Synthesis of the glycosaminoglycan-protein linkage tetraosyl peptide moieties of betaglycan, which serve as a hexosamine acceptor for enzymatic glycosyl transfer

Article abstract of DOI:10.1016/j.carres.2010.06.019

Betaglycan, also known as TGF-β type III receptor, is a membrane-anchored proteoglycan, which has two glycosaminoglycan (GAG) attachment sites (Lo?pez-Casillas, F.; Payne, H. M.; Andres, J. L.; Massague?, J. J. Cell Biol. 1994, 124, 557-568). Chondroitin sulfate (CS) or heparan sulfate (HS) can attach to the first site, Ser⁵³⁵, whereas only CS attaches to the second, Ser⁵⁴⁶. Although the mechanism behind the assembly of CS and HS is not fully understood, it has been reported that the assembly of HS requires not only a cluster of acidic residues but also hydrophobic residues located near the Ser-Gly attachment sites (Esko, J. D. Zhang, L. Curr. Opin. Struct. Biol. 1996, 6, 663-670). To further understand the effects of amino acids close to the Ser residues of the GAG-attachment sites on the glycosyltransferases, two tetraosyl peptides derived from the CS attachment sites of betaglycan, GlcA-Gal-Gal-Xyl-SerGlyAspAsnGly (1) and GlcA-Gal-Gal-Xyl-SerGlyAspAsnGlyPheProGly (2), were synthesized, and used as donor substrates for β1,4-N-acetylgalactosaminyltransferase-I (β4GalNAcT-I) and α1,4-N-acetylglucosaminyltransferase-I (α4GlcNAcT-I). Both the chemically synthesized linkage region tetrasaccharides were far better acceptors for β4GalNAcT-I than for α4GlcNAcT-I in vitro, although they also showed appreciable acceptor activity for α4GlcNAcT-I.

Full text of DOI:10.1016/j.carres.2010.06.019

Carbohydrate Research 345 (2010) 2115–2123
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of the glycosaminoglycan–protein linkage tetraosyl peptide
moieties of betaglycan, which serve as a hexosamine acceptor for
enzymatic glycosyl transfer
a
a
b
b
Jun-ichi Tamura a, , Tomomi Nakamura-Yamamoto , Yuko Nishimura , Shuji Mizumoto , Jun Takahashi ,
*
Kazuyuki Sugahara b
a Department of Regional Environment, Faculty of Regional Sciences, Tottori University, Tottori 680-8551, Japan
b Laboratory of Proteoglycan Signaling and Therapeutics, Frontier Research Center for Post-Genomic Science and Technology, Faculty of Advanced Life Science,
Hokkaido University Graduate School of Life Science, Sapporo 001-0021, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Betaglycan, also known as TGF-b type III receptor, is a membrane-anchored proteoglycan, which has two
glycosaminoglycan (GAG) attachment sites (López-Casillas, F.; Payne, H. M.; Andres, J. L.; Massagué, J.
J. Cell Biol. 1994, 124, 557–568). Chondroitin sulfate (CS) or heparan sulfate (HS) can attach to the first
site, Ser535, whereas only CS attaches to the second, Ser546. Although the mechanism behind the assembly
of CS and HS is not fully understood, it has been reported that the assembly of HS requires not only a clus-
ter of acidic residues but also hydrophobic residues located near the Ser-Gly attachment sites (Esko, J. D.
Zhang, L. Curr. Opin. Struct. Biol. 1996, 6, 663–670). To further understand the effects of amino acids close
to the Ser residues of the GAG-attachment sites on the glycosyltransferases, two tetraosyl peptides
derived from the CS attachment sites of betaglycan, GlcA-Gal-Gal-Xyl-SerGlyAspAsnGly (1) and GlcA-
Gal-Gal-Xyl-SerGlyAspAsnGlyPheProGly (2), were synthesized, and used as donor substrates for b1,4-
Received 23 February 2010
Received in revised form 10 June 2010
Accepted 28 June 2010
Available online 31 August 2010
Keywords:
Chondroitin sulfate
Heparan sulfate
Glycoconjugate synthesis
Glycosyl transfer
N-acetylgalactosaminyltransferase-I
(b4GalNAcT-I)
and
a1,4-N-acetylglucosaminyltransferase-I
N-Acetylgalactosaminyltransferase-I
a1,4-N-Acetylglucosaminyltransferase-I
(a
4GlcNAcT-I). Both the chemically synthesized linkage region tetrasaccharides were far better acceptors
for b4GalNAcT-I than for
a4GlcNAcT-I in vitro, although they also showed appreciable acceptor activity
for 4GlcNAcT-I.
a
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
nyltransferase-I (b4GalNAcT-I) or
a1,4-N-acetylglucosaminyltrans-
ferase-I ( 4GlcNAcT-I), which initiates the synthesis of a CS or HS
a
Glycosaminoglycans (GAGs) are linear polysaccharide side
chains of proteoglycans (PGs), and are composed of two parts, a
linkage tetrasaccharide, glucuronic acid-galactose-galactose-xy-
lose (GlcAb1-3Galb1-3Galb1-4Xylb1-O-), which is covalently
linked to the respective core-protein through the hydroxyl group
of the serine residue(s), and a repeating disaccharide region formed
by hexosamine and hexuronic acid. GAGs are classified into a chon-
droitin sulfate (CS) type and a heparan sulfate (HS) type based on
the hexosamine residues of the repeating disaccharides (Fig. 1).
In the endoplasmic reticulum and Golgi apparatus, the tetrasaccha-
ride of the GAG–protein linkage region is elongated by stepwise
additions of monosaccharides to the non-reducing terminal by
the corresponding glycosyltransferases from uridine diphosphate
(UDP)-monosaccharides.1 Following completion of the synthesis
of the linkage region, GlcAb1-3Galb1-3Galb1-4Xylb1-O-Ser, the
first GalNAc or GlcNAc residue is transferred to the non-reducing
terminal GlcA in the linkage region by b1,4-N-acetylgalactosami-
chain, respectively. Then, the repeating disaccharide region of CS
and HS is further elongated by the chondroitin synthase family
and exostosin (EXT) family, respectively.2–4 Thus, the first transfer
of the hexosamine residue,
a-GlcNAc or b-GalNAc, which is the
fifth saccharide from the reducing terminal, is crucial in determin-
ing the type of GAG, HS, or CS.
Betaglycan, also known as transforming growth factor (TGF)-b
type III receptor, contains two Ser residues bearing a CS and/or
HS chain in most mammalian cells.5 Ser535 can have either CS or
HS, whereas Ser546 only bears CS.5–7 Zhang and Esko demonstrated
that the synthesis of a HS chain on betaglycan requires a cluster of
acidic amino acids and tryptophan residues located near the Ser-
Gly of the GAG-attachment site using betaglycan chimeras, created
by fusing short peptides to Protein A and/or polyhistidine tags and
expression in Chinese hamster ovary cells.7 However, the molecu-
lar mechanism behind the assembly of HS and CS at GAG-attach-
ment sites is still unclear. The use of synthetically available
substrates is one of the best approaches to elucidating the regula-
tion of the enzymatic elongation of glycan. To further understand
the differential assembly of HS and CS chains, this paper reports
* Corresponding author. Tel./fax: +81 857 31 5108.
E-mail address: jtamura@rstu.jp (J. Tamura).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.06.019

2116
J. Tamura et al. / Carbohydrate Research 345 (2010) 2115–2123
Figure 1. Structure of glycosaminoglycans.
the facile synthesis of partial structures of betaglycan having the
GAG–protein linkage tetrasaccharide attached to Ser546 with pep-
tides of different lengths, GlcA-Gal-Gal-Xyl-SerGlyAspAsnGly (1)
and GlcA-Gal-Gal-Xyl-SerGlyAspAsnGlyPheProGly (2). In addition,
converted to the corresponding imidate (10) in 88% yield (two
steps) in the same manner as mentioned above.
The serylglycine allyl ester protected with Fmoc at the N-termi-
nal (11)16 was glycosylated with 10 in a TMSOTf-mediated manner
to give 12 in 92% yield. The allyl group of 12 was removed with
Pd(PPh3)4 and N-methylmorpholine to obtain the corresponding
carboxylic acids (13) in 93% yield.
enzymatic transfers of b-GalNAc and
a-GlcNAc to the non-reduc-
ing terminus of not only these linkage region tetrasaccharide-pep-
tides but also GlcA-Gal-Gal-Xyl-Ser535GlyTrpProAspGly (3),8 which
was synthesized previously, were performed to produce acceptor
As shown in Scheme 2, a commercially available Boc-asparagine
was coupled with glycine methyl ester in the presence of HOBt,
WSCDÁHCl, and Et3N to give the dipeptide (14) in 39% yield. The
Bocgroup of 14 was quantitatively removedwithTFA to give the cor-
responding amine (15) which was elongated with Fmoc-(O-tBu)-
aspartic acid in the same manner as for the synthesis of 14 to afford
the tripeptide (16) in 63% yield. The Fmoc group of 16 was quantita-
tively removed with morpholine to give 17, which was used for the
coupling with 13 in the presence of HOBt and HBTU in DMF. Subse-
quent TFA treatment afforded the tetraosyl pentapeptide (18) in 98%
yield (two steps). Mild saponification was required for the deprotec-
tion of 18 to avoid the elimination of the glycan from the peptide: (1)
aq LiOH in THF for 1 h, (2) 0.25 M NaOH in MeOH for 6 h, then (3)
0.1 M NaOMe in aq MeOH for 5 days. Purification was performed
by gel-permeation (LH-20) to give 1 in 80% yield.
Another target, 2, was synthesized on the Sieber amide resin
(Scheme 3). The elongation was performed manually by the Fmoc
procedure employing HOBt, HBTU, and DIPEA in NMP. Fmoc was re-
moved with piperidine in NMP. The N-terminal of the hexapeptide
on the resin was coupled with 13 in the same manner as for the pep-
tide elongation. Then the glycosyl peptide on resin was treated with
a TFA cocktail to give the corresponding tetraosyl octapeptide with-
out the tert-butyl and trityl groups. Careful saponification as de-
scribed for the synthesis of 1 furnished the desired tetraosyl
octapeptide 2, which was purified by gel-permeation (LH-20) and
HPLC (C8). Tetraosyl penta- and octapeptides (1 and 2) were com-
pletely assigned by 1H NMR spectroscopy and by MALDI-TOFMS.
To study the mechanism behind the assembly of CS and HS, the
glycosyltransferase activities were assayed using the tetraosyl
peptides (1, 2, and 3) as the sugar acceptors and either UDP-GalNAc
substrates for b4GalNAcT-I and
a4GlcNAcT-I using human CS N-
acetylgalactosaminyltransferase 2 (CSGalNAcT2)9 and exostosin-
like 3 (EXTL3)4,10 as enzyme sources, respectively.
2. Results and discussion
To obtain the linkage tetrasaccharide in fewer steps than that
previously reported,8 GlcA-Gal + Gal-Xyl glycosylation was
adopted. As depicted in Scheme 1, the GlcA-Gal moiety was pre-
pared by coupling glucuronosyl trichloroacetimidate (4)11 with
the galactosyl acceptor (5)12 in the presence of TMSOTf in CH2Cl2
to give the b-linked disaccharide (6) in 24% yield together with
the recovery of 5 in 60% yield. The coupling yield to obtain GlcA-
Gal depends on the substrates.13,14 Some efforts have been made
to increase the yield by protecting the acceptors with benzyl
ether15 or silylene acetal.14 Although these modifications are effec-
tive, here, importance was attached to shortening steps for imme-
diate synthesis. The disaccharide (6) was converted to the
corresponding hemiacetal by the removal of the 4-methoxyphenyl
group with CAN, and subsequent trichloroacetimidation with
CCl3CN and DBU afforded 7 in 69% and 87% yields, respectively.
Compound 7 was coupled with the disaccharide acceptor (8)8 in
the presence of TMSOTf to give the desired tetrasaccharide (9b)
in 47% yield. Even with the acetyl group at the 2-O position of
the glycosyl donor, the
Starting from methyl tetra-O-acetyl-b-
4-methoxyphenyl 2,4,6-tri-O-acetyl-3-O-allyl-b-
a
-isomer (9
a
) was obtained in 24% yield.
-glucopyranosyl uronate,
-galactopyrano-
D
D
side, and 8, tetrasaccharide (9b) was obtained in seven steps, while
11 steps were needed previously.8 The tetrasaccharide (9b) was

J. Tamura et al. / Carbohydrate Research 345 (2010) 2115–2123
2117
Scheme 1. Reagents and conditions: (a) TMSOTf, MSAW300, CH2Cl2, À20 to À10 °C; (b) CAN, CH3CN, H2O, 0 °C; (c) CCl3CN, DBU, CH2Cl2, 0 °C; (d) Pd(PPh3)4, N-methylaniline,
THF, 2 h. Abbreviations: MP, 4-MeOC6H4; MBz, 4-MeC6H4CO; All, CH2@CHCH2.
or UDP-GlcNAc as a sugar donor. The enzymes used were soluble
forms of CSGalNAcT29 and EXTL34 secreted into the culture medium
by COS-7 cells transfected with the respective expression vectors.
Gal-Xyl-SGDNGFPG (2) (Table 1). Therefore, a4GlcNAcT-I of EXTL3
may not always require tryptophan and/or a hydrophobic residue
near the GAG-attachment site although they appear to be enhancer
elements.6 Alternatively or in addition, other EXT family members
such as EXT1 and EXT2 may be involved in the initiation/elongation
of a HS chain at this particular site. In fact, the EXT1/EXT2
complex, HS-polymerase, could synthesize heparan polymers
in vitro on GlcA-Gal-Gal-Xyl-SGWPDG (3) but not on GlcA-Gal-
Gal-Xyl-(G)SGE, which corresponds to the CS attachment site of
CSGalNAcT2 and EXTL3 transfer b-GalNAc and
terminal to elongate the linkage tetrasaccharide toward CS and HS
via the glycosyltransferase activities of b4GalNAcT-I and 4GlcN-
AcT-I, respectively. It is worth noting that b-GalNAc and -GlcNAc
a-GlcNAc at the GlcA
a
a
were transferred to both the tetraosyl oligopeptides, GlcA-Gal-Gal-
Xyl-S(546)GDNG and GlcA-Gal-Gal-Xyl-S(546)GDNGFPG (1 and 2),
though with remarkably higher activity for b-GalNAc than for
a
-Glc-
a
-thrombomodulin.3
NAc (Table 1 and Fig. 2). These results were compatible with the fact
that the naturally occurring betaglycan has only CS at Ser546. The
next issue addressed was whether the tetraosyl oligopeptide,
GlcA-Gal-Gal-Xyl-S(535)GWPDG (3), in which the Ser residue corre-
sponds to a potential GAG-attachment site for both HS and CS,5,7
3. Conclusions
Partial sequences of betaglycan having a linkage tetrasaccharide
at Ser546, the site of attachment of CS but not HS in vivo, were syn-
thesized with peptides of different lengths. The newly synthesized
PG primers 1 and 2 proved to be superior acceptors for b-GalNAc
than for a-GlcNAc transfers. However, they also showed apprecia-
ble activity as an acceptor for
serves as an acceptor substrate for a4GlcNAcT-I and b4GalNAcT-I.
Similar b4GalNAc transferase activity of CSGalNAcT2 was observed
using 1, 2, or 3 as an acceptor, suggesting b4GalNAcT-I to be little
influenced by the amino acid sequences around the GAG-attach-
ment site, consistent with previous reports.7,9,17 It has been pro-
a4GlcNAc.
4. Experimental
posed that
the core-protein in the acceptor substrate, which enhanced HS bio-
synthesis and 4GlcNAcT-I activity in a cell system and in vitro,
respectively.6,7,17 Unexpectedly, the amount of
4GlcNAc trans-
a4GlcNAcT-I requires a hydrophobic aglycon moiety of
4.1. General methods
a
a
Optical rotations were measured at 22 3 °C with a JASCO
ferred by EXTL3 to GlcA-Gal-Gal-Xyl-SGWPDG (3) was comparable
to that transferred to GlcA-Gal-Gal-Xyl-SGDNG (1) and GlcA-Gal-
polarimeter DIP-18-1. 1H NMR assignments were confirmed by

2118
J. Tamura et al. / Carbohydrate Research 345 (2010) 2115–2123
Scheme 2. Reagents and conditions: (a) WSCDÁHCl, HOBt, Et3N, CH2Cl2, À20 °C to rt, overnight; (b) TFA, CH2Cl2; (c) morpholine, CH2Cl2; (d) HBTU, HOBt, i-Pr2EtN, DMF,
À20 °C to rt, 2 h; (e) 1.25 M LiOH, THF, H2O, 0 °C, 1 h, then 1 M AcOH; (f) 0.25 M NaOH, MeOH, H2O, overnight, then 1 M AcOH; (g) 0.1 M NaOMe, MeOH, H2O, 5 days, then 1 M
AcOH.
two-dimensional HH COSY experiments with a JEOL ECP 500 MHz
spectrometer. As an example of signal assignments, 1III stands for a
proton at C-1 of sugar residue III. Silica gel chromatography, ana-
lytical TLC, and preparative TLC (PTLC) were performed on a col-
umn of Silica Gel 60 (E. Merck), Silica Gel 60N (spherical neutral)
(Kanto Kagaku), and glass plates coated with Silica Gel F254 (E.
Merck), respectively. The gel for size-exclusion chromatography
(Sephadex LH-20) was from GE Healthcare. Molecular sieves
(MS) were from GL Science, Inc. and activated at 200 °C under
diminished pressure prior to use. All reactions in organic solvents
were performed under a dry Ar-containing atmosphere. As a usual
work-up, the organic phase of the reaction mixture was washed
with aq NaHCO3 and brine, and dried over anhyd MgSO4. 3H-La-
UDP-GlcNAc, p3XFLAG-CMV™-8, and anti-FLAGÒ M2 affinity resin
were from Sigma (St. Louis, MO). COS-7 cells were from Japanese
Collection of Research Bioresources-Cell Bank (Osaka, Japan).
4.2. 4-Methoxyphenyl O-[methyl 2,3,4-tri-O-acetyl-b-D-
glucopyranosyl uronate]-(1?3)-O-2,4,6-tri-O-acetyl-b-D-
galactopyranoside (6)
To a solution of methyl 2,3,4-tri-O-acetyl-
trichloroacetimidate (4,11 9.82 g, 20.5 mmol) and 4-methoxy-
phenyl 2,4,6-tri-O-acetyl-b-
-galactopyranoside (5,16 3.30 g,
D-glucopyranuronosyl
D
8.00 mmol) in CH2Cl2 (220 mL) was added MS AW300 (25 g). This
mixture was stirred for 50 min at room temperature and cooled to
À20 °C. To this solution was added TMSOTf (1.12 mL, 6.19 mmol)
with stirring and the reaction temperature was gradually raised
to À10 °C for 2 h. Then, an excess amount of satd NaHCO3 was
added. Insoluble materials were filtered on Celite. The organic
phase was treated as described in Section 4.1. The volatiles were
beled uridine diphosphate N-acetyl-
NAc) (20 Ci/mmol) and 3H-labeled uridine diphosphate N-acetyl-
-glucosamine (UDP-GlcNAc) (34.6 Ci/mmol) were purchased from
D-galactosamine (UDP-Gal-
D
American Radiolabeled Chemicals (St. Louis, MO) and PerkinElmer
Life Sciences (Boston, MA), respectively. Unlabeled UDP-GalNAc,

J. Tamura et al. / Carbohydrate Research 345 (2010) 2115–2123
2119
Scheme 3. Reagents and conditions: (a) piperidine, NMP; Fmoc-AA, HBTU, HOBt, i-Pr2EtN, CH2Cl2; (c) TFA, CH2Cl2; (d) 1.25 M LiOH, THF, H2O, 0 °C, 1 h, then 1 M AcOH; (e)
0.25 M NaOH, MeOH, H2O, 8.5 h, then 1 M AcOH; (g) 0.1 M NaOMe, MeOH, H2O, overnight, then 1% AcOH. Fmoc-AA: Fmoc-Gly, Fmoc-Pro, Fmoc-Phe, Fmoc-Gly, Fmoc-
Asn(Trt), Fmoc-Asp(O-tBu), and 13.
column of silica gel (5:1–4:1–3:1–2:1 toluene–EtOAc) to give 6
Table 1
(1.40 g) in 24% yield as a syrup. Acceptor (5, 1.97 g) was recovered
Comparison of the acceptor specificity of the recombinant CSGalNAcT2 and EXTL3
in 60% yield. [a]
D +2.9 (c 1.77, CHCl3); 1H NMR (CDCl3): d 6.94–6.92
Acceptor
Activitya (pmol/mL medium/h)
(m, 2H, Ar H), 6.82–6.79 (m, 2H, Ar H), 5.46 (d, 1H, J3,4 = 3.4 Hz, H-
4III), 5.43 (dd, 1H, J1,2 = 8.0 Hz, J2,3 = 10.1 Hz, H-2III), 5.20 (dd, 1H,
J3,4 = 8.7 Hz, J4,5 = 9.6 Hz, H-4IV), 5.19 (dd, 1H, J2,3 = 8.9 Hz, H-3IV),
4.94 (dd, 1H, H-2IV), 4.80 (d, 1H, H-1III), 4.70 (d, 1H, J1,2 = 7.8 Hz,
H-1IV), 4.19 (dd, 1H, J5,6b = 5.4 Hz, Jgem = 11.6 Hz, H-6bIII), 4.13
(dd, 1H, J5,6a = 7.4 Hz, H-6aIII), 4.02 (d, 1H, J4,5 = 9.6 Hz, H-5IV),
3.96 (dd, 1H, H-3III), 3.94 (m, 1H, H-5III), 3.78, 3.77 (2s, 3H Â 2,
MeOPh, COOMe), 2.15, 2.13, 2.07, 2.04, 2.01, 2.01 (6s, 3H Â 6,
6MeCO). Anal. Calcd for C32H40O19Á0.5H2O: C, 52.09; H, 5.61.
Found: C, 52.02; H, 5.43.
GalNAcT-I
GlcNAcT-I
(EXTL3b)
(CSGalNAcT2b)
1
2
3
110
57
99
3
2
4
The recombinant CSGalNAcT2 and EXTL3 were assayed using UDP-[3H]GalNAc and
UDP-[3H]GlcNAc as the sugar donors, respectively, and tetraosyl peptides as the
sugar acceptors (5 nmol) as described in Section 4. The reaction products were
separated from UDP-[3H]GalNAc or UDP-[3H]GlcNAc by gel-permeation chroma-
tography on a Superdex Peptide column (Fig. 2B and C). The radioactivity of each
fraction was measured by liquid scintillation counting.
a
4.3. 4-Methoxyphenyl O-[methyl 2,3,4-tri-O-acetyl-b-D-
No detectable glycosyltransferase activities were detected toward tetraosyl
peptides tested when the empty vector (mock) was used for expression as an
enzyme source.
glucopyranosyl uronate]-(1?3)-O-2,4,6-tri-O-acetyl-a-D-
galactopyranosyl trichloroacetimidate (7)
b
GalNAcT-I and GlcNAcT-I activities were measured using CSGalNAcT2 and
EXTL3 as the enzyme sources, and UDP-[3H]GalNAc and UDP-[3H]GlcNAc as the
sugar donors, respectively.
To a solution of 6 (257.3 mg, 0.353 mmol) in CH3CN (10 mL) and
H2O (2.5 mL) was added cerium(IV) ammonium nitrate (CAN)
(773.8 mg, 1.411 mmol) at 0 °C with stirring. After 1 h, additional
CAN (207.0 mg, 0.378 mmol) was supplied and the reaction mix-
removed under diminished pressure and the residue was subjected
to gel-permeation (LH-20, 1:1 CHCl3–MeOH) and applied to a

2120
J. Tamura et al. / Carbohydrate Research 345 (2010) 2115–2123
ther purification. 1H NMR (CDCl3): d 8.67 (s, 1H, NH), 6.53 (s, 1H,
J1,2 = 3.67 Hz, H-1), 5.58 (d, 1H, J3,4 = 2.75 Hz, H-4III), 5.33 (dd, 1H,
J2,3 = 10.31 Hz, H-2III), 5.21 (m, 2H, H-3IV,4IV), 4.96 (m, 1H, H-2IV),
4.76 (d, 1H, J1,2 = 7.33 Hz, H-1III), 4.37 (br t, 1H, J = 5.19 Hz, H-5III),
4.27 (dd, 1H, H-3III), 4.21 (dd, 1H, J5,6b = 5.04 Hz, Jgem = 11.69 Hz,
H-6bIII), 4.05 (m, 1H, H-5IV), 3.98 (dd, 1H, J5,6a = 7.33 Hz, H-6aIII),
3.78 (s, 3H, COOMe), 2.14, 2.07, 2.03, 2.02, 2.01 (5s, 3H Â 6,
6MeCO).
4.4. 4-Methoxyphenyl O-(methyl 2,3,4-tri-O-acetyl-b-
glucopyranosyl uronate)-(1?3)-O-(2,4,6-tri-O-acetyl-
galactopyranosyl)-(1?3)-O-(2,4,6-tri-O-acetyl-b-
galactopyranosyl)-(1?4)-2,3-di-O-(4-methylbenzoyl)-b-
xylopyranoside (9 and 9b)
D-
ab-D-
D
-
D-
a
To a solution of 6 (1.48 g, 1.93 mmol) and 4-methoxyphenyl O-
(2,4,6-tetra-O-acetyl-b- -galactopyranosyl)-(1?4)-2,3-di-O-(4-
methylbenzoyl)-b-
-xylopyranoside (8,8 2.46 g, 3.15 mmol) in
D
D
CH2Cl2 (137 mL) was added MS AW300 (2.53 g). This mixture
was stirred for 20 min at room temperature and cooled to
À20 °C. To this solution was added TMSOTf (138
lL, 762 lmol)
with stirring and the reaction temperature was gradually raised
to À10 °C for 1 h. An excess amount of satd NaHCO3 was added.
Insoluble materials were filtered on Celite. The organic phase
was treated as described in Section 4.1. The volatiles were removed
under diminished pressure and the residue was subjected to gel-
permeation (LH-20, 1:1 CHCl3–MeOH) and applied to a column of
silica gel (1:2–1:3–1:10 n-hexane–EtOAc) to give 9b (1.28 g) and
9
a
(650 mg) in 47% and 24% yields, respectively, as syrups. Com-
pound 9b: [
a]
+16.5 (c 1.20, CHCl3); 1H NMR (CDCl3): d 7.95 (m,
D
4H, Ar H), 7.24 (m, 4H, Ar H), 6.96 (m, 2H, Ar H), 6.81 (m, 2H, Ar
H), 5.61 (t, 1H, J = 5.96 Hz, H-3I), 5.42 (dd, 1H, J1,2 = 5.04 Hz,
J2,3 = 5.93 Hz, H-2I), 5.37 (d, 1H, J3,4 = 3.67 Hz, H-4III), 5.30 (d, 1H,
H-1I), 5.25 (d, 1H, J3,4 = 3.67 Hz, H-4II), 5.20 (t, 1H,
J3,4 = J4,5 = 9.62 Hz, H-4IV), 5.16 (t, 1H, J2,3 = 9.62 Hz, H-3IV), 5.14
(dd, 1H, J1,2 = 7.79 Hz, J2,3 = 10.54 Hz, H-2III), 5.02 (dd, 1H,
J1,2 = 7.79 Hz, J2,3 = 10.08 Hz, H-2II), 4.88 (br t, 1H, J = 8.25 Hz, H-
2IV), 4.62 (d, 1H, J1,2 = 7.33 Hz, H-1IV), 4.54 (d, 1H, H-1III), 4.39 (d,
1H, H-1II), 4.23 (dd, 1H, J4,5eq = 3.67 Hz, Jgem = 12.37 Hz, H-5eqI),
4.13 (dd, 1H, J5,6a = 6.41 Hz, Jgem = 11.46 Hz, H-6aIII), 4.04 (dd, 1H,
J5,6a = 6.42 Hz, H-6bIII), 3.99 (d, 1H, H-5IV), 3.76 (m, 1H, H-4I),
3.86 (dd, 1H, J5,6a = 5.50 Hz, Jgem = 11.46 Hz, H-6aII), 3.77 (m, 2H,
H-3II, 3III), 3.76 (s, 6H, MeOPh, COOMe), 3.75 (m, 2H, H-5II, 5III),
3.64 (dd, 1H, J4,5ax = 5.96 Hz, H-5axI), 3.54 (dd, 1H, J5,6a = 6.87 Hz,
H-6bII), 2.40, 2.38 (2s, 3H Â 2, 2MePh), 2.12, 2.07, 2.05, 2.03, 2.00,
1.93 (6s, 3H Â 9, 9MeCO). Anal. Calcd for C65H76O33: C, 56.36; H,
5.53. Found: C, 56.13; H, 5.56.
Figure 2. Western blotting of the recombinant CSGalNAcT2 and EXTL3 (A) and gel-
filtration chromatographic analysis of the reaction products individually labeled
with [3H]GalNAc (B) and [3H]GlcNAc (C). (A) The recombinant CSGalNAcT2 and
EXTL3, which were purified and then separated by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis, were detected with the anti-FLAG antibody in
the Western blotting. Mock indicates the conditioned medium transfected with an
empty vector as a negative control. (B) 3H-Labeled GalNAcT-I reaction products
obtained using the recombinant CSGalNAcT2 as the enzyme source, UDP-[3H]Gal-
NAc as the donor substrate, and 1 (solid circles), 2 (solid triangles), and 3 (solid
squares) as the acceptor substrates were analyzed by gel-permeation on a Superdex
Peptide column as described in Section 4, and the radioactivity in the effluent
fractions (0.4 mL each) was analyzed. (C) 3H-Labeled GlcNAcT-I reaction products
obtained using the recombinant EXTL3 as the enzyme source, UDP-[3H]GlcNAc as
the donor substrate, and the tetraosyl peptides described above as the acceptor
substrates were analyzed by gel-permeation on the same column. Arrowheads and
asterisks indicate the elution positions of [3H]GalNAc- or [3H]GlcNAc-labeled
reaction products, and UDP-[3H]GalNAc or UDP-[3H]GlcNAc, respectively. The void
volume and the total volume of the column are shown by Vo and Vt, respectively.
Compound 9a: [a]
+46.5 (c 3.21, CHCl3); 1H NMR (CDCl3): d
D
7.93 (m, 4H, Ar H), 7.22 (m, 4H, Ar H), 6.96 (m, 2H, Ar H), 6.81
(m, 2H, Ar H), 5.66 (br t, 1H, J = 7.0 Hz, H-3I), 5.44 (dd, 1H,
J1,2 = 5.5 Hz, J2,3 = 7.1 Hz, H-2I), 5.43 (m, 1H, H-4III), 5.26 (d, 1H,
H-1I), 5.23 (d, 1H, J3,4 = 2.7 Hz, H-4II), 5.21–5.17 (m, 3H, H-2III, 3IV,
4IV), 5.13 (dd, 1H, J1,2 = 8.0 Hz, J2,3 = 10.4 Hz, H-2II), 5.10 (d, 1H,
J1,2 = 3.4 Hz, H-1III), 4.89 (m, 1H, H-2IV), 4.68 (d, 1H, J1,2 = 7.5 Hz,
H-1IV), 4.52 (d, 1H, H-1II), 4.21 (dd, 1H, J4,5eq = 4.4 Hz, Jgem = 6.2 Hz,
H-5eqI), 4.19 (br t, 1H, J = 5.6 Hz, H-5III), 4.09 (dd, 1H, J5,6a = 6.1 Hz,
Jgem = 11.4 Hz, H-6aIII), 4.09 (d, 1H, J4,5 = 8.7 Hz, H-5IV), 4.05 (dd, 1H,
J2,3 = 10.3 Hz, J3,4 = 3.5 Hz, H-3III), 4.01 (m, 1H, H-4I), 3.94 (dd, 1H,
J5,6a = 6.4 Hz, H-6bIII), 3.76 (m, 1H, H-6aII), 3.76, 3.75 (2s, 3H Â 2,
MeOPh, COOMe), 3.74 (m, 1H, H-3II), 3.68 (br t, 1H, J = 6.8 Hz, H-
5II), 3.63 (dd, 1H, J5,6b = 7.1 Hz, Jgem = 12.3 Hz, H-6bII), 3.60 (dd,
1H, J4,5ax = 10.5 Hz, H-5axI), 2.40, 2.38 (2s, 3H Â 2, 2MePh), 2.09,
2.08, 2.02, 2.01, 2.00, 1.98 (6s, 3H Â 9, 9MeCO). Anal. Calcd for
ture was stirred for another 1 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
(2:1–1:1–1:2 toluene–EtOAc) to give the corresponding hemiace-
tal (151.6 mg, 69%) as a syrup. The hemiacetal was diluted with
CH2Cl2 (4 mL). To the solution were added CCl3CN (1 mL) and a
small amount of DBU at 0 °C. The reaction mixture was stirred
for 1.5 h and directly applied to a column of silica gel (3:1–1:1–
1:3 n-hexane–EtOAc) to give an
a-isomer of 7 (161.7 mg) in 87%
C65H76O33: C, 56.36; H, 5.53. Found: C, 56.12; H, 5.66.
yield as a syrup, which was used for the next reaction without fur-

J. Tamura et al. / Carbohydrate Research 345 (2010) 2115–2123
2121
4.5. 4-Methoxyphenyl O-(methyl 2,3,4-tri-O-acetyl-b-
glucopyranosyl uronate)-(1?3)-O-(2,4,6-tri-O-acetyl-b-
galactopyranosyl)-(1?3)-O-(2,4,6-tri-O-acetyl-b-
galactopyranosyl)-(1?4)-2,3-di-O-(4-methylbenzoyl)-D-
xylopyranosyl trichloroacetimidate (10)
D
-
of 12 (101.4 mg, 60.2
l
mol) in THF (3 mL) with stirring. Two hours
D
-
later, volatiles were removed under diminished pressure and the
residue was applied to columns of gel-permeation (LH-20,
10:10:1 CHCl3–MeOH ÀAcOH) and silica gel (100:1–50:1 EtOAc–
MeOH containing 1% AcOH) to give 13 (91.7 mg) in 93% yield. Com-
plete removal of the allyl ester was confirmed with 1H NMR mea-
surements. The compound (13) was used for the next reaction
without further purification.
D
-
To a solution of 9b (1.28 g, 924 mmol) in CH3CN (178 mL) and
H2O (30 mL) was added CAN (2.03 g, 3.70 mmol) at 0 °C with stir-
ring. After 1.5 h, additional CAN (0.51 g, 0.93 mmol) was supplied
and the reaction mixture was stirred for another 1 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 (2:3–3:7–1:10 toluene–EtOAc) to give the correspond-
ing hemiacetal (1.04 g, 88%) as a syrup. The hemiacetal was diluted
4.8. N-(Butoxycarbonyl)-L-asparaginylglycine methyl ester (14)
Boc-Asn-OH (2.70 g, 11.6 mmol) and H-Gly-O-MeÁHCl (1.46 g,
11.6 mmol) were dissolved in CH2Cl2 (150 mL). To this solution
were added Et3N (1.60 mL, 11.5 mmol) and HOBt (3.15 g,
23.3 mmol) with stirring. The mixture was cooled to À20 °C, and
with CH2Cl2 (19 mL). To the solution were added CCl3CN (816
lL)
and DBU (48 L) at 0 °C. The reaction mixture was stirred for
l
then
a
solution of WSCDÁHCl (2.45 g, 12.8 mmol) in CH2Cl2
1.5 h and applied directly to a column of silica gel (1:2–1:4 tolu-
ene–EtOAc) to give 10 quantitatively as a syrup. The characteristic
anomeric proton and NH signals of 10 were detected with 1H NMR
measurements. The imidate 10 was used for the next reaction
without further purification.
(21 mL) was added. The reaction mixture was stirred overnight
at room temperature. The volatiles were removed under dimin-
ished pressure and the residue was applied to a column of silica
gel (50:1–16:1 EtOAc–MeOH containing 1% AcOH) to give 14
(1.36 g) in 39% yield. This compound was used for the next reaction
without further purification. 1H NMR (CD3OD): d 4.39 (br t, 1H,
4.6. N-(9-Fluorenylmethoxycarbonyl)-O-[(methyl 2,3,4-tri-O-
J = 4.39 Hz, Asna), 3.88, 3.82 (ABq, 2H, J = 17.53 Hz, Gly), 3.62 (s,
acetyl-b-
b- -galactopyranosyl)-(1?3)-O-(2,4,6-tri-O-acetyl-b-
galactopyranosyl)-(1?4)-2,3-di-O-(4-methylbenzoyl)-b-
xylopyranosyl]- -serylglycine allyl ester (12)
D-glucopyranosyl uronate)-(1?3)-O-(2,4,6-tri-O-acetyl-
3H, OMe), 2.60 (dd, 1H, J ,ba = 5.27 Hz, Jgem = 15.35 Hz, Asnba),
2.50 (dd, 1H, J ,bb = 7.79 Hz, Asnbb), 1.35 (s, 9H, tert-Bu).
a
a
D
D
-
D
-
L
4.9. L-Asparaginylglycine methyl ester (15)
To a solution of N-(9-fluorenylmethoxycarbonyl)-
allyl ester (11,16 486.0 mg, 1.14 mmol) and 10 (814.4 mg,
572 mol) in CH2Cl2 (83 mL) was added MS AW300 (2.3 g). This
mixture was stirred for 30 min at room temperature, and then
cooled to À20 °C. To this solution was added TMSOTf (72 L,
397 mol) with stirring for 2 h. The reaction was quenched and
L
-serylglycine
Trifluoroacetic acid (13 mL) was added to a solution of 14
(602 mg, 1.98 mmol) in CH2Cl2 (13 mL) and the mixture was stir-
red for 1 h. The volatiles were removed under diminished pressure
to give 15 quantitatively. All of this compound was used for the
next reaction without further purification. 1H NMR (CD3OD): d
l
l
l
4.17 (dd, 1H, J ,ba = 4.58 Hz, J ,bb = 8.70 Hz, Asna), 3.96, 3.88 (ABq,
a a
treated as described for the synthesis of 9b. The residue was sub-
2H, J = 17.87 Hz, Gly), 3.64 (s, 3H, OMe), 2.84 (dd, 1H,
Jgem = 16.95 Hz, Asnba), 2.70 (dd, 1H, Asnbb).
jected to gel-permeation (LH-20, 1:1 CHCl3–MeOH) to give 12
(884.4 mg) in 92% yield: [
a]
+27.6 (c 1.34, CHCl3); 1H NMR
D
(CDCl3): d 7.85 (m, 4H, Ar H), 7.77 (m, 2H, J = 7.56 Hz, Ar H), 7.57
(br t, 1H, J = 7.43 Hz, Ar H), 7.40 (m, 1H, Ar H), 7.29 (m, 2H, Ar
H), 7.17 (m, 4H, Ar H), 6.90 [m, 1H, NH(Gly)], 5.86 (m, 1H, @CH),
5.80 [m, 1H, NH(Ser)], 5.60 (br t, 1H, J = 8.02 Hz, H-3I), 5.37 (dd,
1H, J3,4 = 3.21 Hz, J4,5 = 0.92 Hz, H-4III), 5.30 (m, 2H, @CH2), 5.23
(m, 2H, H-2I, 4II), 5.20 (br t, 1H, J = 9.40 Hz, H-4IV), 5.16 (br t, 1H,
J = 9.16 Hz, H-3IV), 5.08 (dd, 1H, J1,2 = 8.25 Hz, J2,3 = 9.85 Hz, H-2II),
5.01 (dd, 1H, J1,2 = 8.02 Hz, J2,3 = 10.08 Hz, H-2III), 4.87 (dd, 1H,
J1,2 = 7.56 Hz, J2,3 = 8.94 Hz, H-2IV), 4.74 (m, 1H, H-1I), 4.61 (d, 1H,
H-1IV), 4.51 (m, 2H, OCH2), 4.50–4.31 [m, 8H, OCH2 (Fmoc), H-
4.10. N-(9-Fluorenylmethoxycarbonyl)-O-(tert-butyl)-
L-
aspartyl- -asparaginylglycine methyl ester (16)
L
FmocAsp(O-tBu)-OH (1.39 g, 3.39 mmol) and the dipeptide (15,
1.98 mmol) obtained above were dissolved in CH2Cl2 (13 mL). To
this solution were added Et3N (353 lL, 2.55 mmol) and HOBt
(0.92 g, 6.8 mmol) with stirring. The mixture was cooled to
À20 °C, and then a solution of WSCDÁHCl (1.30 g, 6.78 mmol) in
CH2Cl2 (10 mL) was added. The reaction mixture was stirred over-
night at room temperature, then diluted with CHCl3, and washed
with 1 M HCl and brine. The crude mixture obtained was applied
to a column of silica gel (30:1–25:1 EtOAc–MeOH containing 1%
9(Fmoc), Sera
b, Gly], 4.46 (d, 1H, H-1II), 4.38 (d, 1H, H-1III), 4.19
(m, 1H, H-5eqI), 4.13 (dd, 1H, J5,6a = 5.96 Hz, Jgem = 11.45 Hz, H-
6aIII), 4.05 (dd, 1H, J5,6b = 7.27 Hz, H-6bIII), 4.00 (m, 1H, H-4I), 3.99
(d, 1H, J4,5 = 9.63 Hz, H-5IV), 3.82 (dd, 1H, J5,6a = 5.73 Hz,
Jgem = 11.23 Hz, H-6aII), 3.76 (s, 3H, COOMe), 3.75 (m, 1H, H-3III),
3.73 (m, 2H, H-3II, 5III), 3.66 (br t, 1H, J = 6.66 Hz, H-5II), 3.60 (m,
1H, H-5aI), 3.52 (m, 1H, H-6bII), 2.36, 2.33 (2s, 6H, 2MePh), 2.12,
2.07, 2.05, 2.04, 2.03, 2.00, 2.00, 1.95 (8s, 3H Â 9, 9MeCO). Anal.
Calcd for C81H92N2O37: C, 57.72; H, 5.50; N, 1.66. Found: C,
57.37; H, 5.50; N, 1.63.
AcOH) to give 16 (850.2 mg) in 63% yield. [
a
]
À29.1 (c 0.44,
D
CHCl3); 1H NMR (CD3OD): d 7.70 (d, 2H, J = 7.56 Hz, Ar H), 7.57
(m, 2H, Ar H), 7.29 (br t, 2H, J = 7.45 Hz, Ar H), 7.21 (br t, 2H,
J = 7.45 Hz, Ar H), 4.67 (br t, 1H, J = 6.19 Hz, Asp
H-9(Fmoc)], 4.27 [m, 1H, OCH2 (Fmoc)], 4.14 (br t, 1H,
J = 6.87 Hz, Asn ), 3.83 (s, 2H, Gly), 3.58 (s, 3H, OMe), 2.74 (dd,
1H, J ,ba = 5.50 Hz, Jgem = 16.50 Hz, Asnba), 2.66 (dd, 1H, J
a), 4.40 [m, 1H,
a
=
a,ba
a
5.73 Hz, Jgem = 15.58 Hz, Aspba), 2.62 (dd, 1H,
J ,bb = 6.88 Hz,
a
Aspbb), 2.51 (dd, 1H, J ,bb = 8.25 Hz, Asnbb), 1.33 (s, 9H, tert-Bu).
a
4.7. N-(9-Fluorenylmethoxycarbonyl)-O-[(methyl 2,3,4-tri-O-
Anal. Calcd. for C30H36N4O9: C, 60.40; H, 6.08; N, 9.39. Found: C,
59.90; H, 6.31; N, 9.39.
acetyl-b-
b- -galactopyranosyl)-(1?3)-O-(2,4,6-tri-O-acetyl-b-
galactopyranosyl)-(1?4)-2,3-di-O-(4-methylbenzoyl)-b-
xylopyranosyl]- -serylglycine (13)
D-glucopyranosyl uronate)-(1?3)-O-(2,4,6-tri-O-acetyl-
D
D-
D
-
4.11. O-(tert-Butyl)-L-aspartyl-L-asparaginylglycine methyl ester
(17)
L
Tetrakis(triphenylphosphine)palladium(0) (27.8 mg, 24.1
l
mol)
Morpholine (7.1 mL) was added to a solution of 16 (716.2 mg,
and N-methylaniline (65 L, 0.60 mmol) were added to a solution
l
1.200 mmol) in CH2Cl2 (21.4 mL) and stirred overnight. The crude

2122
J. Tamura et al. / Carbohydrate Research 345 (2010) 2115–2123
mixture was evaporated to dryness with toluene, and the residue
was subjected to gel-permeation (LH-20, 1:1 CHCl3–MeOH) to af-
ford 17 quantitatively. This compound was used for the next reac-
tion without further purification. 1H NMR (D2O): d 4.86 (m, 1H,
moved with toluene under diminished pressure. The residue was
subjected to gel-permeation (LH-20, 1% AcOH) to give 1 (10 mg)
in 80% yield. 1H NMR (D2O): d 4.73 (dd, 1H, J ,ba = 5.27 Hz,
a
J
J
,bb = 8.02 Hz, Asp
a
or Asn
a), 4.69 (dd, 1H, J ,ba = 6.19 Hz,
a
a
Asp
a), 4.35 (br t, 1H, J = 5.95 Hz, Asna), 4.05 (s, 2H, Gly), 3.77 (s,
,bb = 7.79 Hz, Asn
a
or Asp
a
), 4.67 (d, 1H, J1,2 = 8.01 Hz, H-1IV),
a
3H, OMe), 3.07 (dd, 1H, J ,ba = 5.73 Hz, Jgem = 17.64 Hz, Aspba),
4.65 (d, 1H, J1,2 = 7.79 Hz, H-1III), 4.51 (d, 1H, J1,2 = 8.02 Hz, H-1II),
a
3.02 (dd, 1H, J ,bb = 6.42 Hz, Aspbb), 2.90 (dd, 1H, J ,ba = 5.50 Hz,
4.46 (d, 1H, J1,2 = 7.56 Hz, H-1I), 4.40 (br t, 1H, J = 4.35 Hz, Ser
a),
a
a
Jgem = 15.81 Hz, Asnba), 2.79 (dd, 1H, J ,bb = 8.48 Hz, Asnbb), 1.47
(s, 9H, tert-Bu).
4.27 (dd, 1H, Jgem = 11.45 Hz, J ,bb = 5.27 Hz, Serba), 4.17 (2s, 2H,
a
a
H-4II, 4III), 4.11 (dd, 1H, Jgem = 10.31 Hz, J4,5e = 6.19 Hz, H-5eqI),
4.10 (dd, 1H,
J
,bb = 4.59 Hz, Serbb), 4.09, 3.95 (ABq, 2H,
a
4.12. N-(9-Fluorenylmethoxycarbonyl)-O-[(methyl 2,3,4-tri-O-
J = 16.72 Hz, Gly), 3.88 (m, 1H, H-4I), 3.86, 3.83 (ABq, 2H,
J = 17.18 Hz, Gly), 3.83 (m, 1H, H-3II), 3.82 (m, 1H, H-3III), 3.79–
3.69 (m, 6H, H-5II, 6abII, 5III, 6abIII), 3.76 (m, 1H, H-5IV), 3.76 (m,
1H, H-2III), 3.66 (br t, 1H, J = 8.76 Hz, H-2II), 3.60 (br t, 1H,
J = 9.28 Hz, H-3I), 3.52 (m, 2H, H-3IV, 4IV), 3.42 (m, 2H, H-5axI,
2IV), 3.36 (br t, 1H, J = 8.36 Hz, H-2I), 2.88, 2,.6 (2dd, 2H,
Jgem = 8.93 Hz, Aspb or Asnb), 2.85, 2.77 (2dd, 2H, Jgem = 10.31 Hz,
Asnb or Aspb); FAB-MS (positive) m/z: 1103.25 (calcd for
acetyl-b-
b- -galactopyranosyl)-(1?3)-O-(2,4,6-tri-O-acetyl-b-
galactopyranosyl)-(1?4)-2,3-di-O-(4-methylbenzoyl)-b-
xylopyranosyl]- -serylglycyl- -aspartyl- -asparaginylglycine
methyl ester (18)
D-glucopyranosyl uronate)-(1?3)-O-(2,4,6-tri-O-acetyl-
D
D-
D
-
L
L
L
Tetraosyl dipeptide (13, 63.5 mg, 29.5
DMF (170 L), and HOBt (11.9 mg, 38 mol) was added with stir-
ring. This solution was cooled to À20 °C and HBTU (10.2 mg,
26.9 mol) was added. The cooling-bath was removed, the reaction
mixture was stirred at room temperature for 30 min, and 17
(32.9 mg, 87.9 mol) and Hünigs base (15.3 L, 87.8 mol) were
lmol) was dissolved in
l
l
C
38H60N6NaO30 1103.33, [M+Na]+).
l
4.14. General procedure for solid-phase synthesis
l
l
l
Glycopeptides were synthesized manually with 192 mg of Sie-
added. The reaction mixture was stirred overnight. Same amounts
of additional Hünigs base, HOBt, and HBTU were added and the
reaction mixture was diluted with CHCl3 after 2 h. The organic
phase was washed with 1 N HCl, aq NaHCO3, and brine, and dried
over MgSO4. The volatiles were removed under diminished pres-
sure. The crude materials were subjected to gel-permeation (1:1
CHCl3–MeOH) to give a coupling product (60.5 mg) which was di-
luted with CH2Cl2 (5.0 mL), and TFA (5.0 mL) was added to the
solution with stirring. After 1 h all the volatiles were removed un-
der diminished pressure to give 18 (62.5 mg) in 98% yield (two
ber amide resin (100 lmol) as follows. The Fmoc group was re-
moved with 20% of piperidine/NMP (2 mL) (2 Â 3 min and
1 Â 20 min), as monitored with the Kaiser ninhydrin test. After
washing with NMP (2.2 mL) (6 Â 1 min) and CH2Cl2 (2.2 mL)
(3 Â 1 min), it was dried in vacuo. The N-terminal free resin or pep-
tide-on-resin was swollen in NMP (2 mL) and shaken overnight
with the corresponding Fmoc amino acid (250
lmol), HOBt
(250 mol), HBTU (250 mol), and DIPEA (500 mol). Coupling
l
l
l
was monitored by the Kaiser test. The resin was washed with
NMP (2.2 mL) (3 Â 1 min) and CH2Cl2 (2.2 mL) (3 Â 1 min), and
dried in vacuo.
steps). [a]
+49.2 (c 0.13, CHCl3); 1H NMR (CD3OD) (selected): d
D
7.92 [br, 1H, NH(Asp)], 8.82 (d, 4H, J = 7.79 Hz, Ar H), 7.73 [m,
3H, Ar H, NH(Gly)], 7.57 [br, 1H, NH(Asn)], 7.53–7.47 (m, 2H, Ar
H), 7.37 (t, 2H, J = 7.33 Hz, Ar H), 7.28 (m, 2H, Ar H), 7.16–7.09
[m, 5H, Ar H, NH(Gly)], 6.48 [br, 1H, NH(Ser)], 5.57 (br t, 1H,
J = 7.56 Hz, H-3I), 5.36 (d, 1H, J3,4 = 3.43 Hz, H-4III), 5.22 (br s, 1H,
H-4II), 5.21–5.14 (m, 3H, H-2I, 3IV, 4IV), 5.04 (dd, 1H, J1,2 = 8.02 Hz,
J2,3 = 10.08 Hz, H-2II), 5.00 (dd, 1H, J1,2 = 8.02 Hz, J2,3 = 10.08 Hz,
4.15. O-[b-
galactopyranosyl-(1?3)-O-b-
xylopyranosyl]-serylglycyl- -aspartyl-
phenylalanyl- -prolylglycineamide (2)
D
-Glucopyranuronosyl-(1?3)-O-b-
-galactopyranosyl-(1?4)-b-
-asparaginylglycyl-L-
D-
D
D-
L
L
L
Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Phe-OH, Fmoc-Gly-OH,
Fmoc-Asn(Trt)-OH, and Fmoc-Asp(O-t-Bu)-OH were coupled in turn
H-2III), 4.88 (br t, 1H, J = 8.25 Hz, H-2IV), 4.83 (m, 1H, Asp
(d, 1H, H-1I), 4.63 (m, 1H, Ser ), 4.62 (d, 1H, J1,2 = 7.57 Hz, H-1IV),
4.51 (m, 1H, Asn
), 4.46 (d, 1H, H-1II), 4.39 (d, 1H, H-1III), 3.60 (s,
a
), 4.74
a
on the Sieber amide resin (192.0 mg, 100
pled with 13 (245.0 mg, 149.0 mol) as described in the general pro-
cedure, in which case different amounts of HOBt (20.1 mg), HBTU
(56.5 mg), and DIPEA (52 L) were used. The resultant resin was ex-
lmol). The resin was cou-
a
l
3H, COOMe), 2.95–2.57 (m, 4H, Aspb, Asnb),2.33, 2.25 (2s,
3H Â 2, 2MePh), 2.11 (s, 3H, MeCO), 2.05, 2.05, 2.05 (3s, 9H,
3MeCO), 2.00, 2.00, 1.99 (3s, 12H, 4MeCO), 1.92 (s, 3H, MeCO).
Anal. Calcd for C89H104N6O43: C, 54.94; H, 5.39; N, 4.32. Found: C,
54.72; H, 5.63; N, 4.18.
l
posed to 20% TFA in CH2Cl2 (4.5 mL) with shaking for 5.5 h and evap-
orated. The residue was subjected to gel-permeation (LH-20, 1%
AcOH)to give a crude glycopeptide (89.9 mg) stillhaving acylgroups
and 13 (133.5 mg, 54%) was recovered. Some portion of the crude
glycopeptide (31.7 mg) was diluted with 83% aq THF. To the solution
4.13. O-[b-
galactopyranosyl-(1?3)-O-b-
xylopyranosyl]- -serylglycyl-
D
-Glucopyranuronosyl-(1?3)-O-b-
-galactopyranosyl-(1?4]-b-
-aspartyl- -asparaginylglycine (1)
D-
D
D-
was added 1.25 M LiOH (56 lL) with stirring at 0 °C for 1 h and the
reaction was quenched with 1 M AcOH. The volatiles were removed
L
L
L
under diminished pressure. The residue was diluted with 50% aq
To a solution of 18 (27.2 mg, 11.7
water (0.15 mL) was added 1.25 M LiOH (50
for 1 h. The reaction was quenched with 1 M AcOH and the vola-
l
mol) in THF (0.78 mL) and
MeOH (3.4 mL). To this solution was added 0.25 M NaOH (220
dropwise for 8.5 h with the pH kept at 8.5. The reaction mixture
was neutralized with 1 M AcOH (90 L) again and subjected to
lL)
lL) at 0 °C and stirred
l
tiles were removed with toluene under diminished pressure. The
gel-permeation (LH-20, 1% AcOH). The fractions containing glyco-
residue was diluted with 50% MeOH. Next, 0.25 M NaOH (122 lL)
peptides were collected (3.1 mg) and diluted with 50% aq MeOH
was added in portions over 6 h and the solution was stirred over-
night. The reaction was quenched with 1 M AcOH and the reaction
mixture was subjected to gel-permeation (LH-20, 1% AcOH) after
evaporation. The fractions containing the glycopeptides were col-
lected and diluted with 50% MeOH (0.6 mL) again. To this solution
was added 0.1 M NaOMe (0.17 mL) portionwise during 5 days, and
after quenching the reaction with 1 M AcOH the volatiles were re-
(0.6 mL). Methanolic sodium methoxide (0.1 M, 119
to the solution with stirring overnight. Then, the reaction mixture
was neutralized with 1% AcOH (100 L) and evaporated. The residue
was purified by gel-permeation (LH-20, 1% AcOH) and HPLC (C18,
10%CH3CN + 0.1%CF3COOH ꢀ 90%CH3CN + 0.1%CF3COOH) to give 2
(1.7 mg). 1H NMR (D2O): d 7.37–7.28 (m, 5H, Phe), 4.91 (dd, 1H,
lL) was added
l
J
,ba = 6.19 Hz, J ,bb = 8.25 Hz, Phea), 4.66 (d, 1H, J1,2 = 7.79 Hz,
a
a

J. Tamura et al. / Carbohydrate Research 345 (2010) 2115–2123
2123
H-1IV), 4.65 (m, 2H, Asn
4.50 (d, 1H, J1,2 = 8.02 Hz, H-1II), 4.46 (d, 1H, J1,2 = 7.56 Hz, H-1I),
4.39 (m, 2H, Ser Pro ), 4.26 (dd, 1H, Jgem = 11.22 Hz,
,bb = 5.73 Hz, Serba), 4.17 (2s, 2H, H-4II, 4III), 4.10 (m, 2H, H-5eqI,
Serbb), 4.06, 3.96 (ABq, 2H, J = 16.96 Hz, Gly), 3.90, 3.83 (ABq, 2H,
J = 17.64 Hz, Gly), 3.88–3.63 (m, 9H, H-5II, 6abII, 5III, 6abIII, Gly, Pro-
da), 3.88 (m, 1H, H-4I), 3.82 (m, 2H, H-3II, 3III), 3.77 (m, 1H, H-2III),
3.76 (m, 1H, H-5IV), 3.68 (m, 1H, H-2II), 3.60 (t, 1H,
J2,3 = J3.4 = 9.16 Hz, H-3I), 3.51 (m, 3H, H-3IV, 4IV, Prodb), 3.43 (m,
2H, H-5axI, 2IV), 3.36 (dd, 1H, H-2I), 3.13 (dd, 1H, Jgem = 14.43 Hz,
Pheba), 2.98 (dd, 1H, Jgem = 14.43 Hz, Phebb), 2.86–2.67 (m, 4H, Asn-
a
, Asp
a
), 4.64 (d, 1H, J1,2 = 7.79 Hz, H-1III),
method described previously4,9 with slight modifications. Briefly,
the standard reaction mixture (30 L) contained 10 L of the en-
zyme-bound anti-FLAG resin, 50 mM 2-morpholinoethanesulfonic
l
l
a,
a
J
acid-NaOH, pH 6.5, 10 mM MnCl2, 10 mM MgCl2, 167 lM ATP,
a
8.3
l
M UDP-[3H]GalNAc, or UDP-[3H]GlcNAc (ꢀ5 Â 105 dpm) as a
donor substrate, and 5 nmol of each linkage region-peptide as an
acceptor substrate. The reaction mixtures were incubated at
37 °C for 1 or 4 h, and the radiolabeled products were separated
from UDP-[3H]GalNAc or UDP-[3H]GlcNAc by gel-filtration chro-
matography on a Superdex™ Peptide 10/300 GL column (GE
Healthcare, Uppsala, Sweden) equilibrated and eluted with 0.2 M
NH4HCO3.4 Fractions (0.4 mL each) were collected at a flow rate
of 0.4 mL/min, and radioactivity was measured by liquid scintilla-
tion counting in an LS6500 (Beckman coulter Inc., Brea, CA).
bab, Aspbab), 2.25 (m, 1H, Proba), 2.20–1.89 (m, 3H, Probb, cab);
FAB-MS (positive) m/z: 1381.48 (calcd for C54H81N10O32 1381.50,
[M+H]+), 1425.62 (calcd for C54H79N10Na2O32 1425.47,
[M+2NaÀH]+), 1447.63 (calcd for C54H78N10Na3O32 1447.45,
[M+3NaÀ2H]+).
Acknowledgments
4.16. Construction of an expression vector containing a cDNA
fragment encoding the soluble form of CSGalNAcT2 or EXTL3
The authors would like to thank Ms. Miyuki Tanmatsu for per-
forming the elemental analysis measurements (Research Center for
Bioscience and Technology, Division of Instrumental Analysis, Tot-
tori University). This study was supported by Grants-in-aid for Sci-
entific Research (C) (12660098) (J.T.) and (B) (20390019) (K.S.)
from MEXT.
cDNA fragments encoding human chondroitin sulfate N-acety-
lgalactosaminyltransferase 2 (CSGalNAcT29) and exostosin-like 3
(EXTL34) proteins lacking the first N-terminal 36 and 90 amino
acids, respectively, were individually amplified by PCR with IMAGE
clones (ID numbers, 5223190 and 4131101) containing the full-
length forms of CSGalNAcT2 and EXTL3 as templates using KOD-
Plus DNA polymerase (Toyobo, Tokyo, Japan). The PCR fragments
were subcloned into the HindIII and BamHI (or EcoRV) sites of
p3XFLAG-CMV™-8. The resultant vector contained cDNA encoding
a fusion protein that had an NH2-terminal cleavable preprotrypsin
leader peptide and a 3xFLAG tag peptide followed by a truncated
form of either CSGalNAcT2 or EXTL3.
References
1. (a) Sugahara, K.; Kitagawa, H. Curr. Opin. Struct. Biol. 2000, 10, 518–527; (b)
Gesslbauer, B.; Rek, A.; Falsone, F.; Rajkovic, E.; Kungl, A. J. Proteomics 2007, 7,
2870–2880.
2. (a) Kitagawa, H.; Uyama, T.; Sugahara, K. J. Biol. Chem. 2001, 276, 38721–38726;
(b) Kitagawa, H.; Izumikawa, T.; Uyama, T.; Sugahara, K. J. Biol. Chem. 2003, 278,
23666–23671; (c) Izumikawa, T.; Uyama, T.; Okura, Y.; Sugahara, K.; Kitagawa,
H. Biochem. J. 2007, 403, 545–552; (d) Izumikawa, T.; Koike, T.; Shiozawa, S.;
Sugahara, K.; Tamura, J.; Kitagawa, H. J. Biol. Chem. 2008, 283, 11396–11406; (e)
Kitagawa, H.; Shimakawa, H.; Sugahara, K. J. Biol Chem. 1999, 274, 13933–
13937.
4.17. Expression of a soluble form of CSGalNAcT2 and EXTL3,
and enzyme assays
3. Kim, B.-T.; Kitagawa, H.; Tanaka, J.; Tamura, J.; Sugahara, K. J. Biol. Chem. 2003,
278, 41618–41623.
4. Kim, B.-T.; Kitagawa, H.; Tamura, J.; Saito, T.; Kusche-Gullberg, M.; Lindahl, U.;
Sugahara, K. Proc. Natl. Acad. Sci. 2001, 98, 7176–7181.
5. López-Casillas, F.; Payne, H. M.; Andres, J. L.; Massagué, J. J. Cell Biol. 1994, 124,
557–568.
6. Esko, J. D.; Zhang, L. Curr. Opin. Struct. Biol. 1996, 6, 663–670.
7. Zhang, L.; Esko, J. D. J. Biol. Chem. 1994, 269, 19295–19299.
8. (a) Tamura, J.; Yamaguchi, A.; Tanaka, J.; Nishimura, Y. J. Carbohydr. Chem. 2007,
26, 61–82; (b) Tamura, J.; Yamaguchi, A.; Tanaka, J. Bioorg. Med. Chem. Lett
2002, 12, 1901–1903.
The expression plasmids (6.7 lg) were individually introduced
into COS-7 cells (2.2 Â 106), which were cultured on 100-mm
plates in Dulbecco’s modified Eagle’s medium (Wako, Osaka, Japan)
containing 10% fetal bovine serum (Gibco, Carlsbad, CA), using
FuGENE™6 (Roche Diagnostics, Basel, Switzerland) according to
the manufacturer’s instructions. After three days of culture at
37 °C, 0.1 mL of the culture medium was collected and incubated
9. Uyama, T.; Kitagawa, H.; Tanaka, J.; Tamura, J.; Ogawa, T.; Sugahara, K. J. Biol.
Chem. 2003, 278, 3072–3078.
with 10 l
L of anti-FLAGÒ M2 affinity resin at 4 °C for 4 h. The resin
10. Takahashi, I.; Noguchi, N.; Nata, K.; Yamada, S.; Kaneiwa, T.; Mizumoto, S.;
Ikeda, T.; Sugihara, K.; Asano, M.; Yoshikawa, T.; Yamauchi, A.; Shervani, N. J.;
Uruno, A.; Kato, I.; Unno, M.; Sugahara, K.; Takasawa, S.; Okamoto, H.;
Sugawara, A. Biochem. Biophys. Res. Commun. 2009, 383, 113–118.
11. Jacquinet, J.-C. Carbohydr. Res. 1990, 199, 153–181.
12. Lu, Y.-P.; Li, H.; Cai, M.-S.; Li, Z.-J. Carbohydr. Res. 2001, 334, 289–294.
13. Tamura, J.; Miura, Y.; Freeze, H. H. J. Carbohydr. Chem. 1999, 18, 1–14.
14. Thollau, B.; Jacquinet, J.-C. Org. Biomol. Chem. 2004, 2, 434–442.
15. Neumann, K. W.; Tamura, J.; Ogawa, T. Bioorg. Med. Chem. 1995, 3, 1637–1650.
16. Rio, S.; Beau, J.-M.; Jacquinet, J.-C. Carbohydr. Res. 1991, 219, 71–90.
17. Fritz, T. A.; Lugemwa, F. N.; Sarkar, A. K.; Esko, J. D. J. Biol. Chem. 1994, 269, 300–
307.
was washed with 25 mM Tris-buffered saline containing 0.05%
Tween-20 and subjected to sodium dodecyl sulfate–polyacryl-
amide gel electrophoresis. Western blotting was carried out using
anti-FLAG M2 monoclonal antibody (Sigma) and the ECL Advance™
western blotting detection kit (GE Healthcare, Buckinghamshire,
UK).
GalNAcT-I and GlcNAcT-I activities of the recombinant CSGalN-
AcT2 and EXTL3, respectively, toward tetrasaccharide-peptides de-
rived from the GAG–protein linkage region were assayed by a