N-Methyl-phenacyloxycarbamidomethyl (Pocam) group: A novel thiol protecting group for solid-phase peptide synthesis and peptide condensation reactions

Article abstract of DOI:10.1039/c1ob05253e

In the so-called thioester method for the condensation of peptide segments, protecting groups for amino and thiol groups are required for chemoselective ligation. In this study, we developed a novel thiol protecting group, N-methyl-phenacyloxycarbamidomet

Full text of DOI:10.1039/c1ob05253e

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PAPER
N-Methyl-phenacyloxycarbamidomethyl (Pocam) group: a novel thiol
protecting group for solid-phase peptide synthesis and peptide condensation
reactions†
Hidekazu Katayama,* Yoshiaki Nakahara and Hironobu Hojo*
Received 17th February 2011, Accepted 4th April 2011
DOI: 10.1039/c1ob05253e
In the so-called thioester method for the condensation of peptide segments, protecting groups for
amino and thiol groups are required for chemoselective ligation. In this study, we developed a novel
thiol protecting group, N-methyl-phenacyloxycarbamidomethyl (Pocam). We used it for protection of
cysteine side chains, and synthesized Pocam-containing peptides and peptide thioesters. These were
condensed by the thioester method. After the condensation reaction, Pocam groups were cleaved by
Zn/AcOH treatment. At the same time, the azido group, which was used for the protection of lysine
side chains, was also converted to an amino group, demonstrating that this protecting group strategy
simplified the deprotecting reaction after the peptide condensation reaction to only one step.
for example, Zn/AcOH and silver ion treatments for azido and
Introduction
Acm groups, respectively. Since the conditions of the deprotecting
The solid-phase peptide synthesis (SPPS) method is generally
reactions, such as solvent and pH, were quite different from each
limited to a length of approximately less than 50 residues long.
other, purification step(s) would have been necessary between the
To synthesize long peptide chains, methods for the condensation
deprotection steps, and the yield of the final product reduced. If
of peptide segments, such as native chemical ligation,1 traceless
there is a thiol protecting group which is cleaved simultaneously
Staudinger ligation2 and thioester method,3 have been developed.
with amino protecting group, deprotection steps would be simpli-
Although a cysteine residue is usually required at the ligation
fied to only one step and the yield might be higher.
point in the native chemical ligation method, there is no limitation
In our azido-based strategy, an azido group was converted to an
amino group by reduction using Zn/AcOH.4,5 Since this reaction
can reduce disulfide bonds, disulfide-type protecting groups such
as tert-butylthio can be used for such purpose. Several preparation
methods for peptide thioester compatible with Fmoc-SPPS have
been developed.7,8 In these methods however, an excess amount of
thiol compound is usually used, and disulfide bond cannot remain
intact under such conditions. Therefore, a novel thiol protecting
group that is cleaved by Zn/AcOH but stable under reducing
conditions by thiol compounds is required. In this study, we tried
to develop a novel thiol protecting group with such characteristics.
at the ligation point in the thioester method. In return, protecting
groups for amino and thiol groups are required for the chemos-
elective condensation. The tert-butoxycarbonyl (Boc) group was
traditionally used for the amino protecting group.3 Recently, we
demonstrated that the azido group acted as an effective amino
protecting group for SPPS and the peptide condensation reaction
by the thioster method.4 In our previous study, we achieved the
total chemical synthesis of a large glycoprotein using this azido-
based strategy.5 Until now, the acetamidomethyl (Acm) group
was generally used for the protection of a thiol group in peptide
condensation reactions. This protecting group is stable under
acidic and basic conditions used for 9-fluorenylmethoxycarbonyl
(Fmoc)-SPPS method, and specifically removed by silver ion
treatment under weakly basic conditions.6
Results and discussion
Design of the thiol protecting group
After the peptide condensation reaction by the thioester
method, the protecting groups on the amino and thiol groups
must be removed. Until now, two deprotection steps were needed;
We considered that the protected aminomethyl group might act
as a thiol protecting group similarly to the Acm group. The
removal of the amino protecting group generates a thiohemiaminal
structure, and the aminomethyl part is spontaneously cleaved
due to its lability. Since we used an azido group as an amino
protecting group for Lys side chain, it was likely that the azido
group was also valid for the protection of aminomethyl portion.
However, the azidomethyl group on sulfur atom is labile even
under trifluoroacetic acid (TFA) acidic conditions (data not
Department of Applied Biochemistry, Faculty of Engineering, Tokai Uni-
versity, Hiratsuka, Hiratsuka, Kanagawa 259-1292, Japan. E-mail: katay@
tokai-u.jp, hojo@keyaki.cc.u-tokai.ac.jp; Fax: +81 463 50 2075; Tel: +81
463 50 2075
† Electronic supplementary information (ESI) available: RP-HPLC chro-
matograms and 1H-NMR spectra of compounds 1, 5 and 7, and
Supplementary figures. See DOI: 10.1039/c1ob05253e
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shown). Therefore, it is difficult to use it as a thiol protecting group,
and another amino protecting group that could be removed by Zn
reduction was desired.
It is widely known that the phenacyl group is an efficient
protecting group for carboxylic acid and can be removed by the
reducing reaction with Zn powder under acidic conditions.9 It
was likely that the phenacyloxycarbonyl (Poc) group might be a
good candidate for our purpose. To confirm that Poc group acts
efficiently as an amino protecting group, we synthesized Fmoc-
Lys(Poc)-OH and checked its characteristics.
Scheme 2 Proposed mechanism of cyclization at the Poc group.
Synthesis of Na-Fmoc-S-(N-methyl-
phenacyloxycarbamidomethyl)cysteine
Na -Fmoc-S-(N-methyl-phenacyloxycarbamidomethyl)cysteine
[Fmoc-Cys(Pocam)-OH, 5] was synthesized by the procedure
shown in Scheme 3. The carbonyl group of 2 was protected
by ethylene glycol to form ethylene acetal 6 in 55% yield.
Compound 6 was treated with disuccinimidyl carbonate followed
by methylamine, to give compound 7 in 68% yield. The amido
nitrogen of 7 was then hydroxymethylated by HCHO treatment
generating hydroxymethylated compound 8, and then cysteine was
reacted in neat TFA. At this stage, the ethylene acetal structure
was partly decomposed to a carbonyl group, and a mixture of
compound 9 and 10 was obtained. Since these could not be
separated by silica gel chromatography, we used them as a mixture
to the next reaction. The Fmoc group was introduced to the amino
group by Fmoc-OSu treatment, and the product was purified
by silica gel chromatography. The acetal structure was almost
completely lost, and only Fmoc-Cys(Pocam)-OH 5 was obtained.
The yield was 72% over the three steps.
Stability of the phenacyloxycarbonyl group on a Lys side chain
The synthetic route to Fmoc-Lys(Poc)-OH (1) is shown in Scheme
1. 2-Hydroxyacetophenone (2) was treated with disuccinimidyl
carbonate in DMF for 2 h, and then Fmoc-Lys-OH was added to
this solution, giving the desired product 1 in 30% yield.
Scheme 1 Synthesis of Fmoc-Lys(Poc)-OH.
This lysine derivative 1 was introduced to a model peptide, Ser-
Phe-Lys-Tyr-Glu, and the stability of the Poc group was tested.
Starting from Fmoc-Glu(OBut)-Wang resin, the peptide chain
was elongated by the N,N¢-dicyclohexylcarbodiimide (DCC)–1-
hydroxybenzotriazole (HOBt) method. After the peptide chain
assembly, the peptide was cleaved off from the resin by a TFA cock-
tail treatment. On the reversed-phase (RP)-HPLC chromatogram,
two peaks were observed: one (peptide 3) is minor, and the other
(peptide 4) is major with a longer retention time than 3 (See
supplementary Figure†). MALDI-TOF mass analysis revealed
that peptide 3 showed a protonated molecular ion peak at m/z
835.7, which coincided well with the calculated value of the desired
Poc-containing peptide. On the other hand, peptide 4 showed
a protonated ion peak at m/z 817.6, which was smaller than
that of 3 by 18 Da. In the 1H-NMR spectra of 3, a singlet
peak at a chemical shift of 5.33 ppm was observed, which arose
from the methylene group in the phenacyl moiety. On the other
hand, this signal disappeared in the spectrum of 4. These results
suggest that the cyclized product was generated under the acidic
conditions by the proposed mechanism shown in Scheme 2. It
has been demonstrated that a similar reaction was observed in
phenacyloxycarbamide by heating without solvent.10 Although
various TFA cocktails were tested, this undesirable side reaction
could not be suppressed, suggesting that the Poc group is not
suitable for protecting the amino group of primary amines.
Scheme 3 Synthesis of Fmoc-Cys(Pocam)-OH.
Preparation of Pocam-containing peptide
Using this Cys derivative 5, we tried to synthesize growth-blocking
peptide (GBP) by the thioester method. GBP was originally
isolated from the larval hemolymph of the host armyworm,
Pseudaletia separata, whose development is halted in the last
larval instar stage from parasitization by the parasitoid wasp,
Cotesia hariyai.11 To firstly check the characteristics of the Pocam
group, we introduced 5 into the peptide segment GBP(11–25)
(11), by ordinary Fmoc-SPPS. Starting from Fmoc-Gln(Trt)-
CLEAR Acid resin, the peptide chain was elongated manually
by DCC–HOBt strategy. At Lys20 and Cys19 sites, Fmoc-Lys(N3)-
OH and Fmoc-Cys(Pocam)-OH were used as building blocks,
and the protected peptide resin was successfully obtained. When
On the other hand, if the reaction mechanism shown in Scheme
2 is correct, the Poc group should be still valid for protecting
secondary amines, because the nitrogen atom at the intermediate
lacks nucleophilicity and the reaction could not proceed. There-
fore, it was likely that N-methyl-phenacyloxycarbamidomethyl
group might be a good candidate for the thiol protecting group.
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crude peptide was cleaved off from the resin by Reagent K12
treatment at room temperature for 2 h, the Pocam group on
the Cys residue was partly removed (ca. 70%, based on the
peak area on the RP-HPLC chromatogram). To suppress the
removal of the Pocam group, various conditions were tested.
When the TFA cocktail was used at 4 ◦C for 4 h, the Pocam
group almost remained and the other protecting groups, such
as But, Trt, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
(Pbf) and 2,4-dimethoxybenzyl (Dmb) groups, were clearly re-
moved by the TFA treatment (See Supplementary Figure†). The
desired peptide 11 was obtained in 8.9% yield.
To develop the deprotecting conditions for the Pocam group,
peptide 11 was treated with various conditions (Fig. 1). As
expected, the Pocam group was removed by Zn powder treatment
in aqueous AcOH solution, and the azido group of Lys side chain
was also converted to amino group at the same time, giving a
completely deprotected peptide 12. Interestingly, the Pocam group
was easily removed by TFA treatment at 50 ◦C for 1 h without
decomposition of the azido group, generating partly deprotected
azido-peptide 13. In addition, Tris(2-carboxyethyl)phosphine
(TCEP) treatment at a neutral pH reduced only the azido group
without decomposition of the Pocam group, giving peptide 14.
These results suggest that this protecting group might be useful for
site-specific modification of proteins synthesized by the thioester
method.
Synthesis of growth-blocking peptide
GBP was synthesized by the method shown in Scheme 4.
The N-terminal segment of GBP, GBP(1-10) thioester, was
synthesized by Fmoc-SPPS and N-alkylcysteine (NAC)-assisted
thioesterification reaction.8 Fmoc-(Et)Cys(Trt)-OH was intro-
duced to H-Arg(Pbf)-Arg(Pbf)-NH-resin by the DCC–HOBt
method. After Fmoc removal 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. After the peptide chain assembly,
the crude peptide was cleaved from the resin by the TFA cocktail
treatment at 4 ◦C for 4 h, and dissolved in 50% aqueous acetonitrile
solution containing 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 15 in 2.0% yield (Scheme 4).
Scheme 4 Synthetic route to peptide 15.
The peptide segments, 11 and 15, were condensed by the Ag+-
free thioester method (Scheme 5).13 The segments were mixed
and dissolved in dimethyl sulfoxide (DMSO) containing 2% 3-
hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOObt) and 2%
N,N-diisopropylethylamine (DIEA), and the reaction mixture
was held at room temperature. The N-terminal segment 15 was
exhausted within 4 h, and an additional peak corresponding to the
desired product 16 appeared in RP-HPLC chromatogram (Fig. 2).
After the condensation reaction, Fmoc group at the N-terminus
was removed by adding piperidine to give peptide 17. During
condensation and Fmoc-removal steps, no decomposition of the
Fig. 1 (A) RP-HPLC elution profiles of GBP(11–25). (a) Purified peptide
11. Observed: m/z 2093.6; calcd: 2093.9 for (M+H)+. (b) After Zn powder
treatment in 50% aqueous AcOH for 1 h. Observed: m/z 1862.6; calcd:
1862.9 for (M+H)+. (c) After TFA cocktail (TFA/H2O/TIS, 93/5/2)
treatment at 50 ◦C for 1 h. Observed: m/z 1888.4; calcd: 1888.8 for
(M+H)+. (d) After reduction by TCEP in 50 mM phosphate buffer (pH
7.0) for 20 h. Observed: m/z 2067.4; calcd: 2067.9 for (M+H)+. Column:
Mightysil RP-18 GP (4.6 ¥ 150 mm), eluent: 0.1% TFA in aqueous
acetonitrile at a flow rate of 1 ml/min. (B) Structure of peptides 11, 12, 13
and 14.
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Scheme 5 Synthetic route to GBP. (a) 2% HOObt/2% DIEA/DMSO, rt,
4 h. (b) 5% piperidine, rt, 30 min. (c) Zn powder in 50% aqueous AcOH
solution, rt, 1 h. (d) 10% DMSO/50 mM phosphate buffer (pH 7.0), rt,
20 h.
Fig. 3 RP-HPLC elution profiles of oxidation reaction of GBP. (a) 0 h.
(b) 20 h. Column: Mightysil RP-18 GP (4.6 ¥ 150 mm), eluent: 0.1% TFA
in aqueous acetonitrile at a flow rate of 1 ml/min.
Scheme 6 Synthetic route for a-conotoxin SI. (a) 2% HOObt/2%
DIEA/DMSO, rt, 4 h. (b) 5% piperidine, rt, 30 min. (c) Zn powder in
50% aqueous AcOH solution, rt, 1 h. (d) 10% DMSO/50 mM phosphate
buffer (pH 7.0), rt, 20 h. (e) I2 in CH3OH/H2O containing HCl, rt, 15 min.
Fig. 2 RP-HPLC elution profiles of peptide condensation and deprotec-
tion. (a) Coupling reaction mixture of 11 and 15 (0 h). (b) Four hours after
the coupling reaction. (c) Reaction mixture after piperidine treatment. (d)
Reaction mixture after Zn/AcOH treatment. Column: Mightysil RP-18
GP (4.6 ¥ 150 mm), eluent: 0.1% TFA in aqueous acetonitrile at a flow
rate of 1 ml/min. Asterisks indicate non-peptidic components.
(Scheme 6). a-Conotoxin SI is a small peptide neurotoxin isolated
from the venom of fish-hunting cone snail, Conus striatus, and
consists of 13 amino acid residues with four Cys forming two
disulfide bonds.14 This sequence was separated into two segments,
1–8 and 9–13, and these segments were prepared by ordinary
Fmoc-SPPS.
Starting from Fmoc-Rink Amide MBHA resin, the peptide
chain was elongated manually by the DCC–HOBt method, giving
the desired protected peptide resin corresponding to the 9–13
sequence. At Cys13, Fmoc-Cys(Pocam)-OH was used as a building
block. After the TFA cocktail treatment at 4 ◦C for 4 h, the
crude peptide was separated by RP-HPLC, giving the C-terminal
segment 20 in 11% yield.
Pocam groups was observed. The Pocam groups were subsequently
cleaved by Zn powder treatment in an aqueous AcOH solution. At
the same time, the azido group at the Lys side chain was converted
into an amino group without significant side reaction, and the
linear GBP 18 was successfully obtained in 31% yield. Finally,
the disulfide bond was formed by oxidation in a phosphate buffer
containing 10% DMSO, giving the desired product 19 in 52% yield
(Fig. 3).
The N-terminal segment 21 was also synthesized by Fmoc-SPPS
and NAC-assisted thioesterification reaction. As in the synthesis
of peptide 15, two Arg and NAC residues were sequentially
introduced to Rink Amide MBHA resin, and then Fmoc-Gly-
OH was condensed using HATU as a coupling reagent. The
Synthesis of a-conotoxin SI
To demonstrate the usefulness of Pocam group for the selective
disulfide formation, we synthesized a-conotoxin SI as a model
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peptide chain was elongated by the DCC–HOBt method. Fmoc-
Cys(Acm)-OH was used at Cys2 and Cys7, and Fmoc-Cys(Pocam)-
OH was introduced at Cys3. After cleaving the crude peptide
from the resin by the TFA cocktail treatment at 4 ◦C for 4 h,
the thioesterification reaction was carried out in 50% aqueous
acetonitrile solution containing 5% AcOH and 5% MPAA at room
temperature for 20 h, giving the desired peptide thioester21 in 8.0%
yield.
The peptide segments, 20 and 21, were mixed and dissolved in
DMSO containing 2% HOObt and 2% DIEA (Scheme 6). The
N-terminal peptide thioester 21 was almost exhausted within 4
h, and an additional peak corresponding to the desired product
22 appeared in the RP-HPLC chromatogram (Fig. 4). After the
condensation reaction, the Fmoc group at the N-terminus was
removed by adding piperidine to give peptide 23. The Pocam and
azido groups were subsequently cleaved by Zn powder treatment
in an aqueous AcOH solution, and the linear a-conotoxin SI 24
was successfully obtained in 66% yield. During condensation and
deprotection reactions, no significant side reaction was observed,
and the product was generated in good yield. The disulfide bond
between Cys3–Cys13 was formed by oxidation in a phosphate buffer
containing 10% DMSO, giving desired product 25 in 64% yield
(Fig. 5). Finally, the second disulfide bond between Cys2–Cys7 was
formed by iodine oxidation. Peptide 25 was dissolved in water, and
was added dropwise to I2 solution in methanol containing HCl.
After 15 min, the reaction was quenched with ascorbic acid, and
the product was purified by RP-HPLC, to give the final product
26 in 88% yield.
Fig. 5 RP-HPLC elution profiles of disulfide formation reactions of
a-conotoxin SI. (a) Before disulfide formation. (b) After DMSO oxidation
for 20 h. (c) After iodine oxidation. Column: Mightysil RP-18 GP (4.6 ¥
150 mm), eluent: 0.1% TFA in aqueous acetonitrile at a flow rate of
1 ml/min.
Conclusions
We developed a novel thiol protecting group, Pocam. We chem-
ically synthesized Fmoc-Cys(Pocam)-OH and introduced it into
peptides and peptide thioesters, indicating that the Pocam group
was compatible with Fmoc-SPPS and NAC-assisted thioesterifi-
cation reactions. It was useful for peptide condensation by the
thioester method, and also for selective disulfide formation. The
Pocam group was cleaved simultaneously with azido group by
reduction using Zn/AcOH, demonstrating that the deprotection
reaction after peptide condensation by the thioester method, which
conventionally took two or more steps, was simplified to only one
step.
Fig. 4 RP-HPLC elution profiles of peptide condensation and deprotec-
tion. (a) Coupling reaction mixture of 20 and 21 (0 h). (b) Four hours after
the coupling reaction. (c) Reaction mixture after piperidine treatment. (d)
Reaction mixture after Zn/AcOH treatment. Column: Mightysil RP-18
GP (4.6 ¥ 150 mm), eluent: 0.1% TFA in aqueous acetonitrile at a flow
rate of 1 ml/min. Asterisks indicate non-peptidic components.
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N-Methyl-O-phenacylcarbamate ethylene acetal 7
Experimental
2-Hydroxyacetophenone (540 mg, 4.0 mmol) was mixed with ethy-
lene glycol (2.0 ml) in dichloromethane (2 ml) and 4-N HCl/1,4-
dioxane (1 ml). The solution was stirred at room temperature for
2 d. The solvent was removed under reduced pressure, and the
residue was chromatographed on silica gel with toluene/EtOAc
(3/1) to give compound 6 (400 mg, 55%). Then, 6 (290 mg,
1.6 mmol) was mixed with disuccinimidyl carbonate (410 mg,
1.6 mmol), 4-dimethylaminopyridine (20 mg, 0.16 mmol) and
DIEA (280 ml, 1 mmol) in DMSO (3 ml), and the solution was
stirred at room temperature for 1 h. To this solution, methylamine
hydrochloride (110 mg, 1.6 mmol) and DIEA (280 ml, 1.6 mmol)
was added, and the mixture was stirred for 1 h. The solution
was diluted with EtOAc, washed with 1 M HCl and brine, and
dried over Na2SO4. After filtration and concentration in vacuo, the
residue was chromatographed on silica gel with toluene/EtOAc
(3/1) to give compound 7 (260 mg, 68%, colorless amorphous). Rf
0.23 (Toluene/EtOAc, 3/1). Near UV in CH3OH: lmax = 260 nm (e
1,600). 1H-NMR (CDCl3): d 7.52–7.28 (m, 5H, Ar), 4.96 (brs, 1H,
NH), 4.32 [s, 2H, C( O)CH2], 4.07 (m, 2H, -O–CH2–CH2–O-),
3.86 (m, 2H, -O–CH2-CH2–O-), 2.74 (d, 3H, J = 5.2 Hz, CH3).
13C-NMR (CDCl3): d 156.6 (C O), 139.3, 128.5, 128.1, 126.0 and
126.0 [Ph–C(O)2, Ar], 66.8 (>C–CH2–O), 65.2 (O–CH2–CH2–O),
27.5 (N–CH3).
General
Fmoc-(Et)Cys(Trt)-OH8,15 and Fmoc-Lys(N3)-OH4 were prepared
by previously described methods. MALDI-TOF mass spectra
were recorded using a Voyager-DE PRO spectrometer (Applied
Biosystems, CA). 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.
Fmoc-Lys(Poc)-OH 1
2-Hydroxyacetophenone (140 mg, 1.0 mmol), disuccinimidyl
carbonate (260 mg, 1.0 mmol), 4-dimethylaminopyridine (12 mg,
0.10 mmol) and N,N-diisopropylethylamine (DIEA, 170 ml,
1 mmol) were dissolved in N,N-dimethylformamide (DMF, 4 ml),
and stirred at room temperature for 1 h. To this solution, Fmoc-
Lys-OH hydrochloride (370 mg, 1.0 mmol) was added and the
solution was stirred overnight. The reaction mixture was diluted
with EtOAc, washed with 1 M HCl and brine, and dried over
Na2SO4. After filtration and concentration in vacuo, the residue
was chromatographed on silica gel with toluene/EtOAc/AcOH
(50/50/1) to give Fmoc-Lys(Poc)-OH 1 (160 mg, 30%, colorless
solid). Rf 0.15 (Toluene/EtOAc/AcOH, 50/50/1). Near UV in
1
CH3OH: lmax = 251 nm (e 11 900), 264 nm (e 11 800). H-NMR
Fmoc-Cys(Pocam)-OH 5
(CDCl3): d 7.87–7.13 (m, 13H, Ar), 5.91 (d, 1H, J = 8.0 Hz, NaH),
5.48 (t, 1H, J = 5.6, NeH), 5.25 [s, 2H, C( O)CH2], 4.38–4.10
(m, 4H, CaH, >CH–CH2–O-), 3.20 (m, 2H, CeH), 1.87 (m, 1H,
CbH), 1.75 (m, 1H, CbH), 1.54–1.44 (m, 4H, CgH, CdH). 13C-
NMR (CDCl3): d 193.9 (COOH), 175.8 (Ph–CO), 156.4 and 156.1
[NH–C(O)–O], 67.0 (>C–CH2–O), 66.3 (CO–CH2–O), 53.6 (Ca),
47.0 (>C–CH2–O), 40.4 (Ce), 31.4 (Cb), 29.0 (Cd), 22.0 (Cg).
MALDI-TOF mass, found: m/z 552.9, calcd: 553.2 for (M+Na)+.
Compound
7
(260 mg, 1.1 mmol) was dissolved in
dimethoxyethane (1.5 ml), and 36% aqueous formaldehyde so-
lution (0.50 ml) and Na2CO3 (120 mg1.1 mmol) were added. After
stirring at room temperature for 2 h, the mixture was diluted
with EtOAc, washed with H2O and brine, and dried over Na2SO4.
After filtration and concentration, hydroxymethylated product
8 was used in the next reaction without further purification.
Compound 8 was mixed with cysteine hydrochloride (350 mg,
2.0 mmol) and dissolved in TFA (2 ml). The reaction mixture was
stirred at room temperature for 30 min. After TFA was removed
under reduced pressure, the residue was chromatographed on silica
gel with CHCl3/CH3OH/AcOH (90/10/1). During the reaction
and purification steps, ethylene acetal portion was decomposed
in part, and the cysteine derivatives 9 and 10 were obtained as
a mixture. Since these could not be separated on the silica gel
chromatography, we used them in the next reaction as a mixture.
The yield of the mixture was 370 mg. To the mixture of 9 and
10, Fmoc-OSu (370 mg, 1.1 mmol) was added in DMF (5 ml)
containing DIEA (170 ml, 1.0 mmol), and the solution was stirred
at room temperature for 2 h. The solution was then diluted with
EtOAc, washed with 1 M aqueous HCl and brine, and dried over
Na2SO4. After filtration and concentration in vacuo, the residue
was chromatographed on silica gel with toluene/EtOAc/AcOH
(50/50/1). During the reaction and purification steps, the ethylene
acetal portion was almost completely decomposed, and only
Fmoc-Cys(Pocam)-OH 5 was obtained (440 mg, 73% in 3 steps) as
colorless solid. Rf 0.18 (Toluene/EtOAc/AcOH, 50/50/1). Near
Synthesis of the model peptide containing Poc group
Fmoc-Glu(OBut)-Wang resin (0.55 mmol/g, 36 mg, 20 mmol)
was swelled in N-methylpyrrolidone (NMP) for 30 min, and was
treated with 20% piperidine/NMP for 5 and 15 min. After washing
with NMP, Fmoc-Tyr(But)-OBt, which was prepared by mixing
Fmoc-Tyr(But)-OH (0.1 mmol), 1 M DCC/NMP (100 ml) and 1
M HOBt/NMP (100 ml) at room temperature for 30 min, was
added and the reaction mixture was vortexed at 50 ◦C for 1
h. The resin was washed with NMP and 50% dichloromethane
(DCM)/CH3OH, treated with 10% Ac2O/5% DIEA/NMP for
5 min, and washed with NMP. Fmoc-Lys(Poc)-OH, Fmoc-Phe-
OH and Fmoc-Ser(But)-OH were sequentially introduced to the
resin by the same manner as for Tyr residue, and H-Ser(But)-Phe-
Lys(Poc)-Tyr(But)-Glu(OBut)-OCH2-resin (49 mg) was obtained.
The resin was treated with 95% aqueous TFA solution (1 ml)
at room temperature for 2 h. TFA was removed under nitrogen
stream and the peptide was precipitated with diethyl ether. After
washing twice with ether, the precipitant was dried under vacuum.
The crude peptide was separated by RP-HPLC on a Mightysil
RP-18 GP column with a linear gradient of acetonitrile containing
0.1% TFA.
1
UV in CH3OH: lmax = 261 nm (e 6,200), 277 nm (e 5,400) H-
NMR was measured as a mixture of conformers in DMSO-d6: d
7.93–7.29 (m, 13H, Ar), 5.43 [s, 1.2H, C( O)CH2], 5.38 [s, 0.8H,
C( O)CH2], 4.71–4.45 (m, 2H, N–CH2–S), 4.29–4.19 (m, 4H,
4658 | Org. Biomol. Chem., 2011, 9, 4653–4661
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CaH, >CH–CH2–O-), 3.35 (brs, 3H, CH3), 3.12–2.83 (m, 2H,
CbH). 13C-MNR (DMSO-d6): d 193.6 and 193.3 (COOH), 172.3
(Ph–C O), 67.5 (CO–CH2–O), 65.9 (>CH–CH2–O), 54.0 (Ca),
51.3 and 50.3 (N–CH2–S), 46.7 (>CH–CH2–O), 33.1 and 32.8(N–
CH3), 31.8 and 31.5 (Cb). MALDI-TOF mass, found: m/z 571.3,
calcd: 571.2 for (M+Na)+
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) were activated by mixing with 1 M DCC/NMP
(0.2 ml) and 1 M HOBt/NMP (0.2 ml) at room temperature
for 30 min, and the coupling reaction was carried out at 50 ◦C
for 1 h, except that Fmoc-Asp(OBut)-(Dmb)Gly-OH dipeptide
unit (52 mg, 0.1 mmol) was used as a building block at Asp16-
Gly17 site. After elongation, H-Tyr(But)-Met-Arg(Pbf)-Thr(But)-
Pro-Asp(OBut )-(Dmb)Gly-Arg(Pbf)-Cys(Pocam)-Lys(N3 )-Pro-
Thr(But)-Phe-Tyr(But)-Gln(Trt)-OCH2-resin (260 mg) was ob-
tained. A part of the resin (20 mg) was treated with the
TFA cocktail (500 ml) at 4 ◦C for 4 h. TFA was removed
under nitrogen stream and the peptide was precipitated with
diethyl ether. After washing twice with ether, the precipitant
was dried under vacuum. The crude peptide was separated by
RP-HPLC on a Mightysil RP-18 GP column with a linear
gradient of acetonitrile containing 0.1% TFA, to give pep-
tide 11 (340 nmol, 8.9% yield). MALDI-TOF mass, found:
m/z 2094.2, calcd: 2093.9 for (M+H)+. Amino acid analysis:
Fmoc-[Cys(Pocam)7]-GBP(1-10)-SC6H4CH2COOH 15
Fmoc-Rink Amide MBHA resin (0.34 mmol/g, 150 mg, 50 mmol)
was swelled in NMP for 30 min, and was treated with 20%
piperidine/NMP for 5 and 15 min. After washing with NMP,
Fmoc-Arg(Pbf)-OBt, which was prepared by mixing Fmoc-
Arg(Pbf)-OH (0.2 mmol), 1 M DCC/NMP (200 ml) and 1 M
HOBt/NMP (200 ml) at room temperature for 30 min, was
added and the reaction mixture was vortexed at 50 ◦C for 1
h. The resin was washed with NMP and 50% dichloromethane
(DCM)/CH3OH, treated with 10% Ac2O/5% DIEA/NMP for
5 min, and washed with NMP. Another Arg residue was introduced
to the resin by the same manner, and the resin was washed with
NMP. After Fmoc group was removed by 20% piperidine/NMP
treatment at room temperature for 5 and 15 min, the resin was
washed with NMP. Fmoc-(Et)Cys(Trt)-OBt, which was prepared
by mixing Fmoc-(Et)Cys(Trt)-OH (0.1 mmol), 1 M DCC/NMP
(150 ml) and 1 M HOBt/NMP (150 ml) at room temperature for
30 min, was added to the resin, and the mixture was vortexed
at 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. After Fmoc group was removed
by 20% piperidine/NMP treatment at room temperature for 5
and 15 min, the resin was washed with NMP. Fmoc-Gly-OH
(0.5 mmol) and HATU (0.5 mmol) was dissolved in NMP (1 ml)
containing DIEA (170 ml, 1 mmol), and the mixture was added
to the resin. The reaction mixture was vortexted at 50 ◦C for 1
h. After washing with NMP, the peptide chain was elongated by
ordinary Fmoc-based SPPS. The amino acids (0.2 mmol each)
were activated by mixing with 1 M DCC/NMP (0.2 ml) and 1
M HOBt/NMP (0.2 ml) at room temperature for 30 min, and
the coupling reaction was carried out at 50 ◦C for 1 h. Af-
ter elongation, Fmoc-Glu(OBut)-Asn(Trt)-Phe-Ser(But)-Gly-Gly-
Cys(Pocam)-Val-Ala-Gly-(Et)Cys(Trt)-Arg(Pbf)-Arg(Pbf)-NH-
resin (280 mg) was obtained. A part of the resin (20 mg) was
treated with a TFA cocktail (TFA/thioanisole/H2O/phenol/
triisopropylsilane, 82.5/5/5/5/2.5, 500 ml) at 4 ◦C for 4 h. TFA was
removed under nitrogen stream and the peptide was precipitated
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 (1 ml), and MPAA (50 mg) was added to
the solution. After the overnight reaction at room temperature, the
crude peptide was separated by RP-HPLC on a Mightysil RP-18
GP column with a linear gradient of acetonitrile containing 0.1%
TFA, to give peptide thioester 15 (74 nmol, 2.0% yield). MALDI-
TOF mass, found: m/z 1839.5, calcd: 1839.5 for (M+Na)+. Amino
Asp0.98Thr1.94Glu1.08Pro1.84Gly1Met0.82Tyr1.96Phe1.00Lys0.50Arg2.02
.
Linear GBP 18
Peptides 15 (74 nmol) and 11 (100 nmol) were mixed and dissolved
in DMSO (40 ml) containing 2% HOObt/2% DIEA, and incubated
at room temperature for 4 h. Piperidine (3 ml) was then added to
this solution and the reaction mixture was kept at room tempera-
ture for 30 min. 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 (500 ml), and an excess amount of Zn powder was added to
the solution. The mixture was vortexed at room temperature for
1 h. After filtration to remove Zn powder, the desired product 18
was purified by RP-HPLC using a Mightysil RP-18 GP column
with a linear gradient of acetonitrile containing 0.1% TFA. The
isolated yield of 18 was 31% (23 nmol). MALDI-TOF mass,
found: m/z 2783.8, calcd: 2784.2 for (M+H)+. Amino acid analy-
sis: Asp2.13Thr2.00Ser1.01Glu2.16Pro2.00Gly4Ala1.08Val1.02Met0.97Tyr2.47
-
Phe2.10Lys1.04Arg2.13
.
GBP 19
Peptide 18 (22 nmol) was dissolved in 400 ml of 10% DMSO/50
mM phosphate buffer (pH 7.0), and the solution was kept
at room temperature for 24 h. The crude peptide was sep-
arated by RP-HPLC using a Mightysil RP-18 GP column
with a linear gradient of acetonitrile containing 0.1% TFA to
give peptide 19 (11 nmol, 52%). MALDI-TOF mass, found:
m/z 2781.9, calcd: 2782.2 for (M+H)+. Amino acid analy-
sis: Asp1.91Thr1.95Ser1.01Glu2.10Pro1.93Gly4Ala0.99Val0.95Met0.73Tyr2.33
Phe2.16Lys1.08Arg2.06
-
.
Fmoc-[Cys(Acm),2,7 Cys(Pocam)3]-a-conotoxin SI(1–8)-SC6-
H4CH2COOH 21
acid analysis: Asp0.92Ser0.86Glu1.08Gly3Ala1.00Val0.92Phe0.91
.
Fmoc-Rink Amide MBHA resin (0.34 mmol/g, 150 mg, 50 mmol)
was swelled in NMP for 30 min, and was treated with 20%
piperidine/NMP for 5 and 15 min. After washing with NMP,
Fmoc-Arg(Pbf)-OBt, which was prepared by mixing Fmoc-
Arg(Pbf)-OH (0.2 mmol), 1 M DCC/NMP (200 ml) and 1 M
[Cys(Pocam)19, Lys(N3)20]-GBP(11-25) 11
Fmoc-Gln(Trt)-CLEAR Acid resin (0.41 mmol/g, 120 mg,
50 mmol) was swelled in NMP for 30 min, and treated with 20 %
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HOBt/NMP (200 ml) at room temperature for 30 min, was
added and the reaction mixture was vortexed at 50 ◦C for 1
h. The resin was washed with NMP and 50% dichloromethane
(DCM)/CH3OH, treated with 10% Ac2O/5% DIEA/NMP for
5 min, and washed with NMP. Another Arg residue was introduced
to the resin by the same manner, and the resin was washed with
NMP. After Fmoc group was removed by 20% piperidine/NMP
treatment at room temperature for 5 and 15 min, the resin was
washed with NMP. Fmoc-(Et)Cys(Trt)-OBt, which was prepared
by mixing Fmoc-(Et)Cys(Trt)-OH (0.1 mmol), 1 M DCC/NMP
(150 ml) and 1 M HOBt/NMP (150 ml) at room temperature for
30 min, was added to the resin, and the mixture was vortexed
at 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. After Fmoc group was removed
by 20% piperidine/NMP treatment at room temperature for 5
and 15 min, the resin was washed with NMP. Fmoc-Gly-OH
(0.5 mmol) and HATU (0.5 mmol) was dissolved in NMP (1 ml)
containing DIEA (170 ml, 1 mmol), and the mixture was added
to the resin. The reaction mixture was vortexted at 50 ◦C for
1 h. After washing with NMP, the peptide chain was elongated
by the ordinary Fmoc-based SPPS. The amino acids (0.2 mmol
each) were activated by mixing with 1 M DCC/NMP (0.2 ml)
and 1 M HOBt/NMP (0.2 ml) at room temperature for 30 min,
and the coupling reaction was carried out at 50 ◦C for 1 h. Af-
ter elongation, Fmoc-Ile-Cys(Acm)-Cys(Pocam)-Asn(Trt)-Pro-
Ala-Cys(Acm)-Gly-(Et)Cys(Trt)-Arg(Pbf)-Arg(Pbf)-NH-resin
(270 mg) was obtained. A part of the resin (20 mg) was treated
with the TFA cocktail (500 ml) at 4 ◦C for 4 h. TFA was removed
under nitrogen stream and the peptide was precipitated 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 (1 ml), and MPAA (50 mg) was added to
the solution. After the overnight reaction at room temperature, the
crude peptide was separated by RP-HPLC on a Mightysil RP-18
GP column with a linear gradient of acetonitrile containing 0.1%
TFA, to give peptide thioester 21 (300 nmol, 8.0% yield). MALDI-
TOF mass, found: m/z 1521.7, calcd: 1521.5 for (M+Na)+. Amino
mass, found: m/z 827.4, calcd: 827.3 for (M+H)+. Amino acid
analysis: Ser0.67Pro1.06Tyr1Lys0.57
.
[Cys(SH),3,13 Cys(Acm)2,7]-a-conotoxin SI 24
Peptides 21 (280 nmol) and 20 (400 nmol) were mixed and
dissolved in DMSO (150 ml) containing 2% HOObt/2% DIEA,
and incubated at room temperature for 4 h. Piperidine (8 ml) was
then added to this solution and the reaction mixture was kept at
room temperature for 30 min. 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 (1.0 ml), and an excess amount of Zn powder
was added to the solution. The mixture was vortexed at room
temperature for 1 h. After filtration to remove Zn powder, the
desired product 24 was purified by RP-HPLC using a Mightysil
RP-18 GP column with a linear gradient of acetonitrile containing
0.1% TFA. The isolated yield of 24 was 66% (190 nmol). MALDI-
TOF mass, found: m/z 1499.8, calcd: 1499.6 for (M+H)+. Amino
acid analysis: Asp0.92Ser0.59Pro1.95Gly1Ala0.97Ile0.58Tyr1.04Lys0.88
.
[Cys(Acm)2,7]-a-conotoxin SI 25
Peptide 24 (190 nmol) was dissolved in 1.0 ml of 10% DMSO/
50 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 a Mightysil RP-18 GP column
with a linear gradient of acetonitrile containing 0.1% TFA to
give peptide 25 (120 nmol, 64%). MALDI-TOF mass, found:
m/z 1497.7, calcd: 1497.6 for (M+H)+. Amino acid analysis:
Asp0.87Ser1.24Pro2.00Gly1Ala0.94Ile0.94Tyr0.57Lys1.10
.
a-Conotoxin SI 26
Peptide 25 (120 nmol) was dissolved in H2O (1.0 ml) and added
dropwise to CH3OH (5.0 ml) containing 20 mM I2/CH3OH
(200 ml) and 6 M HCl (67 ml) within 5 min with mixing. After
the resultant solution was stirred for another 15 min, the reaction
was terminated by adding aqueous ascorbic acid solution. The
product was purified by RP-HPLC using a Mightysil RP-18 GP
column with a linear gradient of acetonitrile containing 0.1% TFA
to give peptide 26 (100 nmol, 88%). MALDI-TOF mass, found:
m/z 1353.3, calcd: 1353.5 for (M+H)+. Amino acid analysis:
acid analysis: Asp0.94Pro0.95Gly1Ala1.00Ile0.50
.
[Cys(Pocam),13 Lys(N3)10]-a-conotoxin SI(9–13) 20
Asp0.84Ser0.81Pro2.07Gly1Ala0.95Ile0.76Tyr1.10Lys0.99
.
Fmoc-Rink Amide MBHA resin (0.34 mmol/g, 88 mg, 30 mmol)
was swelled in NMP for 30 min, and treated with 20 %
piperidine/NMP for 5 and 15 min. After washing NMP, the
peptide chain was elongated by the Fmoc-SPPS. The amino
acids (0.12 mmol each) were activated by mixing with 1 M
DCC/NMP (0.15 ml) and 1 M HOBt/NMP (0.15 ml) at room
temperature for 30 min, and the coupling reaction was carried
out at 50 ◦C for 1 h. After elongation, Boc-Pro-Lys(N3)-Tyr(But)-
Ser(But)-Cys(Pocam)-NH-resin (120 mg) was obtained. A part of
the resin (20 mg) was treated with the TFA cocktail (500 ml) at
Acknowledgements
We thank Mr. Paul Singleton for the critical reading of this
manuscript. This work was supported in part by Grant-in-Aid
for Scientific Research from the Ministry of Education, Sports,
Science and Technology of Japan (Nos. 22880033 and 00209214).
Notes and references
4
◦C for 4 h. TFA was removed under nitrogen stream and the
1 P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. H. Kent, Science,
1994, 266, 776–779.
2 B. L. Nilsson, L. L. Kiessling and R. T. Raines, Org. Lett., 2000, 2,
1939–1941; B. L. Nilsson, L. L. Kiessling and R. T. Raines, Org. Lett.,
2001, 3, 9–12.
3 H. Hojo and S. Aimoto, Bull. Chem. Soc. Jpn., 1991, 64, 111–117; S.
Aimoto, Biopolymers, 1999, 51, 247–265.
peptide was precipitated with diethyl ether. After washing twice
with ether, the precipitant was dried under vacuum. The crude
peptide was separated by RP-HPLC on a Mightysil RP-18 GP
column with a linear gradient of acetonitrile containing 0.1%
TFA, to give peptide 20 (580 nmol, 11% yield). MALDI-TOF
4660 | Org. Biomol. Chem., 2011, 9, 4653–4661
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4 H. Katayama, H. Hojo, T. Ohira and Y. Nakahara, Tetrahedron Lett.,
2008, 49, 5492–5494.
5 H. Katayama, H. Hojo, I. Shimizu, Y. Nakahara and Y. Nakahara,
Org. Biomol. Chem., 2010, 8, 1966–1972.
6 D. F. Veber, J. Milkowski, S. Varga, R. Denkewalter and R.
Hirschmann, J. Am. Chem. Soc., 1972, 94, 5456–5461; M. Yoshida,
K. Akaji, T. Tatsumi, S. Iinuma, Y. Fujiwara, T. Kimura and Y. Kiso,
Chem. Pharm. Bull., 1990, 38, 273–275.
A. Ueki, Y. Nakahara and Y. Nakahara, Org. Lett., 2006, 8, 4465–4468;
T. J. Hogenauer, Q. Wang, A. K. Sanki, A. J. Gammon, C. H. L. Chu, C.
M. Kaneshiro, Y. Kajihara and K. Michael, Org. Biomol. Chem., 2007,
5, 759–762; T. Kawakami and S. Aimoto, Chem. Lett., 2007, 36, 76–77;
J. B. Blanco-Canosa and P. E. Dawson, Angew. Chem., Int. Ed., 2008,
47, 6857–6861; J. Kang, J. P. Richardson and D. Macmillan, Chem.
Commun., 2009, 407–409; S. Tsuda, A. Shigenaga, K. Bando and A.
Otaka, Org. Lett., 2009, 11, 823–826; A. P. Tofteng, K. K. Sorensen, K.
W. Conde-Frieboes, T. Hoeg-Jensen and K. J. Jensen, Angew. Chem.,
Int. Ed., 2009, 48, 7411–7414; J. Kang, N. L. Reynolds, C. Tyrrell, J. R.
Dorin and D. Macmillan, Org. Biomol. Chem., 2009, 7, 4918–4923.
8 H. Hojo, Y. Onuma, Y. Akimoto, Y. Nakahara and Y. Nakahara,
Tetrahedron Lett., 2007, 48, 25–28; H. Hojo, Y. Onuma, Y.
Akimoto, Y. Nakahara and Y. Nakahara, Tetrahedron Lett., 2007, 48,
1299.
9 D. Hagiwara, M. Neya and M. Hashimoto, Tetrahedron Lett., 1990,
31, 6539–6542.
10 R. Melnicky, L. Kvapil, P. Sle´zar, M. Grepl, J. Hlava´c, A. Lycka and P.
Hradil, J. Heterocycl. Chem., 2008, 45, 1437–1443.
11 Y. Hayakawa, J. Biol. Chem., 1990, 265, 10813–10816; Y. Hayakawa,
J. Biol. Chem., 1991, 266, 7982–7984; Y. Hayakawa and Y. Yasuhara,
Insect Biochem. Mol. Biol., 1993, 23, 225–231.
7 S. Futaki, K. Sogawa, J. Maruyama, T. Asahara, M. Niwa and H.
Hojo, Tetrahedron Lett., 1997, 38, 6237–6240; X. Li, T. Kawakami
and S. Aimoto, Tetrahedron Lett., 1998, 39, 8669–8672; Y. Shin, K. A.
Winans, B. J. Backes, S. B. H. Kent, J. A. Ellman and C. R. Bertozzi,
J. Am. Chem. Soc., 1999, 121, 11684–11689; J. Alsina, T. S. Yokumu,
F. Albericio and G. Barany, J. Org. Chem., 1999, 64, 8761–8769; R.
Ingenito, E. Bianchi, D. Fattori and A. Pessi, J. Am. Chem. Soc., 1999,
121, 11369–11374; A. B. Clippingdale, C. J. Barrow and J. D. Wade,
J. Pept. Sci., 2000, 6, 225–234; D. Swinnen and D. Hilvert, Org. Lett.,
2000, 2, 2439–2442; A. R. Mezo, R. P. Cheng and B. Imperiali, J. Am.
Chem. Soc., 2001, 123, 3885–3891; R. R. Flavell, M. Huse, M. Goger,
M. Trester-Zedlitz, J. Kuriyan and T. W. Muir, Org. Lett., 2002, 4, 165–
168; J. Brask, F. Albericio and K. J. Jensen, Org. Lett., 2003, 5, 2951–
2953; J. A. Camarero, B. J. Hackel, J. J. de Yoreo and A. R. Mitchell, J.
Org. Chem., 2004, 69, 4145–4151; J. D. Warren, J. S. Miller, S. J. Keding
and S. J. Danishefsky, J. Am. Chem. Soc., 2004, 126, 6576–6578; P.
Botti, M. Villain, S. Manganiello and H. Gaertner, Org. Lett., 2004, 6,
4861–4864; T. Kawakami, M. Sumida, K. Nakamura, T. Vorherr and S.
Aimoto, Tetrahedron Lett., 2005, 46, 8805–8807; N. Oliver, J.-B. Behr, O.
El-Mahdi, A. Blanpain and O. Melnyk, Org. Lett., 2005, 7, 2647–2650;
Y. Ohta, S. Itoh, A. Shigenaga, S. Shintaku, N. Fujii and A. Otaka, Org.
Lett., 2006, 8, 467–470; F. Nagaike, Y. Onuma, C. Kanazawa, H. Hojo,
12 D. S. King, C. G. Fields and G. B. Fields, Int. J. Pept. Protein Res.,
1990, 36, 255–266.
13 H. Hojo, Y. Murasawa, H. Katayama, T. Ohira, Y. Nakahara and Y.
Nakahara, Org. Biomol. Chem., 2008, 6, 1808–1813.
14 G. C. Zafaralla, C. Ramilo, W. R. Gray, R. Karlstrom, B. M. Olivera
and L. J. Cruz, Biochemistry, 1988, 27, 7102–7105.
15 Y. Nakahara, I. Matsuo, Y. Ito, R. Ubagai, H. Hojo and Y. Nakahara,
Tetrahedron Lett., 2010, 51, 407–410.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 4653–4661 | 4661
©