Synthetic approach toward the partial sequences of betaglycan in the linkage region on solid support and in solution phase

Article abstract of DOI:10.1080/07328300701296810

We have synthesized, for the first time, the partial sequence of the betaglycan composed of the tetraosyl hexapeptide, which was directly usable as a probe for enzymatic glycosyl transfer. Stepwise elongation afforded the corresponding tetraosyl trichloro

Full text of DOI:10.1080/07328300701296810

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: http://www.tandfonline.com/loi/lcar20
Synthetic Approach Toward the Partial Sequences
of Betaglycan in the Linkage Region on Solid
Support and in Solution Phase
Jun‐ichi Tamura , Akihiro Yamaguchi , Junko Tanaka & Yuko Nishimura
To cite this article: Jun‐ichi Tamura , Akihiro Yamaguchi , Junko Tanaka & Yuko Nishimura
(2007) Synthetic Approach Toward the Partial Sequences of Betaglycan in the Linkage Region
on Solid Support and in Solution Phase, Journal of Carbohydrate Chemistry, 26:2, 61-82, DOI:
10.1080/07328300701296810
To link to this article: http://dx.doi.org/10.1080/07328300701296810
Published online: 09 May 2007.
Submit your article to this journal
Article views: 59
View related articles
Citing articles: 4 View citing articles
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=lcar20
Download by: [University of Cambridge]
Date: 05 June 2016, At: 20:02

Journal of Carbohydrate Chemistry, 26:61–82, 2007
Copyright # Taylor & Francis Group, LLC
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1080/07328300701296810
Synthetic Approach Toward
the Partial Sequences of
Betaglycan in the Linkage
Region on Solid Support
and in Solution Phase
Jun-ichi Tamura, Akihiro Yamaguchi, Junko Tanaka, and
Yuko Nishimura
Department of Environmental Sciences, Faculty of Regional Sciences, Tottori
University, Tottori, Japan
We have synthesized, for the first time, the partial sequence of the betaglycan composed
of the tetraosyl hexapeptide, which was directly usable as a probe for enzymatic glycosyl
transfer. Stepwise elongation afforded the corresponding tetraosyl trichloroacetimidate.
The common glycosyl dipeptide:[b-D-GlcA-(1 ! 3)-b-D-Gal-(1 ! 3)-b-D-Gal-(1 ! 4)-b-
D-Xyl-(1 ! O)-Ser-Gly] was synthesized by glycosylation of the corresponding tetraosyl
trichloroacetimidate and Ser-Gly moiety. The glycosyl dipeptide was coupled with other
core peptide parts in solution phase and on a solid support. These glycosyl hexapeptides
were then transformed into the desired target compounds.
Keywords Proteoglycan, Glycosaminoglycan, Betaglycan, Heparin, Heparan sulfate
INTRODUCTION
Glycosaminoglycan (GAG) is the saccharide part of proteoglycans (PGs) cova-
lently linked to the core peptide through the hydroxyl group of the serine
residue. GAG is composed of two parts: the linkage (tetrasaccharide) region
and repeating disaccharide region. Biochemically, GAG elongates by a
stepwise addition with the help of UDP-sugars and the corresponding
glycosyl transferases.[1] GAG is classified into two categories based on the
Received 16 November 2006; accepted 19 February 2007.
Address correspondence to Jun-ichi Tamura, Department of Environmental Sciences,
Faculty of Regional Sciences, Tottori University, Tottori 680-8551, Japan. E-mail:
jtamura@rstu.jp
61

J. Tamura et al.
62
type of the hexosamine residues in the repeating disaccharide region (Fig. 1).
The first transfer of the hexosamine residue, a-GlcNAc or b-GalNAc, which
is the fifth saccharide moiety from the reducing terminal, determines the
type of GAG as either the heparin or chondroitin type. Most of the glycosyl
transferases involved in the GAG biosynthesis have been found; however, the
factors controlling the biosynthetic sorting mechanisms are still unclear.
To the best of our knowledge, two hypotheses on the sorting mechanisms,
which are not consistent with each other, have been supported on the basis of
the environment around the glycan and/or peptide part of PG. The common
tetrasaccharide often possesses sulfates at specific positions. Sugahara and
his coworkers advocated that no sulfate has been detected on the heparin
type of GAG vs. that on the chondroitin type.[2] It seems that the sulfate in
the linkage region of chondroitin might provide an important function for the
b-GalNAc transferase. On the other hand, Esko’s group reported that the hydro-
phobic and acidic amino acid clusters of the core peptide are often flanking the
heparin type of GAG.[3] His group demonstrated the glycan elongation toward
the heparin type of GAG using mutant peptides and glycosyl primers containing
hydrophobic aglycons.[4] These results provided important evidence for the
relationship between the selective biosynthesis of heparin/heparan sulfate
and the specific character of the common tetrasaccharide and/or the core
peptides; however, the transfer mechanism of the first hexosamine residue
remains unclear.
Heparin/heparan sulfates are medically and pharmaceutically indispensa-
ble materials; therefore, the selective and effective biosynthesis of heparin/
heparan sulfate is anticipated. These facts prompted us to elucidate the
biosynthetic mechanisms of heparin/heparan sulfate from a chemical point
of view. Figure 2 shows the partial structure of betaglycan, of which the core
peptide contains acidic and hydrophobic amino acids. It is noteworthy that
the Ser535 of the betaglycan predominantly possesses the heparin type of
GAG.[5] We selected the target compounds (1 and 2), which are partial
sequences of betaglycan containing the above core peptide. We now report
Figure 1: Structure of glycosaminoglycans.

Partial Sequences of Betaglycan
63
Figure 2: Target compounds. A partial sequence of betaglycan.
the detailed synthesis of these betaglycans, which can be the acceptors for the
a-GlcNAc transfer.
RESULTS AND DISCUSSION
Based on a retrosynthetic analysis, we planned (1) the stepwise elongation by
the coupling of each monosaccharide moiety from the reducing terminal, (2) the
subsequent coupling of the obtained tetrasaccharide with the Ser-Gly moiety,
(3) the coupling of the tetraosyl serylglycine with the tetrapeptide synthesized
in the solution phase or on a solid support, and (4) the final deprotection to
obtain the desired compounds.
As depicted in Scheme 1, in the presence of 4-methoxyphenol and TMSOTf,
known tetraacetyl-b-D-xylose (3)[6] was converted into the corresponding 4-meth-
oxyphenyl b-D-xyloside (4) in a 90% yield. The acetyl groups of 4 were removed
with Et3N to quantitatively give 5. The hydroxyl groups of 5 at the 2 and 3 pos-
itions were isopropylidenated to afford 6 in a 75% yield. The galactosyl imidate
(7) was converted from the known 4-methoxyphenyl 2,4,6-tri-O-acetyl-3-O-
allyl-b-D-galactopyranoside[7] by the removal of the 4-methoxyphenyl group
with CAN, then followed by trichloroacetimidation with CCl3CN and DBU in
79% and 71% yields, respectively. The 4-OH of 6 was glycosylated with 7 in the
presence of TMSOTf in CH2Cl2. The following methanolysis afforded the disac-
charide (8) in an 82% yield in two steps. The diol 8 was acylated with 4-methyl-
benzoyl chloride to produce 9 in a 77% yield. The position of the newly formed
glycosidic linkage was evident from the acylation shifts of the H-2 and H-3 of
the xylosyl residue. The allyl group of 9 was isomerized using an iridium
catalyst, and the I2 treatment gave the disaccharide acceptor 10 in a 95% yield
in two steps. The disaccharide acceptor 10 was stereoselectively coupled with 7
in the presence of TMSOTf in CH2Cl2 to afford the trisaccharide 11 in an 86%
yield. The allyl group of 11 was removed in the same manner as above to give
the trisaccharide acceptor 12 in an 85% yield in two steps.

J. Tamura et al.
64
Scheme 1: Reagents and conditions: i) 4-methoxyphenol, TMSOTf, MS4A, CH2Cl2, 2208C, 1 h;
ii) Et3N, MeOH, H2O, rt, overnight; iii) 2-methoxypropene, camphorsulfonic acid, DMF, 608C,
5 h; iv) TMSOTf, MSAW300, CH2Cl2, 2208C, 1 h; v) camphorsulfonic acid, MeOH, CH2Cl2, rt, 2 h;
vi) 4-methylbenzoyl chloride, pyridine, rt, 1.5 h; vii) (1,5-cyclooctadiene)bis(methyldiphenyl-
phosphine)iridium(I) hexafluorophosphate, THF, H2, then I2, H2O, NaHCO3, rt, 2.5 h; viii)
.
BF3 OEt2, MSAW300, CH2Cl2, 2208C ꢀrt, overnight; ix) CuBr2, AgOTf, n-Bu4NBr, MS4A, CH2Cl2,
08C ꢀrt, overnight; x) CAN, CH3CN, H2O, 08C, 4 h; xi) CCl3CN, DBU, CH2Cl2, 08C, 2 h; xii)
(Ph3P)4Pd(0), N-methylaniline, THF, rt, 4 h.
For the following coupling with the glucuronic acid moiety, we prepared two
types of glycosyl donors: methyl thioglycoside 13[8] and trichloroacetimidate
14.[9] Although 13 was used to couple with 12 mediated by the NIS-TfOH
system, no tetrasaccharide was obtained. However, AgOTf-CuBr2-n-Bu4NBr
could afford the desired compound 15 in a 31% yield. The glycosylation of 12
.
with 14 using BF3 OEt2 also afforded 15 in a 22% yield with 69% of the

Partial Sequences of Betaglycan
65
acceptor being recovered. When TMSOTf was used as the promoter, the reaction
afforded 15 in a 32% yield, while the residual acceptor was completely
decomposed.
The obtained tetrasaccharide was converted into the corresponding
hemiacetal 16 with CAN in a 93% yield, and brought to 17 using CCl3CN in
the presence of DBU in an 89% yield. The imidate (17) was coupled with
known serylglycine allyl esters (18[10] and 19[10]) protected with Boc and Fmoc
groups at their N-terminals, with the help of TMSOTf in CH2Cl2 at 2208C to
give 20 and 21 in 72% and 74% yields, respectively. The allyl groups were
removed with Pd(PPh3)4 and N-methylmorpholine to yield the corresponding
carboxylic acids 22 and 23 in 90% and 76% yields, respectively.
Synthesis of the Peptide Part
As shown in Scheme 2, the tetrapeptide Trp-Pro-Asp-Gly was synthesized
in the conventional manner (liquid phase). Commercially available Fmoc-
proline was converted into the corresponding allyl ester (24) in an 86% yield.
The Fmoc group of 24 was removed with morpholine, and coupled with
Fmoc-(Ni-Boc)-tryptophane using HOBt and HBTU in the presence of
Hu¨nig’s base to give 25 in a 56% yield. The allyl ester was converted into the
carboxylic acid 26 with Pd(PPh3)4 and N-methylmorpholine in a 63% yield.
On the other hand, the Z-(O-t-Bu)-aspartic acid and the glycine tert-butyl
ester, both of which were commercially available, were coupled with
.
WSCD HCl and HOBt in the presence of Et3N to yield 27 in a 96% yield.
Scheme 2: Reagents and conditions: i) Cs2CO3, EtOH, rt, 1 h, then AllBr, DMF, rt, overnight;
ii) Morpholine, CH2Cl2, rt, overnight; iii) HOBT, HBTU, CH2Cl2, 2208C ꢀrt, overnight; iv) (Ph3P)4
Pd(0), N-methylaniline, THF, rt, 15 h; v) Pd-C, H2, MeOH, AcOH, rt, 3 h.

J. Tamura et al.
66
Hydrogenolysis of 27 was successfully performed to give the free amine 28 in a
93% yield. The following condensation of 26 and 28 were executed in the same
manner as the synthesis of 27 to furnish the tetrapeptide 29 in an 85% yield.
The Fmoc group of 29 was finally removed with morpholine to give the free
amine 30, which was coupled with 22 in the presence of HOBt and HBTU.
As depicted in Scheme 3, the successful condensation afforded the tetraosyl
hexapeptide 31 in a 63% yield. The following deprotection procedures—(1)
treatment with a TFA cocktail and (2) saponification with M0.107 NaOMe in
50% MeOH—gave the desired compound 1 in an 83% yield after gel permeation
chromatography.
Scheme 4 shows the synthesis of the corresponding glycopeptide on Sieber
amide resin. The elongation was manually performed by the usual Fmoc pro-
cedure employing HOBt, HBTU, and i-Pr2EtN in NMP. The Fmoc group was
removed using piperidine in NMP. The N-terminal of the tetrapeptide on the
resin was coupled with 23 in the same manner as the peptide elongation.
The obtained glycosylated peptide on the resin was subjected to the TFA
cocktail to give the corresponding tetraosyl hexapeptide. Careful saponification
with diluted base (1M NaOH-MeOH 1:100, and the following 5 mM NaOH)[11]
treatment afforded the fully deprotected glycosyl peptide 2 in the amide
Scheme 3: Reagents and conditions: i) HOBT, HBTU, i-Pr2EtN, DMF, 2208C ꢀrt, overnight; ii) TFA,
thioanisole, phenol, H2O, 1,2-ethanedithiol, triisopropylsilane, CH2Cl2, rt, 2 h; iii) NaOMe,
MeOH, H2O, rt, 3 h.

Partial Sequences of Betaglycan
67
Scheme 4: Reagents and conditions: i) piperidine (20%), NMP, ii) HOBT, HBTU, i-Pr2EtN, iii) TFA,
thioanisole, phenol, H2O, 1,2-ethanedithiol, triisopropylsilane, CH2Cl2, rt, 3 h; iv) NaOMe,
MeOH, H2O, rt.
form. Further purification was successfully performed by gel permeation
chromatography and HPLC (C18). The final compounds 1 and 2 were in good
agreement with the calculated value based on the mass spectrum.
It was revealed that compound 1 was a poor substrate for the chondroitin
GalNAc T-1 and T-2[12] but a superior substrate for heparan sulfate elongation
(EXT1/EXT2) as well as the disaccharide primer, b-D-GlcA(1 ! 3) b-D-
Gal(1 ! O)C2H4NHZ having a hydrophobic aglycon.[13]
In summary, we synthesized the partial sequences in the linkage region of
betaglycan as primers for the biosynthesis of heparan sulfate. Both synthetic
strategies in the liquid phase as well as on a solid support are available for
the synthesis of the target tetraosyl hexapeptides: bGlcA-bGal-bGal-bXyl-
SerGlyTrpProAspGly.
EXPERIMENTAL
General Methods
Optical rotations were measured at 22 + 38C with a HORIBA polarimeter
SEPA-200. 1H NMR assignments were confirmed by two-dimensional HH-
COSY experiments with a JEOL ECP 500 MHz spectrometer. Signal

J. Tamura et al.
68
assignments such as 1III stand for a proton at C-1 of sugar residue III.
MALDI-TOF and ESI mass spectra were measured by Daltonics Autoflex II
spectrometer (Bruker) and Q-TOF2 (Micromass) instrument, respectively.
Silica gel chromatography, analytical TLC, and preparative TLC (PTLC)
were done on a column of Silica Gel 60 (Merck), Silica Gel 60 N (spherical
neutral), (Kanto Kagaku) or glass plates coated with Silica Gel F254 (Merck),
respectively. Gels for size-exclusion chromatography (Sephadex LH-20 and
.
Biobeads S-X1) were purchased from Amersham Biosciences and BIO RAD,
respectively. Reversed phase short column (Bond Elutw C8) was purchased
from Varian Inc. Molecular sieves (MS) were purchased from GL Science,
Inc., and activated at 2008C under diminished pressure prior to use. All
reactions in organic solvents were performed under dry Ar atmosphere. As a
usual workup, the organic phase of the reaction mixture was washed with
aq. NaHCO3 and brine and dried over MgSO4.
4-Methoxyphenyl 2,3,4-tri-O-acetyl-b-D-xylopyranoside (4)
To a suspension of 1,2,3,4-tetra-O-acetyl-b-D-xylopyranose[6] (3, 15.5 g,
48.7 mmol) and 4-methoxyphenol (6.70 g, 54.0 mmol) in the presence of
activated MS 4A (2.5 g) in 1,2-dichloroethane (100 mL) was added TMSOTf
(4.1 mL, 2.3 mmol) at 2208C with stirring, and then the reaction temperature
was gradually raised up to 2148C within 1 h. Et3N (15 mL) and an excess
amount of saturated NaHCO3 were added. Insoluble materials were filtered on
Celite. The organic phase was treated as described in the general methods. The
crude materials were subjected to a column of silica gel (5:1–3:1 toluene:EtOAc)
to give 4 (16.8 g) in 90% yield. A small amount of the product was recrystallized
from n-hexane:EtOAc to give white powder: [a]D –41.5 (c 0.66, CHCl3); m.p.
1
150.48C; H NMR (CDCl3): d 6.96–6.94 (m, 2H, Ar H), 6.84–6.81 (m, 2H, Ar
H), 5.23 (dd, 1H, J2,3 ¼ 8.29 Hz, J3,4 ¼ 7.81 Hz, H-3), 5.16 (dd, 1H,
J1,2 ¼ 6.10 Hz, H-2), 5.03 (d, 1H, H-1), 5.01 (m, 1H, H-4), 4.21 (dd, 1H,
J4,5eq ¼ 4.88 Hz, Jgem ¼ 11.96 Hz, H-5eq), 3.77 (s, 3H, OMe), 3.47 (dd, 1H,
J4,5ax ¼ 8.29 Hz, H-5ax), 2.10, 2.08, 2.08 (3s, 3H ꢀ 3, 3MeCO). Anal. Calcd for
C18H22O9: C, 56.53; H, 5.81. Found: C, 56.51; H, 5.81.
4-Methoxyphenyl b-D-xylopyranoside (5)
Triacetate (4) (16.8 g, 44.0 mmol) was diluted with Et3N (75 mL), MeOH
(150 mL), and H2O (75 mL) with stirring overnight. The volatiles were
removed under diminished pressure and the residue was coevaporated with
water and toluene. The residue was purified from a column of silica gel
(1:1–1:5 n-hexane:EtOAc–100:1–40:1–20:1 EtOAc:MeOH) to give 5 quantitat-
1
ively: [a]D –26.7 (c 1.29, MeOH); H NMR (CD3OD): d 6.91–6.89 (m, 2H, Ar
H), 6.74–6.72 (m, 2H, Ar H), 4.61 (d, 1H, J1,2 ¼ 7.56 Hz, H-1), 3.79 (dd, 1H,
J4,5a ¼ 5.37 Hz, Jgem ¼ 11.47 Hz, H-5a), 3.64 (s, 3H, OMe), 3.5–3.2 (m, 4H,
H-2,3,4,5e). Anal. Calcd for C12H16O6: C, 56.24; H, 6.31. Found: C, 56.19; H, 6.31.

Partial Sequences of Betaglycan
4-Methoxyphenyl 2,3-O-isopropylidene b-D-xylopyranoside (6)
69
To a solution of 5 (15.2 g, 59.4 mmol) in DMF (70 mL) were added a camphor
sulfonic acid (2.1 g, 9.0 mmol) and 2-methoxypropene (12.0 mL, 125 mmol) with
stirring at 608C. After 2 h, 6 mL of 2-methoxypropene was added for 30
minutes. Forty minutes later, the reaction mixture was cooled to rt and Et3N
(6.0 mL, 35 mmol) was added. The reaction mixture was extracted with EtOAc.
The organic phase was treated as described in the general methods. The
residue was eluted from a column of silica gel (5:1–4:1–1:1–1:5 toluene:EtOAc)
to give 6 (13.2 g, 75%) as a syrup: [a]D –32.3 (c 1.32, CHCl3); 1H NMR (CDCl3): d
7.04–7.01 (m, 2H, Ar H), 6.84–6.81 (m, 2H, Ar H), 5.18 (d, 1H, J1,2 ¼ 5.86 Hz,
H-1), 4.15 (m, 1H, H-5eq), 4.11 (m, 1H, H-4), 3.77 (s, 3H, OMe), 3.62 (m, 2H,
H-2,3), 3.38 (dd, 1H, J4,5ax ¼ 6.34 Hz, Jgem ¼ 11.47 Hz, H-5ax), 2.49 (d, 1H,
J4,OH ¼ 3.91 Hz, OH-4), 1.51, 1.49 (2s, 3H ꢀ 2, 2CH3). Anal. Calcd for
.
C15H20O6 0.2H2O: C, 60.06; H, 6.87. Found: C, 59.88; H, 6.90.
2,4,6-Tri-O-acetyl-3-O-allyl-a-D-galactopyranosyl trichloroacetimidate
(7)
To a solution of 4-methoxyphenyl 2,4,6-tri-O-acetyl-3-O-allyl-b-D-galacto-
pyranoside[7] (77.3 g, 171 mmol) in CH3CN (400 mL) and H2O (100 mL) was
added cerium (IV) ammonium nitrate (CAN) (88.4 g, 161 mmol) at 08C with
stirring. After 3 h, additional CAN (175.57 g, 320.3 mmol) was supplied and
the reaction mixture was stirred for another 2.5 h, then diluted with CHCl3
and brine and extracted with CHCl3. The organic phase was washed with
brine and the residue was eluted from a column of silica gel (2:1–3:2–
1:1–1:3 n-hexane:EtOAc) to give the corresponding hemiacetal (47.0 g, 79%)
as a syrup. The hemiacetal was diluted with CH2Cl2 (350 mL). To the
solution were added CCl3CN (22 mL) and DBU (8.0 mL, 54 mmol) at 08C.
The reaction mixture was stirred for 1.5 h and directly subjected to a column
of silica gel (3:1ꢀ3:2ꢀ1:1 n-hexane-EtOAc) to give 7 (47.4 g, 71%) as a
1
syrup, which was used for the next reaction without further purification: H
NMR (CDCl3): d 8.63 (s, 1H, NH), 6.56 (s, 1H, H-1), 5.88–5.78 (m, 1H, CH55),
5.58 (d, 1H, J3,4 ¼ 2.54 Hz, H-4), 5.31–5.17 (m, 3H, H-2, 55CH2), 4.35 (brt,
1H, J ¼ 6.47 Hz, H-5), 4.23–3.97 (m, 5H, H-3, 6, CH2O), 2.16, 2.05, 2.04 (3s,
3H ꢀ 3, 3MeCO).
4-Methoxyphenyl O-(2,4,6-tri-O-acetyl-3-O-allyl-b-D-galactopyranosyl)-
(1 ! 4)-b-D-xylopyranoside (8)
To a solution of 6 (321.0 mg, 1.083 mmol) and 7 (977.5 mg, 1.992 mmol) in
CH2Cl2 (15 mL) was added MS AW300 (300 mg). This mixture was stirred for
30 min at rt and cooled to 2158C. To this solution was added a TMSOTf
(70 mL, 0.38 mmol) with stirring and the reaction temperature was gradually
raised to 2108C for 1 h. Then, Et3N (110 mL, 0.79 mmol) and an excess

J. Tamura et al.
70
amount of aqueous NaHCO3 were added. Insoluble materials were filtered on
Celite. The organic phase was treated as described in the general methods.
To a solution of the crude products in MeOH and CH2Cl2 (1:1, 46 mL) was
added a camphor sulfonic acid (251.6 mg, 1.08 mmol) and the mixture stirred
for 2 h and quenched with Et3N (150 mL, 1.08 mmol). The volatiles were
removed under diminished pressure and the residue was subjected to a
column of silica gel (4:1–2:1–1:1–2:3 toluene:EtOAc) to give 8 (518.1 mg) in
82% yield as a syrup: [a]D –3.9 (c 1.50, CHCl3); 1H NMR (CDCl3): d
7.01–6.97 (m, 2H, Ar H), 6.85–6.82 (m, 2H, Ar H), 5.82–5.73 (m, 1H, CH55),
5.42 (d, 1H, J3,4 ¼ 3.42 Hz, H-4II), 5.34–5.17 (m, 2H, 55CH2), 5.10 (dd, 1H,
J1,2 ¼ 8.05 Hz, J2,3 ¼ 10.00 Hz, H-2II), 4.90 (d, 1H, J1,2 ¼ 6.59 Hz, H-1I), 4.48
(d, 1H, H-1II), 4.24 (dd, 1H, J5,6a ¼ 4.88 Hz, Jgem ¼ 11.47 Hz, H-6aII), 4.14–
4.07 (m, 1H, one of OCH2), 4.09 (m, 1H, H-6bII), 3.95 (dd, 1H,
J4,5eq ¼ 4.64 Hz, Jgem ¼ 11.95 Hz, H-5eqI), 3.92–3.88 (m, 2H, H-5II, one of
CH2O), 3.89 (m, 1H, OH-3I), 3.78 (s, 3H, OMe), 3.78–3.73 (m, 1H, H-3I), 3.67
(m, 2H, H-2I,4I), 3.53 (dd, 1H, H-3II), 3.41 (dd, 1H, J4,5ax ¼ 8.54 Hz, H-5axI),
2.78 (d, 1H, J2,OH ¼ 3.66 Hz, OH-2I), 2.14, 2.12, 2.12, (3s, 3H ꢀ 3, 3MeCO).
.
Anal. Calcd for C27H36O14 0.5 H2O: C, 54.62; H, 6.30. Found: C, 54.68; H, 6.02.
4-Methoxyphenyl O-(2,4,6-tri-O-acetyl-3-O-allyl-b-D-galactopyranosyl)-
(1 ! 4)-2,3-di-O-(4-methylbenzoyl)-b-D-xylopyranoside (9)
To a solution of 8 (1.71 g, 2.92 mmol) in pyridine (10 mL) was added a
4-methylbenzoyl chloride (1.50 mL, 11.6 mmol) dropwise with stirring. After
1.5 h MeOH (0.5 mL) was added to the reaction mixture at 08C and diluted
with CHCl3. The organic phase was treated as described in general methods.
The crude materials were subjected to a column of silica gel (10:1–7:1–
6:1–4:1–1:1 toluene:EtOAc) to give 9 (1.85 g, 77%) as a syrup: [a]D þ33.9 (c
0.945, CHCl3); 1H NMR (CDCl3): d 7.97–7.95 (m, 4H, Ar H), 7.24–7.22
(m, 4H, Ar H), 6.98–6.96 (m, 2H, Ar H), 6.82–6.80 (m, 2H, Ar H), 5.84–5.71
(m, 1H, CH55), 5.68 (brt, 1H, J ¼ 6.30 Hz, H-3I), 5.43 (dd, 1H, J1,2 ¼ 5.04 Hz,
J2,3 ¼ 6.18 Hz, H-2I), 5.29 (brd, 2H, J ¼ 4.58 Hz, H-1I,4II), 5.23–5.14 (m, 2H,
55CH2), 5.05 (dd, 1H, J1,2 ¼ 8.02 Hz, J2,3 ¼ 10.08 Hz, H-2II), 4.57 (d, 1H,
H-1II), 4.23 (dd, 1H, J4,5eq ¼ 3.90 Hz, Jgem ¼ 12.37 Hz, H-5eqI), 4.10–4.06
(m, 1H, one of OCH2), 3.98 (m, 1H, H-4I), 3.87–3.84 (m, 1H, one of CH2O),
3.83 (dd, 1H, J5,6a ¼ 6.19 Hz, Jgem ¼ 10.99 Hz, H-6aII), 3.76 (s, 3H, OMe),
3.71 (brt, 1H, J ¼ 6.53 Hz, H-5II), 3.65 (dd, 1H, J4,5ax ¼ 6.65 Hz, H-5axI), 3.59
(dd, 1H, J5,6b ¼ 6.65 Hz, H-6bII), 3.46 (dd, 1H, J3,4 ¼ 3.44 Hz, H-3II), 2.40,
2.38 (2s, 3H ꢀ 2, 2PhMe), 2.03, 2.02, 1.96 (3s, 3H ꢀ 3, 3MeCO). Anal. Calcd
.
for C43H48O16 H2O: C, 61.56; H, 6.02. Found: C, 61.72; H, 5.87.
4-Methoxyphenyl O-(2,4,6-tri-O-acetyl-b-D-galactopyranosyl)-(1 ! 4)-
2,3-di-O-(4-methylbenzoyl)-b-D-xylopyranoside (10)
A suspension of a catalytic amount of (1,5-cyclooctadiene)bis(methyldiphe-
nylphosphine)iridium(I) hexafluorophosphate in THF (10 mL) was stirred

Partial Sequences of Betaglycan
71
under H2 atmosphere, which was then replaced with argon. This manipulation
was repeated a few times. Then, a solution of 9 (2.01 g, 2.45 mmol) in THF
(20 mL) was added to the above solution of the iridium complex. After stirring
for 2 h, H2O (5 mL), NaHCO3 (3.92 g, 46.7 mmol), and I2 (1.72 g, 6.78 mmol)
were added to the reaction mixture, and the stirring was continued for 2.5 h at
08C. The reaction mixture was then diluted with CHCl3. The organic phase
was washed with aq. NaHCO3, aq. Na2S2O3, and brine. The volatiles were
removed under diminished pressure, and the crude materials obtained were
eluted from a column of silica gel (4:1–3:1–2:1–1:1–1:2 toluene:EtOAc) to give
10 (1.82 g, 95%) as a syrup: [a]D þ22.6 (c 1.54, CHCl3); 1H NMR (CDCl3): d 7.94
(m, 4H, Ar H), 7.29–7.21 (m, 4H, Ar H), 6.98–6.96 (m, 2H, Ar H), 6.82–6.80
(m, 2H, Ar H), 5.69 (t, 1H, J2,3 ¼ J3,4 ¼ 6.34 Hz, H-3I), 5.43 (dd, 1H,
J1,2 ¼ 4.88 Hz, H-2I), 5.30 (d, 1H, H-1I), 5.20 (d, 1H, J3,4 ¼ 3.42 Hz, H-4II),
4.90 (dd, 1H, J1,2 ¼ 8.05 Hz, J2,3 ¼ 10.00 Hz, H-2II), 4.60 (d, 1H, H-1II), 4.26
(dd, 1H, J4,5eq ¼ 3.66 Hz, Jgem ¼ 12.20 Hz, H-5eqI), 4.00 (m, 1H, H-4I), 3.85
(dd, 1H, J5,6a ¼ 6.34 Hz, Jgem ¼ 10.98 Hz, H-6aII), 3.80–3.73 (m, 2H,
H-3II,5II), 3.75 (s, 3H, OMe), 3.68 (dd, 1H, J4,5ax ¼ 6.10 Hz, H-5axI), 3.59 (dd,
1H, J5,6b ¼ 6.83 Hz, H-6bII), 2.51, 2.49 (2s, 3H ꢀ 2, 2PhMe), 2.06, 2.03, 1.94
.
(3s, 3H ꢀ 3, 3MeCO). Anal. Calcd for C40H44O16 H2O: C, 60.14; H, 5.82.
Found: C, 60.00; H, 5.53.
4-Methoxyphenyl O-(2,4,6-tri-O-acetyl-3-O-allyl-b-D-galactopyranosyl)-
(1 ! 3)-O-(2,4,6-tri-O-acetyl-b-D-galactopyranosyl)-(1 ! 4)-2,3-di-O-
(4-methylbenzoyl)-b-D-xylopyranoside (11)
To a solution of 10 (7.40 g, 9.48 mmol) and 7 (7.12 g, 14.5 mmol) in CH2Cl2
(160 mL) was added MS AW300 (4.0 g). This mixture was stirred for 35 min at
rt, and then cooled to 2208C. To this solution was added a TMSOTf (660 mL,
3.65 mmol) with stirring. After 1 h, the reaction was quenched with Et3N
(1.0 mL, 7.0 mmol) and excess amount of aq. NaHCO3. The reaction was
treated as described in the synthesis of 8. The residue was subjected to a
column of silica gel (5:1–4:1–3:1–2:1–1:2 n-hexane:EtOAc) to give 11 (9.06 g)
1
in 86% yield as a syrup: [a]D þ51.5 (c 0.96, CHCl3); H NMR (CDCl3): d 7.98–
7.94 (m, 5H, Ar H), 7.24–7.23 (m, 3H, Ar H), 6.97 (m, 2H, Ar H), 6.82 (m, 2H,
Ar H), 5.80–5.70 (m, 1H, 55CH), 5.68 (t, 1H, J2,3 ¼ J3,4 ¼ 6.10 Hz, H-3I), 5.42
(dd, 1H, J1,2 ¼ 4.88 Hz, H-2I), 5.37 (d, 1H, J3,4 ¼ 3.42 Hz, H-4III), 5.30 (d, 1H,
H-1I), 5.27 (d, 1H, J3,4 ¼ 3.42 Hz, H-4II), 5.19 (m, 2H, 55CH2), 5.16 (dd, 1H,
J1,2 ¼ 7.81 Hz, J2,3 ¼ 9.51 Hz, H-2II), 4.95 (dd, 1H, J1,2 ¼ 8.05 Hz,
J2,3 ¼ 10.00 Hz, H-2III), 4.55 (d, 1H, H-1II), 4.45 (d, 1H, H-1III), 4.23 (dd, 1H,
J4,5eq ¼ 3.66 Hz, Jgem ¼ 12.44 Hz, H-5eqI), 4.10 (m, 2H, OCH2), 4.10 (dd,
J5,6b ¼ 5.12 Hz, Jgem ¼ 12.69 Hz, H-6bIII), 3.96 (m, 1H, H-4I), 3.89–3.85 (m, 2H,
H-6aIII, 6bII), 3.84–3.75 (m, 3H, H-3II, 5II, 5III), 3.64 (dd, 1H, J4,5ax ¼ 6.10 Hz,
H-5axI), 3.51 (dd, 1H, J5,6a ¼ 6.83 Hz, H-6aII), 3.41 (dd, 1H, H-3III), 2.40, 2.39

J. Tamura et al.
72
(2s, 3H ꢀ 2, 2MePh), 2.14, 2.08, 2.06, 2.06, 2.04, 1.94 (6s, 3H ꢀ 6, 6MeCO).
ESI-MS (positive) Calcd for C55H64O24Na [M þ Na]þ, 1131.4. Found: 1130.9.
4-Methoxyphenyl O-(2,4,6-tri-O-acetyl-b-D-galactopyranosyl)-(1 ! 3)-O-
(2,4,6-tri-O-acetyl-b-D-galactopyranosyl)-(1 ! 4)-2,3-di-O-(4-
methylbenzoyl)-b-D-xylopyranoside (12)
The iridium catalyst was suspended in THF and activated as described in
the synthesis of 10. Then, a solution of 11 (95.9 mg, 86.5 mmol) in THF (3 mL)
was added to the iridium complex solution. After stirring for 45 min, H2O
(1 mL), NaHCO3 (145.3 mg, 1.730 mmol), and I2 (43.9 mg, 0.174 mmol) were
added to the reaction mixture at 08C. The solution was stirred for 45 min at
08C and treated as described in the synthesis of 10. The obtained crude
material was eluted from a column of silica gel (9:1–7:1–5:1–3:1–2:1–1:5
toluene:EtOAc) to give 12 (78.5 mg, 85%) as a syrup: [a]D þ26.3 (c 1.01,
1
CHCl3); H NMR (CDCl3): d 7.97–7.94 (m, 5H, Ar H), 7.24–7.22 (m, 3H, Ar
H), 6.97 (m, 2H, Ar H), 6.81 (m, 2H, Ar H), 5.68 (t, 1H, J2,3 ¼ J3,4 ¼ 6.10 Hz,
H-3I), 5.43 (dd, 1H, J1,2 ¼ 4.89 Hz, H-2I), 5.30–5.27 (m, 3H, H-1I, 4II, 4III),
5.16 (dd, 1H, J1,2 ¼ 8.05 Hz, J2,3 ¼ 10.00 Hz, H-2II), 4.83 (dd, 1H,
J1,2 ¼ 8.05 Hz, J2,3 ¼ 10.00 Hz, H-2III), 4.58 (d, 1H, H-1II), 4.47 (d, 1H, H-1III),
4.23 (dd, 1H, J4,5eq ¼ 3.66 Hz, Jgem ¼ 12.44 Hz, H-5eqI), 4.17–4.08 (m, 2H,
H-6III), 3.97 (m, 1H, H-4I), 3.86 (dd, 1H, J5,6b ¼ 5.37 Hz, Jgem ¼ 11.22 Hz,
H-6bII), 3.79–3.71 (m, 3H, H-3II, 3III, 5II), 3.64 (dd, 1H, J4,5ax ¼ 6.34 Hz,
H-5axI), 3.51 (dd, 1H, J5,6a ¼ 7.07 Hz, H-6aII), 2.41, 2.39 (2s, 3H ꢀ 2, 2MePh),
2.18, 2.12, 2.07, 2.04, 2.04, 1.94 (6s, 3H ꢀ 6, 6MeCO). TOF-MS (positive)
Calcd for C52H60O24Na [M þ Na]þ, 1091.3. Found: 1091.5.
4-Methoxyphenyl O-[methyl 2,3,4-tri-O-(4-methylbenzoyl)-b-D-
glucopyranosyl uronate]-(1 ! 3)-O-(2,4,6-tri-O-acetyl-b-D-
galactopyranosyl)-(1 ! 3)-O-(2,4,6-tri-O-acetyl-b-D-
galactopyranosyl)-(1 ! 4)-2,3-di-O-(4-methylbenzoyl)-b-D-
xylopyranoside (15)
Method A: A suspension containing CuBr2 (1.37 g, 6.14 mmol), AgOTf
(1.58 g, 6.14 mmol), n-Bu4NBr (327 mg, 1.01 mmol), and MS 4A (5.0 g) in
CH2Cl2 (31 mL) was stirred in the dark at rt and cooled to 08C after 30 min.
Then, CH2Cl2 (60 mL) and a solution of 13 (798.3 mg, 1.347 mmol) and 12
(713.0 mg, 0.667 mmol) were added dropwise to the suspension and stirred over-
night, gradually raised to rt. The reaction was quenched with aq. NaHCO3 and
brine. Then, the reaction mixture was diluted with EtOAc and filtered on Celite.
The organic phase was treated as usual. The obtained crude materials were
eluted from a column of silica gel (3:1–3:2–1:1–1:10 n-hexane:EtOAc) to give
15 (338.8 mg, 31%) as a syrup together with 288.6 mg (41%) of recovered 12.
Method B: To a solution of 14 (1.11 g, 1.57 mmol) and 12 (675.1 mg,
0.631 mmol) in CH2Cl2 (28 mL) was added MS AW300 (300 mg). This

Partial Sequences of Betaglycan
73
mixture was stirred for 10 min at rt, and then cooled to 2208C. To this solution
was added a TMSOTf (85 mL, 0.47 mmol) with stirring, and the same amount of
TMSOTf was added after 2 h. One hour later, the reaction was quenched with
Et3N (260 mL, 1.88 mmol) and an excess amount of aq. NaHCO3 was added.
The reaction was treated as described in the synthesis of 8. The residue was
purified as above to give 15 (323.7 mg) in 32% yield..
Method C: To a solution of 14 (147.9 mg, 209.2 mmol) and 12 (96.5 mg,
90.3 mmol) in toluene (17.5 mL) and CH2Cl2 (0.2 mL) was added MS AW300
(480 mg). This mixture was stirred for 30 min at rt, and then cooled to
.
2208C. To this solution was added a BF3 OEt2 (8 mL, 0.09 mmol) with
.
stirring and the same amount of BF3 OEt2 was added after 1.5 h. The
reaction mixture was stirred overnight while the temperature gradually was
raised to rt. The reaction was worked up with an excess amount of aq.
NaHCO3 and treated as above to give 15 (31.4 mg) in 22% yield together
with 66.4 mg (69%) of recovered 12: [a]D þ26.9 (c 1.01, CHCl3); 1H NMR
(CDCl3): d 7.97–7.93 (m, 4H, Ar H), 7.81–7.70 (m, 6H, Ar H), 7.23–7.07
(m, 10H, Ar H), 6.96 (m, 2H, Ar H), 6.81 (m, 2H, Ar H), 5.80 (t, 1H,
J2,3 ¼ J3,4 ¼ 9.27 Hz, H-3IV), 5.65 (t, 1H, J2,3 ¼ J3,4 ¼ 6.10 Hz, H-3I), 5.64
(brt, 1H, J ¼ 9.76 Hz, H-4IV), 5.47 (d, 1H, J3,4 ¼ 3.45 Hz, H-4III), 5.41 (dd,
1H, J1,2 ¼ 4.88 Hz, H-2I), 5.37 (dd, 1H, J1,2 ¼ 7.56 Hz, H-2IV), 5.29 (d, 1H,
H-1I), 5.22 (d, 1H, J3,4 ¼ 3.42 Hz, H-4II), 5.08 (dd, 1H, J1,2 ¼ 8.05 Hz,
J2,3 ¼ 10.00 Hz, H-2II), 5.02 (dd, 1H, J1,2 ¼ 8.05 Hz, J2,3 ¼ 10.24 Hz, H-2III),
4.91 (d, 1H, H-1IV), 4.51 (d, 1H, H-1II), 4.37 (d, 1H, H-1III), 4.26 (d, 1H,
J4,5 ¼ 10.01 Hz, H-5IV), 4.21 (dd, 1H, J4,5eq ¼ 3.91 Hz, Jgem ¼ 12.19 Hz,
H-5eqI), 4.16 (dd, 1H, J5,6b ¼ 5.85 Hz, Jgem ¼ 11.71 Hz, H-6bIII), 4.03 (dd, 1H,
J5,6a ¼ 6.34 Hz, H-6aIII), 3.93 (m, 1H, H-4I), 3.85–3.81 (m, 2H, H-6bII, 3III),
3.79–3.73 (m, 3H, H-3II, 5II, 5III), 3.76 (s, 3H, MeOPh), 3.70 (s, 3H, COOMe),
3.61 (dd, 1H, J4,5ax ¼ 5.61 Hz, H-5axI), 3.51 (dd, 1H, J5,6a ¼ 7.07 Hz,
Jgem ¼ 11.47 Hz, H-6aII), 2.40, 2.37, 2.37, 2.36, 2.30 (5s, 3H ꢀ 5, 5MePh),
2.16, 2.09, 2.02, 1.93, 1.89, 1.65 (6s, 3H ꢀ 6, 6MeCO). Anal. Calcd for
.
C83H88O33 4H2O: C, 59.13; H, 5.57. Found: C, 59.01; H, 6.13. ESI-MS
(positive) Calcd for C83H88O33Na [M þ Na]þ, 1635.5. Found: 1635.3.
O-[Methyl 2,3,4-tri-O-(4-methylbenzoyl)-b-D-glucopyranosyl uronate]-
(1 ! 3)-O-(2,4,6-tri-O-acetyl-b-D-galactopyranosyl)-(1 ! 3)-O-(2,4,6-
tri-O-acetyl-b-D-galactopyranosyl)-(1 ! 4)-2,3-di-O-(4-
methylbenzoyl)-a-D-xylopyranosyl trichloroacetimidate (17)
To a solution of 15 (445.0 mg, 0.276 mmol) in CH3CN (36 mL) and H2O (9 mL)
was added CAN (453.6 mg, 0.827 mmol) at 08C with stirring. After 3 h, additional
CAN (150.0 mg, 0.274 mmol) was supplied. The reaction mixture was stirred for
another 1.5 h and treated as the synthesis of 7. The residue was eluted from a
column of silica gel (3:1–2:1–1:1–1:5–1:8 n-hexane:EtOAc) to give 16
(385.8 mg, 93%) as a syrup. The obtained hemiacetal (16) was diluted with

J. Tamura et al.
74
CH2Cl2 (17 mL). To the solution were added CCl3CN (260 mL) and 1 drop of DBU
at 08C. The reaction mixture was stirred for 2 h and directly subjected to a column
of silica gel (3:1–1:1–1:3–1:4 n-hexane:EtOAc) to give 17 (377.2 mg, 89%) as a
syrup, which was used for the glycosylation without further purification.
N-Butoxycarbonyl-O-f[methyl 2,3,4-tri-O-(4-methylbenzoyl)-b-D-
glucopyranosyl uronate]-(1 ! 3)-O-(2,4,6-tri-O-acetyl-b-D-
galactopyranosyl)-(1 ! 3)-O-(2,4,6-tri-O-acetyl-b-D-
galactopyranosyl)-(1 ! 4)-2,3-di-O-(4-methylbenzoyl)-b-D-
xylopyranosylg-L-serylglycine allyl ester (20)
To a solution of 17 (409.7 mg, 248.0 mmol) and 18 (225.2 mg, 744.9 mmol) in
CH2Cl2 (47 mL) was added MS AW300 (1.8 g). This mixture was stirred for
30 min at rt, and then cooled to 2208C. To this solution was added a TMSOTf
(9 mL, 0.05 mmol) with stirring, and the same amount of TMSOTf was added
after 1 h, and stirring was continued for another 1.5 h. Then, the reaction was
treated as described in the synthesis of 8. The residue was subjected to the
columns of gel permeation (LH-20, 1:1 CHCl3:MeOH) and silica gel (5:1–3:1–
2:1–1:1–1:2 toluene:EtOAc) to give 20 (318.7 mg) in 72% yield: [a]D þ31.6 (c
1.26, CHCl3); 1H NMR (CDCl3): d7.80–7.62 (m, 10H, Ar H), 7.12–7.00 (m, 10H,
Ar H), 6.82 [m, 1H, NH(Gly)], 5.80 (m, 1H, 55CH), 5.72 (dd, 1H, J2,3 ¼ 9.03 Hz,
J3,4 ¼ 9.76 Hz, H-3IV), 5.57 (t, 1H, H-4IV), 5.49 (t, 1H, J2,3 ¼ J3,4 ¼ 7.81 Hz,
H-3I), 5.40 (d, 1H, J3,4 ¼ 3.17 Hz, H-4III), 5.36 [m, 1H, NH(Ser)], 5.28 (dd, 1H,
J1,2 ¼ 7.08 Hz, H-2IV), 5.20 (m, 2H, 55CH2), 5.14 (d, 1H, J3,4 ¼ 1.71 Hz, H-4II),
5.12 (dd, 1H, J1,2 ¼ 6.10 Hz, H-2I), 4.95 (dd, 1H, J1,2 ¼ 8.05 Hz, J2,3 ¼ 7.81 Hz,
H-2II), 4.92 (dd, 1H, J1,2 ¼ 7.81 Hz, J2,3 ¼ 10.49 Hz, H-2III), 4.84 (d, 1H, H-1IV),
4.65 (d, 1H, H-1I), 4.46 (m, 2H, OCH2), 4.37 (d, 1H, H-1II), 4.29 (d, 1H, H-1III),
4.19 (d, 1H, H-5IV), 4.15 (m, 1H, Sera), 4.09 (dd, 1H, J5,6b ¼ 6.10 Hz,
Jgem ¼ 10.97 Hz, H-6bIII), 4.06 (dd, 1H, J4,5eq ¼ 5.61 Hz, Jgem ¼ 13.17 Hz,
H-5eqI), 3.97 (dd, 1H, J5,6a ¼ 5.86 Hz, H-6aIII), 3.89 (m, 1H, H-4I), 3.89 (dd, 1H,
Ja,NH ¼ 5.37 Hz, Jgem ¼ 18.54 Hz, Glya), 3.79 (dd, 1H, Jb,NH ¼ 5.37 Hz, Glyb),
3.76 (m, 1H, H-3III), 3.73 (dd, 1H, J5,6b ¼ 5.85 Hz, Jgem ¼ 11.22 Hz, H-6bII),
3.68 (brt, 1H, J ¼ 6.10 Hz, H-5III), 3.63 (m, 1H, H-3II), 3.63 (s, 3H, COOMe),
3.57 (brt, 1H, J ¼ 6.10 Hz, H-5II), 3.48 (m, 2H, Serb), 3.45 (m, 2H, H-5aI, 6aII),
2.29 (s, 6H, 2MePh), 2.28 (s, 6H, 2MePh), 2.22 (s, 3H, MePh), 2.07, 2.01, 1.94,
1.85, 1.83, 1.59 (6s, 3H ꢀ 6, 6MeCO), 1.36 (s, 9H, tert-Bu). Anal. Calcd for
.
C89H102N2O37 H2O: C, 59.06; H, 5.80; N, 1.55. Found: C, 59.08; H, 5.97; N, 1.43.
N-(9-Fluorenylmethoxycarbonyl)-O-f[methyl 2,3,4-tri-O-(4-
methylbenzoyl)-b-D-glucopyranosyl uronate]-(1 ! 3)-O-(2,4,6-tri-O-
acetyl-b-D-galactopyranosyl)-(1 ! 3)-O-(2,4,6-tri-O-acetyl-b-D-
galactopyranosyl)-(1 ! 4)-2,3-di-O-(4-methylbenzoyl)-b-D-
xylopyranosylg-L-serylglycine allyl ester (21)
To a solution of 17 (434.7 mg, 263.2 mmol) and 19 (335.1 mg, 789.5 mmol)
in CH2Cl2 (50 mL) was added MS AW300 (2.3 g). This mixture was stirred

Partial Sequences of Betaglycan
75
for 30 min at rt and then cooled to 2208C. To this solution was added a
TMSOTf (15 mL, 83 mmol) with stirring. Additional TMSOTf (15 and
10 mL) were added after 45 and 100 min, respectively, and stirring was con-
tinued for another 1 h. The reaction was quenched and treated as described
in the synthesis of 8. The residue was subjected to the columns of gel per-
meation (LH-20, 1:1 CHCl3:MeOH) and silica gel (3:1–1:2–1:4 n-hexane:E-
tOAc) to give 21 (427.3 mg) in 74% yield: [a]D þ37.1 (c 0.41, CHCl3); 1H
NMR (CDCl3): d 7.98–7.56 (m, 11H, Ar H), 7.70 (d, 2H, J ¼ 8.02 Hz, Ar
H), 7.57 (brt, 1H, J ¼ 7.45 Hz, Ar H), 7.40 (brt, 1H, J ¼ 7.10 Hz, Ar H),
7.31–7.22 (m, 2H, Ar H), 7.20–7.14 (m, 9H, Ar H), 7.08 (d, 2H,
J ¼ 8.02 Hz, Ar H), 6.91 [brs, 1H, NH(Gly)], 5.87–5.79 (m, 1H, 55CH), 5.79
(brt, 1H, J¼9.28 Hz, H-3IV), 5.64 (t, 1H, J3,4 ¼ J4,5 ¼ 9.63 Hz, H-4IV), 5.58
(t, 1H, J2,3 ¼ J3,4 ¼ 8.02 Hz, H-3I), 5.51 [m, 1H, NH(Ser)], 5.47 (d, 1H,
J3,4 ¼ 3.43 Hz, H-4III), 5.35 (m, 1H, H-2IV), 5.31–5.23 (m, 2H, 55CH2), 5.21
(m, 2H, H-2I, 4II), 5.00 (m, 2H, H-2II, 2III), 4.91 (d, 1H, H-1IV), 4.73
(m, 1H, H-1I), 4.54–4.38 (m, 1H, Sera), 4.51 (m, 2H, OCH2), 4.42 (m, 1H,
H-1II), 4.37–4.31 (m, 1H, Serba), 4.36 (m, 1H, H-1III), 4.26 (d, 1H, H-5IV),
4.17 (m, 1H, H-6bIII), 4.12 (dd, 1H, J4,5eq ¼ 7.10 Hz, Jgem ¼ 14.44 Hz,
H-5eqI), 4.06–3.68 [m, 3H, OCH2 (Fmoc), H-9(Fmoc)], 4.04 (dd, 1H,
J5,6a ¼ 6.64 Hz, Jgem ¼ 11.91 Hz, H-6aIII), 3.99 (m, 1H, H-4I), 3.93 (m, 1H,
Glyb), 3.90–3.54 (m, 1H, Serbb), 3.89 (m, 1H, Glya), 3.82 (m, 1H, H-3III),
3.77 (m, 1H, H-6bII), 3.75 (brt, 1H, J ¼ 5.96 Hz, H-5III), 3.70 (s, 3H,
COOMe), 3.68 (m, 1H, H-3II), 3.63 (brt, 1H, J ¼ 6.30 Hz, H-5II), 3.53
(m, 2H, H-5aI, 6aII), 2.36 (s, 12H, 4MePh), 2.29 (s, 3H, MePh), 2.15, 2.08,
2.05, 2.01, 1.92, 1.91 (6s, 3H ꢀ 6, 6MeCO). ESI-MS (positive) Calcd for
C99H105N2O37 [M þ H]þ, 1913.6. Found: 1913.2, Calcd for C99H104N2O37Na
[M þ Na]þ, 1935.6. Found: 1935.1.
N-Butoxycarbonyl-O-f[methyl 2,3,4-tri-O-(4-methylbenzoyl)-
b-D-glucopyranosyl uronate]-(1 ! 3)-O-(2,4,6-tri-O-acetyl-b-D-
galactopyranosyl)-(1 ! 3)-O-(2,4,6-tri-O-acetyl-b-D-
galactopyranosyl)-(1 ! 4)-2,3-di-O-(4-methylbenzoyl)-b-D-
xylopyranosylg-L-serylglycine (22)
Tetrakis(triphenylphosphine)palladium(0) (22.0 mg, 19.0 mmol) and
N-methylaniline (105 mL, 969 mmol) were added to the solution of 20
(173.5 mg, 96.6 mmol) in THF (4 mL) with stirring. Additional palladium
catalysts (22 and 16 mg) were supplied to the solution after 2 and 3 h, respect-
ively. One hour later, volatiles were removed under diminished pressure and
the residue was subjected to the columns of gel permeation (LH-20, 10:10:1
CHCl3:MeOH:AcOH) and silica gel (1:1–1:3 n-hexane:EtOAc) to give 22
(151.7 mg) in 90% yield. This compound was used for the next reaction
without further purification.

J. Tamura et al.
76
N-(9-Fluorenylmethoxycarbonyl)-O-f[methyl 2,3,4-tri-O-(4-
methylbenzoyl)-b-D-glucopyranosyl uronate]-(1 ! 3)-O-(2,4,6-tri-O-
acetyl-b-D-galactopyranosyl)-(1 ! 3)-O-(2,4,6-tri-O-acetyl-b-D-
galactopyranosyl)-(1 ! 4)-2,3-di-O-(4-methylbenzoyl)-b-D-
xylopyranosylg-L-serylglycine (23)
Tetrakis(triphenylphosphine)palladium(0) (45 mg, 39 mmol) and N-methy-
laniline (211 mL, 1.95 mmol) were added to the solution of 21 (427.3 mg,
195.1 mmol) in THF (8.2 mL) with stirring. Additional palladium catalyst
(22 mg) was supplied to the solution after 1.5 h. One hour later, volatiles
were removed under diminished pressure and the residue was subjected to
the column of silica gel (1:1–1:2–1:3 n-hexane:EtOAc–100:1:0.3–50:1:0.3–
30:1:0.3 EtOAc:MeOH:AcOH) to give 23 (320.0 mg) in 76% yield. This
compound was used for the next reaction without further purification.
N-(9-Fluorenylmethoxycarbonyl)-proline allyl ester (24)
To a solution of Fmoc-Pro-H (2.00 g, 5.93 mmol) in ethanol (100 mL) was
added a 6 mL of aq. Cs2CO3 (0.965 g, 2.96 mmol) with stirring at 208C. After
50 min, the reaction mixture was evaporated with toluene and dried in vacuo
for 3 h. DMF (50 mL) and 3-bromopropene (0.57 mL, 6.75 mmol) were added
to the residue and stirred overnight at rt. The reaction mixture was diluted
with EtOAc and treated as described in general methods. The crude materials
were subjected to the column of silica gel (11:1–7:1–5:1–4:1 n-hexane:EtOAc)
to give 24 (1.93 g, 5.11 mmol) in 86% yield: [a]D –54.4 (c 1.76, CHCl3); 1H NMR
(CDCl3): d 7.78–7.75 (m, 2H, Ar H), 7.64–7.54 (m, 2H, Ar H), 7.42–7.26 (m, 4H,
Ar H), 5.89 (m, 1H, 55CH), 5.28 (m, 2H, 55CH2), 4.65 (d, 2H, OCH2), 4.41
(m, Proa), 4.44 [m, 1H, one of CH2 (Fmoc)], 4.27 [m, 2H, one of CH2 (Fmoc),
H-9], 3.67 (m, 1H, Prodb), 3.55 (m, 1H, Proda), 2.28 (m, 1H, Probb), 2.05
.
.
(m, 1H, Proba), 1.90 (m, 2H, Prog). Anal. Calcd for C23H23NO4 0.3H2O: C,
72.21; H, 6.17; N, 3,66. Found: C, 72.64; H, 6.45; N, 3.64.
N-(9-Fluorenylmethoxycarbonyl)-Ni-(butoxycarbonyl)-L-tryptophanyl-
L-proline allyl ester (25)
Morpholine (4.9 mL) was added to a solution of 24 (429.4 mg, 1.137 mmol)
in CH2Cl2 (20 mL) and stirred overnight. The crude mixture was evaporated to
dryness with toluene and diluted with CH2Cl2 (22 mL). To this solution was
added HOBt (278 mg, 2.08 mmol) with stirring. This solution was cooled to
2208C and HBTU (430 mg, 1.13 mmol) was added. The cooling bath was
removed, and the reaction mixture was stirred at rt for 30 min. Then,
Fmoc-Trp(Boc)-H (544.6 mg, 1.034 mmol) was added. The reaction mixture
was stirred overnight and diluted with CHCl3. Organic phase was washed
with 1M HCl and aq. NaHCO3, brine and dried over MgSO4. The volatiles
were removed under diminished pressure. The crude materials were subjected

Partial Sequences of Betaglycan
77
to the column of silica gel (7:1–6:1–5:1–3:1–1:1 n-hexane:EtOAc) to give 25
1
(391.4 mg) in 56% yield: [a]D –26.3 (c 1.12, CHCl3); H NMR (CDCl3): d 8.14
(m, 1H, Ar H), 7.78–7.23 (m, 11H, Ar H), 5.93 (m, 1H, 55CH), 5.65 [d, 1H,
Ja,NH ¼ 8.54 Hz, NH(Trp)], 5.30 (m, 2H, 55CH2), 4.87 (ddd, Ja,ba ¼ 7.08 Hz,
Ja,bb ¼ 5.86 Hz, Trpa), 4.66 (d, 2H, OCH2), 4.55 (dd, Ja,ba ¼ 4.88 Hz,
Ja,bb ¼ 8.05 Hz, Proa), 4.37–4.15 [m, 3H, CH2 (Fmoc), 9H], 3.61 (m, 1H.
Prodb), 3.25 (m, 1H, Proda), 3.22 (dd, 1H, Jgem ¼ 14.64 Hz, Trpbb), 3.11 (dd,
1H, Trpba), 2.20 (m, 1H, Probb), 1.97 (m, 1H, Proba), 1.91 (m, 2H, Prog),
.
1.63 (s, 9H, t-Bu). Anal. Calcd for C39H41N3O7 2.5H2O: C, 66.07; H, 6.55; N,
5.93. Found: C, 65.94; H, 6.17; N, 5.71. ESI-MS (positive) Calcd for
C39H42N3O7 [M þ H]þ, 664.3. Found: 664.5, Calcd for C39H41N3O7Na
[M þ Na]þ, 686.3. Found: 686.4.
N-(9-Fluorenylmethoxycarbonyl)-Ni-(butoxycarbonyl)-L-tryptophanyl-
L-proline (26)
Tetrakis(triphenylphosphine)palladium(0) (247.3 mg, 0.241 mmol) and N-
methylaniline (2.3 mL, 21 mmol) were added to the solution of 25 (1.447 g,
2.135 mmol) in THF (18 mL) with stirring. After 15 h, volatiles were removed
under diminished pressure and the residue was subjected to a column of
silica gel (5:1 toluene:EtOAc–100:1:0.3–50:1:0.3 EtOAc:MeOH:AcOH) to give
26 (861.9 mg) in 63% yield. This compound was used for the next reaction
without further purification: 1H NMR (CDCl3): d 8.11–8.09 (d, 1H, Ar H),
7.74–7.21 (m, 11H, Ar H), 5.81 [d, 1H, Ja,NH ¼ 8.78 Hz, NH(Trp)], 4.85
(dd, Ja,ba ¼ 7.32 Hz, Ja,bb ¼ 7.08 Hz, Trpa), 4.57 (dd, Ja,ba ¼ 3.66 Hz,
Ja,bb ¼ 8.29 Hz, Proa), 4.36–4.14 [m, 3H, CH2 (Fmoc), 9H], 3.59 (m, 1H,
Prodb), 3.16 (m, 3H, Proda, Trpb), 2.22 (m, 1H, Probb), 2.03 (m, 1H, Proba),
1.85 (m, 2H, Prog), 1.62 (s, 9H, t-Bu).
N-(Benzyloxycarbonyl)-O-(tert-butyl)-L-aspartylglycine tert-butyl ester
(27)
.
Z-Asp(O-t-Bu)-H (200.0 mg, 0.586 mmol) and H-Gly-t-Bu HCl (98.2 mg,
0.586 mmol) were dissolved in CH2Cl2 (18 mL). To this was added Et3N
(81 mL, 0.59 mmol) and HOBt (158.4 mg, 1.172 mmol) with stirring. This
.
solution was cooled to 2208C, and then, a solution of WSCD HCl (146 mg,
0.762 mmol) in CH2Cl2 (5 mL) was added. The reaction mixture was stirred
overnight and gradually raised to rt, diluted with CHCl3, and treated as
described in the synthesis of 25. The crude materials were subjected to a
column of silica gel (5:1–1:2 n-hexane:EtOAc) to give 27 (246.1 mg) in 96%
1
yield: [a]D þ15.0 (c 1.45, CHCl3); H NMR (CDCl3): d 7.37–7.27 (m, 5H, Ar
H), 6.96 [m, 1H, NH(Gly)], 5.98 [d, 1H, Ja,NH ¼ 8.25 Hz, NH(Asp)], 5.14
(s, 2H, PhCH2), 4.57 (brs, 1H, Aspa), 3.92 (m, 2H, Gly), 2.92 (brdd, 1H,
J ¼ 4.38 and 16.95 Hz, Aspba), 2.64 (dd, 1H, Ja,bb ¼ 6.42, Jgem ¼ 13.95 Hz,

J. Tamura et al.
78
Aspbb), 1.46, 1.43 (2s, 9H ꢀ 2, 2t-Bu). Anal. Calcd. for C22H32N2O7: C, 60.54; H,
7.39; N, 6.42. Found: C, 60.31; H, 7.59; N, 6.27.
N-(9-Fluorenylmethoxycarbonyl)-Ni-(butoxycarbonyl)-L-tryptophanyl-
L-prolyl-O-(tert-butyl)-L-aspartylglycine tert-butyl ester (29)
A solution of 27 (246.1 mg, 563.8 mmol) in MeOH (5 mL) and AcOH
(0.1 mL) containing a catalytic amount of Pd-C was stirred vigorously for 3 h
under H2 atmosphere. The reaction mixture was filtered on Celite and the
volatiles were removed under diminished pressure to afford 28 (191.2 mg,
93%), which was dissolved in CH2Cl2 (20 mL). To this solution were added 26
(404.1 mg, 633.6 mmol), Et3N (73 mL, 0.53 mmol), and HOBt (171 mg,
1.27 mmol) with stirring. This solution was cooled to 2208C, and then a
.
solution of WSCD HCl (157 mg, 819 mmol) in CH2Cl2 (6 mL) was added.
The reaction mixture was stirred overnight gradually raised to rt, diluted
with CHCl3, and treated as described in the synthesis of 25. The crude
materials were subjected to a column of silica gel (5:1–3:1–1:1–1:3–1:5 n-hex-
1
ane:EtOAc) to give 29 (414.6 mg) in 85% yield: [a]D –20.7 (c 1.09, CHCl3); H
NMR (CDCl3): d 7.79–7.21 [m, 13H, Ar H, NH (Asp)], 7.22 [m, 1H, NH
(Gly)], 5.76 (d, 1H, Ja,b ¼ 5.50 and 8.02 Hz, Proa), 4.30 [dd, 1H, one of CH2
(Fmoc)], 4.23 [dd, 1H, one of CH2 (Fmoc)], 4.14 [m, 1H, H-9 (Fmoc)], 4.11
(m, 1H, Glya), 3.82 (m, 1H, Glyb), 3.79 (m, 1H, Prodb), 3.54 (m, 1H, Proda),
3.28 (d, 2H, Trpb), 3.00 (dd, 1H, Ja,ba ¼ 4.35 Hz, Jgem ¼ 16.95 Hz, Aspba), 2.59
(dd, 1H, Ja,bb ¼ 5.96 Hz, Aspbb), 2.18 (m, 3H, Probb, Trpba, Progb), 2.03
(m, 1H, Proga), 1.66, 1.61, 1.46, (3s, 9H ꢀ 3, 3t-Bu). Anal. Calcd for
.
C50H61N5O11 3H2O: C, 62.41; H, 7.03; N, 7.28. Found: C, 62.22; H, 6.77; N, 7.07.
N-Butoxycarbonyl-O-f[methyl 2,3,4-tri-O-(4-methylbenzoyl)-b-D-
glucopyranosyl uronate]-(1 ! 3)-O-(2,4,6-tri-O-acetyl-b-D-
galactopyranosyl)-(1 ! 3)-O-(2,4,6-tri-O-acetyl-b-D-
galactopyranosyl)-(1 ! 4)-2,3-di-O-(4-methylbenzoyl)-b-D-
xylopyranosylg-L-serylglycyl-Ni-(butoxycarbonyl)-L-tryptophanyl-L-
prolyl-O-(tert-butyl)-L-aspartylglycine tert-Butyl Ester (31)
Morpholine (0.9 mL) was added to a solution of 29 (110.5 mg, 119.8 mmol) in
CH2Cl2 (3.5 mL) and stirred overnight. The crude mixture was evaporated to
dryness with toluene, and the residue was subjected to a column of silica gel
(1:1 n-hexane:EtOAc–10:1:0.3 EtOAc:MeOH:Et3N) to afford 30 (83.6 mg)
quantitatively. A part of 30 (41.3 mg, 59.0 mmol) was dissolved in DMF
(130 mL), and HOBt (7.9 mg, 58 mmol) was added to the solution with
stirring. This solution was cooled to 2208C and HBTU (12.0 mg, 31.6 mmol)
was added. The cooling bath was removed, and the reaction mixture was
stirred at rt for 30 min. Then, 22 (47.8 mg, 27.2 mmol) and i-Pr2EtN (10.3 mL,
59.1 mmol) in DMF (132 mL) were added. The reaction mixture was stirred
overnight and diluted with EtOAc. Organic phase was treated as described

Partial Sequences of Betaglycan
79
in the synthesis of 25. The volatiles were removed under diminished pressure.
The crude materials were subjected to a column of gel permeation (1:1
1
CHCl3:MeOH) to give 31 (41.5 mg) in 63% yield: [a]D þ78 (c 0.1, CHCl3); H
NMR (CDCl3): d 8.13–7.05 (m, 25H, Ar H), 7.45 [d, 1H, Ja,NH ¼ 8.25 Hz, NH
(Asp)], 7.18 [m, 1H, NH (Gly)], 6.94 [br, 1H, NH (Gly)], 6.78 [br, 1H, NH
(Trp)], 5.80 (t, 1H, J2,3 ¼ J3,4 ¼ 9.39 Hz, H-3IV), 5.64 (brt, 1H, J ¼ 9.26 Hz,
H-4IV), 5.53 (dd, 1H, J2,3 ¼ 7.79 Hz, J3,4 ¼ 8.25 Hz, H-3I), 5.47 (d, 1H,
J3,4 ¼ 2.75 Hz, H-4III), 5.37 [m, 1H, NH (Ser)], 5.36 (dd, 1H, J1,2 ¼ 7.10 Hz,
H-2IV), 5.20 (d, 1H, J3,4 ¼ 3.21 Hz, H-4II), 5.17 (d, 1H, J1,2 ¼ 6.18 Hz, H-2I),
5.06 (m, 1H, Trpa), 4.99 (m, 2H, H-2II, 2III), 4.92 (d, 1H, H-1IV), 4.79 (m, 1H,
Aspa), 4.66 (d, 1H, H-1I), 4.49 (t, 1H, J ¼ 6.42 Hz, Proa), 4.40 (d, 1H,
or
or
J1,2 ¼ 8.02 Hz, H-1III
II), 4.37 (d, 1H, J1,2 ¼ 8.02 Hz, H-1II
III), 4.28
(m, 1H, Sera), 4.27 (d, 1H, J4,5 ¼ 9.85 Hz, H-5IV), 4.16 (dd, 1H, J5,6b
¼
5.73 Hz, Jgem ¼ 11.62 Hz, H-6bIII), 4.06 (m, 1H, H-5eqI), 4.03 (dd, 1H,
J5,6a ¼ 5.27 Hz, H-6aIII), 4.00 (m, 1H, Glya), 3.92 (m, 1H, H-4I), 3.83
(dd, J2,3 ¼ 10.31 Hz, H-3III), 3.81 (m, 1H, Glyb), 3.8–3.5 (m, 2H, H-6II), 3.76
(brt, J ¼ 6.56 Hz, H-5III), 3.75 (m, 2H, Gly), 3.70 (s, 3H, COOMe), 3.69
(m, 1H, H-3II), 3.68 (m, 1H, Prodb), 3.61 (brt, 1H, J ¼ 7.10 Hz, H-5II), 3.55
(m, 2H, Serb), 3.47 (dd, 1H, J4,5ax ¼ 8.01 Hz, Jgem ¼ 12.14 Hz, H-5axI), 3.37
(m, 1H, Proda), 3.24 and 3.15 (m, 2H, Trpb), 2.90 (dd, 1H, Ja,ba ¼ 4.81 Hz,
Jgem ¼ 16.96 Hz, Aspba), 2.62 (dd, 1H, Ja,bb ¼ 5.95 Hz, Aspbb), 2.36, 2.35 (2s,
3H ꢀ 2, 2MePh), 2.34 (s, 6H, 2MePh), 2.29 (s, 3H, MePh), 2.15 (s, 6H,
2MeCO), 2.12 (m, 2H, Prob), 2.09, 2.02, 1.93, 1.91 (4s, each 3H, 4MeCO),
1.92 (m, 2H, Prog), 1.66, 1.44, 1.40, 1.39 (4s, each 9H, 4t-Bu). ESI-MS
(positive) Calcd. for C121H148N7O45 [M þ H]þ, 2418.9. Found: 2418.8.
O-fb-D-Glucopyranuronosyl-(1 ! 3)-O-b-D-galactopyranosyl-(1 ! 3)-O-
b-D-galactopyranosyl-(1 ! 4)-b-D-xylopyranosylg-L-serylglycyl-L-
tryptophanyl-L-prolyl-L-aspartylglycine (1)
To a solution of 31 (30.4 mg, 12.5 mmol) in CH2Cl2 (0.75 mL) was added a
TFA cocktail (40.7:2.5:2.5:2.5:1.3:50 trifluoroacetic acid:thioanisole:phenol:
H2O:1,2-ethanedithiol:triisopropylsilane:CH2Cl2) (0.75 mL) and stirred for
2 h. Volatiles were removed with toluene under diminished pressure and the
residue was eluted from Bond Elutw C8 (H2O–1:1–1:2 H2O:MeOH–MeOH–
CH2Cl2). The crude product was diluted with 50% MeOH (3 mL) and 0.107M
NaOMe (1.4 mL) was added to the solution during 3 h keeping less than pH
9. Then, the solution was neutralized with 50% AcOH and the volatiles were
removed under diminished pressure. The crude materials were subjected to a
column of gel permeation (LH-20, 1% AcOH) to give 1 (14.8 mg) in 83% yield.
1H NMR (D2O): d (selected) 7.71–7.20 (m, 5H, Ar H), 4.96 (t, J ¼ 6.9 Hz,
Trpa), 4.75 (dd, J ¼ 6.0 and 7.0 Hz, Aspa), 4.70 (d, J1, 2¼ 7.9 Hz, H-1IV), 4.66
(dd, J ¼ 5.8 and 7.8 Hz, Aspa0), 4.59 (d, J1,2 ¼ 7.9 Hz, H-1III), 4.57 (m, Trpa0),
4.56 (d, J1,2 ¼ 7.5 Hz, H-1II), 4.46 (d, J1,2 ¼ 7.8 Hz, H-1I), 4.42

J. Tamura et al.
80
(d, J1,2 ¼ 7.3 Hz, H-10 I), 4.41 (m, Proa, Sera), 4.33 (dd, J ¼ 5.0 and 11.0 Hz,
Serba), 4.30 (t, J ¼ 4.1 Hz, Sera0), 4.24 (m, Serba0), 4.14 (s, H-4IIorIII), 4.10
(s, H-4IIIorII), 4.10 (m, Serbb), 4.04 (m, Serbb0), 3.78 (m, H-3IIorIII), 3.73
(m, H-2II, 2III, Proda), 3.63 (m, H-3IIIorII), 3.42 (m, H-2IV), 3.37 (m, H-21,
I
Prodb), 3.33 (m, H-20 ), 3.31 (m, Trpba), 3.30 (m, Prodb0), 3.14 (m, Trpbb),
2.94 (dd, Jgem ¼ 19.1 Hz, Aspba), 2.90 (dd, Jgem ¼ 19.1 Hz, Aspba0), 2.79 (dd,
Aspbb), 2.77 (dd, Aspbb0), 2.22 (m, Proba), 1.92 (m, Probb, Prog), 1.64, 1.44
(m, Prob0, Proga0), 0.97 (m, Progb0); FAB-MS (positive) Calcd. for
C50H69N7O30Na3
[M þ H]þ,
1316.4.
Found:
1316.3.
Calcd.
for
C50H68N7O30Na4 [M þ Na]þ, 1338.4. Found: 1338.4.
General Procedure for Solid-Phase Synthesis
Glycopeptides were synthesized manually for 100 mg of Sieber amide resin
(52 mmol) as follows. The Fmoc group was removed with 20% of piperidine/
NMP (2 mL) (2 ꢀ 3 min and 1 ꢀ 20 min), and was monitored by Kaiser ninhy-
drin test. After washing with NMP (2.2 mL) (6 ꢀ 1 min) and CH2Cl2 (2.2 mL)
(3 ꢀ 1 min), it was dried in vacuo. The N-terminal free resin or peptide-on-
resin was swollen in NMP (2 mL) and shaken overnight with corresponding
Fmoc amino acid (130 mmol), HOBt (130 mmol), HBTU (130 mmol), and i-
Pr2EtN (260 mmol). Coupling was monitored by Kaiser test. The resin was
washed with NMP (2.2 mL) (3 ꢀ 1 min) and CH2Cl2 (2.2 mL) (3 ꢀ 1 min) and
dried in vacuo.
O-fb-D-Glucopyranuronosyl-(1 ! 3)-O-b-D-galactopyranosyl-(1 ! 3)-O-
b-D-galactopyranosyl-(1 ! 4)-b-D-xylopyranosylg-L-serylglycyl-L-
tryptophanyl-L-prolyl-L-aspartylglycineamide (2)
Fmoc-Gly-H (46.4 mg, 156 mmol), Fmoc-Asp (O-t-Bu)-H (64.1 mg, 156 mmol),
Fmoc-Pro-H (52.6 mg, 156 mmol), and Fmoc-Trp(Boc)-H (82.1 mg, 156 mmol)
were coupled in turn on Sieber amide resin (300 mg, 156 mmol) with HOBt
(21.3 mg, 156 mmol), HBTU (53.3 mg, 156 mmol), and i-Pr2EtN (55 ml,
312 mmol) in NMP (2.7 mL). A part of the N-terminal of H-Trp(Boc)-Pro-Asp(O-
t-Bu)-Gly-resin (67.4 mg, 27 mmol) was coupled with 23 (116 mg, 54 mmol) as
described in the general procedure. The resultant resin was exposed to TFA
cocktail (3.5 mL) with shaking for 7.5 h and evaporated. The residue was
eluted from Bond Elutw C8 (1:0–0:1 H2O–MeOH) and subjected to a column of
gel permeation (LH-20, 1% AcOH) to give crude glycopeptide (26.5 mg) still
having acyl groups. Saponification was performed with 1 N NaOH-MeOH
(1:100, 6.2 mL) overnight. The reaction mixture was diluted with H2O (6 mL),
neutralized with 0.1 N AcOH after 5 h, and evaporated to dryness. The residue
was subjected to further saponification with 5 mM NaOH (5 mL) for 4 days.
The reaction mixture was neutralized in the same manner. The crude products
were purified by HPLC (C18, AꢀB ¼ 10%CH3CN þ 0.1%CF3COOHꢀ

Partial Sequences of Betaglycan
81
90%CH3CN þ 0.1%CF3COOH) to give 2. TOF-MS (positive) Calcd. for
C50H71N8O29Na [M þ Na]þ, 1271.4. Found: 1271.4, Calcd. for C50H70N8O29Na2
[M–H þ 2Na]þ, 1293.4. Found: 1293.4.
ACKNOWLEDGEMENT
This study was supported by a Grant-in-Aid for Scientific Research (C),
12660098, from the Ministry of Education, Science, Culture and Sport of
Japan. The authors thank Ms. Miyuki Tanmatsu for the elemental analysis
measurements (Research Center for Bioscience and Technology, Division of
Instrumental Analysis, Tottori University).
REFERENCES
[1] (a) Sugahara, K.; Kitagawa, H. Recent advances in the study of the biosynthesis
and functions of sulfated glycosaminoglycans. Curr. Opin. Struct. Biol. 2000, 10,
518–521; (b) Sugahara, K.; Kitagawa, H. Heparin and heparan sulfate biosyn-
thesis. IUBMB Life 2002, 54, 163–175; (c) Silbert, J.E.; Sugumaran, G. Biosyn-
thesis of chondroitin/dermatan sulfate. IUBMB Life 2002, 54, 177–186;
(d) Kitagawa, H.; Izumikawa, T.; Uyama, Y.; Sugahara, K. Molecular cloning of a
chondroitin polymerizing factor that cooperates with chondroitin synthase
for chondroitin polymerization. J. Biol. Chem. 2003, 278, 23666–23671;
(e) Funderbruch, L.L. Keratan sulfate biosynthesis. IUBMB Life 2002, 54,
187–194; (f) DeAngelis, P.L. Microbial glycosaminoglycan glycosyltransferases. Gly-
cobiology 2002, 12, 9R–16R.
[2] (a) Sugahara, K.; Yamada, S.; de Yoshida, K.; Waard, P.; Vliegenthart, J.F.G. A
novel sulfated structure in the carbohydrate-protein linkage region isolated
from porcine intestinal heparin. J. Biol. Chem. 1992, 267, 1528–1533;
(b) Sugahara, K.; Tsuda, H.; Yoshida, K.; Yamada, S.; de Beer, T.; Vliegenthart, J.F.G.
Structure determination of the octa- and decasaccharide sequences isolated from the
carbohydrate-protein linkage region of porcine intestinal heparin. J. Biol. Chem.
1995, 270, 22914–22923.
[3] Esko, J.D.; Zhang, L. Influence of core protein sequence on glycosaminoglycan
assembly. Curr. Opin. Struct. Biol. 1996, 6, 663–670.
[4] Zhang, L.; Esko, J.D. Amino acid determinants that drive heparan sulfate
assembly in a proteoglycan. J. Biol. Chem. 1994, 269, 19295–19299.
´
´
[5] Lopez-Casillas, F.; Payne, H.M.; Andres, J.L.; Massague, J. Betaglycan can act as a
dual modulator of TGF-beta access to signaling receptors: mapping of ligand
binding and GAG attachment sites. J. Cell. Biol. 1994, 124, 557–568.
[6] Hudson, C.C.; Johnson, J.M. The isomeric tetracetates of xylose, and observations
regarding the acetates of melibiose, trehalose and sucrose. J. Am. Chem. Soc. 1915,
37, 2748–2753.
[7] Tamura, J.; Nishihara, J. Synthesis of phosphorylated and sulfated glycosyl serines
in the linkage region of the glycosaminoglycans. J. Org. Chem. 2001, 66,
3074–3083.
[8] Nakano, T.; Ito, Y.; Ogawa, T. Total synthesis of a sulfated glucuronyl glycosphin-
golipid, IV3GlcA(3-SO3)nLcOse4Cer, a carbohydrate epitope of neural cell adhesion
molecules. Tetrahedron Lett. 1990, 31, 1597–1600.

J. Tamura et al.
82
[9] Goto, F.; Ogawa, T. Synthesis of sulfated glycohexaose of linkage region of
chondroitin 4-sulfate: b-D-GlcA-(1 ! 3)f(SO3Na ! 4)g-b-D-GalNAc-(1 ! 4)-b-D-
GlcA-(1 ! 3)-b-D-Gal-(1 ! 3)-b-D-Gal-(1 ! 4)-D-Xyl. Tetrahedron Lett. 1992, 33,
6841–6844.
[10] Rio, S.; Beau, J.-M.; Jacquinet, J.-C. Synthesis of glycopeptides from the carbo-
hydrate-protein linkage region of proteoglycans. Carbohydr. Res. 1991, 219,
71–90.
[11] Liebe, B.; Kunz, H. Solid-phase synthesis of a sialyl-Tn-glycoundecapeptide of the
MUC1 repeating unit. Helv. Chim. Acta. 1997, 80, 1473–1482.
[12] Uyama, T.; Kitagawa, H.; Tanaka, J.; Tamura, J.; Ogawa, T.; Sugahara, K. Molecu-
lar cloning and expression of a second chondroitin N-acetylgalactosaminyl trans-
ferase involving in the initiation and elongation of chondroitin/dermatan sulfate.
J. Biol. Chem. 2003, 278, 3072–3078.
[13] Kim, B.-T.; Kitagawa, H.; Tanaka, J.; Tamura, J.; Sugahara, K. In vitro heparan
sulfate polymerization. J. Biol. Chem. 2003, 278, 41618–41623.