Site-specific labeling of synthetic peptide using the chemoselective reaction between N-methoxyamino acid and isothiocyanate

Article abstract of DOI:10.1002/psc.2803

Site-specific labeling of synthetic peptides carrying N-methoxyglycine (MeOGly) by isothiocyanate is demonstrated. A nonapeptide having MeOGly at its N-terminus was synthesized by the solid-phase method and reacted with phenylisothiocyanate under various conditions. In acidic solution, the reaction specifically gave a peptide having phenylthiourea structure at its N-terminus, leaving side chain amino group intact. The synthetic human β-defensin-2 carrying MeOGly at its N-terminus or the side chain amino group of Lys¹⁰ reacted with phenylisothiocyanate or fluorescein isothiocyanate also at the N-methoxyamino group under the same conditions, demonstrating that this method is generally useful for the site-specific labeling of linear synthetic peptides as well as disulfide-containing peptides.

Full text of DOI:10.1002/psc.2803

Research article
Received: 9 June 2015
Revised: 1 July 2015
Accepted: 1 July 2015
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2803
Site-specific labeling of synthetic peptide
using the chemoselective reaction
between N-methoxyamino acid and
isothiocyanate
Toshiaki Hara,a Euis Maras Purwati,b Akira Tainosyo,b Toru Kawakami,b
Hironobu Hojob and Saburo Aimotob*
Site-specific labeling of synthetic peptides carrying N-methoxyglycine (MeOGly) by isothiocyanate is demonstrated. A
nonapeptide having MeOGly at its N-terminus was synthesized by the solid-phase method and reacted with phenyliso-
thiocyanate under various conditions. In acidic solution, the reaction specifically gave a peptide having phenylthiourea
structure at its N-terminus, leaving side chain amino group intact. The synthetic human β-defensin-2 carrying MeOGly at its
N-terminus or the side chain amino group of Lys10 reacted with phenylisothiocyanate or fluorescein isothiocyanate also at
the N-methoxyamino group under the same conditions, demonstrating that this method is generally useful for the
site-specific labeling of linear synthetic peptides as well as disulfide-containing peptides. Copyright © 2015 European Peptide
Society and John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: site-specific labeling; N-methoxyamino acid; isothiocyanate; human β-defensin
modified with fluorescent probes without disrupting the tertiary
structure. One of the unique advantage of N-methoxyglycine
Introduction
Site-specifically modified peptides and proteins are important for
the elucidation of their biochemical and biological function. In
particular, fluorescent labeling is an often used technique to study
the interaction between a peptide or a protein with their binding
partner, to analyze the cellular localization, and to create fluores-
cent biosensors for application [1]. However, one of the problems
in these studies has been the difficulty in the site-specific labeling
by fluorophores in the polypeptide. Although the chemical syn-
thesis can realize this, the presence of highly hydrophobic probe
sometimes makes the folding reaction at the final stage of the
synthesis difficult. In particular, peptides with multiple disulfide
bonds tend to misfold, when tagged with hydrophobic probes,
resulting in the formation of disulfide bond isomers. Therefore,
a method to realize site-specific modification after the chemical
synthesis and folding is desirable. Along this line, the
chemoselectivity of the N-alkylaminooxy group with electro-
philes, such as succinimidyl ester, activated haloalkanes, has
been examined [2,3]. In addition, N-alkylaminooxy groups have
been extensively studied for the chemoselective glycosylation
to create glycoconjugates and glycopeptides [4–11]. These
functional groups retain pKa (4–5) significantly lower than that
of the α-amino group (about 9.1) and the ε-amino group of Lys
residue (about 10.5), which is the basis of the chemoselectivity.
over another N-methoxyamino group is that the α-thiourea
structure, which is formed by reaction between N-methoxy-
glycine and arylisothiocyanates such as phenylisothiocyanate
(PITC) and fluorescein isothiocyanate (FITC), can be selectively
removed through Edman degradation reaction. In this paper,
the idea is demonstrated by the selective modification of
human β-defensin-2 (hBD2) at the side chain amino group of
Lys10 as well as terminal amino group.
*
Correspondence to: Saburo Aimoto, Institute for Protein Research, Osaka
University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
E-mail: aimoto@protein.osaka-u.ac.jp
a Department of Chemistry, Graduate School of Science, Osaka University,
Toyonaka, Osaka, 560-0043, Japan
b Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka,
565-0871, Japan
Abbreviations: Boc, t-butoxycarbonyl; DIC, diisopropylcarbodiimide; DIEA, N,
N-diisopropylethylamine; DODT, 3,6-dioxa-1,8-octanedithiol; ESI-MS, electrospray-
ionization mass spectrometry; Fmoc, 9-fluorenylmethoxycarbonyl; FITC, fluores-
cein isothiocyanate; GSH, glutathione reduced form; GSSG, glutathione oxidized
form; hBD2, human β-defensin-2; HBTU, O-(benzotriazol-1-yl)-N, N, N′, N′-
tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; NMP,
1-methyl-2-pyrrolidinone; NMR, nuclear magnetic resonance; RPHPLC, reversed-
phase high-performance liquid chromatography; SPPS, solid-phase peptide syn-
thesis; TFA, trifluoroacetic acid; TIS, triisopropylsilane; PITC, phenylisothiocyanate.
Here, we examined the use of N-methoxyglycine as
a
specificity-generating group to introduce probes after the chem-
ical synthesis. If a peptide with N-methoxyglycine is synthesized
and folded, the N-methoxyamino group can be selectively
J. Pept. Sci. 2015; 21: 765–769
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HARA ET AL.
The reaction was terminated after 14 h. Peptide 2: ESI-MS, found:
m/z 1098.6, calcd: 1098.5 for (M+H)+. Amino acid analysis:
Materials and Method
The NMR spectra were recorded using an AV400 spectrometer at
400 MHz (Bruker, MA, USA). The chemical shifts are expressed in
ppm downfield from the signal for the internal Me4Si for solutions
in the deuterated solvents. The SPPS of peptide 1 was carried out
using a Liberty Blue peptide synthesizer (CEM, NC, USA). The pep-
tide chain assembly for hBD 6 and 7 was carried out by 433A pep-
tide synthesizer (Applied Biosystems, CA). The ESI mass spectra
Glu1.05Gly2.13Ala1Leu1.00Phe1.00Lys1.08Arg0.97
obs: 1233.6 (deconvoluted), calcd: 1233.6 for (M+H)+. Amino acid
analysis: Glu1.07Gly2.16Ala1Leu1.01Phe1.02Lys1.02Arg1.05
. Peptide 3: ESI-MS,
.
TFA Treatment of Peptide 2
Peptide 2 (2.0nmol) was dissolved in TFA containing 5% water
(20 μl), and the solution was kept for 10 min at room temperature.
After TFA was removed by the nitrogen stream, the residue was pu-
rified by RPHPLC. The peak of peptide 2 disappeared, and two new
peaks appeared. One peak had the mass number of 876.7, which
corresponds well to the theoretical value of Gly-Leu-Glu-Phe-Lys-
Ala-Gly-Arg-NH2 4 (876.5, [M+H]+). The other peak did not show
an intense signal, which might show that this peak was derived
from thiohydantoin derivative of methoxyglycine.
were recorded using
a LCQ DECA XP plus spectrometer
(ThermoFisher Scientific, MA, USA). The amino acid composition
was determined using a LaChrom amino acid analyzer (Hitachi,
Tokyo, Japan) after hydrolysis with 6 M HCl at 150 °C for 2 h in an
evacuated sealed tube. The content of the peptides in the powders
was estimated based on the amino acid analysis. Authentic hBD2
9b was purchased from Peptide Institute (Minoh, Japan).
Synthesis of Peptide 1
Starting from Fmoc-CLEAR amide resin (0.48 mmo/g, 0.1 mmol),
peptide chain was elongated by Liberty Blue to give Gly-Leu-Glu
(OBut)-Phe-Lys(Boc)-Ala-Gly-Arg(Pbf)-NH-CLEAR amide resin. Boc-
MeOGly-OPfp [10] (0.2mmol) in DMF was added to the resin, and
the mixture was vortexed for 30 min at room temperature. The
obtained resin was washed three times with NMP, CH2Cl2, ether,
respectively, and dried in vacuo to give protected peptide resin
(312 mg). The whole resin was treated with TFA containing 2.5%
of TIS and 2.5% water (3.0ml) for 2 h at room temperature. The
reaction mixture was filtered, and the filtrate was concentrated by
nitrogen stream. The product was precipitated by adding ether
and purified by RPHPLC to give peptide 1 (34 μmol, 34% yield).
ESI-MS, found: m/z 963.8, calcd: 963.5 for (M+H)+. Amino acid
Fmoc-Lys(Boc-MeOGly)-OH 5
Fmoc-Lys-OH hydrochloride (740 mg, 1.8mmol) and Boc-MeOGly-
OPfp (450 mg, 1.2 mmol) were dissolved in DMF-CH2Cl2 (1: 1,
10 ml), and DIEA (0.32 ml, 1.8 mmol) was added. After the reaction
mixture was stirred for 4 h at room temperature, the solvent was
removed in vacuo. The residue was dissolved in ethyl acetate,
successively washed with 1 M HCl, H2O and brine, and dried over
anhydrous Na2SO4. The solvent was evaporated, and the residue
was purified by silica gel chromatography using CHCl3-methanol-
acetic acid (95 : 5 : 1) to give compound 5 (670 mg, 1.2 mmol). Rf
0.19 (CHCl3-methanol-acetic acid 95 : 5 : 1). [α]D +9.86 (c 1.0,
CHCl3). Anal. calcd for C29H37N3O8 1/2CH3COOH: C, 61.53; H,
1
6.71; N, 7.18, found: C, 61.40; H, 6.41; N, 7.04. H-NMR (CDCl3) δ:
analysis: Glu0.98Gly2.13Ala1Leu1.00Phe0.99Lys1.02Arg1.00
.
7.73 (d, 2H, J = 7.5 Hz), 7.59 (m, 2H, Ar), 7.37 (t, 2H, J = 7.4 Hz,
Ar), 7.28 (t, 2H, J = 7.4 Hz, Ar), 6.48 (brt, 1H, J = 5.2 Hz, Lys εΝH),
5.79 (d, 1H, J = 8.0 Hz, Lys αNH), 4.48–4.35 (m, 3H, Ser αH, Fmoc
CH2-CH), 4.19 (t, 1H, J = 6.9 Hz, Fmoc CH2-CH), 4.11 (s, 2H, Gly
CH2), 3.68 (s, 3H, OCH3), 3.26 (m, 2H, Lys εH), 1.87 (brs, 1H, Lys βH),
1.74 (brs, 1H, Lys βH), 1.52 (m, 2H, Lys δH), 1.47 (s, 9H, t-Bu), 1.40
(m, 2H, Lys γH). 13C-NMR (CDCl3) d: 127.71 (Ar), 127.09 (Ar), 125.15
(Ar), 119.96 (Ar), 67.03 (Fmoc CH2-CH), 62.13 (OCH3), 53.60 (Lys
Cα), 52.76 (Gly CH2), 47.13 (Fmoc CH2-CH), 39.01 (Lys Cε), 31.70
(Lys Cβ), 28.89 (Lys Cδ), 28.17 (C(CH3)3), 22.21 (Lys Cγ).
Reaction of Peptide 1 with PITC
Peptide 1 (0.50 μmol) was dissolved in various buffers shown in
Scheme 1 (100 μl each), and PITC (1.2 μl, 10 μmol) was added. The
solution was kept at room temperature, and an aliquot of the
solution was analyzed by RPHPLC at appropriate reaction times.
Synthesis of hBD2
Synthesis of peptides was carried out using 433A Peptide Synthe-
sizer by the Fmoc method with HBTU/DIEA activation procedures
for preloaded Fmoc-Pro-TrtA-PEG resin (1.0g, 0.21 mmol/g)
(Watanabe Chemical Co., Hiroshima). The coupling of Boc-N-
methoxyglycine to the N-terminus or Fmoc-Lys(Boc-MeOGly)-OH
5 at Lys10 was carried out by pentafluorophenyl ester or
DIC/HOBt method, respectively. After the chain assembly, the resin
was treated with TFA–TIS-DODT–H2O (93/2.5/2.0/2.5) 10 ml/g resin
for 2 h. After the removal of TFA by a nitrogen stream, ether was
added to form a precipitate, which was washed three times with
ether and dried in vacuo. The crude peptide was purified by
RPHPLC to obtain the desired reduced form of hBD2. The folding
reaction was carried out in 0.1 M ammonium acetate (pH 7.8) in
the presence of GSH/GSSG (10 : 100 molar ratio to reduced form
of hBD2) and stirred for 24h at room temperature [12]. After the
purification by RPHPLC, the desired hBD2s were obtained.
Scheme 1. Reaction of nonapeptide with PITC in various buffers.
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SITE-SPECIFIC LABELING OF SYNTHETIC PEPTIDE
Lys(MeOGly)10-hBD-2 6; yield 6.4%, ESI-MS, obs: 4416.1
(deconvoluted), calcd: 4416.3 for (M+H)+ (average). Amino acid
analysis: Asp0.94Thr2.46Ser0.81Glu1.02Pro5.21 Gly6Ala1.01Val1.95Ile2.66
Results and Discussion
Modification of Model Peptide Carrying MeOGly at its
N-Terminus
Leu1.88Tyr0.92Phe0.94Lys4.74 His1.13Arg2.01
.
MeOGly-hBD2 7; yield 3.4% based on Pro content on the initial
resin, ESI-MS, obs: 4416.1 (deconvoluted), calcd: 4416.3 for (M+H)+
(average). Amino acid analysis: Asp0.97Thr2.51Ser0.84Glu1.01Pro5.21
In order to find the optimum condition for the specific labeling of N-
methoxyamino group, a nonapeptide carrying MeOGly at its N-
terminus, MeOGly-Gly-Leu-Glu-Phe-Lys-Ala-Gly-Arg-NH2, 1 was
used as a model. For the introduction of fluorescent probe, an
isothiocyanate [13] of corresponding fluorophore has often been
used. Thus, we first examined the reaction of peptide 1 with PITC
as the model reagent. The synthesis of the nonapeptide 1 was
carried out using the Fmoc SPPS. The terminal MeOGly was intro-
duced using corresponding pentafluorophenyl ester [10]. After
TFA deprotection and purification by RPHPLC, peptide 1 was
obtained in 34% yield.
Peptide 1 was then reacted with PITC (20 eq to peptide 1) as
shown in Scheme 1 in various buffers. Under highly basic condition
of buffer A, the starting peptide was almost consumed within 8 h
and gave peptide 3, in which both methoxyamino and ε-amino
groups were modified by PITC, as a major product (Figure 1). The
yield of peptide 3 decreased by decreasing the pH of the buffer,
while that of the desired product 2 increased from buffer A to C.
Still, the complete selectivity to peptide 2 was not attained under
the basic conditions. In contrast, under the acidic conditions (buffer
D and E), the complete selectivity to peptide 2 was achieved. The
coupling of PITC at the methoxyamino group in peptide 2 was
proved by the TFA treatment of peptide 3 for 10 min, which
removed the phenylthiocarbamoylmethoxyglycine residue at
the N-terminus and gave Gly-Leu-Glu-Phe-Lys-Ala-Gly-Arg-NH2
4 through the Edman degradation reaction (refer to experimental
Gly6Ala1.00Val1.91Ile2.74Leu1.91Tyr0.93Phe0.90 Lys4.82His0.96 Arg1.97
.
Selective Labeling of hBD2 with PITC
Synthetic hBD2 carrying MeOGly 6 or 7 was dissolved in 0.5 M
acetic acid in 80% CH3CN aq. at a concentration of 5 mM and PITC
(20 eq to hBD2) was added. After the solution was kept overnight at
room temperature, the reaction mixture was purified by RPHPLC to
obtain the desired PITC-labeled hBD2s.
Lys[PITC-MeOGly]10-hBD2 8; yield 71%, ESI-MS, obs: 4551.4
(deconvoluted), calcd: 4551.5 for (M+H)+(average). Amino acid
analysis: Asp0.97Thr2.57Ser0.87Glu1.05 Pro4.96Gly6 Ala0.98Val1.90
Ile2.73Leu1.93Tyr0.94Phe1.04Lys4.91His0.97Arg2.05
.
PITC-MeOGly-hBD2 10; yield 78%, ESI-MS, obs: 4551.4
(deconvoluted), calcd: 4551.5 for (M+H)+(average). Amino acid
analysis: Asp0.98Thr2.53Ser0.85Glu1.02Pro5.01 Gly6Ala0.98 Val1.94Ile2.72
Leu1.94Tyr0.94Phe1.00Lys4.89His0.96Arg1.98
.
TFA Treatment of PITC-Labeled hBD2 8 and 10
Peptide 8 or 10 was dissolved in 95% aq TFA at a concentration of
1 mM, and the solution was kept at room temperature for 10 min.
After the solution was concentrated by nitrogen stream, the residue
was precipitated by ether and purified by RPHPLC to obtain native
hBD2.
hBD2 (from peptide 8) 9a; yield 70%, ESI-MS, obs: 4329.4
(deconvoluted), calcd: 4329.2 for (M+H)+ (average). Amino acid
analysis: Asp1.01Thr2.69Ser0.87Glu1.04Pro5.22 Gly6Ala1.03Val2.02Ile2.86
Leu2.02Tyr0.99Phe1.15Lys4.95His0.96Arg2.03
.
hBD2 (from peptide 10) 9c; yield 82%, ESI-MS, obs: 4328.8
(deconvoluted), calcd: 4329.2 for (M+H)+ (average). Amino acid
analysis: Asp1.02Thr2.64Ser0.83Glu1.03Pro5.01 Gly6Ala1.02Val1.99Ile2.82
Leu1.99Tyr0.96Phe1.15Lys4.94His0.98Arg1.94
.
Selective Labeling of hBD2 by FITC
Synthetic hBD2 carrying MeOGly 6 or 7 was dissolved in 80%
CH3CN aq containing 5 mM acetic acid, and 20 eq of FITC dissolved
in DMF was added. After the solution was kept room temperature
for 8 h, CH3CN was briefly removed by a N2 stream. Ether was added
to extract a part of the FITC, and the aqueous phase was purified by
RPHPLC to give FITC-labeled hBD2.
Lys[FITC-MeOGly]10-hBD2 11; yield 72%, ESI-MS, obs: 4805.5
(deconvoluted), calcd: 4805.7 for (M+H)+(average). Amino acid
analysis: Asp0.98Thr2.50Ser0.87Glu1.09 Pro5.37 Gly6Ala1.01Val1.95Ile2.71
Leu1.92Tyr0.97Phe1.47Lys4.89His0.93Arg1.97
.
FITC-MeOGly-hBD2 12; yield 75%, ESI-MS, obs: 4805.5
(deconvoluted), calcd: 4805.7 for (M+H)+(average). Amino acid
analysis: Asp0.98Thr2.48Ser0.85Glu1.05Pro5.17 Gly6Ala1.00Val1.95Ile2.73
Leu1.93Tyr0.96Phe1.33Lys4.88His0.92Arg1.92
.
Figure 1. HPLC profiles of the reaction of peptide 1 with PITC in various
buffers shown in Scheme 1: 1) buffer A, 2) buffer B, 3) buffer C, 4) buffer D,
and 5) buffer E after overnight. Elution conditions: column, Cosmosil 5C18-
1H-NMR Measurement of hBD2
AR-II (4.6 × 150 mm, Nacalai Tesque, Japan) at a flow rate of 1 ml minÀ1
;
hBD2s were dissolved in 5 mM sodium acetate (pH 6.0) containing
10% D2O at a concentration of 1 mg/ml. The spectrum was
obtained suppressing the signal of water by presaturation method.
eluent, A, 0.1% aqueous TFA, B, 0.1% TFA in acetonitrile. Asterisked peaks
are nonpeptides.
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HARA ET AL.
section). If the ε-amino group was modified by PITC, the structure of
the peptide would not have been altered. These data demonstrate
the high chemoselectivity between the isothiocyanate and the
methoxyamino group under acidic conditions.
(MeOGly)10-hBD2 6 in 6.4% yield after RPHPLC purification. As a
same manner, the hBD2 carrying MeOGly at its N-terminus 7 was
also obtained in 3.4% yield.
The site-specific labeling by PITC was performed in buffer D of
Scheme 1, which retains sufficient specificity to methoxyamino
group with milder acidity compared to buffer E. As shown in
Figure 2, the reaction proceeded efficiently, giving the desired Lys
[PITC-MeOGly]10-hBD-2 8 after overnight reaction. To confirm
whether the Lys10 was specifically labeled without disrupting
tertiary structure, peptide 2 was treated with 95% TFA-H2O for
10 min and the obtained native hBD2 9a was compared on RPHPLC
and 1H-NMR with the authentic hBD2 9b. As shown in Figure 2 4)
and 5), both hBD2 showed the same retention time on RPHPLC. In
Application to hBD2
Defensins, diverse members of a large family of cationic host de-
fense peptides, are cysteine-rich peptides and vary in their length,
the spacing of their cysteine residues, and their disulfide connectiv-
ity [14,15]. In this research, human hBD2 [16] was chosen as a model
regarding to properties of having multiple disulfide bonds and
lysine residues.
Based on the results in Figure 1, specific modification of the N-
terminal and side chain amino group of Lys10 of hBD2 was per-
formed. In the tertiary structure, Lys10 points outward and locates
at the end of the helix, which would not cause structural change
even after the modification [17]. Scheme 2 shows the procedure
in the case of the Lys10 modification. Starting from Fmoc-Pro resin,
the machine-assisted chain elongation was carried out. Lys10 was
introduced using Fmoc-Lys(Boc-MeOGly)-OH 5 by DIC-HOBt
method. After completion of the chain assembly, the peptide was
deprotected by TFA and purified by RPHPLC. The obtained reduced
form of hBD2 was then oxidized in the presence of reduced and
oxidized form of glutathione (GSH and GSSG) according to the re-
ported procedure [12] for overnight to obtain the desired Lys
1
addition, they showed quite similar H-NMR spectrum (Figure 3),
Figure 2. HPLC profiles of the reaction of [Lys(MeOGly)9]-hBD2 7 and [Lys
(MeO(PITC)Gly)9]-hBD2 8: 1) 7 with PITC 0 min, 2) 30 min, 3) overnight, 4) 8
with TFA 10 min, 5) authentic sample of hBD2 9b, and 6) 7 with FITC 4 h.
Elution conditions: column, Cosmosil 5C18-AR-II (4.6 × 150 mm, Nacalai
Tesque, Japan) at a flow rate of 1 ml minÀ1; eluent, A, 0.1% aqueous TFA,
B, 0.1% TFA in acetonitrile.
Scheme 2. Synthetic route of PITC-modified hBD2 9a.
Figure 3. 1H-NMR spectra of hBD2 9a and authentic hBD2 9b.
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J. Pept. Sci. 2015; 21: 765–769

SITE-SPECIFIC LABELING OF SYNTHETIC PEPTIDE
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demonstrating that the specific labeling was realized without
disrupting its tertiary structure.
Finally, the specific labeling of hBD2 carrying MeOGly with fluo-
rescent dye, FITC, was performed under the same conditions as in
the case of PITC labeling (see experimental and supporting infor-
mation). The reaction also proceeded specifically and efficiently
giving the desired FITC-labeled hBD2s in comparable yields as
those of PITC-labeled ones. 1H-NMR spectrum (supporting informa-
tion) showed that the FITC-labeling does not disturb the tertiary
structure of hBD2, as the signals were well dispersed as in the case
of PITC-labeling, indicating the success of specific labeling. These
FITC-labeled peptides were stable during RPHPLC purification using
aqueous acetonitrile containing 0.1% TFA as an eluent and also
stable to neutral phosphate buffer. These peptides will be used
for the analysis of the interaction between hBD2 and biological
membrane as well as for the functional analysis of hBD2.
Conclusions
The result in this study shows that if MeOGly residue is introduced
during the SPPS, the obtained peptide can be specifically modified
by isothiocyanate reagent at the N-methoxyamino group. From the
results of PITC and FITC modification of hBD2, it is clear that the
peptides with multiple disulfide-bonds can be selectively modified
without disrupting their tertiary structure. In principle, this method
realizes that peptides with various modifications and fluorescent
probes can be synthesized from a single peptide with MeOGly res-
idue, which will be valuable for the functional analysis of various
bioactive peptides.
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utilization of fluorescent synthetic peptides. Biochim. Biophys. Acta
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structural properties. Curr. Pharm. Des. 2007; 13: 3096–3118.
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Acknowledgement
We thank Dr Ayako Sato of A Rabbit Science Japan Co. Ltd. for the
elemental analysis.
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