Solid-phase synthesis of the B-chain of human α2HS glycoprotein

Article abstract of DOI:10.1016/S0008-6215(98)00142-6

The B-chain of human α2HS glycoprotein 1, a heptacosapeptide carrying a trisaccharide (sialyl T) side chain, was synthesized. Prior to the Fmoc-based solid-phase synthesis of the glycopeptide, the benzyl-protected glycosyl serine building block 6 was prep

Full text of DOI:10.1016/S0008-6215(98)00142-6

Carbohydrate Research 309 (1998) 287±296
Solid-phase synthesis of the B-chain of human
ꢀ2HS glycoprotein
Yoshiaki Nakaharaa,*, Yuko Nakaharab, Yukishige Itob,
Tomoya Ogawab
a Department of Industrial Chemistry, Tokai University, Kitakaname 1117, Hiratsuka, Kanagawa,
259-1292, Japan
b The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama,
351-0198, Japan
Received 24 March 1998; accepted 25 May 1998
Abstract
The B-chain of human ꢀ2HS glycoprotein 1, a heptacosapeptide carrying a trisaccharide (sialyl
T) side chain, was synthesized. Prior to the Fmoc-based solid-phase synthesis of the glycopeptide,
the benzyl-protected glycosyl serine building block 6 was prepared via ꢁ-stereoselective glycosyla-
tion of the 2-azido-2-deoxygalactosyl serine 11 with the sialyl galactosyl trichloroacetimidate 9. An
automated peptide synthesizer was eciently used for the elongation of the entire peptide chain
except for the coupling with 6. The synthesized glycopeptide was cleaved from the resin by the
TFA method. The resultant mixture of the benzylated glycopeptides was treated with TMSOTf±
thioanisole in TFA and then with aq NaHCO3 and 1,4-dithiothreitol to give 1. # 1998 Elsevier
Science Ltd. All rights reserved
Keywords: ꢀ2HS glycoprotein; Glycopeptide synthesis; Solid-phase synthesis
1. Introduction
syntheses of glycopeptides, especially those posses-
sing sialic acid as a component of the oligo-
saccharide side chains, and we have accomplished
the synthesis of a sialooligosaccharide-clustering
fragment of human glycophorin Am, utilizing
solution-phase Fmoc peptide chemistry based on
the benzyl protecting-group strategy [4]. Since it
was of great interest to apply the strategy to the
solid-phase synthesis of a glycopeptide with a
longer peptide back bone, B-chain of ꢀ2HS-glyco-
protein was next chosen as a synthetic target.
There has been a growing demand for chemically
or chemoenzymatically synthesized samples of N-
and O-glycans, and their peptide conjugates as
useful probes to obtain insight into the glycopro-
tein-mediated biologial reactions [1]. Despite the
current developments of the technologies in this
®eld [2], there are only a few papers reporting the
synthesis of large oligopeptides carrying a complex
carbohydrate side chain [3]. We have been studying
ꢀ2HS-Glycoprotein is a normal human plasma
globulin involved in a variety of signi®cant biologi-
cal events, such as bone mineralization, endocytosis,
* Corresponding author. Fax: 81-463-50-2075; e-mail:
yonak@postman.riken.go.jp
0008-6215/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00142-6

288
Y. Nakahara et al./Carbohydrate Research 309 (1998) 287±296
and opsonization [5]. Decreases in its plasma con-
centration are frequently correlated with malignant
diseases. The glycoprotein is composed of two
polypeptide subunits, the structures of which have
been well established and include the inter- and the
intra-chain disul®de linkages as well as the
attached N- and O-linked oligosaccharide side-
chains [6].
of Pd(0) [11] to a carboxylic acid 5 (95%). Desul-
furization of 5 was performed according to the
previously established procedure using Ph3SnH in
re¯uxing benzene [12]. Under these conditions,
compound 5 was readily lactonized, but not
smoothly desulfurized. The desired product 6 was
isolated only in 28% yield due to formation of the
polar byproducts (Scheme 1).
B-Chain, the minor subunit split from ꢀ2HS-
glycoprotein, was characterized as a heptacosa-
peptide carrying a sialotrisaccharide (1) as depicted
in Fig. 1. In a preliminary investigation using the
solid-phase technique, we have synthesized the
model glycopeptide of B-chain, an asialo-[Ala18]-
analog, in which we omitted the labile cysteine and
sialic acid residues [7]. The successful utilization of
solid-phase synthesis led us to adopt similar tactics
for the native B-chain of ꢀ2HS-glycoprotein. This
paper describes the full details of the investigation.
Preliminary accounts of this work have appeared
earlier [8].
This unsatisfactory result led us to search for a
more practical synthetic route to the compound 6.
To this end, we investigated a new route involving
glycosylation of a 2-azidogalactosyl serine deriva-
tive with a sialyl galactose unit, since compound 7
(Scheme 2) related to the latter had previously been
synthesized through a smooth desulfurization of
the corresponding 3-phenylthio derivative followed
by lactonization [4]. Oxidative cleavage of 4-
methoxyphenyl group from 7 with CAN (ceric
ammonium nitrate) a€orded an anomeric mixture
of hemiacetal 8 (77%), which was converted into
the corresponding trichloroacetimidate 9 by treat-
ment with trichloroacetonitrile and DBU (1,8-dia-
zabicyclo[5.4.0]undec-7-ene) in 1,2-dichloroethane.
The imidate 9 was thus obtained as an ꢀ:ꢁ mixture
(ꢀ:ꢁ=3:1) in 97% yield. On the other hand, com-
pound 10 [13] and its sterically less-hindered ana-
logue 11 were chosen as the glycosyl acceptors, the
latter being prepared in two steps (1. debenzylide-
nation, 2. regioselective silylation, 88%) from 10.
Glycosylation of 11 (3 equiv) with 9 (ꢀ:ꢁ=3:1) was
2. Results and discussion
In the course of the studies on the oligosacchar-
ide fragment of glycophorin AM, we have synthe-
sized as an intermediate a trisaccharide-linked
serine derivative 2 [9] that was thought to be read-
ily converted into the building block 6 suitable for
the solid-phase synthesis of the B-chain glycopep-
tide. Benzylidenation of 2, followed by reductive
conversion of the azide functionality into acet-
amide with thioacetic acid±pyridine [10], gave 4
(72%, 2 steps), which was deallylated by catalysis
promoted by BF3 OEt2 (0.3 equiv) at 15ꢀ±5 ꢀC in
.
2:1 toluene±CH2Cl2 to give the ꢁ-glycoside 12
(53%) and the ꢀ-isomer 13 (16%). A glycosyl
¯uoride 14 (ꢀ:ꢁ=1:1) was also produced in 10%
yield. The 1:3 ꢀ:ꢁ-isomeric ratio of the glycosyl-
ation products seemed to suggest that the glycosy-
lation might proceed via inversion of the
stereoisomeric trichloroacetimidate [14]. However,
the ꢀ-isomer 13 (7%) was formed (ꢀ:ꢁ=1:8), even
when the same reaction was carried out with the
pure ꢀ-trichloroacetimidate 9. The use of TMSOTf
as an alternative promoter for the above reaction
was also examined in toluene±CH2Cl2 or in
CH3CN, but no coupling product was produced.
The compound 12 was desilylated with 80% aq
CF3CO2H (15, 88%) and benzylidenated with 1,1-
dimethoxytoluene and p-TsOH in acetonitrile to
give 16 (95%).
In contrast, the reaction of ꢀ-9 and 10 (3 equiv)
predominated the formation of ꢀ-glycoside 18
(ꢀ:ꢁ=4.6:1) probably due to a mismatch of the
substrate pair for ꢁ-glycosidation [15].
Fig. 1. Structure of the B-chain of human ꢀ2HS glycoprotein.

Y. Nakahara et al./Carbohydrate Research 309 (1998) 287±296
289
Scheme 1.
Treatment of 16 with thioacetic acid±pyridine
(17, 77%) and Pd(0)-catalyzed cleavage of the allyl
ester a€orded the desired compound 6 (91%).
Solid-phase synthesis of the glycopeptide based
on the strategy employed in a previous study was
performed [7]. According to the ready-made pro-
tocol, an Fmoc protected henicosapeptide (residues
7±27) was synthesized on HMP resin (4-hydroxy-
methylphenoxymethyl-copolystylene-1% divinyl-
benzene) with an automated peptide synthesizer.
In the program the Fmoc-amino acids were acti-
vated with DCC (dicyclohexylcarbodiimide)±HOBt
(hydroxybenztriazole) in NMP (N-methylpyrrolid-
one) before condensation, while N-terminal Fmoc
group was removed with piperidine in NMP. The
side-chain functional groups of the amino acids
were masked with Trt (triphenylmethyl) groups for
cysteine, glutamine, and histidine, Boc (tert-butox-
ycarbonyl) group for lysine, and Pmc (2,2,5,7,8-
pentamethylchroman-6-sulfonyl) group for argi-
nine. These protecting groups were concomitantly
removed at the stage of TFA treatment.
The henicosapeptide-linked resin was obtained
after twenty condensations. The eciency of
condensation in each step was monitored by nin-
hydrin test, and the overall yield thus estimated
was 88%.
In a model study, we employed a mechanical
shaker only at the coupling step with the dis-
accharide±serine, to facilitate the recovery of
unreacted glycoserine [7]. However, the glycoserine
unit-deleting hexacosapeptide was eventually
formed in a substantial quantity (45%), indicating
that the coupling was incomplete even after 64 h of
shaking. The unreacted glycoserine was recovered
from the reaction mixture in a reasonable amount.
Based on this result, we were convinced that more
ecient mixing of the resin with the activated gly-
coserine would be necessary to increase the cou-
pling yield.
In this study, we utilized a vortexing tube-mixer
which provided more vigorous mixing. The pep-
tide-resin (14.9 ꢂmol) was treated with piperidine
to remove the Fmoc group and reacted with the
activated trisaccharide±serine (6, 38.3 ꢂmol) in
NMP using the mixer. The reaction was run for
24 h, the resin was then washed and transfered to
the automated peptide synthesizer to complete the
peptide chain with the ®ve N-terminal amino acid
residues. The resulting glycopeptide was cleaved
from the resin with a solution of aq TFA contain-
ing alkyl cation scavengers. Reversed-phase HPLC
showed that the glycopeptide thus obtained con-
sisted of three major products (in 14, 17, and 52%).
MALDI-TOF mass spectra indicated the third
product to be the glycopeptide with expected
molecular weight ((M+1)+, 3920), whereas the
other two were its mono-debenzylated congeners
((M+1)+, 3830). In the minor fractions were also
detected di-debenzylated glycopeptides (5%).
In contrast to the previous experiment [7] little of
the non-glycosylated peptide (hexacosapeptide)

290
Y. Nakahara et al./Carbohydrate Research 309 (1998) 287±296
Scheme 2.
was produced. The combined benzylated glyco-
peptides were treated with 1 M TMSOTf/TFA [16]
in the presence of thioanisole at 0 C for 1.5 h and
6756). The second fraction was the monomer
(3363 [M+1]+, calcd 3380). Those fractions were
further puri®ed by reversed-phase HPLC. The
NMR spectrum of the dimeric product exhibited,
not only the characteristic lower ®eld (ꢃ 5.30 ppm)
signal for the 4-O-acylated (lactonized) Gal H-4,
but also the two doublet-of-doublet signals for the
equatorial protons of NeuAc H-3 at ꢃ 2.58 and
2.75 ppm (ratio about 1:1). Therefore, it was con-
cluded that 50% of the molecule kept the lactonic
linkage, but the other half had been hydrolyzed
ꢀ
then with aq ammonium ¯uoride to realize the
complete deprotection, although hydrogenolytic
deprotection was unsuccessful because of simulta-
neous desulfurization of the cysteine residue. The
debenzylated products were separated as two frac-
tions (58:42) by gel-permeation chromatography.
The ®rst fraction consisted of the dimeric product
with the molecular ion of 6760 ([M+1]+, calcd

Y. Nakahara et al./Carbohydrate Research 309 (1998) 287±296
291
during a series of deprotective procedures. On the
other hand, the monomeric fraction had no such
signals characteristic for the lactone structure, and
was the target compound 1. The dimer was treated
with NaHCO3 in D2O (pH 7.5) for 3 days, then
with dithiothreitol overnight, and chromato-
graphed (gel-permeation) to a€ord the compound
1 (85% yield for deprotection). The structure was
and p-TsOH (cat.) in dry CH3CN (5 mL) was stir-
red at room temperature for 1 h. The reaction was
quenched with aq NaHCO3, and the mixture was
concentrated in vacuo. The residue was extracted
with 1:1 ether±EtOAc, washed with water and
brine, dried (Na2SO4), and concentrated in vacuo.
The crude product was chromatographed on silica
gel with 7:3 toluene±EtOAc to give 3 (95 mg, 85%).
Rf 0.47 (7:3 toluene±EtOAc); [ꢀ]d +69.9ꢀ (c 1.0);
1H NMR (270 MHz): ꢃ 7.7±7.1 (m, 53 H, Ar), 6.01
(d, 1 H, J 8.2 Hz, NH), 5.88 (m, 1 H, -CH=CH2),
5.38 [s, 1 H, PhCH(O)2], 5.31 (brd, 1 H, J 17.0 Hz,
=CH2), 5.23 ( brd, 1H, J 10.4 Hz, =CH2), 5.19
and 5.01 (2d, 2 H, J 12.2 Hz, -CO2CH2Ph), 4.99 (d,
1 H, J 3.1 Hz, H-1a), 4.85 (d, 1 H, J 11.3 Hz,
PhCH2), 3.35 (d, 1 H, J 8.9 Hz, H-3c), 1.57 (s, 3 H,
1
established by H NMR and ESI mass spectral
data (see Experimental section). It is noteworthy
that the glycopeptide 1 is readily oxidized to form
the dimer in the absence of antioxidizing agent.
In conclusion, total synthesis of B-chain of
ꢀ2HS-glycoprotein was achieved by solid-phase
synthesis. TFA-based conditions for cleavage of
the synthesized glycopeptide from the resin was
accompanied by partial debenzylation (ꢁ30%).
Full deprotection was successfully performed with
TMSOTf±thioanisole in TFA. The lactone hydro-
lysis and reduction with dithiothreitol a€orded the
target compound 1 in good yield.
.
Ac); Anal. Calcd for C106H107N5O22S H2O: C,
68.70; H, 5.93; N, 3.78. Found: C, 68.50; H, 5.83;
N, 3.51.
N-(9-Fluorenylmethoxycarbonyl)-O-{[benzyl (5-
acetamido-4,7,8,9-tetra-O-benzyl-5-deoxy-3-S-phenyl-
3-thio-d-erythro-a-l-gluco-2-nonulopyranosyl)onate]-
(2!3)-(2,6-di-O-benzyl-b-d-galactopyranosyl)-(1!3) -
2-acetamido-4,6-O-benzylidene-2-deoxy-a-d-galacto-
pyranosyl}-l-serine allyl ester (4). To a solution of
3 (84 mg, 46 ꢂmol) in pyridine (1.2 mL) was added
freshly distilled AcSH (2.4 mL). The mixture was
stirred at room temperature for 1 day, then con-
centrated in vacuo, and the residue was chromato-
graphed on silica gel with 3:2 toluene±EtOAc to
give 4 (72 mg, 85%). Rf 0.40 (1:1 toluene±EtOAc);
[ꢀ]d +57.9ꢀ (c 1.0); 1H NMR (270 MHz): ꢃ 7.7±7.1
(m, 53 H, Ar), 6.07 (d, 1 H, J 8.3 Hz, NH), 5.86 (m,
1 H, -CH=CH2), 5.57 (d, 1 H, J 6.9 Hz, NH), 5.38
[s, 1 H, PhCH(O)2], 5.29 (brd, 1 H, J 18.2 Hz,
3. Experimental
General.ÐOptical rotations were determined
with a Jasco DIP-370 polarimeter for solutions in
CHCl3, unless noted otherwise. Column chroma-
tography was performed on Silica Gel-60 (E.
Merck 70±230 mesh or 230±400 mesh). TLC and
HPTLC were performed on Silica Gel 60 F254 (E.
1
Merck). H and 13C NMR spectra were recorded
with either a JEOL ꢀ600 [1H (600 MHz)] or EX270
[1H (270 MHz), 13C (68 MHz)] spectrometer. Che-
mical shifts are expressed in ppm down®eld from
the signal for internal Me4Si for solutions in
CDCl3. MALDI-TOF mass spectra were obtained
with a Bruker REFLEX (using 2,5-dihydrox-
ybenzoic acid as matrix). ESI mass spectra were
measured with a Finnigan MAT TSQ 700. Peptide
synthesis was performed with an Applied Biosys-
tems Model 431A peptide synthesizer. Fmoc Val-
preloaded HMP resin, Fmoc amino acids in car-
tridges, and the reagents for the peptide synthesis
were purchased from Applied Biosystems, Inc.
N-(9-Fluorenylmethoxycarbonyl)-O-{[benzyl (5-
acetamido-4,7,8,9-tetra-O-benzyl-5-deoxy-3-S-phenyl-
3-thio-d-erythro-a-l-gluco-2-nonulopyranosyl)onate]-
(2!3)-(2,6-di-O-benzyl-b-d-galactopyranosyl)-(1!3)-
2-azido-4,6-O-benzylidene-2-deoxy-a-d-galactopyr-
anosyl}-l-serine allyl ester (3). A mixture of 2
(107 mg, 61 ꢂmol), 1,1-dimethoxytoluene (100 mL)
1
=CH2), 5.23 ( brd, H, J 10.6 Hz, =CH2), 5.15
(brs, 1 H, H-1a), 5.12 and 5.00 (2d, 2 H, J 11.9 Hz,
-CO2CH2Ph), 3.39 (d, 1 H, J 8.3 Hz, H-3c), 1.59
and 1.37 (2s, 6 H, 2 Ac); Anal. Calcd for
C108H111N3O23S: C, 70.08; H, 6.04; N, 2.27.
Found: C, 69.70; H, 6.05; N, 2.17.
N-(9-Fluorenylmethoxycarbonyl)-O-{[benzyl (5-
acetamido-4,7,8,9-tetra-O-benzyl-5-deoxy-3-S-phenyl-
3-thio-d-erythro-a-l-gluco-2-nonulopyranosyl)onate]-
(2 !3)-(2,6-di-O-benzyl-b-d-galactopyranosyl)-(1!3)-
2-acetamido-4,6-O-benzylidene-2-deoxy-a-d-galacto-
pyranosyl}-l-serine (5). A mixture of 4 (67 mg,
36 ꢂmol), Pd(PPh3)4 (22 mg, 19 ꢂmol), and N-
methylaniline (220 ꢂL, 2 mmol) in dry THF
(1.5 mL) was stirred under Ar at room temperature
for 1 day and concentrated in vacuo. The residue
was extracted with EtOAc, washed with 0.1 N aq

292
Y. Nakahara et al./Carbohydrate Research 309 (1998) 287±296
HCl at pH 3, water, and brine, dried (Na2SO4),
and concentrated in vacuo. The crude product was
chromatographed on silica gel with 96.5:3:0.5
CHCl3±EtOH±AcOH to give 5 (62 mg, 95%). Rf
0.26 (92.5:7:0.5 CHCl3±EtOH±AcOH); [ꢀ]d +55.3ꢀ
(s, 3 H, Ac). ꢁ-9; Rf 0.43 (7:3 toluene±EtOAc); [ꢀ]d
+23.5ꢀ (c 1.2); 1H NMR (270 MHz): ꢃ 8.70 (s, 1 H,
=NH), 7.4±7.1 (m, 30 H, Ar), 5.59 (d, 1 H, J
7.6 Hz, H-1a), 5.28 (d, 1 H, J 4.0 Hz, H-4a), 2.27
(dd, 1 H, J 5.3, 13.5 Hz, H-3b eq), 1.74 (s, 3 H, Ac).
Anal. (ꢀ:ꢁ-imidate) Calcd for C61H63Cl3N2O13: C,
64.35; H, 5.58; N, 2.46. Found: C, 64.10; H, 5.63;
N, 2.41.
1
(c 0.8); H NMR (270 MHz): ꢃ 7.7±7.1 (m, 53 H,
Ar), 6.07 (br, 2 H, 2 NH), 5.30 [s, 1 H, PhCH(O)2],
5.13 (brs, 1 H, H-1a), 5.13 and 5.00 (2d, 2 H, J
11.9 Hz, -CO2CH2Ph), 3.39 (d, 1 H, J 7.9 Hz, H-
3c), 1.58 and 1.45 (2s, 6 H, 2 Ac); Anal. Calcd for
C105H107N3O23S: C, 69.64; H, 5.96; N, 2.32.
Found: C, 69.58; H, 5.92; N, 2.06.
N-(9-Fluorenylmethoxycarbonyl)-O-(2-azido-6-
O-tert-butyldimethylsilyl-2-deoxy-a-d-galactopyr-
anosyl)-l-serine allyl ester (11). A mixture of 10
(265 mg, 0.41 mmol), 80% aq CF3CO2H (3.5 mL),
ꢀ
5-Acetamido-4,7,8,9-tetra-O-benzyl-3,5-dideoxy-
d-glycero-a-d-galacto-2-nonulopyranosylonic acid-
(2!3)-2,6-di-O-benzyl-d-galactopyranose-(1b!4a)-
lactone (8). A mixture of 7 (235 mg, 0.21 mmol)
and CAN (1.2 g, 2.19 mmol) in 3:4:3 toluene±
CH3CN±H2O (54 mL) was stirred at room tem-
perature for 2.5 h. The organic layer was separated,
the aqueous layer was diluted with water and
extracted with EtOAc, the organic layer and the
extract were combined, washed successively with
water, dil aq NaHCO3, and brine, dried (Na2SO4),
and concentrated in vacuo. Chromatography of
the crude product on silica gel with 4:1±3:2
toluene±EtOAc gave 8 (163 mg, 77%) as an
anomeric mixture. Rf 0.28 and 0.26 (7:3 toluene±
and CH2Cl2 (1 mL) was stirred at 0 C for 3 h,
diluted with water and toluene, and concentrated
in vacuo. The residue was extracted with EtOAc,
washed with water and brine, dried (Na2SO4), and
concentrated in vacuo. The crude product was
puri®ed on a short column of silica gel with 19:1
CHCl3±MeOH to a€ord a triol (222 mg) that was
dissolved in dry DMF (3.8 mL) and stirred with
tert-BuMe2SiCl (78 mg, 0.52 mmol) and imidazole
(70 mg, 1.04 mmol) at room temperature for 1 h.
The reaction was quenched with water and the
mixture was concentrated in vacuo. The residue
was extracted with 1:1 ether±EtOAc, washed with
water and brine, dried (Na2SO4), and concentrated
in vacuo. The product was chromatographed on
silica gel with 13:7 toulene EtOAc to give 11
(244 mg, 88%). Rf 0.53 (1:1 toluene±EtOAc); [ꢀ]d
1
EtOAc); H NMR (270 MHz): ꢃ 7.4±7.1 (m, 30 H,
Ar), 5.26 [d, 0.6 H, J 4.0 Hz, H-1a (ꢀ)], 5.16 (d, 1
H, J 3.6 Hz, H-4a), 2.35 [dd, 0.6 H, J 5.3, 13.5 Hz,
H-3b eq (ꢀ)], 2.26 [dd, 0.4 H, J 5.0, 13.2 Hz, H-3b
eq (ꢁ)], 1.80 (m, 1 H, H-3b ax), 1.74 (ꢀ) and 1.72
(ꢁ) (2s, 3 H, Ac); Anal. Calcd for C59H63-
+74.3ꢀ (c 1.9); H NMR (270 MHz): ꢃ 7.76 (d, 2
1
H, Ar), 7.58 (m, 2 H, Ar), 7.40 (t, 2 H, J 7.3 Hz,
Ar), 7.31 (t, 2 H, J 7.3 Hz, Ar), 5.93 (m, 1 H, -
CH=CH2), 5.89 (d, 1 H, J 7.9 Hz, NH), 5.35 (brd,
1 H, J 17.2 Hz, =CH2), 5.27 (brd, 1 H, J 10.6 Hz,
=CH2), 4.91 (d, 1 H, J 3.3 Hz, H-1), 3.53 (dd, 1 H,
J 3.3, 10.2 Hz, H-2), 0.87 (s, 9 H, t-Bu) , 0.06 (s, 6
H, 2 Me). Combustion analysis of 11 did not give
the correct CHN values because of decomposition
at 80 ꢀC.
N-(9-Fluorenylmethoxycarbonyl)-O-[(5-acetamido-
4,7,8,9-tetra-O-benzyl-3,5-dideoxy-d-glycero-a-d-
galacto-2-nonulopyranosylonic acid)-(2!3)-(2,6-
di-O-benzyl-b- and a-d-galactopyranosyl)-(1!3)-
2-azido-6-O-tert-butyldimethylsilyl-2-deoxy-a-d-gal-
actopyranosyl-(1c!4b)-lactone]-l-serine allyl ester
(12) and its a anomer (13). A mixture of 9
(ꢀ:ꢁ=3:1, 47 mg, 41.3 ꢂmol), 11 (92 mg, 137.6 ꢂmol,
3.3 eq), and dried molecular sieves (AW 400 pow-
der, 0.7 g) in dry 2:1 toluene±CH2Cl2 (3 mL) was
stirred under Ar at room temperature for 1 h, and
then cooled on an ice±MeOH bath. To the mixture
.
NO13 0.5H2O: C, 70.64; H, 6.43; N, 1.40. Found:
C, 70.82; H, 6.34; N, 1.33.
5-Acetamido-4,7,8,9-tetra-O-benzyl-3,5-dideoxy-
d-glycero-a-d-galacto-2-nonulopyranosylonic acid-
(2!3)-2,6-di-O-benzyl-a- and b-d-galactopyrano-
syl trichloroacetimidate-(1b!4a)-lactone (9). To a
stirred mixture of 8 (160 mg, 0.16 mmol) and DBU
(2.8 ꢂL, 18.7 ꢂmol) in dry 1,2-dichloroethane
(2.8 mL), was added CCl3CN (170 ꢂL, 1.7 mmol)
ꢀ
at 0 C. After stirring for 2 h, the mixture was
chromatographed on silica gel with 9:1±17:3
toluene±EtOAc to give 9 as three fractions of pure
ꢀ-imidate, ꢀ:ꢁ-mixture and ꢁ-imidate (100, 60, and
17 mg, respectively, 97%). ꢀ-9; Rf 0.47 (7:3
toluene±EtOAc); [ꢀ]d +44.6ꢀ (c 1.1); H NMR
1
(270 MHz): ꢃ 8.61 (s, 1 H, =NH), 7.4±7.1 (m, 30
H, Ar), 6.46 (d, 1 H, J 3.6 Hz, H-1a), 5.41 (d, 1 H,
J 3.0 Hz, H-4a), 2.34 (dd, 1 H, J 4.9, 13.5 Hz, H-3b
eq), 1.84 (dd, 1 H, J 10.5, 13.5 Hz, H-3b ax), 1.75
.
was added 0.8 M BF3 OEt2±CH2Cl2 (17 ꢂL,

Y. Nakahara et al./Carbohydrate Research 309 (1998) 287±296
293
ꢀ
ꢀ
13.6 ꢂmol) at 15 C, and stirring was continued
was stirred at 0 C for 45 min, then diluted with
water, neutralized with NaHCO3, and extracted
with EtOAc. The extract was washed with water
and brine, dried (Na2SO4), and concentrated in
vacuo. Chromatography of the residue on silica gel
with 3:2 toluene±EtOAc gave 15 (24 mg, 88%). Rf
at 15 to 5 ꢀC for 1.5 h. The mixture was diluted
with EtOAc, and ®ltered through Celite. The ®l-
trate was washed with dil aq NaHCO3, water, and
brine, dried (Na2SO4), and concentrated in vacuo.
The residue was chromatographed on Bio-beads
SÂ1 with 1:1 toluene±EtOAc to a€ord a fraction
(55 mg) containing 12 and 13. Further elution gave
a fraction (82 mg) containing unreacted 11 and 14.
The former fraction was puri®ed by preparative
TLC developed 6 times with 3:2 hexane EtOAc to
give 13 (11 mg, 16%) and 12 (36 mg, 53%). From
the latter, ¯uoride 14 (ꢀ:ꢁ=1:1, 4 mg, 10%) and 11
(72 mg, 78% recovery) were obtained. Compound
12: Rf 0.43 (7:3 toluene±EtOAc); [ꢀ]d +58.7ꢀ (c
2.2); 1H NMR (270 MHz): ꢃ 7.75 (d, 2 H, J 7.3 Hz,
Ar), 7.61 (d, 2 H, J 7.3 Hz, Ar), 7.4±7.1 (m, 34 H,
Ar), 5.93 (m, 1 H, -CH=CH2), 5.83 (d, 1 H, J
8.6 Hz, NH), 5.34 (brd, 1 H, J 17.2 Hz, =CH2),
5.26 (brd, 1 H, J 10.2 Hz, =CH2), 5.21 (d, 1 H, J
4.0 Hz, H-4b), 4.95 (d, 1 H, J 3.3 Hz, H-1a), 2.18
(dd, 1 H, J 4.6, 11.9 Hz, H-3c eq), 1.71 (s, 3 H, Ac),
0.85 (s, 9 H, t-Bu), 0.03 and 0.02 (2 s, 6 H, 2 Me);
13C NMR (68 MHz): ꢃ 103.0 (C-1b), 99.4 (C-1a),
ꢀ
1
0.18 (7:3 toluene±EtOAc); [ꢀ]d +72.6 (c 1.0); H
NMR (270 MHz): ꢃ 7.76 (d, 2 H, J 7.5 Hz, Ar),
7.61 (d, 2 H, J 6.9 Hz, Ar), 7.4±7.1 (m, 34 H, Ar),
6.06 (d, 1 H, J 8.3 Hz, NH), 5.92 (m, 1 H,
±CH=CH2), 5.34 (brd, 1 H, J 16.8 Hz, =CH2),
5.26 (brd, 1 H, J 10.2 Hz, =CH2), 5.19 (d, 1 H, J
4.0 Hz, H-4b), 4.94 (d, 1 H, J 3.3 Hz, H-1a), 2.19
(dd, 1 H, J 4.6, 13.5 Hz, H-3c eq), 1.68 (s, 3 H, Ac).
Combustion analysis of azide 15 did not give the
correct CHN values because of partial decomposi-
tion on vacuum drying at 90 ꢀC.
N-(9-Fluorenylmethoxycarbonyl)-O-[(5-ace-
tamido-4,7,8,9-tetra-O-benzyl-3,5-dideoxy-d-glycero-
a-d-galacto-2-nonulopyranosylonic acid)-(2!3)-
(2,6-di-O-benzyl-b-d-galactopyranosyl)-(1!3)-2-
azido-4,6-O-benzylidene-2-deoxy-a-d-galactopyr-
anosyl-(1c!4b)-lactone]-l-serine allyl ester (16).
A mixture of 15 (65 mg, 42.5 ꢂmol), ꢀ,ꢀ-dimethox-
ytoluene (55 ꢂL, 0.37 mmol), and p-TsOH (cat.) in
CH3CN (2.5 mL) was stirred at room temperature
for 0.5 h, and the reaction was quenched with a few
drops of pyridine before concentration in vacuo.
The residue was chromatographed on silica gel
with 4:1 toluene±EtOAc to give 16 (65 mg, 95%).
Rf 0.42 (7:3 toluene±EtOAc); [ꢀ]d +91.4ꢀ (c 1.0);
1H NMR (270 MHz): ꢃ 7.76 (d, 2 H, J 7.2 Hz, Ar),
7.59 (d, 2 H, J 7.6 Hz, Ar), 7.53 ( brd, 2 H, J
7.3 Hz, Ar), 7.4±7.1 (m, 37 H, Ar), 6.01 (d, 1 H, J
8.2 Hz, NH), 5.90 (m, 1 H, -CH=CH2), 5.44 [s, 1
H, PhCH(O)2], 5.35 (brd, 1 H, J 17.2 Hz, =CH2),
5.27 (brd, 1 H, J 10.6 Hz, =CH2), 5.17 (d, 1 H, J
4.0 Hz, H-4b), 5.04 (d, 1 H, J 3.3 Hz, H-1a), 2.14
(brd, 1 H, J 12.2 Hz, H-3c eq), 1.69 (s, 3 H, Ac);
Anal. Calcd for C93H95N5O21: C, 69.00; H, 5.92;
N, 4.33. Found: C, 69.33; H, 5.97; N, 4.28.
.
95.3 (C-2c); Anal. Calcd for C92H105N5O21Si H2O:
C, 66.48; H, 6.43. Found: C, 66.52; H, 6.32.
1
Compound 13: Rf 0.45 (7:3 toluene±EtOAc); H
NMR (270 MHz): ꢃ 7.75 (d, 2 H, J 7.3 Hz, Ar),
7.59 (d, 2 H, J 7.3 Hz, Ar), 7.4±7.1 (m, 34 H, Ar),
5.90 (m, 1 H, -CH=CH2), 5.80 (d, 1 H, J 8.6 Hz,
NH), 5.36 (d, 1 H, J 4.0 Hz, H-4b), 5.32 (brd, 1 H,
J 17.2 Hz, =CH2), 5.23 (brd, 1 H, J 10.2 Hz,
=CH2), 4.92 and 4.91 ( 2 d, 2 H, J 3.0 Hz, H-1a
and H-1b), 2.38 (dd, 1 H, J 4.6, 13.2 Hz, H-3c eq),
1.85 (dd, 1 H, J 10.2, 13.0 Hz, H-3c ax), 1.78 (s, 3
H, Ac), 0.88 (s, 9 H, t-Bu), 0.05 and 0.04 (2 s, 6 H,
2 Me).Compound 14: Rf 0.22 (4:1 toluene±EtOAc);
1H NMR (270 MHz): ꢃ 7.4±7.1 (m, 30 H, Ar), 5.50
[dd, 0.5 H, J 2.3, 52.5 Hz, H-1a (ꢀ)], 5.37 [d, 0.5 H,
J 3.3 Hz, H-4a (ꢀ)], 5.23 [br, 0.5 H, H-4a (ꢁ)], 4.96
[dd, 0.5 H, J 6.9, 52.1 Hz, H-1a (ꢁ)], 2.37 [dd, 0.5
H, J 4.6, 12.9 Hz, H-3b eq (ꢀ)], 2.32 [dd, 0.5 H, J
4.4, 12.5 Hz, H-3b eq (ꢁ)], 1.81 [m, 1 H, H-3b ax (ꢀ
and ꢁ)], 1.70 and 1.69 (2 s, 3 H, Ac).
N-(9-Fluorenylmethoxycarbonyl)-O-[(5-ace-
tamido-4,7,8,9-tetra-O-benzyl-3,5-dideoxy-d-glycero-
a-d-galacto-2-nonulopyranosylonic acid)-(2!3)-
(2,6-di-O-benzyl-b-d-galactopyranosyl)-(1!3)-2-
azido-2-deoxy-a-d-galactopyranosyl-(1c!4b)-lact-
one]-l-serine allyl ester (15). To an ice-cooled soln
of 12 (29 mg, 17.6 mmol) in CH2Cl2 (0.5 mL) was
added 80% aq CF3CO2H (0.5 mL). The mixture
N-(9-Fluorenylmethoxycarbonyl)-O-[(5-Ace-
tamido-4,7,8,9-tetra-O-benzyl-3,5-dideoxy-d-glycero-
a-d-galacto-2-nonulopyranosylonic acid)-(2!3)-
(2,6-di-O-benzyl-b-d-galactopyranosyl)-(1!3)-2-
acetamido-4,6-O-benzylidene-2-deoxy-a-d-galacto-
pyranosyl-(1c!4b)-lactone]-l-serine allyl ester
(17). Compound 16 (62 mg, 38.3 ꢂmol) was trea-
ted with AcSH in pyridine as described for the
synthesis of 4. The crude product was chromato-
graphed on silica gel with 1:1 toluene±EtOAc to
give 17 (48 mg, 77%). Rf 0.23 (1:1 toluene±EtOAc);

294
Y. Nakahara et al./Carbohydrate Research 309 (1998) 287±296
[ꢀ]d +78.2ꢀ (c 1.3); 1H NMR (270 MHz): ꢃ 7.76 (d,
2 H, J 7.6 Hz, Ar), 7.56 (m, 4 H, Ar), 7.4±7.1 (m,
37 H, Ar), 5.88 (m, 2 H, NH and -CH=CH2), 5.65
(d, 1 H, NH), 5.45 [s, 1 H, PhCH(O)2], 5.33 (brd, 1
H, J 17.3 Hz, =CH2), 5.28 (dd, 1 H, J 1.0, 10.2 Hz,
=CH2), 5.16 (d, 1 H, J 4.0 Hz, H-4b), 5.00 (brs, 1
H, H-1a), 2.13 (dd, 1 H, J 5.0, 12.9 Hz, H-3c eq),
1.77 and 1.71 ( 2 s, 6 H, 2 Ac); Anal. Calcd for
The combined ®ltrate and washings were con-
centrated in vacuo. The residue was chromato-
graphed on silica gel with 96.5:3:0.5±92:7:1 CHCl3±
EtOH±AcOH to a€ord 6 (15 mg, 28%). (b) Deal-
lylation of 17: Compound 17 (72 mg, 44.0 ꢂmol)
was deallylated as described for the synthesis of 5.
The crude product was chromatographed on silica
gel with 93.5:6:0.5 CHCl3±EtOH±AcOH, and then
on a C18 reversed-phase column with 95% aq
CH3CN containing AcOH (0.1%) to a€ord 6
(64 mg, 91%). Rf 0.33 (92.5:7:0.5 CHCl3±EtOH±
AcOH); [ꢀ]d +91.2 (c 0.9); H NMR (270 MHz):
ꢃ 7.72 (d, 2 H, J 7.6 Hz, Ar), 7.54 (m, 4 H, Ar), 7.4±
7.1 (m, 37 H, Ar), 6.12 (brd, 1 H, J 6.9 Hz, NH),
5.93 (d, 1 H, J 7.3 Hz, NH), 5.39 [brs, 1 H,
PhCH(O)2], 5.12 (d, 1 H, J 3.6 Hz, H-4b), 5.04 (brs,
1 H, H-1a), 2.13 (brd, 1 H, J 13.0 Hz, H-3c eq),
1.82 and 1.72 ( 2 s, 6 H, 2 Ac); Anal. Calcd for
C92H95N3O22: C, 69.29; H, 6.00; N, 2.63. Found:
C, 69.58; H, 6.08; N, 2.67.
l-Threonyl-l-valyl-l-valyl-l-glutaminyl-l-prolyl-
[(5-Acetamido-3,5-dideoxy-d-glycero-a-d-galacto-
2-nonulopyranosylonic acid)-(2!3)-(b-d-galacto-
pyranosyl)-(1!3)-2-acetamido-2-deoxy-a-d-galacto-
pyranosyl]-l-seryl-l-valyl-l-glycyl-l-alanyl-l-alanyl-
l-alanyl-l-glycyl-l-prolyl-l-valyl-l-valyl-l-prolyl-l-
prolyl-l-cysteinyl-l-prolyl-l-glycyl-l-arginyl-l-iso-
leucyl-l-arginyl-l-histidyl-l-phenylalanyl-l-lysyl-l-
valine (1). 1. Synthesis of the benzylated glyco-
heptacosapeptide. Starting from commercial Fmoc-
Val-preloaded HMP resin (357 mg, 250 ꢂmol),
an Fmoc-protected henicosapeptide-linked resin
(953 mg) was obtained after twenty cycles of the
standard synthesizer program of condensation with
the DCC±HOBt-activated Fmoc amino acids
(1 mmol each) in NMP. A cycle of the program
involved a coupling step for 71 min and a depro-
tection step for 21 min. Eciency of the condensa-
tion at each step was monitored by utilizing the
ninhydrin test, and the overall yield of the henico-
sapeptide was estimated as 88.0%.
A part of this resin (68 mg, 14.9 ꢂmol) was stir-
red with 20% piperidine±NMP (2 mL) for 45 min,
washed with dry CH2Cl2, and dried to give Fmoc
deprotected peptide-resin (63 mg), while 6 (61 mg,
38.3 ꢂmol) was activated with DCC±NMP (1 M,
0.25 mL, 250 ꢂmol) and HOBt±NMP (1 M,
0.25 mL, 250 ꢂmol) in a polypropylene test tube
using a vortexing test tube mixer at room tem-
perature for 1 h. The resin and NMP (0.3 mL) were
added to the activated 6. Vortex mixing of the
resultant suspension was continued for 24 h at
.
C95H99N3O22 0.5H2O: C, 69.41; H, 6.13; N, 2.56.
Found: C, 69.38; H, 6.07; N, 2.48.
N-(9-Fluorenylmethoxycarbonyl)-O-[(5-ace-
tamido-4,7,8,9-tetra-O-benzyl-3,5-dideoxy-d-glycero-
a-d-galacto-2-nonulopyranosylonic acid)-(2!3)-
(2,6-di-O-benzyl-a-d-galactopyranosyl)-(1!3)-2-
azido-4,6-O-benzylidene-2-deoxy-a-d-galactopyr-
anosyl-(1c!4b)-lactone]-l-serine allyl ester (18).
(a) Glycosylation of 10 with 9: Reaction of 10
(43 mg, 66.9 ꢂmol) and 9 (ꢀ-isomer, 25 mg, 22 ꢂmol)
was performed in a similar manner as described for
12. The crude product was chromatographed on
Bio-beads SÂ1 and then on silica gel to give 18
(24 mg, 68%) as an ꢀ:ꢁ mixture (ꢀ:ꢁ=4.6 : 1).
Conversion of 13.ÐAccording to the procedure
described for the synthesis of 16, compound 13
(10 mg, 6.1 ꢂmol) was desilylated and benzyliden-
ated to a€ord 18 (8 mg, 86%). Rf 0.38 (7:3 toluene±
ꢀ
1
ꢀ
1
EtOAc); [ꢀ]d +84.9 (c 0.5); H NMR (270 MHz):
ꢃ 7.76 (d, 2 H, J 7.3 Hz, Ar), 7.56 (d, 2 H, J 7.6 Hz,
Ar), 7.4±7.1 (m, 39 H, Ar), 5.88 (d, 1 H, J 8.2 Hz,
NH), 5.86 (m, 1 H, -CH=CH2), 5.34±5.20 [m, 4 H,
PhCH(O)2, =CH2, and H-4b], 5.13 and 5.01 (2 d,
2 H, J 3.3 Hz, H-1a and H-1b), 2.39 (dd, 1 H, J 4.6,
13.5 Hz, H-3c eq), 1.87 (dd, 1 H, J 10.6, 13.5 Hz,
H-3c ax), 1.75 (s, 3 H, Ac). Combustion analysis of
azide 18 did not give the correct CHN values
because of partial decomposition on vacuum dry-
ꢀ
ing at 90 C.
N-(9-Fluorenylmethoxycarbonyl)-O-[(5-ace-
tamido-4,7,8,9-tetra-O-benzyl-3,5-dideoxy-d-glycero-
a-d-galacto-2-nonulopyranosylonic acid)-(2!3)-
(2,6-di-O-benzyl-b-d-galactopyranosyl)-(1!3)-2-
acetamido-4,6-O-benzylidene-2-deoxy-a-d-galacto-
pyranosyl-(1c!4b)-lactone]-l-serine
(6).
(a)
Desulfurization of 5: A mixture of 5 (60 mg,
33.1 ꢂmol), M Ph3SnH-benzene (1.5 mL, 1.5 mmol),
and 5% ꢀ,ꢀ0-azobis(isobutyronitrile) (AIBN)±ben-
zene (0.2 mL, 60.9 ꢂmol) in dry benzene (1.5 mL)
was heated under re¯ux in an atmosphere of Ar for
13 h. During this period, 5% AIBN solution
(0.1 mL each) was repeatedly (every two hours)
added to the mixture. After cooling, the pre-
cipitate was ®ltered o€ and washed with EtOAc.

Y. Nakahara et al./Carbohydrate Research 309 (1998) 287±296
295
room temperature. The resin was removed by ®l-
tration, washed with NMP and dry CH2Cl2, and
dried in vacuo (83 mg), before being transfered to
the automated synthesizer. Pro, Gln, Val, Val, and
Thr residues were sequentially coupled, and ®nally
the N-terminal Fmoc group was removed accord-
ing to the small-scale program (37 min for coupling
and 14 min for deprotection) to a€ord glycohepta-
cosapeptide-resin (86 mg, 94%).
(TOF MS M + 1; 6760, calcd 6756), which kept lac-
tonic structure partly (ca. 50%). The second (2.1 mg)
was the monomeric glycopeptide fraction (M + 1:
3363, calcd 3379), which possessed no lactone. Both
fractions were further puri®ed on C18 silica gel with
a gradient elution of aq CH3CN containing 0.1%
TFA (concentration of CH3CN: 0!20 min;
16!32%, 20!30 min; 32%) to give the dimer
(2.8 mg) and the monomer (1: 1.0 mg, 0.3 ꢂmol),
respectively. The dimer fraction was dissolved in
D2O (0.7 mL), and to the solution was added 0.2 M
NaHCO3±D2O (0.15 mL, 30 ꢂmol). The mixture
(pH 7.5) was left at room temperature for 3 days,
the progress of lactone hydrolysis being monitored
by NMR. Then a solution of 1,4-dithiothreitol
(1.2 mg) in D2O was added to the mixture, which
was allowed to stand overnight. The mixture was
chromatographed on the Superdex 30 pg column in
the same manner to give 1 (2.8 mg, 0.8 ꢂmol, total
yield 85%). The isolated monomer was prone to
dimerize easily in the solution without antioxidant.
2. Cleavage from the resin: The above glyco-
heptacosapeptide-resin (25.9 mg, 4.2 ꢂmol) was
stirred with a mixture of TFA (0.85 mL), deionized
H2O (40 ꢂL), thioanisole (40 ꢂL), 1,2-ethanedithiol
(40 ꢂL), and phenol (65 mg) at room temperature
for 1.5 h. The mixture was ®ltered and the resin was
washed with 80% aq TFA (0.5 mL). The combined
®ltrate and washings were diluted with water (20mL)
and extracted three times with ether (20mL). The
separatedꢀaqueous layer was concentrated in vacuo
below 40 C. The residue was dissolved in 30% aq
CH3CN and the insoluble material was ®ltered o€
through a membrane ®lter before chromatography
on a gel-permeation column (Pharmacia Biotech.
Superdex peptide HR 10/30) with 30% aq CH3CN
containing 0.1% TFA as the eluent. The most
mobile fractions were collected and concentrated in
vacuo. The residue (15.8 mg) was then fractionated
by preparative HPLC on C18 silica gel (Kanto
Chemical Co, Mightysil RP-18, 250±10 (5 ꢂm))
with a gradient elution of aq CH3CN containing
0.1% TFA (concentration of CH3CN: 0!10 min;
24!32%, 10!20 min; 32!64%). The fractions
including peaks 1,2,4,5, and 6 were collected and
concentrated in vacuo to give a mixture of desired
glycopeptide, mono-, and di-debenzylated products
(14.4 mg, 3.7 ꢂmol, 88%).
3. Deprotection: The mixture of the benzylated
glycopeptides (5.2 mg, 1.3 ꢂmol) was stirred with
TMSOTf±TFA solution (1 M, 200 ꢂL, 200 ꢂmol)
and thioanisole (20 ꢂL, 169 ꢂmol) at room tem-
perature for 1.5 h. The mixture was added drop-
wise into dry ether (7 mL) to precipitate
glycopeptidic substances, and ethereal layer was
pipetted out after centrifugation. The precipitate
was washed twice with ether, dryed in vacuo, and
then stirred with NH4F (5 mg) in H2O (0.5 mL) at
room temperature. The reaction mixture was
chromatographed on a gel-permeation column
(Pharmacia Biotech. Hi-load 26/60 superdex 30 pg)
with 30% aq CH3CN containing 0.1% TFA to
give two major fractions. The ®rst fraction (2.9 mg)
mainly consisted of the dimerized glycopeptide
,
ESIMS; m/z 1133.1 [(M+3)/3]3+ 1H NMR
[600 MHz; D2O, 25 ꢀC (or 60 ꢀC), t-BuOH (ꢃ 1.23)]:
d 7.62 (brs, 1 H, His), 7.31 (brt, 2 H, Phe), 7.27
(brt, 1 H, Phe), 7.19 (d, 2 H, J 7.3 Hz, Phe), 6.85
(brs, 1 H, His), 4.89 (d, 1 H, J 2.4 Hz, H-1:Gal-
NAc, 60 ꢀC), 4.45 (d, 1 H, J 7.3 Hz, H-1:Gal), 4.19
(brd, 1 H, J 2.4 Hz, H-4:GalNAc), 3.51 (brt, 1 H,
H-2:Gal), 2.74 (dd, 1 H, J 4.3, 12.2 Hz, H-3eq:
NeuAc), 2.02 and 1.99 (2s, 6 H, 2Ac:GalNAc and
NeuAc), 1.77 (t, 1 H, J 12.2 Hz, H-3ax: NeuAc).
Acknowledgements
This work was ®nancially supported by the
Grant-in-Aid for Scienti®c Research on Priority
Areas No. 06240105 from the Ministry of Educa-
tion, Science, Sports, and Culture, Japan and
partly by the CREST program of Japan Science
and Technology Corporation.
We are grateful to Drs. J. Uzawa, H. Koshino
and Ms T. Chijimatsu for NMR, Drs. K. Takio
and N. Dohmae for MALDI-TOF MS, Dr. S.
Kurono for ESIMS measurements. We thank Ms
M. Yoshida and her sta€ for elemental analyses,
and Ms A. Takahashi for technical assistance.
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