The phenacyl group as an efficient thiol protecting group in a peptide condensation reaction by the thioester method

Article abstract of DOI:10.1039/c3ob40644j

One of the condensation methods for the preparation of long-chain peptides, the so-called thioester method requires protecting groups for amino and thiol groups for regioselective ligation. In this study, we demonstrated that the phenacyl (Pac) group acts as an efficient protecting group of cysteine side chains. We synthesized a cysteine derivative carrying the Pac group at the side chain sulfur atom, and Pac-containing peptides and peptide thioesters were synthesized using it by the ordinary 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis strategy. Pac-containing peptide segments could be condensed by the thioester method. After the condensation reaction, Pac groups could be removed by Zn/AcOH treatment. In addition, the azido group, which was used for the protection of lysine side chains, was simultaneously converted into an amino group, demonstrating that this protecting group scheme simplified the deprotecting reaction after the peptide condensation reaction to a single step. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob40644j

Organic &
Biomolecular Chemistry
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PAPER
The phenacyl group as an efficient thiol protecting
group in a peptide condensation reaction by the
thioester method†
Cite this: Org. Biomol. Chem., 2013, 11,
4405
Hidekazu Katayama*a and Hironobu Hojo*a,b
One of the condensation methods for the preparation of long-chain peptides, the so-called thioester
method requires protecting groups for amino and thiol groups for regioselective ligation. In this study, we
demonstrated that the phenacyl (Pac) group acts as an efficient protecting group of cysteine side chains.
We synthesized a cysteine derivative carrying the Pac group at the side chain sulfur atom, and Pac-contain-
ing peptides and peptide thioesters were synthesized using it by the ordinary 9-fluorenylmethoxycarbonyl
(Fmoc)-based solid-phase peptide synthesis strategy. Pac-containing peptide segments could be condensed
by the thioester method. After the condensation reaction, Pac groups could be removed by Zn/AcOH treat-
ment. In addition, the azido group, which was used for the protection of lysine side chains, was simul-
taneously converted into an amino group, demonstrating that this protecting group scheme simplified the
deprotecting reaction after the peptide condensation reaction to a single step.
Received 2nd April 2013,
Accepted 9th May 2013
DOI: 10.1039/c3ob40644j
www.rsc.org/obc
functionality of the N-terminal segment is activated by silver
ions, and allowed to condense with a free N-terminal amino
Introduction
Peptide condensation methods enable protein chemical syn- group of the other segment in the presence of 3,4-dihydro-3-
thesis with long peptide chains. Especially, the native chemical hydroxy-4-oxo-1,2,3-benzotriazine (HOObt). Recently, we found
ligation (NCL) method developed by Dawson et al. in 19941 is that an aryl thioester could be directly activated by HOObt,
one of the most general methods for protein synthesis. Among and the condensation reaction proceeded without silver ions.5
the advantages of this method, no protecting group is required
In the thioester condensation method, protecting groups at
for forming a native peptide bond regioselectively; instead, the Lys and Cys side chains are necessary to achieve regioselective
cysteine residue should be positioned at the ligation site. condensation reaction. Until now, tert-butoxycarbonyl (Boc)
However, the synthesis of proteins often requires condensation and acetamidomethyl (Acm) groups have been generally used
at a non-Cys site. For overcoming this limitation, various for protecting Lys and Cys residues, respectively. However,
approaches, such as a thiol group directly attached at the beta- since the Boc group is labile in trifluoroacetic acid (TFA) solu-
carbon atom of an N-terminal amino acid/de-sulfurization tion which is usually used to cleave peptides from the solid
strategy2 and ligation auxiliary groups,3 have been developed. support after 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-
However, specific amino acid derivatives are generally used in phase peptide synthesis (SPPS), the Boc group should be re-
these methods, and complicated synthetic procedures are introduced into peptide segments after purification steps. In
required for preparing those compounds. The thioester order to overcome this inconvenience, an alternative amino
method, which is an alternative peptide condensation method protecting group that is stable under the various conditions
developed by Hojo and Aimoto in 1991,4 has an advantage that used in Fmoc-SPPS was required. Along this line, we have
no specific residue is required at the condensation position. In demonstrated that the azido group acted efficiently as an
the classical thioester method, C-terminal alkyl thioester amino protecting group in the Fmoc-SPPS and peptide con-
densation reactions.6 Using this azido-based strategy, we have
achieved the total chemical synthesis of a glycoprotein.7
aDepartment of Applied Biochemistry, School of Engineering, Tokai University,
4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan. E-mail: katay@tokai-u.jp,
hojo@keyaki.cc.u-tokai.ac.jp; Tel: +81-463-50-2075
The azido group can be easily converted to an amino group
by reduction using Zn powder in an acetic acid solution. On
the other hand, the Acm group attached to Cys side chains is
stable under reducing reaction conditions. Indeed, an
additional deprotection step for cleaving the Acm group after
the reduction is usually required in order to obtain
bInstitute of Glycoscience, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa
259-1292, Japan
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c3ob40644j
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unprotected polypeptides. In our previous study, we aimed Acm and Pac groups were used for the protection of Cys side
to simplify the deprotection to a single step, and a novel chains at positions 2, 12 and 13, respectively. Starting from
protecting group, the N-methyl-phenacyloxycarbamidomethyl Fmoc-Arg(Pbf)-Wang resin, the peptide chain was elongated
(Pocam) group, has been developed.8 This protecting group manually by the ordinary Fmoc-SPPS strategy using N,N′-
can be removed by Zn reduction in an acetic acid solution dicyclohexylcarbodiimide (DCC)–1-hydroxybenzotriazole (HOBt)
simultaneously with the conversion of an azido group into an as condensation reagents. After chain assembly, the crude
amino group. Using this protecting group, we have achieved peptide was cleaved from solid supports by treatment with a
the synthesis of several peptides by the thioester method. The TFA cocktail, and purified by reversed-phase (RP)-HPLC, giving
Pocam group is, however, not completely stable under TFA peptide 3. However, during peptide chain assembly, the intro-
acidic conditions, and careful manipulation was required to duction efficiency of the amino acid residue at the N-terminal
prepare Pocam-containing peptides.
side of Cys(Pac) was low, and the isolated yield of 3 was low
It is widely known that the phenacyl (Pac) group is an (9.4%). It may be partly due to the cyclization at Cys(Pac) by
efficient protecting group for carboxylic acid, and can be imine formation between the α-amino group and the carbonyl
removed by reducing reaction with Zn powder under acidic group of Pac. The imine structure is generally formed under
conditions.9 Recently, we found that the Pac group attached weakly acidic conditions, and the condensation conditions are
directly to a sulfur atom is also cleavable by Zn treatment in an weakly acidic due to the acidity of HOBt. In order to keep the
acetic acid solution. In this paper, we demonstrate that the Pac weakly basic conditions in the condensation reaction, 2-(1H-
group acts as an efficient thiol protecting group in the Fmoc- benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluoro-
SPPS and peptide condensation reactions by the thioester phosphate (HBTU)–N,N-diisopropylethylamine (DIEA) was
method. Using this Pac-based strategy, we synthesized several used as a condensation reagent instead of DCC–HOBt. As
model peptides as will be described below.
expected, the reaction efficiency was improved, and the desired
peptide 3 was obtained by RP-HPLC purification in higher
yield (19%) (Fig. 1).
The stability of the Pac group under the various deprotec-
tion conditions for Cys-protecting groups was examined. When
peptide 3 was treated with Zn powder in an acetic acid
aqueous solution, the Pac group was cleaved without the
removal of the Acm or MeOBn group, and peptide 4 was
obtained as a single peak in RP-HPLC (Fig. 2). On the other
hand, when the MeOBn group of peptide 3 was removed by
treatment with a 1 M trifluoromethanesulfonic acid (TfOH)–
TFA solution, MeOBn-deprotected peptide 5 was obtained as
the major product, although cleavage of the Pac group in part
was observed as a minor peak in RP-HPLC analysis. When the
Acm group was removed by treatment with silver nitrate, the
desired Acm-deprotected product 7 was mainly obtained,
although Pac-deprotected peptide 8 was also obtained as a
minor product. These results indicated that the reducing
Results and discussion
Orthogonality of the Pac group with other known Cys
protecting groups
A cysteine derivative carrying the Pac group at the side chain
was synthesized at first. Starting from (Fmoc-Cys-OBut)2 (1),
N-Fmoc-S-Pac-cysteine [Fmoc-Cys(Pac)-OH] (2) was synthesized
as shown in Scheme 1. The disulfide bond of 1 was reduced
by treatment with Zn powder in an acetic acid solution,
and then the Pac group was attached using phenacyl bromide
in dichloromethane (DCM). Subsequently, the tert-butyl
group was removed using TFA, giving the desired product 2 in
80% yield.
In order to investigate the orthogonality of the Pac group
with other Cys protecting groups, a model peptide having
three Cys residues with various protecting groups generally
used for regioselective disulfide formation in peptide
chemical synthesis was synthesized by Fmoc-SPPS. The
peptide YCRVNGARYVRCCSRR was used as a model sequence.
In the Fmoc-SPPS of the peptide p-methoxybenzyl (MeOBn),
Scheme 1 Synthesis of Fmoc-Cys(Pac)-OH. Reaction conditions: (a) Zn powder
in 50% AcOH–dichloromethane, RT, 2.5 h; (b) Pac-Br (1.1 eq.), DIEA (2 eq.) in
dichloromethane, RT, 1 h; (c) 2% H2O–TFA, RT, 1 h (80% yield in 3 steps).
Fig. 1 RP-HPLC elution profiles of crude model peptide 3. Column: Inertsil
ODS-3 (4.6 × 150 mm), eluent: 0.1% TFA in aqueous acetonitrile at a flow rate
of 1 mL min−1
.
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Synthesis of the growth-blocking peptide
In order to demonstrate the utility of the Pac group in the
peptide condensation reaction by the thioester method, insect
growth-blocking peptide (GBP) 9 from the armyworm, Pseuda-
letia separata, was used as a model sequence. GBP consists of
25 amino acid residues containing two Cys residues that form
an intrachain disulfide bond.10 The peptide chain was divided
into two segments, (1–10) 10 and (11–25) 11, and these were
synthesized by the Fmoc-SPPS strategy and condensed by the
thioester method as shown in Scheme 2.
The N-terminal segment 10 should have a thioester func-
tionality at the C-terminus. To obtain the peptide thioester, we
used the N-alkylcysteine (NAC)-assisted thioesterification
method.11 Fmoc-(Et)Cys(Trt)-OH was introduced into H-Arg-
(Pbf)-Arg(Pbf)-NH-resin by the DCC–HOBt method. After
removal of the Fmoc group by piperidine treatment, Fmoc-Gly-
OH was condensed using O-(7-azabenzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU) as a conden-
sation reagent, and then the peptide chain was elongated
manually by the DCC–HOBt method except for the amino acid
residue next to Cys(Pac) which was introduced by the HBTU–
DIEA method. After the peptide chain assembly, the crude
peptide was cleaved from the resin by TFA cocktail treatment,
and dissolved in a 50% aqueous acetonitrile solution contain-
ing 5% AcOH. 4-Mercaptophenylacetic acid (MPAA) was added
to the solution at a concentration of 5%. The thioesterification
reaction was almost complete within 20 h, giving the desired
peptide thioester 10 in 5.3% yield. During thioesterification
reaction using an excess amount of the thiol compound under
weakly acidic conditions, no significant side reaction, such as
thioketal formation at the carbonyl group of the Pac moiety, was
observed. These results indicated that the Pac group was fully
compatible with a NAC-assisted thioesterification reaction.
The C-terminal segment 11 was also synthesized by the
ordinary Fmoc-SPPS strategy. Starting from Fmoc-Gln(Trt)-
CLEAR acid resin, the peptide chain was elongated manually
Fig. 2 (A) Deprotection conditions and structures of the obtained peptides. (a)
Zn powder in 50% AcOH–H2O, RT, 1 h; (b) TfOH–TFA–m-cresole–thioanisole
(3/14/1/2), 0 °C, 20 min; AgNO3 in 5% H2O–0.1% DIEA–DMSO, RT, 2 h. (B)
RP-HPLC elution profiles of model peptide 3 and crude samples after deprotec-
tion. (a) Purified peptide 3; (b) the crude sample after Zn powder treatment
under acidic conditions; (c) the crude sample after treatment with 1 M TfOH in
TFA; (d) the crude sample after treatment with AgNO3 in an aqueous DMSO
solution. Numbers in the chromatograms indicate compounds shown in panel
A. Column: Inertsil ODS-3 (4.6 × 150 mm), eluent: 0.1% TFA in aqueous aceto-
nitrile at a flow rate of 1 mL min−1
.
reaction using Zn/AcOH cleaved only the Pac group and did
not affect the other Cys-protecting groups; on the other hand,
the Pac group was slightly labile under strongly acidic con-
ditions and silver ion treatment.
Scheme 2 Synthesis of GBP. Reaction conditions: (a) 1% HOObt–1% DIEA–
DMSO, RT, 4 h; (b) 10% piperidine, RT, 30 min; (c) Zn powder in 50% AcOH–
H2O, RT, 1 h (84% in 3 steps); (d) 10% DMSO–6 M guanidine–HCl–50 mM phos-
phate buffer (pH 7.0), RT, o/n (50%).
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by the DCC–HOBt method or the HBTU–DIEA method, and
the protected peptide resin corresponding to the GBP (11–25)
sequence was obtained. A crude peptide was cleaved from the
solid support by TFA cocktail treatment, and purified by
RP-HPLC, giving the desired peptide segment 11 in 14% yield.
The two segments, 10 and 11, were condensed by the silver
ion-free thioester method. These were dissolved in anhydrous
dimethyl sulfoxide (DMSO) containing 1% DIEA and 1%
HOObt, and the mixture was kept at room temperature. The
reaction proceeded without significant side reaction, and was
complete within 4 h. In the RP-HPLC analysis, a new peak
corresponding to the desired product 12 appeared after the
reaction (Fig. 3). The N-terminal Fmoc group of 12 was cleaved
by adding piperidine at a concentration of 10%, giving peptide
13. After precipitation of the crude peptide by addition of
ether, Pac groups were removed by reduction using Zn powder
in an AcOH aqueous solution. An azido group was simul-
taneously converted into an amino group by Zn reduction, and
the desired linear GBP 14 was obtained in 84% yield. During
deprotection steps, no significant side reaction was observed.
These results clearly indicated that the Pac group acted
efficiently as a protecting group of Cys side chains in the
peptide condensation reaction by the thioester method. The
disulfide bond was formed by oxidation in a phosphate buffer
Fig. 4 RP-HPLC elution profiles in oxidation reaction of GBP. (a) 0 h. (b) 20 h.
Column: Inertsil ODS-3 (4.6 × 150 mm), eluent: 0.1% TFA in aqueous acetonitrile
at a flow rate of 1 mL min−1
.
containing 10% DMSO, and GBP 9 was successfully obtained
in 50% yield (Fig. 4).
Synthesis of tachyplesin
In order to investigate the applicability of the Pac group for
regioselective disulfide formation in Cys-rich peptide syn-
thesis, we tried to synthesize tachyplesin 15, an antimicrobial
peptide originally isolated from the hemocytes of the horse-
shoe crab Tachypleus tridentatus, as a model (Scheme 3). Tachy-
plesin consists of 17 amino acid residues including four Cys
that form two intrachain disulfide bonds and an amidated
Fig. 3 RP-HPLC elution profiles in peptide condensation and deprotection of
GBP. (a) Coupling reaction mixture of 10 and 11 (0 h). (b) At 4 hours after the
coupling reaction. (c) Reaction mixture after piperidine treatment. (d) Reaction
mixture after Zn/AcOH treatment. Column: Inertsil ODS-3 (4.6 × 150 mm),
eluent: 0.1% TFA in aqueous acetonitrile at a flow rate of 1 mL min−1. *Non-
peptidic components; **11-derived peptide.
Scheme 3 Synthesis of tachyplesin. Reaction conditions: (a) 1% HOObt–1%
DIEA–DMSO, RT, 4 h; (b) 10% piperidine, RT, 30 min; (c) Zn powder in 50%
AcOH–H2O, RT, 1 h (30% in 3 steps); (d) 10% DMSO–6 M guanidine–HCl–
50 mM phosphate buffer (pH 7.0), RT, o/n (59%); (e) I2–HCl–CH3OH–H2O, RT,
60 min (70%).
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Fig. 6 RP-HPLC elution profiles in oxidation reaction of GBP. (a) 0 h. (b) 20 h.
(c) I2 oxidation. Column: Inertsil ODS-3 (4.6 × 150 mm), eluent: 0.1% TFA in
aqueous acetonitrile at a flow rate of 1 mL min−1
.
Fig. 5 RP-HPLC elution profiles in peptide condensation and deprotection of
tachyplesin. (a) Coupling reaction mixture of 10 and 11 (0 h). (b) After overnight
reaction. (c) Reaction mixture after piperidine treatment. (d) Reaction mixture
after Zn/AcOH treatment. Column: Inertsil ODS-3 (4.6 × 150 mm), eluent: 0.1%
stable under various conditions used for the ordinary Fmoc-
SPPS, and was cleavable with Zn reduction. Using this protect-
ing group, we successfully synthesized GBP and tachyplesin by
the thioester method. The azido group was simultaneously
converted into an amino group by Zn-reduction, demonstrat-
ing that this Pac/azido-strategy simplifies the deprotection
steps after the peptide condensation reaction to a single step.
It is likely that this Pac-based strategy is applicable for synthe-
sizing larger proteins with or without modifications. Along
this line, we are currently trying to synthesize glycoproteins by
this strategy.
TFA in aqueous acetonitrile at a flow rate of 1 mL min−1
. *Non-peptidic
components.
C-terminus.12 The tachyplesin sequence was divided into two
segments, (1–10) 16 and (11–17) 17, and these were separately
synthesized by the ordinary Fmoc-SPPS and NAC-assisted
thioesterification reactions.
Segments 16 and 17 were condensed by the thioester
method (Fig. 5). These segments had Acm groups, and the
Ag+-free thioester method rather than the classical Ag+-cata-
lyzed condensation method facilitated efficient ligation
without undesirable cleavage of the Acm groups. After the con-
densation reaction and the removal of the N-terminal Fmoc
group, the Pac and azido groups were subsequently cleaved by
treatment with Zn powder in an aqueous AcOH solution, giving
the linear tachyplesin 20 in 30% yield. The first disulfide bond
between Cys7 and Cys12 was formed by oxidation in a phosphate
buffer containing 10% DMSO, giving the desired product 21 in
59% yield (Fig. 6). Finally, the second disulfide bond between
Cys3 and Cys16 was formed by iodine oxidation, to give the final
product 15 in 70% yield. All these results clearly indicated that
the Pac group could be used for regioselective disulfide for-
mation in Cys-rich peptide synthesis.
Experimental
General
Fmoc-(Et)Cys(Trt)-OH11 and Fmoc-Lys(N3)-OH6 were prepared
by the previously described methods. Specific rotation values
were determined with a Jasco P-2200 polarimeter (Jasco,
Tokyo, Japan) at 20 2 °C for solutions in CHCl3. The NMR
spectra were recorded by a Jeol AL-400 spectrometer (400 MHz
in 1H-NMR, JEOL, Tokyo, Japan) using CDCl3 as a solvent.
MALDI-TOF mass spectra were recorded using a Voyager-DE
PRO spectrometer (Applied Biosystems, CA). The amino acid
composition was determined using a LaChrom amino acid
analyzer (Hitachi, Tokyo, Japan) after hydrolysis with a 6 M
HCl solution at 150 °C for 2 h in a vacuum-sealed tube.
Conclusion
N-(9-Fluorenylmethoxycarbonyl)-S-phenacyl-cysteine 2
We demonstrated that the Pac group could be used as a Cys- (Fmoc-Cys-OBut)2 (1, 400 mg, 0.50 mmol) was dissolved in
protecting group in peptide chemistry. The Pac group was 50% AcOH–dichloromethane (DCM) (4 mL), and Zn powder
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(0.30 g) was added to the solution. After stirring at room temp- found: m/z 2270.7, calcd: 2271.1 for (M + H)+. Amino acid ana-
erature for 2.5 h, insoluble material was removed by filtration, lysis: Asp1Ser1.22Gly1.08Ala0.96Cys0.19Val1.75Tyr1.76Arg4.77
and the solvent was evaporated. The residue was dissolved in For cleavage of the Pac group, peptide 3 (20 nmol) was dis-
.
EtOAc, washed with a saturated NaHCO3 aqueous solution and solved in a 50% AcOH aqueous solution (100 μL), and an
brine, and dried over Na2SO4. After filtration and concen- excess amount of Zn powder was added. After mixing with vor-
tration, the crude material containing Fmoc-Cys-OBut was texing at room temperature for 1 h, the reaction mixture was
used in the following steps without further purification. The analyzed by RP-HPLC on an Inertsil ODS-3 column with a
obtained sample was dissolved in DCM (2 mL), and phenacyl linear gradient of acetonitrile containing 0.1% TFA.
bromide (220 mg, 1.1 mmol) and DIEA (0.35 mL, 2.0 mmol)
were added. After stirring at room temperature for 1 h, the dissolved in
For cleavage of the MeOBn group, peptide 3 (20 nmol) was
TfOH solution (TfOH–TFA–m-cresole–thio-
a
solvent was removed under reduced pressure. The residue was anisole, 3/14/1/2, 20 μL), and the mixture was kept at 0 °C for
dissolved in EtOAc, washed with 1 M HCl, H2O and brine, and 20 min. The crude peptide was precipitated with diethyl ether,
dried over Na2SO4. After filtration and concentration, the washed twice with ether, and dried under vacuum. The result-
crude material containing Fmoc-Cys(Pac)-OBut was used in the ing material was analyzed by RP-HPLC.
next step without further purification. The obtained material
For cleavage of the Acm group, peptide 3 (20 nmol) was dis-
was dissolved in 2% H2O–TFA (10 mL), and stirred at room solved in a 95% DMSO–0.1% DIEA aqueous solution (20 μL)
temperature for 1 h. After the solvent was removed under containing AgNO3 (0.20 μmol), and the mixture was kept at
reduced pressure, the residue was chromatographed on silica room temperature for 2 h. Then, the reaction mixture was ana-
gel with toluene–EtOAc–AcOH (50/50/1) to give Fmoc-Cys(Pac)- lyzed by RP-HPLC.
OH 2 (370 mg, 0.80 mmol, 80% in three steps). Rf 0.19
(toluene–EtOAc–AcOH, 50/50/1). [α]D +14.1 (c, 1.0). H-NMR: δ
1
Fmoc-[Cys(Pac)7]-GBP (1–10)-SC6H4CH2COOH 10
7.93–7.22 (m, 13H, Ar), 6.05 (d, 1H, J = 8.0 Hz, NH), 4.66 (m, Fmoc-Rink amide MBHA resin (0.34 mmol g−1, 147 mg,
1H, CαH), 4.36 (m, 2H, >CH–CH2–O–), 4.20 (t, 1H, J = 7.0 Hz, 50 μmol) was swelled in NMP for 30 min, and was treated with
>CH–CH2–O–), 3.92 [s, 2H, C(vO)CH2], 3.12–3.01 (m, 2H, 20% piperidine–NMP for 5 and 15 min. After washing with
CβH). 13C-NMR: δ 195.0, 174.5, 156.1 (CvO), 143.7, 143.6, NMP, Fmoc-Arg(Pbf)-OBt, which was prepared by mixing
141.2, 134.9, 133.7, 128.7, 127.7, 127.0, 125.1, 119.9 (Ar), 67.4 Fmoc-Arg(Pbf)-OH (0.20 mmol), 1 M DCC–NMP (300 μL) and
(>CH–CH2–O–), 53.9 (Cα), 46.9 (>CH–CH2–O–), 38.0 (C(vO) 1 M HOBt–NMP (300 μL) at room temperature for 30 min, was
CH2), 34.2 (Cβ). MALDI-TOF MS, found: m/z 484.2, calcd: 484.1 added and the reaction mixture was mixed with vortexing at
for (M + Na)+.
50 °C for 1 h. The resin was washed with NMP and 50% DCM–
CH3OH, treated with 10% Ac2O–5% DIEA–NMP for 5 min, and
washed with NMP. Another Arg residue was introduced into
Synthesis of model peptide 3 and deprotection
Fmoc-Arg(Pbf)-Wang resin (0.28 mmol g−1, 179 mg, 50 μmol) the resin in the same manner, and the resin was washed with
was swelled in 1-methyl-2-pyrrolidinone (NMP) for 30 min, and NMP. After the Fmoc group was removed by 20% piperidine–
was treated with 20% piperidine–NMP for 5 and 15 min. After NMP treatment at room temperature for 5 and 15 min, the
washing with NMP, Fmoc-Arg(Pbf)-OBt, which was prepared by resin was washed with NMP. Fmoc-(Et)Cys(Trt)-OBt, which was
mixing Fmoc-Arg(Pbf)-OH (0.20 mmol),
1 M DCC–NMP prepared by mixing Fmoc-(Et)Cys(Trt)-OH (0.1 mmol), 1 M
(300 μL) and 1 M HOBt–NMP (300 μL) at room temperature for DCC–NMP (150 μL) and 1 M HOBt–NMP (150 μL) at room
30 min, was added and the reaction mixture was mixed with temperature for 30 min, was added to the resin, and the
vortex at 50 °C for 1 h. The resin was washed with NMP and mixture was vortexed at 50 °C for 1 h. The resin was washed
50% DCM–CH3OH, treated with 10% Ac2O–5% DIEA–NMP for with NMP and 50% DCM–CH3OH, treated with 10% Ac2O–5%
5 min, and washed with NMP. The peptide chain was DIEA–NMP for 5 min, and washed with NMP. After the Fmoc
elongated in essentially the same manner as described above, group was removed by 20% piperidine–NMP treatment at
except for Fmoc-Cys(Acm)-OH which was activated by HBTU room temperature for 5 and 15 min, the resin was washed with
(0.20 mmol) and DIEA (0.3 mmol) in NMP. After the chain NMP. Fmoc-Gly-OH (0.50 mmol) and HATU (0.50 mmol) were
assembly,
H-Tyr(But)-Cys(MeOBn)-Arg(Pbf)-Val-Asn(Trt)-Gly- dissolved in NMP (1 mL) containing DIEA (170 μL, 1.0 mmol),
Ala-Arg(Pbf)-Tyr(But)-Val-Arg(Pbf)-Cys(Acm)-Cys(Pac)-Ser(But)- and the mixture was added to the resin. The reaction mixture
Arg(Pbf)-Arg(Pbf)-OCH2-resin (304 mg) was obtained. A part of was mixed with vortex at 50 °C for 1 h. After washing with
the resin (10 mg) was treated with the TFA cocktail (TFA–thio- NMP, the peptide chain was elongated by the ordinary Fmoc-
anisole–H2O–phenol–triisopropylsilane, 82.5/5/5/5/2.5, 200 μL) based SPPS. The amino acids (0.20 mmol each) except for Gly
at room temperature for 2 h. TFA was removed under a nitro- just after Cys(Pac) were activated by mixing with 1 M DCC–
gen stream and the peptide was precipitated with diethyl NMP (0.30 mL) and 1 M HOBt–NMP (0.30 mL) at room temp-
ether. After washing twice with ether, the precipitant was dried erature for 30 min, and the coupling reaction was carried out
under vacuum. The crude peptide was separated by RP-HPLC at 50 °C for 1 h. At the glycine position just after Cys(Pac),
on an Inertsil ODS-3 column (GL Science, Tokyo, Japan) with a Fmoc-Gly-OH (0.2 mmol) was activated by mixing HBTU
linear gradient of acetonitrile containing 0.1% TFA to give (0.2 mmol) and DIEA (0.3 mmol) in NMP (0.5 mL), and the
model peptide 3 (310 nmol, 19% yield). MALDI-TOF mass, coupling reaction was carried out at 50 °C for 1 h. After
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elongation, Fmoc-Glu(OBut)-Asn(Trt)-Phe-Ser(But)-Gly-Gly-Cys- acetonitrile containing 0.1% TFA. The isolated yield of 14 was
(Pac)-Val-Ala-Gly-(Et)Cys(Trt)-Arg(Pbf )-Arg(Pbf )-NH-resin 84% (79 nmol). MALDI-TOF mass, found: m/z 2784.0, calcd:
(247 mg) was obtained. A part of the resin (10 mg) was treated 2784.2 for (M + H)+. Amino acid analysis: Asp2.01Thr1.87Ser0.89
-
with a TFA cocktail (200 μL) at room temperature for 2 h. TFA Glu1.98Pro2.60Gly3.77Ala1Val0.95Met0.74Tyr2.38Phe2.07Lys0.93Arg1.94
.
was removed under a nitrogen stream and the peptide was pre-
cipitated with diethyl ether. After washing twice with ether, the GBP 9
precipitant was dried under vacuum. The crude peptide was
Peptide 14 (40 nmol) was dissolved in 0.6 mL of 10% DMSO–
100 mM phosphate buffer (pH 7.0), and the solution was kept
at room temperature for 24 h. The crude peptide was separated
by RP-HPLC using an Inertsil ODS-3 column with a linear gra-
dient of acetonitrile containing 0.1% TFA to give peptide 9
(20 nmol, 50%). MALDI-TOF mass, found: m/z 2781.7, calcd:
dissolved in 50% CH3CN–5% AcOH–H2O (0.50 mL), and MPAA
(25 mg) was added to the solution. After overnight reaction at
room temperature, the crude peptide was separated by
RP-HPLC on an Inertsil ODS-3 column with a linear gradient
of acetonitrile containing 0.1% TFA to give peptide thioester
10 (110 nmol, 5.3% yield). MALDI-TOF mass, found: m/z
1452.6, calcd: 1452.5 for (M + Na)+. Amino acid analysis:
2782.2 for (M + H)+. Amino acid analysis: Asp1.91Thr1.89Ser1.03
-
Glu2.15Pro1.74Gly4.02Ala1Val0.92Met0.54Tyr1.97Phe1.95Lys0.94Arg1.97
.
Asp0.90Ser0.74Glu0.96Gly2.87Ala1Val0.93Phe0.93
.
Fmoc-[Lys(N3)1, Cys(Acm)3, Cys(Pac)7]-tachyplesin
(1–10)-SC6H4CH2COOH 16
[Cys(Pac)19, Lys(N3)20]-GBP (11–25) 11
Fmoc-Gln(Trt)-CLEAR acid resin (0.41 mmol g−1, 122 mg,
50 μmoL) was swelled in NMP for 30 min, and treated with
20% piperidine–NMP for 5 and 15 min. After washing with
NMP, the peptide chain was elongated by Fmoc-SPPS. The
amino acids (0.20 mmol each) except for Arg just after Cys(Pac)
were activated by mixing with 1 M DCC–NMP (300 μL) and 1 M
HOBt–NMP (300 μL) at room temperature for 30 min, and the
coupling reaction was carried out at 50 °C for 1 h. At the argi-
nine position next to Cys(Pac) residue, Fmoc-Arg(Pbf)-OH
(0.2 mmol) was activated by mixing HBTU (0.2 mmol) and
DIEA (0.3 mmol) in NMP (0.5 mL), and the coupling reaction
was carried out at 50 °C for 1 h. After elongation, H-Tyr(But)-
Met-Arg(Pbf)-Thr(But)-Pro-Asp(OBut)-Gly-Arg(Pbf)-Cys(Pac)-Lys-
(N3)-Pro-Thr(But)-Phe-Tyr(But)-Gln(Trt)-OCH2-resin (201 mg)
was obtained. A part of the resin (10 mg) was treated with the
TFA cocktail (200 μL) at room temperature for 2 h. TFA was
removed under a nitrogen stream and the peptide was precipi-
tated with diethyl ether. After washing twice with ether, the
precipitant was dried under vacuum. The crude peptide was
separated by RP-HPLC on an Inertsil ODS-3 column with a
linear gradient of acetonitrile containing 0.1% TFA to give
peptide 11 (360 nmol, 14% yield). MALDI-TOF mass, found:
m/z 2006.9, calcd: 2006.9 for (M + H)+. Amino acid analysis:
Fmoc-Gly-(Et)Cys(Trt)-Arg(Pbf)-Arg(Pbf)-Rink amide MBHA
resin (50 μmol equivalent) was obtained in the same manner
as described above. After washing with NMP, the peptide chain
was elongated by the ordinary Fmoc-based SPPS. The amino
acids (0.20 mmol each) except for Val just after Cys(Pac) were
activated by mixing with 1 M DCC–NMP (300 μL) and 1 M
HOBt–NMP (300 μL) at room temperature for 30 min, and the
coupling reaction was carried out at 50 °C for 1 h. At the valine
position next to Cys(Pac) residue, Fmoc-Val-OH (0.2 mmol)
was activated by mixing HBTU (0.2 mmol) and DIEA
(0.3 mmol) in NMP (0.5 mL), and the coupling reaction was
carried out at 50 °C for 1 h. After elongation, Fmoc-Lys(N3)-
Trp(Boc)-Cys(Acm)-Phe-Arg(Pbf)-Val-Cys(Pac)-Tyr(But)-Arg(Pbf)-
Gly-(Et)Cys(Trt)-Arg(Pbf)-Arg(Pbf)-NH-resin (280 mg) was
obtained. A part of the resin (10 mg) was treated with a TFA
cocktail (200 μL) at room temperature for 2 h. TFA was
removed under a nitrogen stream and the peptide was precipi-
tated with diethyl ether. After washing twice with ether, the
precipitant was dried under vacuum. The crude peptide was
dissolved in 50% CH3CN–5% AcOH–H2O (0.4 mL), and MPAA
(20 mg) was added to the solution. After the overnight reaction
at room temperature, the crude peptide was separated by
RP-HPLC on an Inertsil ODS-3 column with a linear gradient
of acetonitrile containing 0.1% TFA to give peptide thioester
16 (112 nmol, 6.2% yield). MALDI-TOF mass, found: m/z
1904.8, calcd: 1904.8 for (M + Na)+. Amino acid analysis:
Gly1.08Val0.92Tyr1.01Lys0.34Arg2.
Asp0.91Thr1.88Glu1.08Pro2.10Gly1Met0.72Tyr1.74Phe0.97Lys0.13Arg1.79
.
Linear GBP 14
Peptides 10 (94 nmol) and 11 (140 nmol) were mixed and dis-
solved in DMSO (50 μL) containing 1% HOObt–1% DIEA, and
incubated at room temperature for 4 h to give peptide 12.
Piperidine (5 μL) was then added to this solution and the reac-
[Cys(Pac)12, Cys(Acm)16]-tachyplesin (11–17)-NH2 17
tion mixture was kept at room temperature for 30 min, yielding Fmoc-Rink amide MBHA resin (0.34 mmol g−1, 147 mg,
peptide 13. The crude peptide was precipitated by addition of 50 μmoL) was swelled in NMP for 30 min, and treated with
20 times volume of diethyl ether, washed twice with ether and 20% piperidine–NMP for 5 and 15 min. After washing with
dried under vacuum. The precipitant was dissolved in 50% NMP, the peptide chain was elongated by Fmoc-SPPS. The
aqueous AcOH (1 mL), and an excess amount of Zn powder amino acids (0.20 mmol each) except for Ile just after Cys(Pac)
was added to the solution. The mixture was mixed with vortex- were activated by mixing with 1 M DCC–NMP (300 μL) and 1 M
ing at room temperature for 1 h. After filtration to remove Zn HOBt–NMP (300 μL) at room temperature for 30 min, and the
powder, the desired product 14 was purified by RP-HPLC coupling reaction was carried out at 50 °C for 1 h. At the iso-
using an Inertsil ODS-3 column with a linear gradient of leucine position next to Cys(Pac) residue, Fmoc-Ile-OH
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(0.2 mmol) was activated by mixing HBTU (0.2 mmol) and calcd: 2263.1 for (M + H)+. Amino acid analysis: Gly1.02Cys0.86
-
DIEA (0.3 mmol) in NMP (0.5 mL), and the coupling reaction Val0.95Ile0.84Tyr2.07Phe1.07Lys1.09Arg5.
was carried out at 50 °C for 1 h. After elongation, H-Ile-Cys-
(Pac)-Tyr(But)-Arg(Pbf)-Arg(Pbf)-Cys(Acm)-Arg(Pbf)-NH-resin
(240 mg) was obtained. A part of the resin (10 mg) was treated
with the TFA cocktail (200 μL) at room temperature for 2 h.
TFA was removed under a nitrogen stream and the peptide was
Notes and references
precipitated with diethyl ether. After washing twice with ether,
the precipitant was dried under vacuum. The crude peptide
was separated by RP-HPLC on an Inertsil ODS-3 column with a
linear gradient of acetonitrile containing 0.1% TFA to give
peptide 17 (390 nmol, 19% yield). MALDI-TOF mass, found:
m/z 1157.5, calcd: 1157.6 for (M + H)+. Amino acid analysis:
Cys0.51Ile0.57Tyr0.99Arg3.
1 P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. H. Kent,
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2 (a) L. Z. Yan and P. E. Dawson, J. Am. Chem. Soc., 2001, 123,
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( j) A. Saporito, D. Marasco, A. Chambery, P. Botti,
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687; (l) Y.-Y. Yang, S. Ficht, A. Brik and C.-H. Wong, J. Am.
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(o) R. Okamoto and Y. Kajihara, Angew. Chem., 2008, 120,
5482, (Angew. Chem., Int. Ed., 2008, 47, 5402); (p) H. Hojo,
C. Ozawa, H. Katayama, A. Ueki, Y. Nakahara and
Y. Nakahara, Angew. Chem., Int. Ed., 2010, 49, 5318.
[Cys(Acm)3,16, Cys(SH)7,12]-tachyplesin 20
Peptides 16 (110 nmol) and 17 (160 nmol) were mixed and dis-
solved in DMSO (55 μL) containing 1% HOObt–1% DIEA, and
incubated at room temperature overnight to give peptide 18.
Piperidine (6 μL) was then added to this solution and the reac-
tion mixture was kept at room temperature for 30 min, yielding
peptide 19. The crude peptide was precipitated by addition of
20 times volume of diethyl ether, washed twice with ether and
dried under vacuum. The precipitant was dissolved in 50%
aqueous AcOH (1 mL), and an excess amount of Zn powder
was added to the solution. The mixture was mixed with vortex
at room temperature for 1 h. After filtration to remove
Zn powder, the desired product 20 was purified by RP-HPLC
using an Inertsil ODS-3 column with a linear gradient of
acetonitrile containing 0.1% TFA. The isolated yield of 20
was 30% (32 nmol). MALDI-TOF mass, found: m/z 2409.0,
calcd: 2409.2 for (M
+
H)+. Amino acid analysis:
Gly1.09Cys0.71Val1.00Ile0.87Tyr1.95Phe1.06Lys1.00Arg5.
[Cys(Acm)3,16]-tachyplesin 21
Peptide 20 (32 nmol) was dissolved in 2 mL of 10% DMSO–
100 mM phosphate buffer (pH 7.0), and the solution was kept
at room temperature for 24 h. The crude peptide was separated
by RP-HPLC using an Inertsil ODS-3 column with a linear
gradient of acetonitrile containing 0.1% TFA to give peptide
21 (19 nmol, 59%). MALDI-TOF mass, found: m/z 2407.1,
calcd: 2407.2 for (M
+
H)+. Amino acid analysis:
Gly1.10Cys0.69Val0.93Ile0.88Tyr2.00Phe1.03Lys0.94Arg5.
Tachyplesin 15
Peptide 21 (18 nmol) was dissolved in distilled water (150 μL),
and the solution was added dropwise to CH3OH (0.6 mL)
containing 20 mM I2–CH3OH (20 μL) and 1 M HCl (36 μL)
within 5 min with mixing. After the resulting solution
was stirred for another 55 min at room temperature, the
reaction was quenched by adding an ascorbic acid aqueous
solution. The reaction mixture was separated by RP-HPLC
on an Inertsil ODS-3 column with a linear gradient of
acetonitrile containing 0.1% TFA to give the desired product
15 (13 nmol, 70% yield). MALDI-TOF mass, found: m/z 2262.8,
4 (a) H. Hojo and S. Aimoto, Bull. Chem. Soc. Jpn., 1991, 64,
111; (b) S. Aimoto, Biopolymers, 1999, 51, 247.
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Paper
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This journal is © The Royal Society of Chemistry 2013
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