Chemoenzymatic synthesis of a trisaccharide-serine conjugate, gal(β1-3)gal(β1-4)xyl(β1-o)-l-ser, use of galactosyl fluoride as a donor for transglycosylation

Article abstract of DOI:10.1246/bcsj.74.1123

The trisaccharide - serine conjugate [Gal(β1-3)Gal(β1-4)Xyl(β-Ser], constituting the linkage region between the glycosaminoglycan and protein in proteoglycan, was synthesized by using enzymatic transglycosylation with a β-D-galactosidase as a key reaction. The yields of transglycosylation were much improved by using galactosyl fluoride as a donor in comparison with those obtained by a conventional p-nitrophenyl (PNP) galactoside. After enzymatic synthesis of Gal(β1-3)Gal(β1-4)Xyl(β)-PNP, cleavage of the PNP group of the fully acetylated trisaccharide, chemical coupling with serine, and final deprotection afforded Gal(β1-3)Gal(β1-4)Xyl(β)-Ser.

Full text of DOI:10.1246/bcsj.74.1123

c. Jpn.
© 2001 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 74, 1123—1128 (2001) 1123
e Chemical
ciety of Ja-
n
e Chemical
ciety of Ja-
n
Chemoenzymatic Synthesis of a Trisaccharide–Serine Conjugate,
β
β
β
Gal( 1-3)Gal( 1-4)Xyl( 1-O)–L-Ser, Use of Galactosyl Fluoride
as a Donor for Transglycosylation
Chemoenzymatic Synthesis of Gal–Gal–Xyl–l-Ser
K. Fukase et al.
01
23
Koichi Fukase,* Takashi Yasukochi, and Shoichi Kusumoto*
28
Department of Chemistry, Graduate School of Science, Osaka University,
Machikaneyama 1-1, Toyonaka, Osaka 560-0043
SJA8
09-2673
402
(Received November 22, 2000)
vember
00
01
The trisaccharide–serine conjugate [Gal(β1-3)Gal(β1-4)Xyl(β)-Ser], constituting the linkage region between the
glycosaminoglycan and protein in proteoglycan, was synthesized by using enzymatic transglycosylation with a β-D-ga-
lactosidase as a key reaction. The yields of transglycosylation were much improved by using galactosyl fluoride as a do-
nor in comparison with those obtained by a conventional p-nitrophenyl (PNP) galactoside. After enzymatic synthesis of
Gal(β1-3)Gal(β1-4)Xyl(β)–PNP, cleavage of the PNP group of the fully acetylated trisaccharide, chemical coupling with
serine, and final deprotection afforded Gal(β1-3)Gal(β1-4)Xyl(β)–Ser.
.4
.5
4
Enzymatic synthesis is of increasing importance for synthe-
sis of oligosaccharides, since it allows facile preparation of de-
sired oligosaccharides in short steps.1,2 Among enzymatic syn-
thesis, transglycosylation catalyzed by glycosidases has
apparent advantages in view of the availability of enzymes and
rather low specificity for both glycosyl donors and acceptors,
allowing the application of this method to a variety of com-
pounds. The major issues in transglycosylation have been how
to improve the regioselectivity and the yield which are not al-
glycosyl transfer to acceptors within short reaction times to
suppress hydrolysis of the glycosylated products. Their solu-
bilities in water are, however, not high enough because of the
hydrophobicity of the nitrophenyl group. Since the reaction at
high concentrations of substrates is advantageous for glycosy-
lation rather than hydrolysis of the donors, glycosyl donors
possessing high reactivity and solubility are advantageous for
transglycosylation. We therefore examined the use of glycosyl
fluorides as glycosyl donors for transglycosylation. Enzymatic
glycosylation using glycosyl fluorides as donors was already
reported by several authors.5–10 Shoda et al. extensively stud-
ied the enzymatic synthesis of cellulose and their derivatives
using cellobiosyl fluoride or its derivatives.6 In the present
study, β-galactosyl fluoride (Gal–F: 2) was used for β-galac-
tosidase-catalyzed transglycosylation. Application of this
method to the improved synthesis of Gal(β1-3)Gal(β1-
4)Xyl(β)–Ser (1) is described (Fig. 1).
Disaccharide, Gal(β1-4)Xyl(β)–PNP (5), was prepared by
transgalactosylation with Gal–F (2) to Xyl–PNP (4) (Scheme
1). The higher concentration of the phosphate buffer (200
mM, pH 7.3) was required for the reaction using Gal–F (100
mM) as compared to the concentration (50 mM, pH 7.3) for
the reaction with Gal–PNP (3). This was important to neutral-
ize HF liberated during the reaction mixture at the optimum
pH range of the enzyme. The desired transglycosylated prod-
uct 5 was obtained in 18.2% yield (Table 1, entry 1), which
was similar to that obtained with Gal–PNP (Table 1, entry 4).
When the reaction was carried out at a higher concentration of
the substrate, i.e., under saturated conditions of Gal–F (2) (400
mM) at room temperature, a better yield (27.7%) was obtained
as expected (Table 1, entry 2). The concentration of 2 was then
increased to 600 mM by warming the solution to 50 ˚C, where
a higher buffer-concentration (1.0 M) was required to maintain
the solution at pH 7.3. Although the enzymatic activity de-
creased at the present high buffer-concentration, the glycosyla-
ways high.
In our previous studies, Gal(β1-3)Gal(β1-
4)Xyl(β)–Ser (1), Gal(β1-3)Gal(β1-4)Xyl(β)–MU [MU ꢀ 4-
methyl-2-oxo-2H-chromen-7-yl (4-methylumbelliferyl)], and
GlcA(β1-3)Gal(β1-3)Gal(β1-4)Xyl(β)–MU, corresponding to
the linkage region between the glycosaminoglycan (GAG)
chain and protein in proteoglycan, were synthesized from Xyl–
PNP (PNP ꢀ p-nitrophenyl) or Xyl–MU by stepwise enzymat-
ic transglycosylation using β-galactosidase (Escherichia coli)
and β-glucuronidase (bovine liver) as enzymes and the corre-
sponding PNP glycosides as donors.3,4 Regioselective trans-
glycosylation at the 3′-position of Gal(β1-4)Xyl(β)–PNP or
Gal(β1-4)Xyl(β)–MU was achieved by partial protection of
the primary hydroxy group of the disaccharide intermediates.
The yields of transglycosylation were, however, low, since
competitive hydrolysis of a glycosyl donor occurred in prefer-
ence to the desired transglycosylation.
PNP glycosides have been frequently used as glycosyl do-
nors for transglycosylation, since they are efficiently recog-
nized by glycosidases and their high reactivity enables facile
Fig. 1.
The structure of trisaccharide–serine conjugate 1.

1124 Bull. Chem. Soc. Jpn., 74, No. 6 (2001)
Chemoenzymatic Synthesis of Gal–Gal–Xyl–L-Ser
Scheme 1.
Table 1.
The Yields of Disaccharide 5 and Its Regioisomer 6 in Transgalactosylation of Xyl–PNP (4)
with Gal–F (2)a) or Gal–PNP (3)a) by the Use of β-Galactosidase (E. coli)b)
Concentration
of Donor/mM
Concentration
of Buffer/M
Yieldsc)/%
Entry
Donor
Temp/˚C
Time/h
5
6
1
2
3
4
2
2
2
3
100
400
600
100
25
25
50
25
0.2
0.8
1.0
0.5
2.5
4.8
0.6
18.2
27.7
34.9
16.9
2.1
3.0
4.1
2.0
0.05
a) All reactions were carried out using 3 molar amounts of the donor to Xyl–PNP. b) 30 mU per 1 µmol
of the donor was used. c) Based on HPLC.
tion proceeded effectively to give the desired disaccharide 5 in
acetyl group.3,4 As in our previous works, the regioselective
introduction of an acetyl group to the 6′-position of 5 was
readily carried out by the use of lipase (celite-immobilized
Amano PS) in vinyl acetate and THF (1:1) to give the 6′-O-
monoacetylated disaccharide 7 in 87.4% yield. Regioselective
transglycosylation to 3′-position of 7 was then effected by the
use of 5 mol amt. of Gal–F (2) in a phosphate buffer at 50 ˚C to
give the desired trisaccharide 8 in 30.6% yield with 60.5% re-
covery of 7. The yield was much improved in comparison with
that of Gal–PNP (3) (23.1%).3
The trisaccharide 8 was then subjected to chemical glycosy-
lation with L-serine derivative 13 as described in our prelimi-
nary communication (Scheme 3).3 After complete O-acetyla-
tion, the PNP group of the resultant 9 was removed as
follows.11 The PNP group was changed into p-acetamidophe-
nyl (AAP) group by catalytic hydrogenolysis in Ac2O. The
AAP group was then removed by ammonium cerium(IV) ni-
a yield as high as 34.9% (entry 3). In a preparative scale syn-
thesis where the reaction was carried out under the same con-
ditions, the desired 5 was obtained in 32.9% yield after HPLC
purification. The structures of both 5 and its regioisomer 6
were confirmed by NMR spectrum (1H–1H COSY, HMQC,
and HMBC).
Enzymatic galactosylation of the hydroxy group of the 3′-
position of the disaccharide 5 was then investigated to form the
trisaccharide, Gal(β1-3)Gal(β1-4)Xyl(β)–PNP (Scheme 2).
Since the acceptor 5 itself in this reaction is a good substrate of
galactosidase, facile enzymatic hydrolysis was expected when
direct glycosylation to 5 would be attempted. Furthermore,
glycosylation at the more reactive 6′-position rather than the
3′-position would occur preferentially.2t In our previous stud-
ies, we already overcame these problems by selective protec-
tion of the primary hydroxy group of its Gal residue with an
Scheme 2.

K. Fukase et al.
Bull. Chem. Soc. Jpn., 74, No. 6 (2001) 1125
Scheme 3.
matograms. FAB-MS spectra were obtained using a JEOL SX-
102 mass spectrometer. Positive-ion MALDI-MS were performed
using a Voyager Elite XL time-of-flight mass spectrometer
equipped with a delayed-extraction system (PerSeptive Biosys-
tem).16 α-Cyano-4-hydroxycinnamic acid was used as a matrix.
NMR spectra were recorded on a JEOL JNM-GSX 270 (270
MHz), VARIAN-UNITY 600 NMR (600 MHz), or JEOL LA 500
(500 MHz) spectrometer, using D2O or CDCl3 as a solvent. The
chemical shifts of the protons are given in δ values as determined
either with TMS in CDCl3 or HDO (δ 4.65) in D2O as the internal
standard. The galactose residue connected to the xylose in the
compound 1, 8, 9, 11, or 14 is designated GalA and the other ga-
lactose is designated GalB, respectively, in the NMR assignment.
trate [NH4Ce(NO3)6, (CAN)] oxidation to give the trisaccha-
ride 11 in 73.4% yield from 9. The reaction of 11 with
CCl3CN in the presence of Cs2CO3 gave glycosyl trichloroace-
timidate 12,12 which was then coupled with Z–L-Ser–OBn (13)
by the use of trimethylsilyl triflate (TMSOTf) as a catalyst in
1,2-dichloroethane to give the protected serine conjugate 14.
Catalytic hydrogenolysis of 14 and subsequent hydra-
zinolysis13 afforded the desired Gal(β1-3)Gal(β1-4)Xyl(β1-
O)–L-Ser (1) in 88% yield. The structure of 1 was confirmed
by the NMR spectra (1H–1H COSY, HMQC, and HMBC) and
the matrix-assisted laser desorption/ionization (MALDI) mass
spectrum.
The combination of chemical and enzymatic methods de-
scribed above provides a facile overall procedure for synthesis
of trisaccharide derivatives with a minimum use of protecting
groups. The use of p-nitrophenyl glycoside as a temporary
protection of reducing-end sugar moiety enables not only fac-
ile purification of the products by reversed phase HPLC owing
to its strong UV absorption and appropriate lipophilicity but
also easy transformation from a simple sugar chain to a com-
plex glycoconjugate. Transglycosylation by using galactosyl
fluoride at high concentrations proved to be effective to im-
prove the yields of glycosylated product.
-D-Galactopyranosyl Fluoride15 (2). To the solution of ga-
β
lactose pentaacetate (2.15 g, 5.51 mmol) in CH2Cl2 (20 mL) was
added HBr–AcOH (6.0 mL) at 0 ˚C. The mixture was stirred at 0
˚C for 30 min and at r.t. for 2 h and then concentrated in vacuo. To
the solution of the residue in anhydrous CH3CN (150 mL) was
added AgF (0.801 g, 6.31 mmol) and the mixture was stirred at r.t.
for 12 h. The insoluble materials were removed by filtration
through celite. The filtrate was concentrated in vacuo and the res-
idue was purified by silica-gel column chromatography [toluene–
acetone (7:1)] to give the fully acetylated galactosyl fluoride (1.40
g, 72.5%, m.p.: 98 ˚C). To the solution of this compound in anhy-
drous MeOH (20 mL) was added NaOMe (0.1 mL, 1 M MeOH
solution) and the mixture was stirred at 0 ˚C for 10 h. The reac-
tion was quenched with Dowex 5W-X8 (Hꢂ form). After the resin
was removed, the solution was concentrated in vacuo to give β-ga-
Experimental
β-Galactosidase (E. coli, EC 3.2.1.23) was purchased from Sig-
ma Chemical Co. Lipase (EC 3.1.1.3), Amano PS was a generous
gift from Amano Pharmaceutical Co., Ltd. (Aichi, Japan). Xyl–
PNP (3) was purchased from Sigma Chemical Co. Gal–F (2) was
prepared according to the literature.14,15 Kieselgel 60 F254 (E.
Merck), 0.5 mm, was used for silica-gel preparative TLC. HPLC
was performed using Shimadzu LC-6AD liquid chromatograph
fitted with a YMC-GEL ODS-M 120 S-5 column, 20 ꢁ 250 mm
(designated column A), or a Cosmosil 5C18AR column, 20 ꢁ 250
mm (column B). The yields and regioselectivities of the products
were determined from the peak areas in the analytical HPLC chro-
25
lactosyl fluoride (2) as a solid: Yield 0.712 g (97.8%); [α]D
ꢃ32.6 (c 1.5, H2O); 1H NMR (400 MHz, D2O) δ 6.33 (1H, d, J ꢀ
7.2 Hz, H-1: Gal), 4.11 (1H, dd, J ꢀ 12, 5.2 Hz, H-5: Gal), 3.77
(1H, d, J ꢀ 3.1 Hz, H-4: Gal), 3.64–3.43 (2H, m), 3.38–3.21 (2H,
m, H-6: Gal); FAB-MS [positive, matrix: glycerol (Gro)] m/z
183.2 [(M ꢂ H)ꢂ].
β
β
p-Nitrophenyl O- -D-Galactopyranosyl-(1 → 4)– -D-xylo-
β
pyranoside (5) and p-Nitrophenyl O- -D-Galactopyranosyl-(1
→ 3)- -D-xylopyranoside (6). To a solution of Gal–F (2)
β

1126 Bull. Chem. Soc. Jpn., 74, No. 6 (2001)
Chemoenzymatic Synthesis of Gal–Gal–Xyl–L-Ser
(141.0 mg, 0.774 mmol) and Xyl–PNP (4) (70.0 mg, 0.258 mmol)
in a phosphate buffer (1.0 M, pH 7.3, 1.30 mL) was added β-ga-
lactosidase (50 U17) at 50 ˚C. After the mixture was allowed to
stand at 50 ˚C for 2 h, the reaction was stopped by heating the
mixture at 80 ˚C for 1 min. The mixture was filtered and the fil-
trate concentrated in vacuo. The residue was purified by HPLC
[column A, H2O–CH3CN (18%), flow rate: 8 mL minꢃ1, detection:
UV at 320 nm, retention time: 16.9 min (5) and 27.1 min (6)] to
give Gal(β1-4)Xyl(β)–PNP (5) (36.8 mg, 32.9%) and Gal(β1-
3)Xyl(β)–PNP (6) (3.6 mg, 3.2%) as colorless powder after lyo-
philization with recovery of 4 (38.6 mg, 55.1%). 5: [α]D25 ꢃ64.9
rate: 8 mL minꢃ1, gradient: 20–38% (0–30 min), detection: UV at
300 nm, retention time: 17.1 min] to give 8 (8.2 mg, 30.6%) as
colorless powder after lyophilization with recovery of 7 (12.1 mg,
60.5%). 8: [α]D25 ꢃ49.0 (c 1.51, H2O); 1H NMR (500 MHz, D2O)
δ 8.16 (2H, d, J ꢀ 11 Hz, H-3: PNP), 7.14 (2H, d, J ꢀ 9 Hz, H-2:
PNP), 5.15 (1H, d, J ꢀ 7.5 Hz, H-1: Xyl), 4.49–4.46 (2H, m, H-1:
GalA; H-1: GalB), 4.24–4.16 (2H, m), 4.14 (1H, d, J = 3.0 Hz, H-4:
GalA), 4.09 (1H, dd, J ꢀ 5.1, 12.1 Hz, H-5eq: Xyl), 3.86–3.80
(2H, m, H-5: Gal; H-4: Xyl), 3.82 (1H, d, J ꢀ 3.0 Hz, H-4: GalB),
3.72 (1H, dd, J ꢀ 3.5, 9.5 Hz, H-3: GalA), 3.68–3.48 (9H, m), 2.04
(3H, s, Ac); FAB-MS (positive, Matrix: Gro) m/z 638.1 [(M ꢂ
H)ꢂ]. Found: C, 46.98; H, 5.50; N, 2.15%. Calcd for
C25H35NO18: C, 47.10; H, 5.53; N, 2.20%.
1
(c 4.95, H2O); H NMR (500 MHz, D2O) δ 8.14 (2H, d, J ꢀ 9.2
Hz, H-3: PNP), 7.12 (2H, d, J ꢀ 9.4 Hz, H-2: PNP), 5.13 (1H, d, J
ꢀ 7.6 Hz, H-1: Xyl), 4.37 (1H, d, J ꢀ 7.8 Hz, H-1: Gal), 4.07 (1H,
dd, J ꢀ 12, 5.2 Hz, H-5ex: Xyl), 3.84 (1H, m, H-4: Xyl), 3.79
(1H, d, J ꢀ 3.2 Hz, H-4: Gal), 3.64–3.55 (6H, m), 3.51 (1H, dd, J
ꢀ 7.2, 11.0 Hz, H-2: Gal); 13C NMR (125 MHz, D2O) δ 160.346
(C-4: PNP), 141.439 (C-1: PNP), 124.872 (C-3: PNP), 116.265
(C-2: PNP), 100.980 (C-1: Gal), 98.633 (C-1: Xyl), 75.000 (C-4:
Xyl), 74.120 , 72.385, 71.200, 69.432 (C-2: Gal), 67.400 (C-4:
Gal), 61.988 (C-5: Xyl), 59.890 (C-6: Gal); FAB-MS [positive,
Matrix: Glycerol (Gro)] m/z 434.2 [(M ꢂ H)ꢂ]. Found: C, 47.11;
H, 5.31; N, 3.29%. Calcd for C17H23NO12: C, 47.12; H, 5.35; N,
β
p-Nitrophenyl O-(2,3,4,6-Tetra-O-acetyl- -D-galactopyrano-
β
syl)-(1 → 3)-O-(2,4,6-tri-O-acetyl- -D-galactopyranosyl)-(1 →
β
4)-2,3-di-O-acetyl- -D-xylopyranoside (9). To a solution of
trisaccharide 8 (40.2 mg, 0.0631 mmol) in pyridine (2 mL) was
added Ac2O (1.5 mL) at 0 ˚C. After the mixture was stirred at r.t.
for 12 h, the mixture was concentrated in vacuo. The residue was
purified by silica-gel preparative TLC [CHCl3-acetone (10:1)] to
give colorless powder after lyophilization: Yield 58.3 mg (94.9%);
25
[α]D ꢂ16.3 (c 4.92, CHCl3); FAB-MS [positive, Matrix: p-ni-
trobenzyl amine (NBA)] m/z 974.4 [(M ꢂ H)ꢂ]. Found: C, 50.62;
H, 5.20; N, 1.40%. Calcd for C41H51NO26: C, 50.57; H, 5.28; N,
1.44%.
1
3.23%. 6: H NMR (600 MHz, D2O) δ 7.71 (1H, d, J ꢀ 8.8 Hz,
H-3: PNP), 7.21 (2H, d, J ꢀ 8.8 Hz, H-2: PNP), 5.21 (1H, d, J ꢀ
7.4 Hz, H-1: Xyl), 4.33 (1H, d, J ꢀ 7.7 Hz, H-1: Gal), 4.15 (1H,
dd, J ꢀ 4.9, 10.9 Hz, H-5eq: Xyl), 3.62 (1H, d, J ꢀ 2.9 Hz, H-4:
Gal), 3.80–3.73 (2H, m, H-4: Xyl; H-2; Xyl), 3.70 (1H, dd, J ꢀ
3.4, H-3: Xyl) 3.61–3.55 (6H, m); 13C NMR (125 MHz, D2O) δ
162.447 (C-4: PNP), 143.627 (C-1: PNP), 127.019 (C-3: PNP),
117.436 (C-2: PNP), 104.136 (C-1: Gal), 100.730 (C-1: Xyl),
84.485 (C-3: Xyl), 76.251 , 73.512, 73.092, 72.154, 69.530,
68.659, 65.788 (C-5: Xyl), 62.004 (C-6: Gal); FAB-MS (positive,
Matrix: Gro) m/z 434.2 [(M ꢂ H)ꢂ].
β
O-(2,3,4,6-Tetra-O-acetyl- -D-galactopyranosyl)-(1→ 3)-O-
β
(2,4,6-tri-O-acetyl- -D-galactopyranosyl)-(1→ 4)-2,3-di-O-
acetyl-D-xylopyranose (11). Compound 9 (52.1 mg, 0.0535
mmol) was hydrogenated with Pd (30 mg, 0.28 mmol) in Ac2O (4
mL) under 7 kg cmꢃ2 of hydrogen at r.t. for 2 days. The catalyst
was removed by filtration and the filtrate concentrated in vacuo.
To the solution of the resulting 10 in CH3CN–H2O (10:1) (2 mL)
was added CAN (358 mg, 0.53 mmol) at 0 ˚C. After being stirred
for 20 min, the mixture was diluted with EtOAc (10 mL) and
washed with water. The organic layer was dried over Na2SO4 and
concentrated in vacuo. The residue was purified by silica-gel pre-
parative TLC [CHCl3-acetone (4:1)] to give colorless powder af-
β
p-Nitrophenyl O-(6-O-Acetyl- -D-galactopyranosyl)-(1 →
β
4)- -D-xylopyranoside (7). To a suspension of disaccharide 5
(200 mg, 0.462 mmol) in THF (50 mL) and vinyl acetate (50 mL)
was added immobilized lipase18 (Amano PS) (2.1 g). The mixture
was stirred at r.t. for 23 h and then filtered. After the filtrate was
concentrated in vacuo, the residue was purified by HPLC [column
B, solvent: H2O–CH3CN, flow rate: 8 mL minꢃ1, gradient: 20–
50% (0–30 min), detection: UV at 320 nm, retention time: 17.8
min] to give colorless powder after lyophilization: Yield 192 mg
(87.4%); [α]D25 ꢃ58.9 (c 1.92, DMF); 1H NMR (500 MHz, D2O)
δ 8.13 (2H, d, J ꢀ 9.3 Hz, H-3: PNP), 7.11 (2H, d, J ꢀ 9.6 Hz, H-
2: PNP), 5.13 (1H, d, J ꢀ 7.5 Hz, H-1: Xyl), 4.38 (1H, d, J ꢀ 8.8
Hz, H-1: Gal), 4.17 (2H, d, J ꢀ 5.0 Hz, H-6: Gal), 4.05 (1H, dd, J
ꢀ 5.3, 13 Hz, H-5eq: Xyl), 3.83 (1H, d, J ꢀ 3.5 Hz, H-4: Gal),
3.81–3.78 (1H, m, H-4: Xyl), 3.61 (1H, dd, J ꢀ 9.1, 9.1 Hz),
3.56–3.48 (3H, m), 3.41 (1H, dd, J ꢀ 7.5, 10.0 Hz, H-5ax: Xyl),
2.07 (3H, s, Ac); FAB-MS (positive, Matrix: Gro) m/z 476.2 [(M
ꢂ H)ꢂ]. Found: C, 48.09; H, 5.32; N, 3.00%. Calcd for
C19H25NO13: C, 48.00; H, 5.30; N, 2.95%.
25
ter lyophilization: Yield 33.5 mg (73.4%); [α]D ꢃ3.64 (c 3.50,
CHCl3); FAB-MS (positive, Matrix: NBA) m/z 853.3 [(M ꢂ H)ꢂ].
Found: C, 49.01; H, 5.60%. Calcd for C35H48O24: C, 49.30; H,
5.67%.
β
N-Benzyloxycarbonyl-O-(2,3,4,6-tetra-O-acetyl- -D-galac-
β
topyranosyl)-(1 → 3)-O-(2,4,6-tri-O-acetyl- -D-galactopyrano-
β
syl)-(1 → 4)-O-(2,3-di-O-acetyl- -D-xylopyranosyl)-(1 → 3)-L-
serine Benzyl Ester (14). To a solution of compound 11 (37.4
mg, 0.0439 mmol) and Cs2CO3 (7.0 mg, 0.21 mmol) in dry
CH2Cl2 (1 mL) was added CCl3CN (57 µL, 0.57 mmol) at r.t.. Af-
ter being stirred for 1.5 h, the mixture was diluted with CH2Cl2
and washed with saturated aqueous NaHCO3 and brine. The or-
ganic layer was dried over Na2SO4 and concentrated in vacuo.
The residue was dissolved in benzene and lyophilized to give the
trichloroacetimidate 12 as colorless powder. To a mixture of 12,
N-benzyloxycarbonyl-L-serine benzyl ester (13) (17.3 mg, 0.0525
mmol), and molecular sieves 4A (200 mg) in dry 1,2-dichloroet-
hane (1 mL) was added a solution of TMSOTf (3.5 µL, 0.023
mmol) in 1,2-dichloroethane (0.2 mL) under argon atmosphere at
ꢃ20 ˚C. After being stirred for 3 h, the mixture was diluted with
CH2Cl2, filtered, and washed with saturated aqueous NaHCO3 and
water. The organic layer was dried over Na2SO4 and concentrated
in vacuo. The residue was purified by silica-gel preparative TLC
[CHCl3–acetone (7:1)] to give colorless powder after lyophiliza-
β
p-Nitrophenyl O- -D-Galactopyranosyl-(1→ 3)-O-(6-O-ace-
β
β
tyl- -D-galactopyranosyl)-(1 → 4)- -D-xylopyranoside (8).
To a solution of disaccharide 7 (20.0 mg, 0.0421 mmol) and Gal–
F (2) (37.9 mg, 0.208 mmol) in a phosphate buffer (1.0 M, pH 7.3,
0.35 mL) was added β-galactosidase (5 U) at 50 ˚C. After the so-
lution was allowed to stand at 50 ˚C for 1.5 h, the reaction was
stopped by addition of AcOH (0.1 mL). The mixture was filtered
and the filtrate concentrated in vacuo. The residue was purified by
HPLC [column B, solvent: 0.1% aqueous AcOH–CH3CN, flow
25
1
tion: Yield 41.5 mg (81.2%); [α]D ꢃ9.87 (c 3.14, CHCl3); H

K. Fukase et al.
Bull. Chem. Soc. Jpn., 74, No. 6 (2001) 1127
NMR (500 MHz, CDCl3) δ 7.45 (10H, m, PhCH2), 5.71 (1H, NH),
5.56 (1H, d, J ꢀ 8.5 H), 5.37 (1H, dd, J ꢀ 0.7, 3.2 Hz, H-4: Gal),
5.35 (1H, dd, J ꢀ 0.9, 3.4 Hz, H-4: GalB), 5.21–5.01 (9H, m), 4.93
(1H, dd, J ꢀ 3.4, 10.5 Hz, H-3: GalB), 4.75 (1H, dd, J ꢀ 6.2, 7.6
Hz, H-2: GalA), 4.54 (2H, m, H-1: Xyl; H-2: Ser), 4.42 (1H, d, J ꢀ
6.0 Hz, H-1: GalB), 4.37 (1H, d, J ꢀ 7.9 Hz, H-1: GalA), 4.24–
4.06 (4H, m), 4.00 (1H, dd, J ꢀ 7.1, 11.5 Hz, H-5ex: Xyl), 3.86–
3.79 (6H, m), 3.66 (1H, m, H-5: Gal), 3.20 (1H, dd, J ꢀ 8.0, 12.1
Hz, H-5: Xyl), 2.41–1.98 (27H, Ac); FAB-MS (positive, Matrix:
NBA) m/z 1164.5 [(M ꢂ H)ꢂ]. Found: C, 54.39; H, 5.60; N,
1.18%. Calcd for C53H65NO28: C, 54.68; H, 5.63; N, 1.20%.
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β
β
O- -D-Galactopyranosyl-(1→ 3)-O- -D-galactopyranosyl-
β
(1 → 4)-O- -D-xylopyranosyl-(1 → 3)-L-serine (1). Com-
pound 14 (14.7 mg, 0.0126 mmol) was hydrogenated with Pd (20
mg, 0.19 mmol) in MeOH (1 mL) under 7 kg cmꢃ2 of hydrogen at
r.t. for 1h. The catalyst was removed by filtration and the filtrate
was concentrated in vacuo. To the solution of the residue in
MeOH (4 mL) was added hydrazine monohydrate (1 mL) at 0 ˚C.
After 5 h, the reaction was stopped by addition of acetone and the
mixture was concentrated in vacuo. The residue was purified by
gel permeation chromatography [LH-20 (Pharmacia Biotech),
H2O] and HPLC [column B, solvent: H2O, flow rate: 8 mL minꢃ1
,
detection: UV at 220 nm, retention time: 7.1 min] to give colorless
powder after lyophilization: Yield 6.2 mg (88%); [α]D25 ꢂ3.71 (c
5.21, H2O); 1H NMR (500 MHz, D2O) δ 4.49 (1H, d, J ꢀ 7.2 Hz,
H-1: Xyl), 4.41 (1H, d, J ꢀ 7.4 Hz, GalB), 4.35 (1H, d, J ꢀ 7.3
Hz, H-1: GalA), 4.15 (1H, dd, J ꢀ 3.8, 10.1 Hz, H-3: Ser), 4.07
(1H, d, J ꢀ 3.2 H, H-4, GalB), 4.00 (1H, dd, J ꢀ 3.1, 8.2 Hz, H-
5ex: Xyl), 3.93–3.89 (2H, m, H-2: Ser; H-3: Ser), 3.80 (1H, d, J ꢀ
3.4 Hz, H-4: Gal), 3.75–3.46 (13H, m), 3.31–3.23 (2H, m, H-5ax:
Xyl; H-2: GalA). 13C NMR (125 MHz, D2O) δ 172.293 (COOH),
105.172 (C-1: Xyl), 103.461 (C-1: GalB), 102.285 (C-1: GalA),
82.922 (C-3: GalA), 77.295 (C-4: Xyl), 75.963, 75.831, 74.606,
73.495 (C-2: GalA), 73.413, 71.932 (C-2: Xyl), 70.723, 69.473,
68.626 (C-3: Ser), 68.838 (H-5: Xyl), 61.946 (H-6: Gal), 61.848
(H-6: Gal), 55.391 (C-2: Ser). MALDI-MS Found: m/z 584.1114.
Calcd for C20H35NO17Na (M ꢂ Na): 584.1803.
3
K. Fukase, T. Yasukochi, Y. Suda, M. Yoshida, and S.
Kusumoto, Tetrahedron Lett., 37, 6763 (1996).
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4
5
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6
a) S. Kobayashi, K. Kashiwa, T. Kawasaki, and S. Shoda,
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The authors are grateful to Dr. Hiroyuki Fukuda of PE Bio-
systems Ltd. for the skillful measurement of high resolution
MALDI-TOF mass spectra. The authors would also like to
thank to Mr. Tamio Mase of Amano Pharmaceutical Co., Ltd.
(Aichi, Japan) for the generous gift of lipase, Amano PS. This
work was supported in parts by Grants-in-Aid for Scientific
Research on Priority Areas No. 06240105, No. 08245229 and
No. 09231228 and by a Grant-in-Aid for JSPS Fellows No.
2247, all from the Ministry of Education, Science, Sports and
Culture.
7
W. Treder, J. Thiem, M. Schlingmann, Tetrahedron Lett.,
27, 5605 (1986).
8
S. Kobayashi, J. Shimada, K. Kashiwa, and S. Shoda, Mac-
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1128 Bull. Chem. Soc. Jpn., 74, No. 6 (2001)
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