S-acyl isopeptide method: Use of allyl-type protective group for improved preparation of thioester-containing S-acyl isopeptides by Fmoc-based SPPS

Article abstract of DOI:10.1002/bip.21410

We have studied the S-acyl isopeptide method for the synthesis of peptides containing difficult sequences. The S-acyl isopeptide, which contains a β-thioester instead of the native N-acyl bond at a Cys residue, can be converted into the target peptide via an S-to-N intramolecular acyl migration reaction. However, the synthesis of the S-acyl isopeptide structure by Fmoc-based SPPS is hampered by repetitive base treatments; decomposition of the thioester and the epimerization of the thioesterified residue are commonly observed. Here, we adopted allyloxycarbonyl (Aloc) protective group to avoid the problem. Catalytic amount of Pd in the presence of scavengers such as PhSiH3 and dimedone selectively removed the Aloc group with neither decomposition of the thioester structure nor epimerization at the thioesterified residue. A model pentapeptide and amylin(1-12) with difficult sequences were efficiently synthesized by the improved S-acyl isopeptides method. Finally, the isolated S-acyl isopeptides were quantitatively converted into the desired peptides via the S-to-N intramolecular acyl migration reaction. The S-acyl isopeptide method will be a useful method to prepare the difficult sequence-containing peptides with Cys residue.

Full text of DOI:10.1002/bip.21410

S-Acyl Isopeptide Method: Use of Allyl-Type Protective Group
S-Acyl Isopeptide Method: Use of Allyl-Type Protective Group
for Improved Preparation of Thioester-Containing S-Acyl
for Improved Preparation of Thioester-Containing S-Acyl
Isopeptides by Fmoc-Based SPPS
Isopeptides by Fmoc-Based SPPS
Taku Yoshiya, Yuka Hasegawa, Wakana Kawamura, Hiroyuki Kawashima, Youhei Sohma,
Tooru Kimura, Yoshiaki Kiso
Department of Medicinal Chemistry, Division of Medicinal Chemical Sciences, Center for Frontier Research in
Medicinal Science, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
Received 12 January 2010; revised 7 February 2010; accepted 8 February 2010
Published online 21 August 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.21410
reaction. The S-acyl isopeptide method will be a useful
ABSTRACT:
method to prepare the difficult sequence-containing
We have studied the ‘‘S-acyl isopeptide method’’ for the
#
peptides with Cys residue. 2010 Wiley Periodicals, Inc.
synthesis of peptides containing difficult sequences. The S-
Biopolymers (Pept Sci) 96: 228–239, 2011.
acyl isopeptide, which contains a b-thioester instead of
Keywords: acyl migration; difficult sequence; isopeptide
method; peptide thioester; peptide synthesis
the native N-acyl bond at a Cys residue, can be converted
into the target peptide via an S-to-N intramolecular acyl
migration reaction. However, the synthesis of the S-acyl
This article was originally published online as an accepted
preprint. The ‘‘Published Online’’ date corresponds to the
isopeptide structure by Fmoc-based SPPS is hampered by
repetitive base treatments; decomposition of the thioester
preprint version. You can request a copy of the preprint by
emailing the Biopolymers editorial office at biopolymers@wiley.
com
and the epimerization of the thioesterified residue are
commonly observed. Here, we adopted allyloxycarbonyl
(Aloc) protective group to avoid the problem. Catalytic
amount of Pd in the presence of scavengers such as
PhSiH3 and dimedone selectively removed the Aloc group
with neither decomposition of the thioester structure nor
epimerization at the thioesterified residue. A model
pentapeptide and amylin(1-12) with difficult sequences
were efficiently synthesized by the improved S-acyl
isopeptides method. Finally, the isolated S-acyl
INTRODUCTION
n 2003, we discovered that replacing the native amide
(N-acyl moiety) by a b-ester (O-acyl moiety) at Ser or
Thr residue within the peptide backbone significantly
changed the secondary structure of the native peptide.1,2
In addition, due to the presence of an additional amino
I
group, the O-acyl isopeptide is generally hydrophilic, which
is advantageous in effective purification by HPLC. The native
peptide is then generated from the corresponding O-acyl iso-
peptide via an O-to-N intramolecular acyl migration reaction
(Figure 1A). These findings led to the development of a novel
method, called the ‘‘O-acyl isopeptide method,’’ for the syn-
thesis of peptides containing difficult sequences.3 To date, the
O-acyl isopeptide method has also been used in various
research fields including peptide synthesis,4–13 ‘‘click pep-
tide’’ (‘‘switch peptide’’) concept,14–20 macromolecule,21 pep-
tide localization,22 protein splicing,23 and proteomics.24
Under these contexts, we recently expanded the concept
for Cys-containing peptides, leading to the S-acyl isopeptide
isopeptides were quantitatively converted into the desired
peptides via the S-to-N intramolecular acyl migration
Additional Supporting Information may be found in the online version of this article.
Correspondence to: Prof. Yoshiaki Kiso; e-mail: kiso@mb.kyoto-phu.ac.jp
Contract grant sponsor: MEXT (Ministry of Education, Culture, Sports, Science
and Technology) of the Japanese Government
C
VV
2010 Wiley Periodicals, Inc.
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PeptideScience Volume 96 / Number 2

Efficient Preparation of the S-Acyl Isopeptide
229
FIGURE 1 (A) O-Acyl isopeptide method and (B) S-acyl isopeptide method.
method.25 In a study with model peptide Ac-Val-Val-Cys- residue according to previous optimization study in the cou-
Val-Val-NH2 (1), its S-acyl isopeptide had 1400-fold higher pling of O-acyl isodipeptide.10
water-solubility than native pentapeptide, and thus the iso-
peptide was readily purified using HPLC. The synthesized S-
Deprotection of Allyloxycarbonyl (Aloc) Group
acyl isopeptide could convert into the native peptide via an
From the Thioester-Containing Peptide
S-to-N intramolecular acyl migration reaction (Figure 1B).
Various deprotection conditions for Aloc group were exam-
However, chemical synthesis of the S-acyl isopeptide by
ined using the Boc-Cys(Aloc-Val)-Val-Val-resin (Table I).
Fmoc SPPS was laborious because of side reactions at the thio-
The reactions were performed in toluene in the presence of
ester part during base treatment for Fmoc deprotection. The
Pd (catalytic amount) and scavengers (PhSiH3, dimedone or
side reactions involved decomposition of the Val-Cys thioester
N-methylaniline) (20 min, 32). The use of PhSiH3 resulted
(by simple aminolysis and/or diketopiperazine formation) bond
in 29% of desired 3 and 70% of starting material (entry 1).
and epimerization at the thioesterified Val residue because of
The use of dimedone increased the ratio of 3 to 58% (entry
high acidity of the a-proton. We herein report that the synthesis
2). In the use of N-methylaniline (entry 3), 5% of 4 (derived
of the S-acyl isopeptide by the Fmoc-based SPPS was remark-
from thioester decomposition) and 14% of 5 (derived from
ably improved by combination of allyl-type protective group.
reduction of the allyl moiety) were observed in addition to 3.
In the use of PhSiH3, presence of 0.1% TFA accelerated the
Aloc deprotection up to 69% (entry 4). In contrast, the start-
RESULTS
ing material was almost retained in dimedone plus 0.1% TFA
(entry 5), indicating that dimedone lost the scavenger activ-
Synthesis of S-Acyl Isodipeptide Unit and the
ity by the addition of TFA.
Incorporation to the Solid Support
The use of DCM (instead of toluene) resulted in complex
Preactivated Aloc-Val-OH by EDC ꢀ HCl was reacted with
mixture including 4 and 5. On the other hand, the use of
Boc-Cys-OH in CHCl3 to obtain S-acyl isodipeptide unit Boc-
PEG-containing resins such as NovaSyn TGR resin and
Cys(Aloc-Val)-OH (2) (see Figure 2). In an HPLC analysis of
NovaPEG Rink Amide resin throughout accelerated the
crude 2, a peak of D-Val derivative, which was confirmed by
deprotection reaction. The reaction proceeded up to 99% in
the synthesis of the authentic sample (Supporting Information
the presence of Pd-PhSiH3-0.1% TFA.
Figure S1), was not detected, indicating that the epimerization
at Aloc-Val did not occur in the reaction (Figure 2C).
The purified 2 was then coupled to peptide-resin by the Synthesis of a Model Pentapeptide Ac-Val-Val-Cys-
DIPCDI-HOAt method in dichloromethane (DCM). We did Val-Val-NH2 by the Optimized
not observe any significant by-product formation in the cou- S-Acyl Isopeptide Method
pling. The coupling condition with the less polar DCM As shown in Scheme 1, after the synthesis of Boc-Cys(Aloc-
would be crucial to suppress a possible deletion of the Cys Val)-Val-Val-NH-NovaSyn TGR resin (7), the Aloc group
Biopolymers (Peptide Science)

230
Yoshiya et al.
FIGURE 2 (A) S-Acyl isodipeptide units, (B) synthesis of S-acyl isodipeptide unit 2, (C) epimeri-
zation-free was confirmed by chiral HPLC: (i) crude 2, (ii) crude 2 + the authentic D-derivative
(eluted at #). Chiral HPLC was performed using Chiralcel OD normal phase column (4.6 3 250
mm2; Daicel Chemical, Tokyo, Japan); mobile phase, n-hexane/ethanol (18:1), 0.1% TFA; flow rate,
1.5 ml minꢁ1 (room temperature), detected at 230 nm. (D) HPLC of purified 2. Analytical HPLC
was performed using a C18 reverse phase column (4.6 3 150 mm2; YMC Pack ODS AM302) with
binary solvent system: a linear gradient of CH3CN (0–100% CH3CN, 40 min) in 0.1% aqueous
TFA at a flow rate of 0.9 ml minꢁ1 (408C), detected at 230 nm.
was deprotected by Pd-PhSiH3-0.1% TFA in toluene. Aloc- rivative was detected in the crude 10 (Supporting Informa-
Val-OH was then coupled by in situ neutralization with tion Figure S2 and Figure 3A, respectively), indicating that
HCTU and DIPEA26,27 to give 8. Finally, the S-acyl isopeptide the coupling of S-acyl isodipeptide unit did not involve any
10 ꢀ TFA was obtained through another Aloc-removal, acetyla- epimerization and formation of mixed anhydride upon the
tion followed by final deprotection of 9 with a TFA cocktail.
activation.10 In addition, H-Cys-Val-Val-NH2 was not
As shown in Figure 3A, the isopeptide 10 was obtained as detected, indicating that the decomposition of the thioester
a major product. In HPLC and MS analysis of crude 10, we bond did not take place during the treatment with Pd-
did not observe any deletion peptide or Aloc-peptide derived PhSiH3-0.1% TFA. Moreover, D-Val2 derivative was not
from the difficult sequence. Neither D-Cys nor des-Cys de- detected in the crude sample (Supporting Information Figure
Table I Deprotection of Aloc Group From a Thioester-Containing Peptide on the Resin in Dry Toluenea
Entry
Reagents
s.m. : 3 : 4 : 5b
1
2
3
4
5
Pd (0.1 eq.)-PhSiH3 (10 eq.)
Pd (0.1 eq.)-dimedone (20 eq.)
70 : 29 : <1 : N.D.c
41 : 58 : <1 : N.D.
33 : 48 : 5 : 14
Pd (0.1 eq.)-N-methylaniline (20 eq.)
Pd (0.1 eq.)-PhSiH3 (10 eq.)-0.1% TFAd
Pd (0.1 eq.)-dimedone (20 eq.)-0.1% TFAd
30 : 69 : <1 : N.D.
98 : N.D. : 2 : N.D.
a The reaction was performed under Ar atmosphere at room temperature (20 min 32). Pd(PPh3)4 was used as Pd(0).
b After the reaction, the peptides were cleaved from the resin using TFA for 90 min and precipitated with ether. Then, the obtained samples were analyzed
by analytical RP-HPLC.
c Not detected.
d Dry toluene containing 0.1% TFA was used as a solvent.
Biopolymers (Peptide Science)

Efficient Preparation of the S-Acyl Isopeptide
231
SCHEME 1 Reagents and conditions: (a) Fmoc-Val-OH (2.5 eq.), DIPCDI (2.5 eq.), HOBt (2.5
eq.), DMF, 2 h; (b) 20% piperidine/DMF, 20 min; (c) 2 (2.5 eq.), DIPCDI (2.5 eq.), HOAt (2.5 eq.),
DCM, 3 h; (d) Pd(PPh3)4 (0.1 eq.), PhSiH3 (10 eq.), 0.1% TFA/toluene, Ar, 20 min (three times);
(e) Aloc-Val-OH (5 eq.), HCTU (5 eq.), DIPEA (10 eq.), DMF, 2 h; (f) Pd(PPh3)4 (0.1 eq.), PhSiH3
(10 eq.), 0.1% TFA/toluene, Ar, 20 min; then, Ac2O (2.5 eq.), TEA (2.5 eq.), DMF, 20 min (two
cycles); (g) TFA:thioanisole:m-cresol:H2O (92.5:2.5:2.5:2.5), 90 min; (h) 10% DMSO/pH 7.6 phos-
phate buffer (10 mM), 4 h, 258C.
S2). This indicated that epimerization at the thioesterified sequence, was synthesized by the optimized S-acyl isopeptide
Val residue did not occur in the treatment of peptide-resin method (Scheme 2). Using conventional stepwise Fmoc SPPS
with Pd-PhSiH3-0.1% TFA.
method, 23 was synthesized with 19% yield (Supporting In-
Finally, 10 ꢀ TFA was purified by RP-HPLC with the isola- formation Figure S3). Boc-Cys(Aloc-Thr(tBu))-OH (20),
tion yield of 44% based on resin-bound Val residue. The which was synthesized in a similar manner to 2 (Supporting
purified 10 was then dissolved in phosphate buffer (pH 7.6) Information Figure S4), was coupled to 11 by the DIPCDI-
containing 10% DMSO, and quantitative conversion to the HOAt method in DCM. After the Aloc group of resulting 12
desired peptide 1 was observed after 4 h reaction.
was removed by Pd-PhSiH3-0.1%TFA in toluene for 20 min
(33), Aloc-Ala-OH was coupled by the HCTU-DIPEA
method to give 13. Fmoc-Thr(tBu)-OH was coupled after the
deprotection of the Aloc group of 13. Then, Fmoc-Asn(Trt)-
OH was coupled after the removal of Fmoc group of Thr4
by a lower nucleophilic base cocktail (1-methylpyrrolidine
Synthesis of Amylin(1-12)
Amylin(1-12) (H–KCNTATCATQRL–NH2 having a disulfide
bond between Cys2–Cys7, 23), which is known as a difficult
FIGURE 3 HPLC profiles of (A) crude isopeptide 10, (B) T ¼ 5 min migration of 10 in pH 7.6
phosphate buffer containing 10% DMSO at room temperature, (C) T ¼ 5 h migration of 10 and
washed with H2O to give pure 1 (see experimental section for detail). Analytical HPLC was per-
formed using the same conditions as described in Figure 2.
Biopolymers (Peptide Science)

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Yoshiya et al.
SCHEME 2 Synthesis of (A) 2,7-di-S-acyl-isoamylin(1-12) (17) and (B) 7-S-acyl isoamylin(1-12)
(18). Reagents and conditions: (a) Fmoc-Xaa-OH (2.5 eq.), DIPCDI (2.5 eq.), HOBt (2.5 eq.), DMF,
2 h; (b) 20% piperidine/DMF, 20 min; (c) 20 (2.5 eq.), DIPCDI (2.5 eq.), HOAt (2.5 eq.), DCM, 3
h; (d) Pd(PPh3)4 (0.1 eq.), PhSiH3 (10 eq.), 0.1% TFA, toluene, Ar, 20 min (3 times); (e) Aloc-Ala-
OH (5 eq.), HCTU (5 eq.), DIPEA (10 eq.), DMF, 2 h; (f) Pd(PPh3)4 (0.1 eq.), PhSiH3 (10 eq.),
0.1% TFA, toluene, Ar, 20 min; then, Fmoc-Thr(tBu)-OH (5 eq.), HCTU (5 eq.), DIPEA (5 eq),
DMF, 40 min (three cycles); (g) 1-methylpyrrolidine (25 v/v%)-hexamethyleneimine (2 v/v%)-
HOBt (3 w/v%) in NMP-DMSO (1:1) (also known as Reagent A28), 10 min; (h) Fmoc-Asn(Trt)-
OH (2.5 eq.), DIPCDI (2.5 eq.), HOBt (2.5 eq.), DMF, 2 h; (i) 21 (2.5 eq.), DIPCDI (2.5 eq.),
HOAt (2.5 eq.), DCM, 3 h; (j) TFA:thioanisole:m-cresol:H2O (92.5:2.5:2.5:2.5), 90 min; (k) Fmoc-
Cys(Trt)-OH (2.5 eq.), DIPCDI (2.5 eq.), HOBt (2.5 eq.), DMF, 2 h; (l) Boc-Lys(Boc)-OH (2.5
eq.), DIPCDI (2.5 eq.), HOBt (2.5 eq.), DMF, 2 h; (m) TFA:ethanedithiol:H2O:triisopropylsilane
(94:2.5:2.5:1), 90 min.
Biopolymers (Peptide Science)

Efficient Preparation of the S-Acyl Isopeptide
233
FIGURE 4 HPLC profiles of crude S-acyl isopeptide of amylin(1–12) obtained in (A) Route A
and (B) Route B. Analytical HPLC was performed using the same conditions as described in Figure
2. Inset: Another HPLC analysis with slower gradient was performed in the condition C (2–32%/40
min).
(25 v/v%)-hexamethyleneimine (2 v/v%)-HOBt (3 w/v%) in peptide structure predominantly occurred during cleavage
NMP-DMSO (1:1), also known as Reagent A28). After the reaction and/or lyophilization, although the reason is not
Fmoc removal of Asn3, Boc-Cys(Boc-Lys(Boc))-OH (21), clear. In the crude mixture, *1% of D-allo-Thr6 derivative
which was synthesized in a similar manner to 2 (Supporting was observed, verified by the synthesis of the authentic sam-
Information Figure S5), was coupled by the DIPCDI-HOAt ple (Supporting Information Figure S6). We also synthesized
method in DCM to give 16 (Route A). Finally, peptide-resin the authentic 7-mono-S-acyl isopeptide 18 containing an S-
16 was treated with TFA cocktail, and the crude sample was acyl moiety at Thr6–Cys7 (Scheme 2, Route B). Fmoc-
analyzed. As shown in Figure 4A, the major product of crude Cys(Trt)-OH and Boc-Lys(Boc)-OH were sequentially
sample was 7-mono-S-acyl isopeptide 18, while a few percent coupled instead of using isodipeptide unit 21. In the crude
of 2,7-di-S-acyl isopeptide 17 was detected. This result indi- sample (Figure 4B), we observed D-allo-Thr derivative with a
cated that the S-to-N acyl migration at Lys1–Cys2 S-acyl iso- yield of *2% in addition to 18 (Supporting Information
FIGURE 5 (A) HPLC profile of pure S-acyl isopeptide 18. (B) Reduced-form amylin(1-12) (22)
was released from the S-acyl isopeptide 18 via the S-to-N intramolecular acyl migration reaction,
and then oxidized to amylin(1-12) (23) by addition of DMSO to the migration buffer. Process was
monitored by analytical RP-HPLC: (i) 0 s, (ii) 1 min, and (iii) 4 h (after the addition of DMSO).
(C, D) HPLC profiles of (C) crude 23 and (D) pure 23. Analytical HPLC was performed using the
same conditions as described in Figure 2 for (A), (C), and (D). The condition C (2–32%/40 min)
was used for (B).
Biopolymers (Peptide Science)

234
Yoshiya et al.
SCHEME 3 Reagents and conditions: (a) pH 7.6 phosphate buffer (10 mM), 10 min, 258C; (b)
then, addition of DMSO (for the concentration of 10%), 4 h.
Figure S7). Finally, isopeptide 18 ꢀ TFA was purified by RP- 1 day at 48C and at least 30 days at -208C as an amorphous
HPLC (Figure 5A).
powder.
Isopeptide 18 ꢀ TFA had two-fold higher water solubility
The resulting pure 18 was dissolved in phosphate buffer
(pH 7.6) and the reaction mixture was shaken at room tem- (>380 mg mlꢁ1) than that of native 23 ꢀ TFA (160 mg mlꢁ1).
perature (Scheme 3). As a result, after a 1-min reaction, We also examined secondary structures of 18, 22, and 23 by
quantitative S-to-N intramolecular acyl migration giving CD measurement (see Figure 6). The CD spectrum of 18 in
reduced amylin(1-12) 22 was observed (Figure 5B). DMSO 12 mM HCl/H2O with 30% TFE showed a negative maxi-
(final conc 10%) was directly added to the solution and the mum at *196 nm, which is characteristic of a random coil
mixture was incubated at 378C for 4 h to form a disulfide structure. In contrast, the CD spectrum of 23 in the same
bond at Cys2–Cys7 (Figures 5B and 5C). After final purifica- condition showed an a-helix-like structure with a negative
tion, the yield of oxidized amylin(1-12) (23) was 30% from maximum at *208 nm. The reduced-form amylin(1-12)
resin-bound Arg residue (Figure 5D).
(22) had a partial a-helix structure. These results indicated
We also examined the stability of 18 ꢀ TFA (i.e., S-to-N that the S-acyl isopeptide structure at Thr6–Cys7 of 18 dis-
acyl migration at Thr6–Cys7 to give 22) under various condi- rupted the formation of the a-helical structure.
tions (Table II). The migration proceeded 13% after 5 min
and >99% after 1 h in water, 5% after 5 min, and 32% after
1 h in pH 2.6 buffer, and 4% after 5 min and 13% after 1 h in
0.1% TFA/H2O. In contrast, only a few percentage of the
rearrangement product was observed in 0.1% HCl/H2O and
1% TFA/H2O after 1 h. In addition, 18 ꢀ TFA was stable for
Table II Stability of S-Acyl Isopeptide 18 ꢀ TFA: S-to-N Acyl
Migrations in Various Conditions
Percentage of 22b
Conditiona
5 min
1 h
Water
13
5
>99
32
—
13
1
pH 2.6 phosphate buffer
pH 7.6 phosphate buffer
0.1% TFA/water
1.0% TFA/water
0.1% HCl/water
>99
4
<1
<1
FIGURE 6 Circular dichroism (CD) spectra of S-acyl isopeptide
18, reduced amylin(1-12) (22) and oxidized amylin(1-12) (23) in 12
mM HCl/H2O containing 30% TFE at 158C. The molar ellipticity
[h] values have been normalized for the number of backbone amide
groups.
2
a S-Acyl isopeptide 18 ꢀ TFA was dissolved in the solvent at the concentra-
tion of 1 mg mlꢁ1
.
b Determined by analytical HPLC.
Biopolymers (Peptide Science)

Efficient Preparation of the S-Acyl Isopeptide
235
lecular acyl migration reaction. The rearrangement pro-
ceeded even in pH 2.6 buffer, indicating that the rate of S-to-
N migration is faster than that of O-to-N migration as
expected, although 18 ꢀ TFA was stable under more acidic
conditions such as 1% TFA/H2O or as a solid state at -208C.
Subsequent one-pot DMSO oxidation of 22 gave desired 23
cleanly. The total yield of oxidized amylin(1-12) (23) by use
of S-acyl isopeptide method was *1.5-fold higher (30%)
than that of conventional SPPS method (19%). Finally, the
CD spectrum indicated that S-acyl isopeptide 18 did not
have an a-helical nature unlike its N-acyl form 22 (see Figure
6), which agrees with the fact that the O-acyl isopeptide
modified the secondary structure of the original peptide.
Additionally, oxidized amylin (1-12) 23 had higher helix con-
tent than 22, suggesting that the disulfide bond stabilized the
a-helical structure as reported.42
DISCUSSION
We previously showed that the S-acyl isopeptide H-Cys(Ac-
Val-Val)-Val-Val-NH2 (10) could be constructed by Fmoc-
based SPPS without the interference of the difficult sequence.
The isopeptide also possessed 1400-fold higher water solubil-
ity than that of parent N-acyl peptide (1), which was advan-
tageous in HPLC purification. The S-acyl isopeptide could be
converted into 1 via the S-to-N intramolecular acyl migra-
tion reaction. As a result, the total yield of 1 by the S-acyl iso-
peptide method was 10%, approximately two-fold higher
than that in the use of conventional Fmoc SPPS (4.7%).
However, base treatment for Fmoc removal caused partial
decomposition of the thioester bond and epimerization at
the thioesterified residue.25 This prompted us to study an al-
ternative procedure for the synthesis of S-acyl isopeptide by
Fmoc-based method. Here we report the use of allyloxycar-
bonyl (Aloc) group29 because the protective group can be
removed under neutral conditions in the presence of Pd and
scavengers such as dimedone,30 stannane,31 PhSiH3,32 or N-
methylaniline.33
SUMMARY
By combination with Aloc protective group, the preparation
of thioester-containing S-acyl isopeptides by Fmoc-based
SPPS was remarkably improved. Catalytic amount of Pd in
the presence of scavengers such as PhSiH3 and dimedone
selectively removed the Aloc group without decomposition
of the thioester structure and epimerization at the thioesteri-
fied residue. These side reactions have been commonly prob-
lematic in the treatments of thioester-containing peptides with
the basic conditions such as 20% piperidine. The model pen-
tapeptide and amylin(1-12) with difficult sequences were effi-
In our studies on Aloc deprotection from thioester-con-
taining peptide (Table I), the use of PhSiH3 or dimedone in
the presence of catalytic amount of Pd in toluene showed
selective deprotection of the Aloc group while retaining
thioester bonds. This suggested that the decomposition of
the thioester by Pd-PhSiH3 giving aldehyde and thiol
(‘‘Fukuyama reduction’’34) did not take place under these
conditions. Moreover, addition of 0.1% TFA accelerated the
Aloc-removal reaction, probably because the TFA increased
the activity of hydride of PhSiH3 as PhSi(trifluoroacetox- ciently synthesized by the improved S-acyl isopeptides
y)nH3-n. In contrast, TFA attenuated the nucleophilicity of method. In the synthesis, (1) the Aloc-type S-acyl isodipeptide
dimedone.
The use of Aloc-type isodipeptide unit 2 and the opti-
unit, synthesized independently in solution phase, was intro-
duced to the peptide-resin without des-Cys and epimerization,
(2) the Aloc group of the dipeptide part was removed by Pd-
PhSiH3-0.1% TFA, and (3) another Aloc-amino acid was used
to avoid diketopiperazine formation. Finally, the isolated S-
acyl isopeptides were quantitatively converted into the desired
peptides via the S-to-N intramolecular acyl migration reac-
tion. The S-acyl isopeptide method will be a useful method to
prepare the difficult sequence-containing peptides.
mized Aloc deprotection conditions (Pd-PhSiH3-0.1% TFA)
remarkably improved the purity of crude 10 (see Figure 3).
The synthesis did not involve the side products derived from
the difficult sequence, indicating that the incorporated iso-
peptide structure disrupted the formation of undesired sec-
ondary structures on the resin. Neither decomposition of the
thioester nor epimerization at thioesterified Val residue,
which were previously observed in the base treatment,25 was
detected by using the Aloc protective group. The Cys-dele-
tion side reaction during the coupling of dipeptide unit did
not proceed because of the use of less polar DCM, which is
EXPERIMENTAL PROCEDURES
compatible with the previous observation in the coupling of General Procedures
O-acyl isodipeptide unit.10
Fmoc-amino acid side-chain protections were selected as fol-
Similarly, the S-acyl isopeptide 18 of amylin(1-12)35–41 lows: tBu (Thr), Boc (Lys), Pmc (Arg), and Trt (Cys, Asn,
was efficiently constructed using the Aloc protective group. Gln). All Fmoc protected amino acids and resins were pur-
Isopeptide 18 was rapidly and quantitatively converted into chased from Calbiochem-Novabiochem Japan (Tokyo). Aloc
N-acyl peptide 22 in neutral buffer via the S-to-N intramo- protected L-amino acids were purchased from Watanabe
Biopolymers (Peptide Science)

236
Yoshiya et al.
Chemical (Hiroshima) as DCHA salts, and used after wash- lution was additionally stirred for 1 h at room temperature.
ing with 10% citric acid. Other chemicals were purchased The solution was condensed in vacuo, diluted in ethyl acetate,
from commercial suppliers, Wako Pure Chemical (Osaka, Ja- and washed successively with 1 N HCl and brine. The organic
pan), Nacalai Tesque (Kyoto, Japan), and Aldrich Chemical layer was dried over Na2SO4 and the solvent was removed in
(Milwaukee, WI), and were used without further purifica- vacuo. The resulting oil was dissolved in MeOH (1.5 ml), and
tion. Analytical HPLC was performed using a C18 reverse purified by RP-HPLC with a 0.1% aqueous TFA-CH3CN sys-
phase column (4.6 3 150 mm; YMC Pack ODS AM302) tem. The desired fractions were collected and immediately ly-
with a binary solvent system: a linear gradient of CH3CN in ophilized to afford pure 2 as a white powder. Yield: 21.5 mg
0.1% aqueous TFA ((A) 0–100%/40 min, (B) 23–43%/40 (45%); HPLC analysis at 230 nm: purity was higher than
min, or (C) 2–32%/40 min) at a flow rate of 0.9 ml minꢁ1 99% (retention time (tR) ¼ 26.0 min on RP-HPLC (condi-
1
(temperature: 408C), detected at 230 nm. Preparative HPLC tion A), 7.4 min on chiral HPLC (condition A)); H NMR
was carried out on a C18 reverse phase column (20 3 250 (CDCl3, 300MHz) d 6.01–5.84 (m, 1H), 5.41–5.18 (m, 2H),
mm; YMC Pack ODS SH343-5) with a binary solvent system: 4.69–4.55 (m, 2H), 4.36 (dd, J ¼ 9.3, 4.8 Hz, 1H), 4.25–4.13
a linear gradient of CH3CN in 0.1% aqueous TFA at a flow (m, 1H), 3.45 (dd, J ¼ 14.1, 4.8 Hz, 1H), 3.36 (dd, J ¼ 14.1,
rate of 5.0 ml minꢁ1 (temperature: 408C), detected at 230 6.6 Hz, 1H), 2.39–2.19 (m, 1H), 1.44 (s, 9H), 1.01–0.85 (m,
nm. Chiral HPLC analysis was performed using Chiralcel1 6H); HRMS (FAB): calcd. for C17H28N2O7SNa (M+Na)+:
OD normal phase column (4.6 3 250 mm; Daicel Chemical, 427.1515, found: 427.1519.
Tokyo, Japan); mobile phase, (A) n-hexane/ethanol (18:1)
with 0.1% TFA or (B) n-hexane/ethanol (32:1) with 0.1%
Aloc-D-Val-OH (24)
TFA; flow rate, 1.5 ml minꢁ1 (room temperature), detected
Compound 24 was synthesized based on the procedure
at 230 nm. Solvents used for HPLC were of HPLC grade.
reported in Ref. 43. The 2-chlorotrityl chloride resin (400
NMR spectra were recorded on a JEOL 300 MHz instrument,
mg, 0.52 mmol) and Fmoc-D-Val-OH (264.7 mg, 0.78
and calibrated using tetramethylsilane (TMS) as an internal
mmol) were mixed and stirred for 2.5 h in the presence of
reference. MALDI-TOF MASS spectra were recorded on Voy-
DIPEA (362.3 ll, 2.08 mmol) in 1,2-dichloroethane (3.0 ml)
ager DE-STR using a-cyano-4-hydroxy cinnamic acid as a
under Ar. Then, Fmoc deprotection was carried out by treat-
matrix. FAB-MS was performed on a JEOL JMS-SX102A
ment with 20% piperidine/DMF (2 ml, 1 min 31 and 20
spectrometer equipped with the JMA-DA7000 data system.
min 31), followed by washing with DMF (1.5 ml, 310) and
ESI-MS was performed using LCMS-2010 EV with LC-20AD,
chloroform (1.5 ml, 35). The liberated amino group on the
and the sample was directly analyzed. Water-solubility was
resin was reacted with Aloc-Cl (164.9 ll, 1.56 mmol) in the
estimated using a similar method reported in Ref. 15.
presence of DIPEA (362.3 ll, 2.08 mmol) in DCM (Ar
atmosphere, 58C, 10 min, twice). Aloc-D-Val-OH was
released by 20% HFIP in DCM for 30 min, and concentrated
CD Spectroscopy
Each peptide or isopeptide (0.1 mg mlꢁ1) was dissolved in 12 in vacuo. Then, 24 was obtained as light yellow oil after dry-
mM HCl/H2O containing 30% TFE, and applied to CD spec- ing in vacuo. Yield: 83.0 mg; HPLC analysis at 230 nm: purity
1
troscopy, respectively. The CD spectra were recorded from was 78% [tR ¼ 16.2 min on RP-HPLC (condition A)]; H
190 to 260 nm at a scanning rate of 20 nm minꢁ1 with a time NMR (CDCl3, 300 MHz) d 6.00–5.80 (m, 1H), 5.41–5.17
constant of 1 s on a J-720WI (JASCO Corporation, Tokyo, (m, 2H), 4.67–4.51 (m, 2H), 4.38–4.25 (m, 1H), 2.33–2.14
Japan) instrument using a 1.0-mm path length quartz cell. (m, 1H), 1.07, 0.94 (2d, J ¼ 6.9, 6.6 Hz, 6H); HRMS (FAB):
Four scans with step resolution of 0.1 nm and bandwidth of calcd. for C9H16NO4 (M+H)+: 202.1079, found: 202.1083.
1.0 nm were averaged for each sample. Photomultiplier-volt-
age was under 500 V in each scan. Averaged blank spectra
Boc-Cys(Aloc-D-Val)-OH (D-Val Derivative of 2)
were subtracted, respectively.
Boc-Cys(Aloc-D-Val)-OH was synthesized in a similar man-
ner to 2. Yield: 25%; HPLC analysis at 230 nm: purity was
Boc-Cys(Aloc-Val)-OH (2)
higher than 99% [tR ¼ 26.0 min on RP-HPLC (condition
To a stirring solution of Aloc-Val-OH (48.0 mg, 0.24 mmol) A)], 9.4 min on chiral HPLC (condition A); 1H NMR
in 4 ml of CHCl3 was added EDC ꢀ HCl (22.9 mg, 0.12 (CDCl3, 300 MHz) d 6.01–5.78 (m, 1H), 5.40–5.17 (m, 2H),
mmol) and collidine (63.2 ll, 0.48 mmol), and the solution 4.72–4.49 (m, 2H), 4.36 (dd, J ¼ 9.3, 4.4 Hz, 1H), 4.23–4.08
was stirred at room temperature for 1 h. Then, Boc-Cys-OH (m, 1H), 3.53–3.12 (m, 2H), 2.36–2.18 (m, 1H), 1.51–1.39
(52.8 mg, 0.24 mmol) was added to the solution, and the so- (m, 9H), 1.00, 0.89 (2d, J ¼ 6.9, 6.3 Hz, 6H); HRMS (FAB):
Biopolymers (Peptide Science)

Efficient Preparation of the S-Acyl Isopeptide
237
calcd. for C17H28N2O7SNa (M+Na)+: 427.1515, found: was 99% [tR ¼ 28.7 min on RP-HPLC (condition A), 23.9
1
427.1519.
min on chiral HPLC (condition B)]; H NMR (CD3OD, 300
MHz) d 4.25 (dd, J ¼ 8.1, 4.6 Hz, 1H), 4.10 (dd, J ¼ 4.2 Hz,
9.3 Hz, 1H), 3.47 (dd, J ¼ 4.6 Hz, 13.8 Hz, 1H), 3.11–2.97
(m, 3H), 1.89–1.71 (m, 2H), 1.68–1.55 (m, 2H), 1.48–1.38
(m, 27H), 1.30–1.18 (m, 2H); HRMS (FAB): calcd. for
C24H43N3O9SNa (M+Na)+: 572.2618, found: 572.2612.
Boc-Cys(Aloc-Thr(tBu))-OH (20)
Compound 20 was synthesized in a similar manner to 2.
Yield: 41%; HPLC analysis at 230 nm: purity was higher than
1
99% [tR ¼ 31.2 min on RP-HPLC (condition A)]; H NMR
(CDCl3, 300 MHz) d 6.04–5.87 (m, 1H), 5.47–5.19 (m, 2H),
4.71–4.55 (m, 2H), 4.54–4.41 (m, 1H), 4.34–4.15 (m, 1H),
3.52–3.20 (m, 3H), 1.45 (s, 9H), 1.20 (d, J ¼ 6.0 Hz, 3H),
1.10 (s, 9H); HRMS (FAB): calcd. for C20H34N2O8SNa
(M+Na)+: 485.1934, found: 485.1927.
H-Cys(Ac-Val-Val)-Val-Val-NH2 (10)
After the preparation of the H-Val-Val-NH-resin (NovaSyn
TGR resin, 140 mg, 0.028 mmol),25 Boc-Cys(Aloc-Val)-OH
(2, 28.3 mg, 0.07 mmol) was coupled in the presence of
DIPCDI (10.8 ll, 0.07 mmol) and HOAt (9.5 mg, 0.07
mmol) for 3 h in dry DCM (1.0 ml). The Aloc group was
cleaved by Pd(PPh3)4 (3.2 mg, 0.0028 mmol) and PhSiH3
(48.8 ll, 0.28 mmol) in 0.1% TFA/dry toluene (1 ml) under
Ar atmosphere for 20 min (three times). Then, Aloc-Val-OH
(28.2 mg, 0.14 mmol) was coupled by the HCTU (57.9 mg,
0.14 mmol)-DIPEA (48.8 ll, 0.28 mmol) method for 2 h in
DMF (1.5 ml). Deprotection of this Aloc group using the
combination of Pd(PPh3)4 and PhSiH3 in toluene containing
0.1% TFA for 20 min, followed by final acetylation with
Ac2O (6.6 ll, 0.07 mmol) in the presence of TEA (9.7 ll, 0.07
mmol) for 20 min. The Aloc deprotection-acetylation was
repeated once. This procedure enabled minimum exposure
of the deprotected amino group to the solvent that could
trigger the diketopiperazine formation, and would attenuate
decomposition of the thioester bond. The resulting protected
peptide-resin was treated with TFA (4.0 ml)-m-cresol (100
ll)-thioanisole (100 ll)-H2O (100 ll) for 90 min, concen-
trated in vacuo, washed with diethyl ether at 08C, and centri-
fuged to give the crude S-acyl isopeptide 10 ꢀ TFA (18.8 mg).
This crude peptide (3.0 mg) was dissolved in MeOH, and im-
mediately injected into preparative HPLC with a 0.1% aque-
ous TFA-CH3CN system. The desired fractions were collected
and immediately lyophilized to afford pure 10 ꢀ TFA as a
white amorphous powder. Yield: 1.3 mg (44%); HPLC analy-
sis at 230 nm: purity was higher than 99% [tR ¼ 17.7 min on
RP-HPLC (condition A)]; TOF-MS: calcd. for (M+Na)+:
581.7, found: 581.8.
Aloc-D-allo-Thr(tBu)-OH (25)
Compound 25 was synthesized in a similar manner to 24.
Yield: 89%; HPLC analysis at 230 nm: purity was 92% [tR ¼
21.3 min on RP-HPLC (condition A)]; 1H NMR (CDCl3,
300 MHz) d 5.99–5.83 (m, 1H), 5.36–5.19 (m, 2H), 4.63–
4.53 (m, 2H), 4.40–4.29 (m, 1H), 4.09–3.97 (m, 1H), 1.28–
1.18 (m, 12H); HRMS (FAB): calcd. for C12H22NO5
(M+H)+: 260.1498, found: 260.1501.
Boc-Cys(Aloc-D-allo-Thr(tBu))-OH (D-allo-Thr
Derivative of 20)
Boc-Cys(Aloc-D-allo-Thr(tBu))-OH was synthesized in a
similar manner to 2. Yield: 31%; HPLC analysis at 230 nm:
purity was higher than 99% [tR ¼ 30.0 min on RP-HPLC
(condition A)]; 1H NMR (CDCl3, 300 MHz) d 6.01–5.81 (m,
1H), 5.40–5.20 (m, 2H), 4.72–4.40 (m, 3H), 4.20–4.04 (m,
1H), 3.69–3.14 (m, 3H), 1.26–1.11 (m, 12H); HRMS (FAB):
calcd. for C20H34N2O8SNa (M+Na)+: 485.1934, found:
485.1938.
Boc-Cys(Boc-Lys(Boc))-OH (21)
Compound 21 was synthesized in a similar manner to 2.
Yield: 40%; HPLC analysis at 230 nm: purity was higher than
99% [tR ¼ 29.1 min on RP-HPLC (condition A), 29.8 min
on chiral HPLC (condition B)]; 1H NMR (CD3OD, 300
MHz) d 4.25 (dd, J ¼ 4.8 Hz, 8.4 Hz, 1H), 4.12 (dd, J ¼ 4.8
Hz, 9.9 Hz, 1H), 3.43 (dd, J ¼ 4.8 Hz, 13.2 Hz, 1H), 3.12
(dd, J ¼ 8.4 Hz, 13.2 Hz, 1H), 3.06–2.96 (m, 2H), 1.88–1.71
(m, 2H), 1.69–1.52 (m, 2H), 1.48–1.38 (m, 27H), 1.30–1.20
(m, 2H); HRMS (FAB): calcd. for C24H43N3O9SNa
(M+Na)+: 572.2618, found: 572.2612.
Ac-Val-Val-Cys-Val-Val-NH2 (1, by the S-Acyl
Isopeptide Method)
Purified S-acyl isopeptide 10 ꢀ TFA (0.15 mg) was dissolved
in 1 ml of 10% DMSO/phosphate buffer (10 mM, pH 7.6),
and stirred for 5 h at room temperature. The mixture was
Boc-Cys(Boc-D-Lys(Boc))-OH (D-Cys Derivative of 21) lyophilized and washed with water followed by lyophilization
Boc-Cys(Boc-D-Lys(Boc))-OH was synthesized in a similar to give 1 as a white powder. Yield: 0.12 mg (99%); HPLC
manner to 2. Yield: 31%; HPLC analysis at 230 nm: purity analysis at 230 nm: purity was higher than 94% [tR ¼ 19.3
Biopolymers (Peptide Science)

238
Yoshiya et al.
min on RP-HPLC (condition A)]; TOF-MS: calcd. for by the DIPCDI-HOBt method after Fmoc deprotection by
(M+Na)+: 581.7, found: 581.4; the retention time on HPLC Reagent A (1 ml) for 10 min. Finally, Boc-Cys(Boc-Lys
(0–100% CH3CN for 40 min, 230 nm) of synthesized prod- (Boc))-OH (55.0 mg, 0.10 mmol) was coupled by the
uct was identical to the authentic sample, which was synthe- DIPCDI-HOAt method in DCM (1 ml) for 3 h. The resulting
sized independently by the conventional method.25
protected peptide-resin was treated with TFA (4.4 mL)-m-
cresol (20 ll)-thioanisole (20 ll)-H2O (20 ll) for 90 min,
concentrated in vacuo, washed with diethyl ether, and centri-
fuged to give the crude S-acyl isopeptide 17 ꢀ TFA (54.0 mg).
However, major product was 7-S-acyl-isoamylin(1-12) (18),
which was confirmed by RP-HPLC with the authentic sample
synthesized as described below. Thus, after purification using
RP-HPLC, 18 ꢀ TFA was obtained as a white amorphous pow-
der. Yield: 20.5 mg (29%); TOF-MS: calcd. for (M+H)+:
1309.5, found: 1309.6; HPLC analysis at 230 nm: purity was
96% [tR ¼ 14.0 min on RP-HPLC (condition A)].
Amylin(1-12) (23, by the Conventional Method)
By the general Fmoc SPPS using NovaSyn TGR resin (200
mg, 0.04 mmol), reduced-form amylin(1-12) (22) was syn-
thesized. After TFA treatment, crude 22 was purified by RP-
HPLC to afford 22 ꢀ TFA as a white powder. Yield: 24.5 mg
(37%); HPLC analysis at 230 nm: purity was 92% [tR ¼ 14.3
min on RP-HPLC (condition A)]; TOF-MS: calcd. for
(M+H)+: 1309.5, found: 1309.4.
Then, pure 22 (3.0 mg) was dissolved in 10% DMSO/H2O
(1 mg mlꢁ1), incubated at 378C for 4 h, and the mixture was
lyophilized. Crude 23 was dissolved in 50% MeCN/H2O, and
immediately injected into preparative HPLC with a 0.1%
aqueous TFA-CH3CN system. The desired fractions were col-
lected and immediately lyophilized to afford pure 23 ꢀ TFA as
a white amorphous powder. Yield: 1.5 mg (50%); HPLC
analysis at 230 nm: purity was higher than 99% [tR ¼ 14.5
min on RP-HPLC (condition A)]; TOF-MS: calcd. for
(M+H)+: 1307.5, found: 1307.4.
7-S-Acyl-Isoamylin(1-12) (18, by the Route B)
The 18 was synthesized in a similar manner to 17 on Nova-
Syn TGR resin (100 mg, 0.02 mmol). N-terminal two amino
acids were introduced using Fmoc-Cys(Trt)-OH and Boc-
Lys(Boc)-OH instead of S-acyl isodipeptide unit 21. The
resulting protected peptide-resin (131.7 mg) was treated with
TFA (2.6 ml)-ethanedithiol (65.9 ll)-H2O (65.9 ll)-triiso-
propylsilane (26.3 ll) for 90 min, concentrated in vacuo,
washed with diethyl ether, and centrifuged to give the crude
S-acyl isopeptide 18 ꢀ TFA (32.0 mg). This crude peptide (5.0
2,7-di-S-Acyl-Isoamylin(1-12) (17, by the Route A)
After the preparation of the H-Ala-Thr(tBu)-Gln(Trt)- mg) was dissolved in 1 ml of 12 mM HCl/H2O, and immedi-
Arg(Pmc)-Leu-NH-resin (NovaSyn TGR resin, 200 mg, 0.04 ately injected into preparative HPLC with a 0.1% aqueous
mmol) according to the general Fmoc-based solid-phase pro- TFA-CH3CN system. The desired fractions were collected
cedure, Boc-Cys(Aloc-Thr(tBu))-OH (20, 46.3 mg, 0.10 and immediately lyophilized to afford pure 18 ꢀ TFA as a
mmol) was coupled in the presence of DIPCDI (15.5 ll, 0.10 white amorphous powder. Yield: 2.0 mg (36%); TOF-MS:
mmol) and HOAt (13.6 mg, 0.10 mmol) for 3 h in dry DCM calcd. for (M+H)+: 1309.5, found: 1309.9; HPLC analysis at
(1.0 ml). The Aloc group was cleaved by Pd(PPh3)4 (4.6 mg, 230 nm: purity was 96% [tR ¼ 13.6 min on RP-HPLC (con-
0.0040 mmol) and PhSiH3 (49.7 ll, 0.40 mmol) in 0.1% dition A)].
TFA/dry toluene (1 ml) under Ar atmosphere for 20 min
(three times). Then, Aloc-Ala-OH (34.6 mg, 0.20 mmol) was
coupled by the HCTU (82.7 mg, 0.20 mmol)-DIPEA (69.7 Amylin(1-12) (23, by the S-Acyl Isopeptide Method)
ll, 0.40 mmol) method for 2 h in DMF (1 ml). Deprotection Pure isopeptide 18 (3.0 mg) was dissolved in 2.7 ml of pH
of this Aloc group by the combination of Pd(PPh3)4 (4.6 mg, 7.6 phosphate buffer (10 mM), and allowed for stand for 10
0.0040 mmol) and PhSiH3 (49.7 ll, 0.40 mmol) for 20 min min. About 300 ll of DMSO was added, and the mixture was
under Ar atmosphere followed by successive coupling of leaved at 378C for 4 h. After lyophilization, 23 was dissolved
Fmoc-Thr(tBu)-OH (79.2 mg, 0.20 mmol) by the HCTU in 50% MeCN/H2O, and immediately injected into prepara-
(82.7 mg, 0.20 mmol)-DIPEA (69.7 ll, 0.40 mmol) method tive HPLC with a 0.1% aqueous TFA-CH3CN system. The
for 40 min, respectively. The Aloc deprotection-acylation was desired fractions were collected and immediately lyophilized
repeated twice (total: three cycles). This procedure enabled to afford pure 23 ꢀ TFA as a white amorphous powder. Yield:
minimum exposure of the deprotected amino group to the 2.5 mg (83%); TOF-MS: calcd. for (M+H)+: 1307.5, found:
solvent that could trigger the diketopiperazine formation, 1307.7; HPLC analysis at 230 nm: purity was 99% [tR ¼ 15.1
and would attenuate decomposition of the thioester bond. min on RP-HPLC (condition A)]; the retention time on
Fmoc-Asn(Trt)-OH (59.7 mg, 0.10 mmol) was introduced HPLC (0–100% CH3CN for 40 min, 230 nm) of synthesized
Biopolymers (Peptide Science)

Efficient Preparation of the S-Acyl Isopeptide
239
18. Mutter, M.; Chandravarkar, A.; Boyat, C.; Lopez, J.; Santos,
`
S. D.; Mandal, B.; Mimna, R.; Murat, K.; Patiny, L.; Saucede, L.;
product was identical to the authentic sample, synthesized by
the conventional method as described above.
Tuchscherer, G. Angew Chem Int Ed 2004, 43, 4172–4178.
19. Kiewitz, S. D.; Kakizawa, T.; Kiso, Y.; Cabrele, C. J Pept Sci 2008,
14, 1209–1215.
The authors thank Dr. J-T. Nguyen for his help in preparing the
manuscript. They thank Ms. W. Takagi for technical assistance.
´
20. Bozso, Z.; Penke, B.; Simon, D.; Laczko, I.; Juhasz, G.; Szegedi,
´
´
V.; Kasza, A.; Soos, K.; Hetenyi, A.; Weber, E.; Tohati, H.; Csete,
´
´
´
´ ´
´
¨ ¨
M.; Zarandi, M.; Fulop, L. Peptides 2010, 31, 248–256.
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