift reactions may occur at a cysteine residue in
peptides to some extent under acidic conditions. However,
the N–S acyl shift reaction has not been studied in detail. In
1966, Sakakibara et al. referred to a possible N–S acyl shift
reaction at a cysteine residue during the treatment of a pro-
tected cysteine-containing peptide with anhydrous hydrogen
fluoride.1
O
peptide 2
peptide 1
N
OCH
3
HS
OCH
3
1
Vizzavona et al. recently reported that a peptide, prepared
by extended chemical ligation using an N-2-mercapto-4,5-di-
methoxybenzyl (Dmmb) group as an auxiliary,2 generated an
earlier eluting compound on reversed-phase (RP) HPLC when
the ligated peptide was treated with a trifluoroacetic acid
(TFA) cleavage solution.3 They attempted to analyze this more
polar compound but failed. Judging from the chemical behav-
ior of the compound, they presumed that it was probably an
S-peptide that formed a thioester bond with the thiol group
in the auxiliary (Fig. 1).
+
+
-H
H
O
peptide 1
peptide 2
S H N
2
H CO
3
Peptide thioesters are used as building blocks in protein syn-
thesis. An N–S acyl shift reaction is a key step in the protein
splicing reaction,4,5 which has been successfully applied to the
biological preparation of peptide thioesters.6 If it were possible
to achieve an N–S acyl shift reaction of a peptide bond trig-
gered by chemical process such as TFA treatment, such a re-
action would represent a new method for preparing a peptide
thioester as well as for the chemical modification of proteins.
In a previous report,7 we described a new procedure for the
preparation of peptide thioesters, in which a TFA-treated pep-
tide resin that contained a Dmmb group-attached amide bond
released a peptide thioester in the presence of a thiol com-
pound, such as 2-mercaptoethanesulfonic acid.
OCH
3
2
Fig. 1. Possible S-peptide formation under acidic conditions.
TFA solution at a cysteine residue and also at the Dmmb
group-attached amide bond in peptides. We also report the
details of the N–S acyl shift reaction at a cysteine residue in
a peptide. Furthermore, we show that a 2-sulfoethyl thioester
is formed in the reaction of the TFA-treated Dmmb group-
attached peptide with sodium 2-mercaptoethanesulfonate.
Results and Discussion
In the present paper, we provide evidence to show that a
thioester bond is formed via an N–S acyl shift reaction in a
Analysis of the Compound Generated from a Cysteine-
Containing Peptide in a TFA Solution. Fmoc–Ile–Ala–
Published on the web November 9, 2006; doi:10.1246/bcsj.79.1773

1774 Bull. Chem. Soc. Jpn. Vol. 79, No. 11 (2006)
N–S Acyl Shift of Peptide Backbone
13
C NMR
RP-HPLC
(A) t = 0 h
(D) t = 0 h
TCEP
(B) t = 24 h
(E) t = 24 h
(C) t = 910 h
(F) t = 910 h
0
10
20
30
210
200
190
180
170
chemical shift / ppm
retention time / min
Fig. 2. Analysis of the product generated from Fmoc–Ile–Ala–Gly(1-13C)–Cys–Arg–NH2 (3) in a TFA solution containing CDCl3
(29%) and TCEP (0.5%) (v=v=w). (A), (B), and (C): 13C NMR spectra of peptide 3 in the TFA solution after 0-h, 24-h, and 910-h
storage, respectively. (D), (E), and (F): RP-HPLC elution profiles of peptide 3 in the TFA solution after 0-h, 24-h, and 910-h stor-
age, respectively. The arrow indicates the peak at the retention time of 18.5 min. HPLC conditions; column: YMC-Pack ProC18
(4:6 ꢁ 150 mm), eluent: linear gradient of acetonitrile in 0.1% TFA from 20 to 50% over 30 min, flow rate: 1.0 mL minꢂ1
peptide 3 with a TFA solution, a new signal appeared at
.
Gly(1-13C)–Cys–Arg–NH2 (3) was prepared by 9-fluorenylme-
thoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS)
and purified by RP-HPLC after cleavage of the peptide using
Reagent B (TFA/phenol/water/triisopropylsilane, 88/5/5/2,
v=v=v=v),8 and the formulation was confirmed by electron
spray ionization mass spectrometry (ESI-MS) and amino acid
analysis after acid hydrolysis. Peptide 3 was dissolved in a
solution containing TFA (71%), CDCl3 (29%), and tris(2-car-
boxyethyl)phosphine hydrochloride (TCEP) (0.5%) (v=v=w).
The progress of the reaction was monitored by using 13C NMR
spectroscopy. An aliquot of the peptide solution was subjected
to RP-HPLC analysis, and the eluted solutions with a UV ab-
sorbance at 220 nm were collected. The molecular mass num-
ber of the material in each fraction was determined by ESI-
MS. In the 13C spectrum, signals were observed at 172.5 and
172.7 ppm corresponding to the carbonyl carbon of the amide
bond just after the peptide was dissolved in a TFA solution, as
shown in panel A in Fig. 2. Panel D shows the RP-HPLC elu-
tion profile for the same sample. A major peak that was eluted
at 22.8 min had exactly the same retention time and molecular
mass number as those of peptide 3. After 24 h treatment of
201.8 ppm. This chemical shift was assigned to the carbonyl
carbon of a thioester. The signal intensity ratio of the carbonyl
carbons between the amide and thioester was about 11 to 1. An
RP-HPLC analysis of the sample that was treated for 24 h
showed a new peak with a retention time of 18.5 min, indicated
by an arrow in panel E. The peptide corresponding to this new
peak had the same mass number as that of peptide 3. However,
the proportion of the peak area toward that of peptide 3 was
less than one fiftieth. The proportion of the 13C-signal intensity
between the amide carbonyl carbon and the thioester reached a
nearly constant value of 1 to 4 after the storage of peptide 3 in
a TFA solution for 910 h. However, the proportion of the new-
ly generated peak area to that of peptide 3 was still less than a
fifth in RP-HPLC. These results indicate that the newly gener-
ated compound is S-peptide 4, as shown in Fig. 3, which
would be formed via an N–S acyl shift reaction, and that pep-
tide 4 is rapidly transformed to the original peptide even in a
solution that contains 0.1 vol % of TFA in aqueous acetonitrile.
Furthermore, 80% of the total peptide was S-peptide 4 at equi-
librium in a mixed solvent of TFA (71%), CDCl3 (29%), and

K. Nakamura et al.
Bull. Chem. Soc. Jpn. Vol. 79, No. 11 (2006) 1775
SH
Arg
O
C
O
C
O
H
H
13
13
N
TFA
N
NH
2
Fmoc-Ile-Ala
N
H
NH
2
Fmoc-Ile-Ala
S
Arg
O
NH
3
3
4
Fig. 3. S-Peptide formation via an N–S acyl shift reaction of peptide 3 in a TFA solution.
The new signals at 204.6 and 205.3 ppm are consistent with
1.0
0.8
0.6
0.4
0.2
the chemical shift of a carbonyl carbon of a thiophenyl ester.
These results indicate that a carbonyl carbon in the amide bond
of peptide 8 formed a thioester with the thiol in the Dmmb
group via an N–S acyl shift reaction. The two signals at
204.6 and 205.3 ppm indicate that two conformers are present
in the NMR time scale, the ratio of which changed depending
on the temperature of the TFA solution.
On RP-HPLC analysis, new peaks with retention times of
12.3 and 14.2 min appeared after storage of the peptide solu-
tion for 24 h (Fig. 6E). On ESI-MS analysis, the molecular
mass number of the compound in the fraction eluting at
12.3 min was the same as that of amide 8. The compound that
was eluted earlier than peptide 8 on RP-HPLC must be the
compound generated via the same reaction as was observed
by Vizzavona et al.3 Thus, the compound must be 9 (Fig. 7).
The molecular mass number of the compound in the fraction
eluting at 14.2 min coincided with that of Fmoc–Gly(1-13C)–
D,L-Ala–OMe (10). This compound was produced via acidoly-
sis of the Dmmb group. The 13C-signal at 173.4 ppm is as-
signed as the signal of the amide carbonyl carbon of 10.
After a 48 h storage, the ratio of 13C signal intensities be-
came nearly constant. Judging from the 13C signal intensities,
the ratio of compounds 8 to 9 in the TFA solution was 1 to 4
(Fig. 6C). The RP-HPLC elution profile of the sample stored
for 48 h showed roughly the same ratio of compounds 8 to 9
(Fig. 6F). This indicates that the thioester 9 is much more sta-
ble than the thioester produced from the –Gly–Cys– sequence.
Reactivity of Compound 9. To obtain additional evidence
that compound 9 is, in fact, a thioester, the fraction at 12.3 min
was isolated, freeze-dried and mixed with sodium 2-mercap-
toethanesulfonate in a neutral buffer. The reaction mixture
was subjected to RP-HPLC after 2 h, and the fraction was an-
alyzed by ESI-MS. Mass analysis indicated that the thioester,
0
0
200
400
600
800
1000
TFA treatment time / h
Fig. 4. Time course of the ratio of S-peptide 4 over the total
peptides in a mixture of TFA (71%), CDCl3 (29%), and
TCEP (0.5%) (v=v=w).
TCEP (0.5%) (v=v=w) as shown in Fig. 4.
Analysis of the Compound Generated from a Dipeptide
Having a Dmmb Group on Amide Bond in a TFA Solu-
tion. Fmoc–Gly(1-13C)–D,L-(Dmmb)Ala–OMe (8) was pre-
pared as a model compound in an attempt to detect the thio-
ester form of the model peptide by 13C NMR and RP-HPLC.
Peptide 8 was prepared according to the scheme shown in
Fig. 5 in an overall yield of 27%.
Peptide 8 was dissolved in a mixture of TFA (86%), CDCl3
(14%), and TCEP (0.5%) (v=v=w). The solution was subjected
to 13C NMR and RP-HPLC analyses. The 13C signal of the gly-
cine(1-13C) in peptide 8 was observed at 172.8 ppm (Fig. 6A).
Peptide 8 was eluted at 21.5 min by RP-HPLC (Fig. 6D). After
storage of the TFA solution for 24 h, new 13C signals at 204.6
and 205.3 ppm appeared (Fig. 6B). After 48 h, the 13C signal
of the amide in peptide 8 had nearly disappeared (Fig. 6C).
O
H
N
NH •HCl
2
OCH
3
13
Fmoc-Gly(1- C)-OH
1) Ph COH, TFA
3
TrtS
CH
3
HS
CHCl
HBTU, DIEA
3
2) CH COCOOCH
3
DMF
3
OCH
OCH
3
3
NaB(OAc) H
3
OCH
OCH
CH Cl
3
3
2
2
5
6
O
C
O
H
H
O
O
13
13
N
N
C
Fmoc
N
Fmoc
N
OCH
OCH
3
3
i
TFA, Pr SiH
3
TrtS
CH
HS
CH
3
3
CH Cl
2
2
OCH
OCH
3
3
OCH
OCH
3
3
7
8
Fig. 5. Synthetic route of 13C-labeled peptide 8.

1776 Bull. Chem. Soc. Jpn. Vol. 79, No. 11 (2006)
N–S Acyl Shift of Peptide Backbone
13
C NMR
RP-HPLC
8
(A) t = 0 h
(D) t = 0 h
8
TCEP
(B) t = 24 h
(E) t = 24 h
9
9
9
10
8
8
10
10
(C) t = 48 h
(F) t = 48 h
9
10
8
210
200
190
180
170
0
10
20
30
chemical shift / ppm
retention time / min
Fig. 6. Analysis of the product generated from peptide 8 in a TFA solution containing CDCl3 (14%) and TCEP (0.5%) (v=v=w).
(A), (B), and (C): 13C NMR spectra of peptide 8 in the TFA solution after 0-h, 24-h, and 48-h storage, respectively. (D), (E), and
(F): RP-HPLC elution profiles of peptide 8 in the TFA solution after 0-h, 24-h, and 48-h storage, respectively. HPLC conditions;
column: YMC-Pack ProC18 (4:6 ꢁ 150 mm), eluent: linear gradient of acetonitrile in 0.1% TFA from 30 to 90% over 30 min, flow
rate: 1.0 mL minꢂ1
.
O
C
O
C
CH
3
H
N
O
H
13
13
N
OCH
3
O
C
CH
3
Fmoc
N
Fmoc
S H N
2
H
OCH
13
3
TFA
N
OCH
3
O
Fmoc
N
H
HS
CH
3
O
H CO
3
OCH
3
OCH
3
OCH
3
8
9
10
Fig. 7. S-Peptide formation from peptide 8 in a TFA solution. The Dmmb group was partly cleaved by acidolysis.
Fmoc–Gly(1-13C)–SCH2CH2SO3H, was present. At the same
time, peptide 8 was detected by RP-HPLC. These observations
indicate that compound 9 is a thioester. Thus, it can be safely
concluded that the Dmmb group mediates thioester-bond for-
mation via an N–S acyl shift reaction in the TFA solution.
Preparation of a Peptide Thioester from a Peptide Hav-
ing a Dmmb Group on Its Backbone Amide. Val–Ala–Val–
Phe–Val–Gly(1-13C)–D,L-(Dmmb)Ala–NH2 (13) was prepared
according to the scheme shown in Fig. 8. Fmoc–Gly(1-13C)–
D,L-[Dmmb(Trt)]Ala–OH (11), obtained by the hydrolysis
of peptide 7, was introduced to 4-(ꢀ-amino-2,4-dimethoxy-
benzyl)phenoxymethyl polyethylene glycol polystyrene resin
(H2N–SAL–PEG resin). Peptide chain elongation was carried
out by using Fmoc SPPS to give Val–Ala–Val–Phe–Val–
Gly(1-13C)–D,L-[Dmmb(Trt)]Ala–NH–SAL–PEG resin (12).
Peptide 13 was obtained by treating resin 12 with Reagent
B, followed by purification by RP-HPLC, in an 18% yield,
which was based on the amino group in the Fmoc–NH–
SAL–PEG resin. Peptide 13 was eluted at 23.8 min as an over-
lapping peak, reflecting D,L-Ala.
Peptide 13 was treated with TFA containing 0.5% TCEP
(w=v) for 30 h (Fig. 9A). The newly generated peptide 14,
which was eluted at 18.9 min, with a molecular mass number
of 844.3 was isolated by injecting the reaction mixture into a
RP-HPLC. After adding sodium 2-mercaptoethanesulfonate
to the isolated fraction, it was lyophilized. The resulting pow-
der was dissolved in a mixture of sodium phosphate buffer
(pH 7.8) and acetonitrile (1:1, v=v), and analyzed by RP-
HPLC after stirring for 2 h (Fig. 9B). The peptide thioester,
Val–Ala–Val–Phe–Val–Gly(1-13C)–SCH2CH2SO3H (15), was

K. Nakamura et al.
Bull. Chem. Soc. Jpn. Vol. 79, No. 11 (2006) 1777
tide was converted to the corresponding 2-sulfoethyl peptide
thioester, which is widely used as a building block in polypep-
tide synthesis. This work will provide deeper insight into the
chemical nature of the cysteine residue and thiol-containing
auxiliary groups in peptides and will help in the development
of a new methodology in peptide and protein chemistry.
H
Fmoc
N SAL PEG
(resin)
1) 20% piperidine
2) 11, HBTU,
HOBt•H O,
2
DIEA
Experimental
O
CH
3
H
H
13
N
C
N
General. Acetonitrile was HPLC grade. Other solvents were
reagent grade. Amino acid derivatives used were of the L-configu-
ration, unless otherwise noted. Fmoc–amino acid derivatives were
purchased from the Peptide Institute Inc. 4-(N-t-Butoxycarbonyl–
alaninyl–oxymethyl)phenylacetoamidemethyl resin (Boc–Ala–
OCH2–Pam resin) were purchased from Applied Biosystems Inc.
4-(ꢀ-9-Fluorenylmethoxycarbonylamino-2,4-dimethoxybenzyl)-
phenoxy resin (Fmoc–Rink amide resin) were purchased from
Novabiochem. The Fmoc–NH–SAL–PEG resin was purchased
from Watanabe Chemical Ind., Ltd. All other chemicals were com-
mercially available and were used without further purification.
Melting points were measured on a Yanaco micro melting point
apparatus and are uncorrected. NMR spectra were recorded on a
Bruker Avanceꢁ 400 (1H, 400 MHz, 13C, 100 MHz) spectrome-
ter, and chemical shifts (ꢁ) are expressed in parts per million rel-
ative to tetramethylsilane. Splitting patterns are designated as: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad.
ESI-MS spectra were recorded on a Thermo Finnigan LCQꢁ
DECA XP spectrometer. Peptide yields were determined by
amino acid analyses, which were performed on a Hitachi L-8500
amino acid analyzer after hydrolysis with constantly boiling point
HCl (Nacalai Tesque) at 110 ꢃC for 24 h in an evacuated sealed
tube. Silica-gel column chromatography was carried out on E.
Merck silica gel 60 (230–400 mesh). HPLC was carried out on a
reversed phase column (YMC-Pack ProC18, (4:6 ꢁ 150 mm)
(YMC Co., Ltd.), Cosmosil 5C18-AR-II (10 ꢁ 250 mm), or
Cosmosil 5C18-AR-II (20 ꢁ 250 mm) (Nacalai Tesque)) using a
linear increasing gradient of acetonitrile in water/0.1% TFA and
peptides were detected by an absorbance measurement at 220
nm. A disposable ODS cartridge, TOYOPACK ODS-M, was pur-
chased from Tosoh.
Fmoc
N
SAL-PEG
CH
O
2
TrtS
OCH
3
OCH
3
Fmoc-SPPS
O
CH
3
H
N
H
13
C
N
Val-Ala-Val-Phe-Val
N
SAL-PEG
CH
O
2
TrtS
OCH
3
OCH
12
3
Reagent B
O
CH
3
H
N
13
C
NH2
Val-Ala-Val-Phe-Val
N
CH
O
2
HS
OCH
3
OCH
3
13
Fmoc–Ile–Ala–Gly(1-13C)–Cys–Arg–NH2 (3). Starting from
the Fmoc–Rink amide resin (0.43 mmol gꢂ1 resin, 465 mg), Fmoc–
Ile–Ala–Gly(1-13C)–Cys(Trt)–Arg(Pbf)–Rink amide resin was
prepared manually following Fmoc SPPS. Each synthetic cycle
consisted of the removal of Fmoc groups with a 20% piperidine
solution of 1-methyl-2-pyrrolidinone (NMP), washing with NMP
(ꢁ5), coupling of the Fmoc–amino acid (0.80 mmol), which was
preactivated by mixing for 3 min with 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (0.72
mmol), 1-hydroxybenzotriazole (HOBt) (0.8 mmol), and N,N-di-
isopropylethylamine (DIEA) (1.6 mmol) in a mixture of DMF
(1.6 mL) and NMP (0.8 mL), washing with NMP (ꢁ3), end cap-
ping with an NMP solution containing 10% acetic anhydride
and 5% DIEA, and washing with NMP (ꢁ3). After each coupling
step, the completion of the coupling reaction was confirmed by
using a ninhydrin color test. The Fmoc–amino acids used were
Fmoc–Arg(Pbf)–OH, where Pbf is 2,2,4,6,7-pentamethyl-2,3-
dihydrobenzofuran-5-sulfonyl group, Fmoc–Cys(Trt)–OH, where
Trt is trityl, Fmoc–Ala–OH and Fmoc–Ile–OH. Fmoc–Gly-
(1-13C)–OH was introduced by mixing Fmoc–Gly(1-13C)–OH
Fig. 8. Synthetic route of Dmmb containing peptide 13.
eluted at 17.5 min, and the formulation was confirmed by ESI-
MS. The peptide thioester was isolated by RP-HPLC, and the
fraction containing 15 was freeze-dried. This peptide thioester
15 readily reacted with cysteine methyl ester in a solution of
Tris-tricine buffer (pH 8.2) and acetonitrile (1:1, v=v) to yield
Val–Ala–Val–Phe–Val–Gly(1-13C)–Cys–OMe (16) (Fig. 9C).
Conclusion
An N–S acyl shift reaction occurs in a cysteine-containing
peptide and peptides with a thiol-containing ligation auxiliary
in a TFA solution. A peptide containing a –Gly–Cys– se-
quence was transformed into the corresponding S-peptide with
a conversion rate of ca. 80% and that a peptide with Dmmb
attached also formed an S-peptide. However, the Dmmb group
was gradually decomposed when the peptide was stored in a
TFA solution, as observed by 13C NMR spectroscopy. The
quantitative estimation of the S-peptide ratio by RP-HPLC
analysis was difficult because of the rate of the S–N acyl shift
reaction. The S-peptide generated from a Dmmb-attached pep-
(72 mg, 0.24 mmol), HBTU (87 mg, 0.23 mmol), HOBt H2O (37
ꢄ
mg, 0.24 mmol), and DIEA (77 mL, 0.44 mmol) in DMF (1.6 mL).

1778 Bull. Chem. Soc. Jpn. Vol. 79, No. 11 (2006)
N–S Acyl Shift of Peptide Backbone
14
(A)
CH
3
13
Val-Ala-Val-Phe-Val-Gly(1- C)
NH2
N
CH
O
2
*
HS
OCH
3
13
OCH
3
13
TFA
(B)
CH
3
13
Val-Ala-Val-Phe-Val-Gly(1- C)
NH2
S
HN
15
O
**
13
**
H CO
3
OCH
3
14
HSCH CH SO Na
2
2
3
(C)
16
13
Val-Ala-Val-Phe-Val-Gly(1- C) SCH CH SO H
2
2
3
15
Cys-OMe
13
Val-Ala-Val-Phe-Val-Gly(1- C)-Cys-OMe
16
0
10
20
30
35
retention time / min
Fig. 9. Generation of peptide 16 from peptide 13 via an N–S acyl shift reaction followed by an intermolecular thiol exchange
reaction (left), and RP-HPLC elution profiles of reaction mixtures (right). (A) Generation of peptide thioester 14 from peptide
13 via an N–S acyl shift reaction; (B) generation of 2-sulfoethyl thioester 15 from peptide thioester 14 via an intermolecular thiol
exchange reaction; (C) generation of peptide 16 from peptide thioester 15 via native chemical ligation.9 Peak ꢀ contained a peptide
with a higher molecular mass number than original peptide 13 by 1.0. Peaks ꢀꢀ were not peptides, whose molecular mass number
were not detected more than 700 by ESI-MS. HPLC conditions; column: YMC-Pack ProC18 (4:6 ꢁ 150 mm), eluent: linear
gradient of acetonitrile in 0.1% TFA from 10 to 40% over 30 min, flow rate: 1.0 mL minꢂ1
.
Fmoc–Ile–Ala–Gly(1-13C)–Cys(Trt)–Arg(Pbf)–Rink amide resin
was obtained in a yield of 658 mg.
capto-4,5-dimethoxybenzylamine hydrochloride (5) (2.00 g, 7.81
mmol) was dissolved in chloroform (35 mL). To the solution, tri-
phenylmethyl alcohol (2.13 g, 8.18 mmol) and TFA (1.16 mL,
15.6 mmol) were added, followed by stirring for 3 h at room tem-
perature. A saturated aq solution of NaHCO3 (40 mL) was then
added. The reaction mixture was concentrated in vacuo. The prod-
uct was extracted with chloroform (50 mL ꢁ 2), and the combined
organic layers were washed with brine (20 mL) and dried over
Na2SO4. The solvent of the organic layer was evaporated under
reduced pressure to give a solid crude material (4.39 g). This
was dissolved in chloroform and precipitated with ether to yield
a white solid (3.17 g). This solid (2.65 g) was dissolved in
dichloromethane (DCM) (30 mL) and methyl pyruvate (674 mg,
6.60 mmol) was added to the solution. After stirring for 15 min,
sodium triacetoxyhydroborate (3.18 g, 15.0 mmol) was added to
An aliquot of the protected peptide resin (195 mg) was mixed
with 2.0 mL of reagent B, consisting of 88% TFA, 5% PhOH,
5% H2O, and 2% iPr3SiH (v=v=v=v), and stirred for 2 h. Ether was
added to the reaction mixture. The precipitate that formed was
washed with ether three times and dissolved in aq acetonitrile. The
resin-containing suspension was passed through a disposable ODS
cartridge, and the filtrate was freeze-dried to give a crude peptide
(40 mg). This crude material was purified by RP-HPLC (column:
Cosmosil 5C18-AR-II (20 ꢁ 250 mm)) to give labeled peptide 3
(24.6 mg, 41% yield based on the amino group on the Rink amide
resin); ESI-MS, m=z 741.6, calcd for C35H50N9O7S: ðM þ HÞþ,
741.4; amino acid analysis: Gly1:2Ala1Cysn.d.Ile0:6Arg1:2
.
D,L-(4,5-Dimethoxy-2-tritylthiobenzyl)Ala–OMe (6). 2-Mer-

K. Nakamura et al.
Bull. Chem. Soc. Jpn. Vol. 79, No. 11 (2006) 1779
the solution. The mixture was stirred for 4 h under Ar at room tem-
perature. A saturated aq solution of NaHCO3 (30 mL) was then
added. The solution was concentrated in vacuo, and the product
was extracted with EtOAc (200 mL) from the solution. The organ-
ic layer was successively washed with a saturated aq solution of
NaHCO3 (50 mL ꢁ 2) and brine (20 mL), and the extract was
dried over Na2SO4. The organic layer was concentrated in vacuo.
The crude product (4.65 g) was chromatographed over silica gel
using a mixture of methanol and chloroform (5:95, v=v) to yield
the product (2.51 g, 79%) as a white solid after evaporating the
eluent. An aliquot of the product was dissolved in dioxane, and
a 1 M HCl solution in dioxane, followed by ether, was added. The
crystals were subjected to analysis: mp 96.8–98.5 ꢃC; 1H NMR
(CDCl3) ꢁ 1.19 (d, 3H, J ¼ 6:9 Hz, –CH(CH3)–), 3.01 (d, 1H, J ¼
13:0 Hz, –CHH–NH2–), 3.08 (d, 1H, J ¼ 13:0 Hz, –CHH–NH2–),
3.19 (q, 1H, J ¼ 6:9 Hz, –CH(CH3)–CO), 3.38 (s, 3H, –COOCH3),
3.68 (s, 3H, –OCH3), 3.87 (s, 3H, –OCH3), 6.63 (s, 1H), 6.81 (s,
1H), 7.20–7.30 (m, 15H); 13C NMR (CDCl3) ꢁ 18.2, 49.2, 51.7,
55.5, 55.8, 56.0, 70.8, 112.0, 118.7, 123.5, 126.8, 127.5, 130.2,
138.9, 144.3, 147.1, 149.8, 175.9; ESI-MS m=z 527.8, calcd for
C32H34NO4S: ðM þ HÞþ, 528.2; Anal. Found: C, 72.80; H, 6.41;
N, 2.62%, calcd for C32H34ClNO4S: C, 72.84; H, 6.30; N, 2.65%.
Fmoc–Gly(1-13C)–D,L-(4,5-Dimethoxy-2-tritylthiobenzyl)-
Ala–OMe (7). To a solution of Fmoc–Gly(1-13C)–OH (58.5 mg,
0.196 mmol) and HBTU (69.3 mg, 0.183 mmol) in DMF (500 mL),
DIEA (66.0 mL, 0.380 mmol) was added, and the solution was
stirred for 5 min. Compound 6 (51.5 mg, 0.0976 mmol) was added
to the reaction mixture, followed by stirring for 17 h under an Ar
atmosphere. The solution was concentrated in vacuo. The solid
residue was dissolved in EtOAc (50 mL), and the organic layer
was successively washed with 5% aq citric acid (20 mL ꢁ 2),
sat. aq NaHCO3 (20 mL ꢁ 2), and brine (5 mL), and dried over
Na2SO4. The filtrate was concentrated in vacuo to give a crude
product (118 mg). The crude material was purified by silica-gel
column chromatography using a mixture of hexane:EtOAc (70:30,
v=v) as an eluent to yield 57.8 mg (73%) of 13C-labeled compound
7 as a white solid: ESI-MS, m=z 830.1, calcd for C4813CH46N2-
NaO7S: ðM þ NaÞþ, 830.3.
11 as a white solid: ESI-MS, m=z 816.6, calcd for C4713CH44N2-
NaO7S: ðM þ NaÞþ, 816.3.
Val–Ala–Val–Phe–Val–Gly(1-13C)–D,L-(Dmmb)Ala–NH2 (13).
Fmoc–NH–SAL–PEG resin (0.26 mmol gꢂ1, 385 mg, 0.10 mmol)
was washed with NMP (2 min ꢁ 3), treated with 20% piperidine
in NMP (v=v) (5 min ꢁ 2, 10 min ꢁ 1) and washed with NMP
(1 min ꢁ 5). The resin was mixed with a solution, which was pre-
pared by mixing dipeptide 11 (83 mg, 0.11 mmol), HBTU (38 mg,
0.10 mmol), HOBt H2O (16 mg, 0.11 mmol), and DIEA (36 mL,
ꢄ
0.21 mmol) in DMF, for 17 h. The obtained resin, Fmoc–Gly-
(1-13C)–D,L-[Dmmb(Trt)]Ala–NH–SAL–PEG resin, was washed
with NMP (1 min ꢁ 5), treated with 10% Ac2O, 5% DIEA in
NMP (v=v), and washed with NMP (1 min ꢁ 3). Then, the peptide
chain elongation was carried out on a model 433A peptide synthe-
sizer (Applied Biosystems, Inc.) using the FastMoc 0.25 mmol
MonPrevPk protocol with end capping with acetic anhydride/
HOBt/DIEA. The amino acids were introduced by a double cou-
pling protocol. After completion of the peptide elongation, the
peptide resin was extensively washed with methanol and dried
in vacuo to give 448 mg of peptide resin 12.
An aliquot of peptide resin 12 (101 mg) was stirred with
Reagent B for 2 h. Ether was added to the reaction mixture, fol-
lowed by stirring for 1 h. The resulting precipitate was washed
with ether three times and then dissolved in 50% aq acetonitrile.
The solution was passed through a disposable ODS cartridge,
and the eluted solution was lyophilized to give a white powder
(18 mg). This compound was purified by RP-HPLC (column:
Cosmosil 5C18-AR-II (10 ꢁ 250 mm)) to give Val–Ala–Val–
Phe–Val–Gly(1-13C)–D,L-(Dmmb)Ala–NH2 (13) (18% based on
Fmoc–NH–SAL–PEG resin); ESI-MS, m=z 844.4, calcd for
C4013CH63N8O9S: ðM þ HÞþ, 844.4; amino acid analysis: Gly1-
Ala1:1Val3:2Phe1:3
.
Val–Ala–Val–Phe–Val–Gly(1-13C)–SCH2CH2SO3H
(15).
Peptide 13 (0.5 mg) was treated with a 200 mL of a TFA solution
containing 0.5% TCEP (v=w) by stirring for 30 h. The reaction
mixture was injected directly onto an RP-HPLC (column: YMC-
PACK ProC18 (4:6 ꢁ 150 mm)). After the addition of sodium 2-
mercaptoethanesulfonate (1 mg, excess) to the fraction at 18.9 min
(Fig. 9), the mixture was freeze-dried to give white powder. The
powder was dissolved in 100 mL of a mixture of 0.2 M sodium
phosphate buffer (pH 7.8) and acetonitrile (1:1, v=v), and the
reaction mixture was stirred for 2 h and subjected to RP-HPLC
to isolate the product, peptide thioester 15; ESI-MS, m=z 716.3,
calcd for C3013CH51N6O9S2: ðM þ HÞþ, 716.3.
Fmoc–Gly(1-13C)–D,L-(4,5-Dimethoxy-2-mercaptobenzyl)-
Ala–OMe (8). To a solution of 7 (40.5 mg, 50.1 mmol) in DCM
(200 mL), triisopropylsilane (51.3 mL, 250 mmol) and TFA (200
mL) were added. The reaction mixture was stirred at room temper-
ature for 1 h and then concentrated in vacuo. The product was
isolated by RP-HPLC (column: Cosmosil 5C18-AR-II (20 ꢁ 250
mm)) and lyophilized to give 12.9 mg (46%) of 8 as a whþite solid:
ESI-MS, m=z 566.2, calcd for C2913CH33N2O7S: ðM þ HÞ , 566.2.
Fmoc–Gly(1-13C)–D,L-(4,5-Dimethoxy-2-tritylthiobenzyl)-
Ala–OH (11). To a stirred ice cold solution of 7 (619 mg, 0.766
mmol) in THF (15 mL), 0.5 M aq solution of lithium hydroxide
(1.53 mL, 0.765 mmol) was added dropwise over 30 min. The re-
action mixture was stirred for 30 min, and a 0.5 M aq solution of
lithium hydroxide (0.770 mL, 0.385 mmol) was then added drop-
wise to the reaction solution, in an ice bath, over 15 min and
1.0 M aq HCl was added until the pH of the solution reached to
4.0. The reaction mixture was concentrated in vacuo, and H2O
(10 mL) was added to dissolve the residue. The product was
extracted from the aqueous layer with chloroform (20 mL ꢁ 2),
and the combined organic layer was washed with brine (5 mL)
and dried over Na2SO4. The organic layer was concentrated in
vacuo, and the solid residue (826 mg) was purified by silica-gel
column chromatography using a mixture of AcOH, hexane, and
EtOAc (1:40:60, v=v=v) as an eluent to give 223 mg (36%) of
Val–Ala–Val–Phe–Val–Gly(1-13C)–Cys–OMe (16). To a stir-
red solution of peptide thioester 15, which was obtained by lyo-
philizing the elution at 17.5 min (Fig. 9B), in 100 mL of a mixture
of 0.1 M Tris-tricine buffer (pH 8.2), containing 6 M guanidine and
0.02 M TCEP, and acetonitrile (1:1, v=v) was added Cys–OMe
ꢄ
HCl (1 mg, excess). The reaction mixture was stirred for 1 h and
then subjected to RP-HPLC to give the product, peptide 16; ESI-
MS, m=z 709.3, calcd for C3213CH54N7O8S: ðM þ HÞþ 709.4.
13C NMR Spectra Measurement of Fmoc–Ile–Ala–Gly-
(1-13C)–Cys–Arg–NH2 (3) in a TFA Solution.
Fmoc–Ile–
Ala–Gly(1-13C)–Cys–Arg–NH2 (4.0 mg) was dissolved in a mix-
ture of TCEP (3.5 mg), CDCl3 (200 mL), and TFA (500 mL). The
13C NMR measurement was carried out at 300 K.
13C NMR Spectra Measurement of Fmoc–Gly(1-13C)–D,L-
(Dmmb)Ala–OMe (8) in a TFA Solution. Fmoc–Gly(1-13C)–
D,L-(Dmmb)Ala–OMe (4.2 mg) was dissolved in a mixture of
TCEP (3.5 mg), CDCl3 (100 mL), and TFA (600 mL). The 13C NMR
measurement was carried out at 300 K.

1780 Bull. Chem. Soc. Jpn. Vol. 79, No. 11 (2006)
N–S Acyl Shift of Peptide Backbone
1403.
3
Lett. 2002, 12, 1963.
This research was supported, in part, by Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture,
Sports, Science and Technology, Japan.
J. Vizzavona, F. Dick, T. Vorherr, Bioorg. Med. Chem.
4
a) R. Hirata, Y. Ohsumi, A. Nakano, H. Kawasaki, K.
Supporting Information
Suzuki, Y. Anraku, J. Biol. Chem. 1990, 265, 6726. b) P. M.
Kane, C. T. Yamashiro, D. F. Wolczyk, N. Neff, M. Geobl,
T. M. Stevens, Science 1990, 250, 651.
5
2000, 39, 450.
Analytical data, such as melting point, chemical shift, elemen-
tal analysis, of non-labeled compounds 70, 80, and 110. The struc-
tures of 13C-labeled compound 7, 8, and 11 were confirmed by
comparing with 70, 80, and 110. This material is available free of
charge on the web at http://www.csj.jp/journals/bcsj/.
C. J. Noren, J. Wang, F. B. Perler, Angew. Chem., Int. Ed.
6
a) T. W. Muir, D. Sondhi, P. A. Cole, Proc. Natl. Acad.
Sci. U.S.A. 1998, 95, 6705. b) T. C. Evans, J. Benner, M.-Q.
Xu, Protein Sci. 1998, 7, 2256.
7 T. Kawakami, M. Sumida, K. Nakamura, T. Vorherr,
S. Aimoto, Tetrahedron Lett. 2005, 46, 8805.
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