O-Acyl isopeptide method: Efficient synthesis of isopeptide segment and application to racemization-free segment condensation

Article abstract of DOI:10.1039/b903624e

We report the establishment of the O-acyl isopeptide method-based racemization-free segment condensation reaction toward future chemical protein synthesis. Peptide segments containing C-terminal O-acyl Ser/Thr residues were successfully synthesized by use

Full text of DOI:10.1039/b903624e

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Taku Yoshiya et al.
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O-Acyl isopeptide method: efficient
synthesis of isopeptide segment
and application to racemization-free
segment condensation
EMERGING AREA
Tobias Seiser and Nicolai Cramer
Enantioselective metal-catalyzed
activation of strained rings

PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
O-Acyl isopeptide method: efficient synthesis of isopeptide segment and
application to racemization-free segment condensation†
Taku Yoshiya, Hiroyuki Kawashima, Youhei Sohma, Tooru Kimura and Yoshiaki Kiso*
Received 20th February 2009, Accepted 7th April 2009
First published as an Advance Article on the web 18th May 2009
DOI: 10.1039/b903624e
We report the establishment of the O-acyl isopeptide method-based racemization-free segment
condensation reaction toward future chemical protein synthesis. Peptide segments containing
C-terminal O-acyl Ser/Thr residues were successfully synthesized by use of a lower nucleophilic base
cocktail for Fmoc removal, and then coupled to an amino group of a peptide-resin without side
reactions or epimerization. We also succeeded in performing the segment condensation in a sequential
manner and in solution phase conditions as well.
resin. To synthesize longer peptides and proteins, many kinds
of convergent synthetic methods have been proposed.1–3 Among
Introduction
them, “segment condensation”,1,2 in which a side-chain protected
peptide carboxylate is coupled with an amino group of another
peptide segment to construct a longer peptide, has attracted
attention as an important method. However, a fundamental
drawback of the segment condensation is that epimerization
at the C-terminal residue of an N-segment occurs during the
condensation reaction with a C-segment (Fig. 1A), limiting the
N-segment to contain either a C-terminal Gly or Pro.2 This
epimerization occurs because, in contrast to urethane-protected
amino acids, peptides easily form chirally labile oxazolones upon
C-terminal carboxyl activation.
We developed an “O-acyl isopeptide method”4 for the synthesis
of peptides containing a difficult sequence. The presence of an
O-acyl instead of a native N-acyl residue at a hydroxyamino
acid residue (e.g. Ser, Thr) in the peptide backbone drastically
changed the secondary structure of the native peptide, and the
Peptides and proteins synthesized by chemical means have been
widely used in biological studies. Moreover, chemical synthesis
of peptides and proteins allows us to incorporate non-native
structures into molecules, thereby enlarging our understanding
of peptide and protein functions. Stepwise solid phase peptide
synthesis is commonly adopted to synthesize ~50 amino acids
long with satisfactory outcomes. However, synthesis of longer
peptides and proteins using the stepwise method is generally
difficult due to a lower reactivity of the constructed peptide-
Department of Medicinal Chemistry, Division of Medicinal Chemical
Sciences, Center for Frontier Research in Medicinal Science, 21st Century
COE program, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto,
607-8412, Japan. E-mail: kiso@mb.kyoto-phu.ac.jp; Fax: +81 75 591 9900;
Tel: +81 75 595 4635
† Electronic supplementary information (ESI) available: HPLC profiles for
isopeptide segments and some intermediates. See DOI: 10.1039/b903624e
Fig. 1 (A) Conventional segment condensation, (B) O-acyl isopeptide method-based segment condensation.
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target peptide was generated via an O-to-N intramolecular acyl
migration. An application of the method to Alzheimer’s disease-
related Ab1–42 revealed that the O-acyl isopeptide of Ab1–42
could be effectively synthesized and stored without spontaneous
self-assembly.5 The intact monomer of Ab1–42 could then be
obtained from the isopeptide under physiological experimental
conditions. The O-acyl isopeptide method has further evolved as
a general method for peptide synthesis with the development of
“O-acyl isodipeptide units” (Fig. 2).6 Isodipeptide units have
enabled routine use of the O-acyl isopeptide method by avoiding
the often difficult esterification reaction on resin. So far, Mutter
et al.,7 Bienert et al.,8 the Merck KGaA group,9 Bo¨rner et al.,10
Otaka et al.,11 Martinez et al.,12 Brik et al.13 and Muir et al.14
have also reported using the O-acyl isopeptide method. We also
expanded the concept to the “S-acyl isopeptide method”.15
observations would be useful information for chemical protein
synthesis based on the segment condensation method.
Results
Synthesis of influenza A virus matrix M1 58–66
Influenza A virus matrix M1 58–6619 (H–GILGFVFTL–OH, 1)
¯
was synthesized via segment condensation with O-acyl isopep-
tide segment 4 (Scheme 1). Segment 4 was constructed by
Fmoc-based SPPS on 2-chlorotrityl resin (Scheme 1A). O-Acyl
isodipeptide unit, Boc–Thr(Fmoc–Phe)–OH6b was loaded onto
the resin in the presence of diisopropylethylamine (DIPEA) in 1,2-
dichloroethane to give 2. The fully protected isopeptide segment
4 was obtained after couplings of Fmoc-amino acids and base
treatments, followed by cleavage from the resin by treating with
20% hexafluoroisopropanol (HFIP) in dichloromethane (DCM).
However, base treatments with 20% piperidine in DMF for Fmoc-
removal caused severe losses of peptides during SPPS, resulting
in low isolated yield (<3%) of 4. This is probably due to ester
decomposition by the base and/or diketopiperazine formation
when the Fmoc group of the amino acid next to the isodipep-
tide unit was removed. Therefore, we adopted 1-methylpyrrolidine
(25 v/v%)–hexamethyleneimine (2 v/v%)–HOBt (3 w/v%) in
NMP–DMSO (1 : 1) (Reagent A)20 as a lower nucleophilic base
developed by Aimoto et al. for an effective preparation of peptide-
thioesters using Fmoc-SPPS. HPLC analysis of crude 4, which
was prepared using Reagent A for Fmoc removal, showed that
any significant side product was not detected, suggesting that the
ester moiety was sufficiently stable under Reagent A treatment
(Fig. 3). HPLC purification gave pure 4 with moderate yield
(60%, Fig. S1A).
Fig. 2 O-Acyl isodipeptide unit.
Under these contexts, we have developed the “racemization-
free segment condensation”16 based on the O-acyl isopeptide
method (Fig. 1B). The idea was that an N-segment with a
C-terminal isopeptide structure (O-acyl Ser/Thr residue) can
be coupled to an N-terminal amino group of the C-segment
without any epimerization, because the amino group of C-terminal
isopeptide part is protected by a urethane-type protective group.
Activation of the carboxyl group of the isopeptide was thus
expected to suppress the formation of racemization-inducing
oxazolone. We have succeeded synthesizing model pentapeptide
Ac–Val–Val–Thr–Val–Val–NH2 to prove the concept.16 An N-
segment, Boc–Thr(Ac–Val–Val)–OH, was successfully coupled
to a H–Val–Val–NH-resin without epimerization. This result
suggested that, toward the synthesis of longer peptides or proteins,
effective segment condensation would be possible for C-terminal
Ser/Thr residues in addition to Gly/Pro. After the disclosure,
Bienert et al.17 and the Merck KGaA group18 also reported the
efficacy of this convergent methodology. Very recently, Martinez
et al. applied this method for the preparation of a cyclic
peptide.12
Toward future protein synthesis, we herein established the
O-acyl isopeptide method-based racemization-free segment con-
densation method by synthesizing three bioactive peptides (in-
fluenza A virus matrix M1 58–66, humanin and orexin-B). The
key segments containing C-terminal O-acyl Ser/Thr residues
were successfully synthesized by use of a lower nucleophilic
base cocktail for the removal of the Fmoc group. The middle-
sized peptide segments were then coupled to the amino group
of the peptide-resin quantitatively under optimized conditions
with no epimerization due to the urethane-protected O-acyl
isopeptide structure. In addition, we successfully performed the
segment condensation in a sequential manner, and also in solution
phase conditions. Finally, the synthesized isopeptides released
their native peptides rapidly (half time: <30 s ~ 10 min) and
quantitatively via O-to-N intramolecular acyl migration. These
Fig. 3 HPLC profile of crude 4 synthesized using Reagent A. Analytical
HPLC was performed using a C18 reverse phase column (4.6 ¥ 150 mm;
YMC Pack ODS AM302) with binary solvent system: a linear gradient of
CH3CN (0–100% CH3CN, 40 min) in 0.1% aqueous TFA with a flow rate
of 0.9 mL min-1 (40 ◦C), detected at 230 nm.
As shown in Scheme 1B, 4 was coupled to H-Leu-resin
(5) using the standard 1,3-diisopropylcarbodiimide (DIPCDI)–
1-hydroxybenzotriazole (HOBt) method in DMF to give 6. Crude
isopeptide 7 was successfully obtained with high purity (90%,
Fig. 4A) after TFA treatment of 6. In crude 7, D-allo-Thr derivative
was not detected (<2.0%), which was confirmed by a comparison
with an authentic sample using the analytical HPLC, indicating
that epimerization at the activated Thr residue did not occur
during segment condensation. In addition, des-Thr derivative
(H–GILGFVFL–OH) was not detected in crude 7, as also
confirmed by a comparison with an authentic sample. This
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Scheme 1 Reagents and conditions: a) Boc–Thr(Fmoc–Phe)–OH, diisopropylethylamine (DIPEA), 1,2-dichloroethane, 2.5 h; b) 20% piperidine in DMF,
20 min; c) 1-methylpyrrolidine (25 v/v%), hexamethyleneimine (2 v/v%), HOBt (3 w/v%), NMP–DMSO (1 : 1) (Reagent A),20 20 min; d) Fmoc–AA–OH
(2.5 eq) or Boc–Gly–OH (2.5 eq), 1,3-diisopropylcarbodiimide (DIPCDI, 2.5 eq), HOBt (2.5 eq), DMF, 2 h; e) 20% hexafluoroisopropanol (HFIP)
in dichloromethane (DCM), 45 min; f) 4 (2.5 eq), DIPCDI (2.5 eq), HOBt (2.5 eq), DMF, 2 h; g) TFA (92.5 v/v%), m-cresol (2.5 v/v%), thioanisole
(2.5 v/v%), water (2.5 v/v%), 90 min; h) pH 7.4 phosphate buffer, 6 h, room temperature.
Fig. 4 (A) HPLC profile of crude 7. (B, C) O-to-N Intramolecular acyl migration reaction released native peptide 1 from pure 7 (B: time = 30 s, C:
time = 30 min). HPLC conditions were similar to those described in Fig. 3.
result indicates that a possible side reaction during condensation
(Scheme S1), in which the HOBt ester of 4 reacts intramolecularly
to form a mixed anhydride of Boc–GILGFVF–OH and Boc–
bMeDAla–OH (eventually H–GILGFVFL–OH should be given),
did not occur. Such a side reaction was previously observed
during an HOBt activation step of O-acyl isodipeptide.6c Finally,
isopeptide 7 was purified (Fig. S1B), and converted to the desired
peptide 1 via an O-to-N intramolecular acyl migration reaction in
pH 7.4 buffered solution (half time: <30 s, Fig. 4B, C).6b After final
purification, peptide 1 was successfully obtained with a yield of
70% based on resin-loaded-Leu, by the racemization-free segment
condensation method (Fig. S1C).
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Scheme 2 Reagents and conditions: a) Reagent A, 20 min; b) Fmoc–AA–OH (2.5 eq), DIPCDI (2.5 eq), HOBt (2.5 eq), DMF, 2 h; c) 10 (2.5 eq), DIPCDI
(2.5 eq), HOAt (2.5 eq), NMP, 2 h; d) 9 (2.6 eq), HATU (2.5 eq), HOAt (2.6 eq), 2,4,6-collidine (2.5 eq), 1,8-bis(dimethylamino)naphthalene (2.5 eq),
DCM–NMP (1 : 4), 4 h; e) TFA (94%), 1,2-ethanedithiol (EDT, 2.5%), water (2.5%), triisopropylsilane (TIS, 1%), 90 min; f) pH 7.4 phosphate buffer,
37 ◦C, overnight.
Synthesis of humanin
by HPLC (Fig. 6A). In the HPLC chart, deprotected/cleaved 13
was obtained as a major product without deprotected/cleaved
12, indicating that the segment condensation reaction of 10
onto peptide-resin 12 was proceeded quantitatively. However,
the following condensation reaction of N1-segment 9 with 13
was not successfully achieved by the DIPCDI–based method,
as verified by an analysis of the deprotected/cleaved compound
after the coupling reaction, probably because of the low reac-
tivity of 13. Hence, we adopted HATU–HOAt–2,4,6-collidine–
1,8-bis(dimethylamino)naphthalene in DCM–NMP (1 : 4) for
the coupling. The resulting peptide-resin 14 was subsequently
treated with a TFA cocktail to give 15. As shown in Fig. 6B,
deprotected/cleaved 13 was not detected in crude 15, suggesting
that segment condensation of 9 under the conditions proceeded
quantitatively. Also, none of D-Ser7, des-Ser7, D-Ser14 or des-
Ser14 derivative was identified (<2.0%, verified by comparison
with authentic samples using the analytical HPLC) in crude
15, indicating that neither epimerization nor intramolecular side
reaction with formation of mixed anhydrides occurred during the
coupling reactions of 9 and 10, respectively. Pure 15 (Fig. S3C)
was then dissolved in pH 7.4 phosphate buffer to afford target
Next, we synthesized humanin (H–MAPRGFSCLLLLT-
¯
¯
SEIDLPVKRRA–OH, 8)21 to examine the advantage of the
method in the synthesis of a longer peptide. Humanin, a
neuroprotective factor in Alzheimer’s disease,21a,b is reported
as a “difficult sequence”-containing peptide because of highly
hydrophobic L9L10L11L12 sequence.21c In our hands, the total
isolated yield of 8 was only 6% using the conventional stepwise
Fmoc-SPPS because of the poor quality of crude 8 (Fig. S2).
Thus, 8 was synthesized via sequential segment condensation
using protected O-acyl isopeptide 9 and 10 (Scheme 2). Both
N1-segment 9 and N2-segment 10 could be efficiently prepared us-
ing Reagent A as an Fmoc deprotection reagent without byproduct
derived from the base-mediated ester decomposition (Fig. 5). Both
segments were purified by RP-HPLC (Fig. S3, 24% yield for 9 and
40% yield for 10). Using the DIPCDI–HOAt method in NMP, N2-
segment 10 was coupled to peptide-resin 12, which was synthesized
by standard Fmoc SPPS on a 2-chlorotrityl resin. To monitor for
the completeness of the reaction, peptide-resin 13 was in part
cleaved by a TFA cocktail, and the obtained peptide was analyzed
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Fig. 5 HPLC profiles of crude isopeptide segments (A) 9 and (B) 10 of humanin synthesis. HPLC conditions were similar to those described in Fig. 3.
Fig. 6 Humanin (8) was synthesized by the racemization-free segment condensation method on resin. (A) To monitor the completeness of the reaction,
peptide-resin 13 was in part cleaved by TFA cocktail, and the crude peptide was analyzed by HPLC; (B) crude 15; (C, D) pure 15 was dissolved in pH 7.4
phosphate buffer at 37 ◦C to afford 8 (C: time = 10 min, D: time = overnight); (E) 8 after purification. HPLC conditions were similar to those described
in Fig. 3.
peptide 8 via an O-to-N intramolecular acyl migration reaction
(half time: ca. 10 min at 37 C, Fig. 6C, D), followed by a final
Synthesis of orexin-B
◦
Solution phase segment condensation might be more suit-
able as compared to solid phase for the synthesis of longer
peptides or proteins, due to the use of less aggregative
(thus more reactive) peptides (without resin). In fact, seg-
ment condensation in the solution phase has successfully been
HPLC purification to give pure 8 (Fig. 6E) with a total yield of
41% based on the resin-bound-Ala residue. These results suggest
that middle-sized peptides could be efficiently synthesized by use
of the O-acyl isopeptide method-based segment condensation
reaction.
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applied to chemical protein synthesis.1i,22 Thus, we studied
the application of the O-acyl isopeptide method to solu-
tion phase segment condensation by synthesizing orexin-B (H–
RSGPPGLQGRLQRLQRLLQASGNHAAGILTM–NH2, 16), a
N2-segment 18 was coupled to peptide-amide 19, which was
prepared using Sieber amide resin with a yield of 37% (Fig. 8A),
using EDC·HCl–HOAt–2,4,6-collidine in DMF–CHCl3 (1 : 1)
to afford crude 20 (Fig. 8B). In crude 20, starting materials
18 and 19 were almost completely consumed, indicating that
segment coupling was quantitatively completed. In addition,
neither D-Ser nor des-Ser derivatives were detected (<2.0%),
which were confirmed by comparison with authentic samples
using the analytical HPLC. After deprotection of the Fmoc
group (Fig. 8C) and purification of 21 by RP-HPLC (Fig. S4C),
a second segment condensation reaction efficiently gave 22 by
the EDC·HCl–HOAt method in DMF–CHCl3 (1 : 1) (Fig. 8D).
Fully deprotected isopeptide 23 was then obtained through a final
deprotection with TFA (Fig. 8E), and isopeptide 23 was purified by
RP-HPLC (Fig. 8F). Pure 23 was treated with dimethyl sulfide
(DMS)–NH4I24c,d in TFA to reduce methionine sulfoxide24 to
methionine, affording isopeptide 24 (Fig. 8G). Finally, crude
24 was dissolved in pH 7.4 phosphate buffer to trigger O-to-
N intramolecular acyl migration reaction (half time: <30 s) to
afford orexin-B 16 (Fig. 8H, I). Purification by RP-HPLC gave
pure 16 with a reasonable total yield (44%, based on isopeptide
¯
¯
28-mer neuropeptide associated with sleeping and feeding.23 N1-
Segment 17 was synthesized with a yield of 34%. O-Acyl isopeptide
N2-segment 18 was efficiently prepared in a similar manner
to 4 with a yield of 42% (Fig. 7). As shown in Scheme 3,
Fig. 7 HPLC profile of crude isopeptide segment 18. HPLC conditions
were similar to those described in Fig. 3.
Scheme 3 Reagents and conditions: a) 18 (0.8 eq), EDC·HCl (1.2 eq), HOAt (2.4 eq), 2,4,6-collidine (1.0 eq), DMF-CHCl3 (1 : 1), 4 h; b) 20% piperidine
in DMF, 5 min; c) 17 (2.4 eq), EDC·HCl (4.2 eq), HOAt (3.6 eq), 2,4,6-collidine (3.0 eq), DMF–CHCl3 (1 : 1), 18 h; d) TFA (95%), TIS (2.5%), water
(2.5%), 90 min; e) dimethyl sulfide (DMS, 20 eq), NH4I (20 eq), TFA–water (20 : 1); f) pH 7.4 phosphate buffer, 3 h, room temperature.
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Fig. 8 Orexin-B (16) was synthesized by the segment condensation in solution phase. The condensation reaction was monitored by HPLC; (A) pure 19,
(B) 1st condensation (crude 20), (C) Fmoc deprotection by 20% piperidine in DMF (crude 21), (D) 2nd condensation (crude 22), (E, F) final deprotection
with TFA cocktail followed by purification using RP-HPLC (E: crude 23, F: pure 23), (G) following reduction of Met(O) gave crude isopeptide 24.
(H, I, J) via O-to-N intramolecular acyl migration (H, half time: <30 s), native peptide 16 was obtained (I: crude 16, J: pure 16). HPLC conditions are in
a same manner described in Fig. 3, except for (H) in which binary solvent system: a linear gradient of CH3CN (15–55% CH3CN, 40 min) in 0.1% aqueous
TFA was used. # = des-Trt-19, $ = deprotected 17.
segment 18, Fig. 8J). The successful synthesis of 16 indicates that
the convergent synthesis using an isopeptide segment in solution
phase could be advantageous to the synthesis of longer peptides
or proteins.
possessing a C-terminal Ser/Thr residue allows an effective
segment condensation without any epimerization, thanks to
the C-terminal O-acyl isopeptide structure with a urethane-
protected Ser/Thr residue. To confirm the applicability of this
racemization-free segment condensation to the synthesis of pro-
teins, we synthesized herein three bioactive peptides (influenza
A virus matrix M1 58–66, humanin and orexin-B) with op-
timization of reaction conditions. Conventional Fmoc SPPS
including piperidine treatment for Fmoc removal gave no de-
sired O-acyl isopeptide segment for the synthesis of influenza
Discussion
We have recently disclosed a novel segment condensation method
based on the O-acyl isopeptide method with synthesizing a
model pentapeptide. In this method, the use of an N-segment
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A virus matrix M1 58–66, however, we found that the use
of Reagent A (1-methylpyrrolidine–hexamethyleneimine–HOBt–
NMP–DMSO) instead of 20% piperidine for Fmoc removal
enabled an effective construction of the O-acyl isopeptide seg-
ments while avoiding a base-mediated ester cleavage. The segment
condensation reactions of middle-sized segments onto peptide-
resin were then completed under the conventional DIPCDI–
HOAt method and the stronger HATU–HOAt–2,4,6-collidine–
1,8-bis(dimethylamino)naphthalene method without side reac-
tions. Additionally, toward future chemical protein synthesis,
sequential segment coupling and solution phase condensation
were also successfully achieved. In each segment condensation
reaction, a side reaction such as epimerization and des-Ser/Thr
formation was not observed. Finally, the synthesized isopeptides
released their native peptides rapidly (half time: <30 s ~ 10 min)
and quantitatively via O-to-N intramolecular acyl migration
reaction in pH 7.4 phosphate buffer.
These results suggested that the racemization-free segment
condensation reaction of middle-sized peptide segments becomes
possible at not only the C-terminal Gly/Pro but also Ser/Thr
residues of the N-segment. Using these segment condensation
reactions in solution as well as on resin would enable a future
chemical protein synthesis. In addition, final deprotected peptides
and proteins were effectively purified by HPLC, because a simple
isomerization to an O-acyl isopeptide remarkably and temporarily
changed the physicochemical properties of the native peptide.
Especially, higher water solubility of O-acyl isopeptide would
enable easy purification of hydrophobic peptides. Finally, an
O-to-N intramolecular acyl migration reaction triggered the native
amide bond formation quantitatively under neutral conditions.
These observations would be useful information for future chemi-
cal protein synthesis based on the segment condensation method.
Institute, Inc. (Osaka, Japan), and were used without further
purification. Analytical HPLC was performed using a C18 reverse
phase column (4.6 ¥ 150 mm; YMC Pack ODS AM302) with
a binary solvent system: a linear gradient of CH3CN in 0.1%
aqueous TFA at a flow rate of 0.9 mL min-1 (temperature: 40 ◦C),
detected at 230 nm. Preparative HPLC was carried out on a C18
reverse phase column (20 ¥ 250 mm; YMC Pack ODS SH343–5)
with a binary solvent system: a linear gradient of CH3CN in 0.1%
aqueous TFA at a flow rate of 5.0 mL min-1 (temperature: 40 ◦C),
detected at 230 nm. Solvents used for HPLC were of HPLC grade.
MALDI-TOF-MS spectra were recorded on Voyager DE-STR
using a-cyano-4-hydroxy cinnamic acid as a matrix. FAB-MS was
performed on a JEOL JMS-SX102A spectrometer equipped with
the JMA-DA7000 data system.
Solid phase peptide synthesis
In general, the peptide chains were assembled on a 2-chloro-
trityl resin by sequential coupling of activated Na-Fmoc-
amino acid (2.5 eq) in DMF (1–2 mL) in the presence of
1,3-diisopropylcarbodiimide (DIPCDI, 2.5 eq) and 1-hydroxy-
benzotriazole (HOBt, 2.5 eq) with a reaction time of 2 h at room
temperature. The resins were then washed with DMF (1.5 mL, ¥ 5)
and the completeness of each coupling was verified by the Kaiser
test.25 Na-Fmoc deprotection was carried out by treatment with
Reagent A19 (2 mL, 1 min ¥ 1 and 20 min ¥ 1), followed by
washing with NMP (1.5 mL, ¥ 5), DMF (1.5 mL, ¥ 10) and CHCl3
(1.5 mL, ¥ 5). After complete elongation of the peptide chains, the
peptide resins were washed with CHCl3 (1.5 mL, ¥ 3) and MeOH
(1.5 mL, ¥ 3), and then dried for at least 2 h in vacuo.
(A) Cleavage of protected peptide segments. The protected
peptides were cleaved from the resin with 20% HFIP in DCM
for 45 min, concentrated in vacuo, dissolved in MeOH or DMSO,
filtered using a 0.45 mm filter unit, and immediately injected into
preparative HPLC with a 0.1% aqueous TFA–CH3CN system.
The peak fractions were collected and immediately lyophilized,
affording the desired isopeptide segment. For the synthesis of
protected peptide amide segment, Sieber amide resin was used,
and 1% TFA in DCM treatment gave a crude protected peptide
amide.
(B) Cleavage of native peptides. The peptides were cleaved from
the resin with TFA in the presence of thioanisole, m-cresol and
distilled water (92.5 : 2.5 : 2.5 : 2.5) for 90 min at room temperature,
concentrated in vacuo, and precipitated with diethyl ether at 0 ◦C
followed by centrifugation at 4,000 rpm for 5 min (¥ 3). In the
synthesis of humanin, a cocktail of TFA : 1,2-ethanedithiol (EDT)
: distilled water : triisopropylsilane (TIS) (94 : 2.5 : 2.5 : 1) was
used, instead of TFA–thioanisole–m-cresol–water. The resultant
peptides were dissolved in water and lyophilized for at least 12 h.
The crude products were purified by preparative reversed-phase
HPLC with 0.1% aqueous TFA–CH3CN system as an eluant,
immediately frozen at -78 ◦C, and lyophilized at least 12 h.
Boc–Thr65(Boc–Gly58–Ile–Leu–Gly–Phe–Val–Phe64)–OH (4).
The 2-chlorotrityl chloride resin (125 mg, 0.188 mmol) and
Boc–Thr(Fmoc–Phe)–OH6b (73.6 mg, 0.125 mmol) were taken to
the manual solid-phase reactor under an argon atmosphere and
stirred for 2.5 h in the presence of DIPEA (13.1 ml, 0.125 mmol) in
1,2-dichloroethane (1.5 ml). After washing with DMF (1.5 ml, ¥
5), capping was performed with MeOH (125 ml) in the presence of
Conclusion
We have described optimized conditions for the preparation of
isopeptide segments and the segment condensation reaction with
synthesizing three bioactive peptides. The use of Reagent A was
suitable for the construction of the isopeptide segments. Sequential
segment coupling reactions both on resin and in solution were
successfully achieved without side reactions. Additionally, the
synthesized isopeptides released their native peptides rapidly via
O-to-N intramolecular acyl migration reaction. Considering these
features, the O-acyl isopeptide method-based segment conden-
sation methodology would contribute to the field of chemical
synthesis of long peptides and proteins.
Experimental
General procedures
Fmoc-amino acid side-chain protections were selected as follows:
tBu (Asp, Glu, Ser, Thr), Boc (Lys), Pmc (Arg), Trt (Cys,
Asn, Gln, His). All protected amino acids and resins were
purchased from Calbiochem-Novabiochem Japan Ltd. (Tokyo,
Japan) and Watanabe Chemical Ind., Ltd. (Hiroshima, Japan).
Other chemicals were purchased from commercial suppliers, Wako
Pure Chemical Ind., Ltd. (Osaka, Japan), Nacalai Tesque (Kyoto,
Japan), Aldrich Chemical Co., Inc. (Milwaukee, WI) and Peptide
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 2894–2904 | 2901
©

DIPEA (32.7 ml, 0.188 mmol) in DMF for 10 min. After washing
with DMF (¥ 5), DMF–water (1 : 1, ¥ 5), CHCl3 (¥ 2) and
MeOH (¥ 2) followed by drying in vacuo, the loading ratio was
determined (0.071 mmol) photometrically from the amount of
Fmoc chromophore liberated upon treatment with 50% piperidine
in DMF for 30 min at 37 ◦C. The following Fmoc-protected
amino acids (0.18 mmol) and Boc–Gly–OH (0.18 mmol) were
manually coupled in the presence of DIPCDI (27.9 ml, 0.18 mmol)
and HOBt (27.6 mg, 0.18 mmol) in DMF (1.5 ml) for 2 h after
removal of each Fmoc group by Reagent A (1.5 mL) for 20 min
(peptide-resin: 179 mg). The protected peptide was then cleaved
from the resin (150 mg) with HFIP (1.0 ml)–DCM (4.0 ml) for
45 min, concentrated in vacuo, dissolved in MeOH (2.0 ml),
filtered using a 0.45 mm filter unit, and immediately injected into
preparative HPLC with a 0.1% aqueous TFA–CH3CN system.
The peak fractions were collected and immediately lyophilized,
affording the desired isopeptide segment 4 as a white amorphous
powder (37.5 mg, 35.5 mmol, 60%). HRMS (FAB): calcd for
C53H80N8O14Na (M + Na)+: 1075.5692, found: 1075.5699; HPLC
analysis at 230 nm: purity was higher than 95%.
in the presence of DIPCDI (10.5 mL, 0.068 mmol) and HOBt
(10.4 mg, 0.068 mmol) for 2 h in DMF (1.0 mL) after removing
each Fmoc group with Reagent A (1 mL) for 20 min. The
resulting protected peptide-resin (120.6 mg) was treated with
TFA (1.85 mL)–EDT (50.0 mL)–TIS (50.0 mL)–water (50.0 mL)
for 90 min, concentrated in vacuo, washed with diethyl ether,
centrifuged, dissolved in water, and lyophilized to give crude
peptide 8 (21.9 mg). Crude 8 (3.0 mg) was dissolved in DMSO,
filtered using a 0.45 mm filter unit, applied to preparative HPLC,
and eluted using 0.1% aqueous TFA-CH3CN. The peak fractions
were collected and immediately lyophilized to afford desired
peptide 8 as a white amorphous powder. Yield: 0.7 mg, 0.21 mmol,
6% based on the resin-bound-Ala; MALDI-MS (TOF): Mcalc
:
2687.2; M + Hfound: 2688.2; HPLC analysis at 230 nm: purity was
higher than 95%; the retention time on HPLC (0–100% CH3CN
for 40 min, 230 nm) of synthesized 8 was identical to that of
commercially available humanin (PEPTIDE INSTITUTE, INC.,
Osaka, Japan).
Boc–Ser7(Boc–Met1–Ala–Pro–Arg(Pmc)–Gly–Phe6)–OH (9).
9 was synthesized (0.128 mmol scale) in a similar manner to
that described for 4 using Boc–Ser(Fmoc–Phe)–OH6b (97.7 mg,
0.170 mmol). Yield: 38.4 mg, 0.031 mmol, 24%; HRMS (FAB):
calcd. for C57H86N10O16S2Na (M + Na)+: 1253.5562, found:
1253.5566; HPLC analysis at 230 nm: purity was higher than 95%.
65-O-Acyl-isoinfluenza A virus matrix M1 58–66 (7). Fmoc–
Leu–resin (2-chlorotrityl resin, 34.4 mg, 0.013 mmol) was prepared
in a similar manner as described above. After Fmoc deprotection,
the protected isopeptide segment (4, 34.8 mg, 0.033 mmol) was
condensed in the presence of DIPCDI (5.2 mL, 0.034 mmol) and
HOBt (5.1 mg, 0.033 mmol) in DMF (400 mL) for 2 h (peptide-
resin 6: 43.4 mg). Then, the peptide was cleaved from the resin
using TFA (0.8 mL) in the presence of thioanisole (21.7 mL),
m-cresol (21.7 mL) and distilled water (21.7 mL) for 90 min at
room temperature, concentrated in vacuo, washed with diethyl
ether, centrifuged, suspended in water, and lyophilized to give the
crude peptide 7 as a white amorphous powder (14.1 mg). Crude
7 (5.0 mg) was dissolved in DMSO (0.7 mL), filtered using a
0.45 mm filter unit, applied to preparative HPLC, and eluted using
0.1% aqueous TFA–CH3CN. The peak fractions were collected
and immediately lyophilized to afford desired peptide 7·TFA as a
white amorphous powder (4.6 mg, 3.9 mmol, 85%). MALDI-MS
(TOF): Mcalc: 966.2; M + Hfound: 967.4; HPLC analysis at 230 nm:
purity was higher than 95%.
Boc–Ser14(Fmoc–Cys8(Trt)–Leu–Leu–Leu–Leu–Thr13(tBu))–
OH (10). 10 was synthesized (0.121 mmol scale) in a similar
manner to that described for 4 using Boc–Ser(Fmoc–Thr(tBu))–
OH6b (99.4 mg, 0.170 mmol). Yield: 66.6 mg, 0.048 mmol, 40%;
HRMS (FAB): calcd. for C77H103N7O14SNa (M + Na)+: 1404.7181,
found: 1404.7170; HPLC analysis at 230 nm: purity was higher
than 95%.
7,14-Di-O-Acyl-isohumanin (15) synthesized by the segment con-
densation method. After H–humanin(15–24)–resin (12, 2-chloro-
trityl resin, 35.0 mg, 0.010 mmol) was constructed, N2-segment 10
(33.2 mg, 0.024 mmol) was coupled in the presence of DIPCDI
(3.7 mL, 0.024 mmol) and HOAt (3.3 mg, 0.024 mmol) in NMP
(0.5 mL) for 2 h. After removing the Fmoc group using Reagent
A (1.0 mL) for 20 min, N1-segment 9 (32.0 mg, 0.026 mmol)
was coupled in the presence of HATU (9.5 mg, 0.025 mmol),
HOAt (3.5 mg, 0.026 mmol), 2,4,6-collidine (3.3 mL, 0.025 mmol)
and 1,8-bis(dimethylamino)naphthalene (5.4 mg, 0.025 mmol)
(preactivated in DCM (0.2 mL) for 30 sec) in DCM–NMP (1 :
4, 1 mL) for 4 h. Resulting peptide-resin 14 (42.0 mg) was
treated with TFA (0.94 mL)–EDT (25 mL)–TIS (10 mL)–water
(25 mL) for 90 min, concentrated in vacuo, washed with diethyl
ether, centrifuged, dissolved in water, and lyophilized to give
crude isopeptide 15 (17.6 mg). MALDI-MS (TOF): Mcalc: 2687.2;
M + Hfound: 2688.2; HPLC analysis at 230 nm: purity was 83%
(on the other hand, Met1(O) derivative (8%) was detected). Crude
15 (4.0 mg) was dissolved in MeOH–DMSO (95 : 5, 500 mL),
filtered using a 0.45 mm filter unit, applied to preparative HPLC,
and eluted using 0.1% aqueous TFA–CH3CN. The peak fractions
were collected and immediately lyophilized to afford 15·TFA as
a white amorphous powder (3.6 mg, 1.0 mmol, 44% based on
Influenza A virus matrix M1 58–66 (1)6b. A solution of pure
isopeptide 7·TFA (2.0 mg, 1.7 mmol) in DMSO (40 mL) was added
to pH 7.4 phosphate buffer (100 mM, 1.96 mL), and the solution
was stirred for 6 h at room temperature. After lyophilization,
crude 1 was washed by water (1 mL, ¥ 2), dissolved in DMSO
(1.2 mL), filtered using a 0.45 mm filter unit, applied to preparative
HPLC, and eluted using 0.1% aqueous TFA–CH3CN. The peak
fractions were collected and immediately lyophilized to afford
desired peptide 1·TFA as a white amorphous powder (1.5 mg,
1.4 mmol, 82%). MALDI-MS (TOF): Mcalc: 966.2; M + Hfound
:
967.7; HPLC analysis at 230 nm: purity was higher than 95%; the
retention time on HPLC (0–100% CH3CN for 40 min, 230 nm) of
synthesized 1 was identical to that of an authentic sample that was
previously synthesized in Ref. 6b.
Humanin (8) synthesized by conventional stepwise manner.
Fmoc–Ala–resin (11, 2-chlorotrityl resin, 100 mg, 0.027 mmol)
was prepared in a similar manner as described above. Sequential
Fmoc-protected amino acids (0.068 mmol) were manually coupled
resin-bound-Ala). MALDI-MS (TOF): Mcalc: 2687.2; M + Hfound
:
2688.1; HPLC analysis at 230 nm: purity was higher than 95%.
2902 | Org. Biomol. Chem., 2009, 7, 2894–2904
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The Royal Society of Chemistry 2009
©

Humanin (8) synthesized by the segment condensation method.
Pure 15 (2.0 mg, 0.57 mmol) was dissolved in pH 7.4 phosphate
buffer (100 mM, 2 mL), stirred overnight at room temperature and
lyophilized to give crude 8. This crude peptide was dissolved in wa-
ter, filtered using 0.45 mm filter unit, and immediately injected into
preparative HPLC with a 0.1% aqueous TFA–CH3CN system.
The desired fractions were collected and immediately lyophilized
to afford desired peptide 8·TFA as a white amorphous powder
(1.8 mg, 0.53 mmol, 93%). MALDI-MS (TOF): Mcalc: 2687.2;
M + Hfound: 2688.2; HPLC analysis at 230 nm: purity was 95%; the
retention time on HPLC (0–100% CH3CN for 40 min, 230 nm)
of synthesized 8 was identical to that of commercially available
humanin (PEPTIDE INSTITUTE, INC., Osaka, Japan).
18-O-Acyl-Met28(O)-isoorexin-B(1–28) (23). To a solution of
pure 17 (1.9 mg, 1.2 mmol), EDC·HCl (0.4 mg, 2.1 mmol) and
HOAt (0.5 mg, 3.7 mmol) in CHCl3 (100 mL) was added a solution
of 21·TFA (4.0 mg, 1.0 mmol) and 2,4,6-collidine (0.2 mL, 1.5 mmol)
in DMF (50 mL). After the solution was stirred for 4 h at room
temperature, another potions of pure 17 (1.9 mg, 1.2 mmol),
EDC·HCl (0.4 mg, 2.1 mmol) and 2,4,6-collidine (0.2 mL, 1.5 mmol)
were added to the mixture, and stirred for more 14 h at room
temperature. The crude mixture was concentrated in vacuo, and
triturated in 1 mL of CH3CN–water (1 : 1) in an ice bath.
After centrifugation, the supernatant was removed. The resulting
peptide was suspended in CH3CN (2 mL) and lyophilized to
afford crude fully protected isopeptide 22 (5.7 mg) as a white
powder. Obtained 22 (5.7 mg) was treated with TFA (0.95 mL)–
TIS (25 mL)–water (25 mL) (92.5 : 2.5 : 2.5) for 90 min at
room temperature, concentrated in vacuo, washed with diethyl
ether, centrifuged, suspended with water, and lyophilized to give
crude 23·TFA (3.5 mg). Crude 23 (3.0 mg) was dissolved in
0.9 mL of MeOH–DMSO (3 : 1) and immediately purified by
preparative RP-HPLC with a 0.1% aqueous TFA–CH3CN system.
The desired fractions were collected and immediately lyophilized
to afford desired 23·TFA as a white amorphous powder. Yield:
2.0 mg, 0.56 mmol, 70%; MALDI-MS (TOF): Mcalc: 2915.3;
M + Hfound: 2916.4; HPLC analysis at 230 nm: purity was higher
than 95%.
Boc–Arg1(Pmc)–Ser(tBu)–Gly–Pro–Pro–Gly–Leu–Gln(Trt)–
Gly9–OH (17). 17 was synthesized (0.089 mmol scale) in a
similar manner to that described for peptide 4. Yield: 45.7 mg,
0.030 mmol, 34%; MALDI-MS (TOF): Mcalc: 1532.4; M + Nafound
1555.8; HPLC analysis at 230 nm: purity was higher than 95%.
:
Boc–Ser18(Fmoc–Arg10(Pmc)–Leu–Gln(Trt)–Arg(Pmc)–Leu–
Leu–Gln(Trt)–Ala17)–OH (18). 18 was synthesized (0.015 mmol
scale) in a similar manner to that described for 4 using Boc–
Ser(Fmoc–Phe)–OH6b (97.7 mg, 0.170 mmol). Yield: 15.0 mg,
6.2 mmol, 42%; HPLC analysis at 230 nm: purity was higher
than 95%. Fully protected isopeptide 18 could not be analyzed by
MALDI-MS (TOF) due to its low ionization property. Once the
N-terminal Fmoc group was removed, the obtained product could
Orexin-B (16). To a solution of pure 23 (2.0 mg, 0.56 mmol) in
TFA–water (2 mL–100 mL) was added NH4I (1.6 mg, 11 mmol) and
dimethyl sulfide (0.8 mL, 11 mmol), and the solution was allowed
to stand for 30 min at 0 ◦C. After concentration in vacuo, the
crude peptide was dissolved in 1 mL of water and washed by
1 mL of CCl4. Then, the aqueous layer was lyophilized to give
a crude isopeptide mixture. Finally, the obtained crude mixture
was dissolved in 1 mL of phosphate buffer (100 mM, pH 7.4) and
stirred for 3 h at room temperature. After lyophilization, the crude
peptide was dissolved in 1 mL DMSO–water (1 : 1), filtered using
a 0.46 mm filter unit, applied to preparative HPLC, and eluted
using a 0.1% aqueous TFA–CH3CN. The desired fractions were
collected and immediately lyophilized to afford desired peptide 16
as a white amorphous powder. Yield: 1.8 mg, 0.54 mmol, 96%;
MALDI-MS (TOF): Mcalc: 2899.3; M + Hfound: 2900.5; HPLC
analysis at 230 nm: purity was higher than 95%. The retention time
on HPLC (0–100% CH3CN for 40 min, 230 nm) of synthesized
16 was identical to that of authentic sample from commer-
cially available orexin-B (PEPTIDE INSTITUTE, INC., Osaka,
Japan).
be analyzed using MALDI-MS (TOF): Mcalc: 2201.7; M + Hfound
:
2203.0. Similarly, after TFA treatment, the obtained product could
be analyzed by MALDI-MS (TOF): Mcalc: 1306.5; M + Hfound
1307.5.
:
H–Gly19–Asn(Trt)–His(Trt)–Ala–Ala–Gly–Ile–Leu–Thr(tBu)–
Met28(O)–NH2 (19). 19 was synthesized using Sieber amide resin
(113 mg, 0.070 mmol) in a similar manner to that described for
peptide 4, cleaved with 1% TFA in DCM for 10 min (¥ 5). Crude 19
was concentrated in vacuo, and purified by RP-HPLC. The peak
fractions were collected and immediately lyophilized to afford
desired peptide 19·TFA as a white amorphous powder. Yield:
42.9 mg, 0.026 mmol, 37%; MALDI-MS (TOF): Mcalc: 1539.3;
M + Nafound: 1562.9; HPLC analysis at 230 nm: purity was higher
than 95%.
Partially protected 18-O-acyl-Met28(O)-isoorexin-B(10–28) (21).
A solution of 19·TFA (8.2 mg, 5.0 mmol) and 2,4,6-collidine
(0.7 mL, 5.2 mmol) in DMF (50 mL) was added to a solution of
18 (10.0 mg, 4.1 mmol), EDC·HCl (1.2 mg, 6.3 mmol) and HOAt
(1.7 mg, 12 mmol) in CHCl3 (150 mL). The mixture was stirred for
4 h at room temperature. After the solvent was removed in vacuo,
the residue was triturated in 1 mL of CH3CN–water (1 : 1) in an ice
bath. After centrifugation, the supernatant was removed to afford
crude 20 as a white precipitate. Then, the Fmoc group of crude
20 was removed using 20% piperidine in DMF for 5 min at room
temperature. The solvent was removed in vacuo and the resulting
peptide was dissolved in DMSO and purified by RP-HPLC with
a 0.1% aqueous TFA–CH3CN system. The peak fractions were
collected and immediately lyophilized to afford desired protected
isopeptide 21·TFA as a white amorphous powder. Yield: 10.3 mg,
Acknowledgements
This research was supported in part by the “Academic Frontier”
Project for Private Universities: matching fund subsidy from
MEXT (Ministry of Education, Culture, Sports, Science and
Technology) of the Japanese Government, and the 21st Century
COE Program from MEXT. T. Y. and Y. S. are grateful for
Research Fellowships of JSPS for Young Scientists. We thank
Ms. A. Fujimoto for technical assistance. We are grateful to
Ms. K. Oda, Mr. T. Hamada and Mr. H-O. Kumada for mass
spectra measurements. We thank Dr. J-T. Nguyen for his English
correction.
2.7 mmol, 66%; MALDI-MS (TOF): Mcalc: 3723.6; M + Nafound
:
3746.0; HPLC analysis at 230 nm: purity was higher than 95%.
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 2894–2904 | 2903
©

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Schmid, G. Tuchscherer, M. Mutter and H. A. Lashuel, ChemBioChem,
2008, 9, 2104–2112.
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