Regioselective synthesis of p-nitrophenyl glycosides of β-D-galactopyranosyl-disaccharides by transglycosylation with β-D-galactosidases

www.elsevier.nl/locate/carres

Carbohydrate Research 325 (2000) 120–131

Regioselective synthesis of p-nitrophenyl glycosides of

b-

D

-galactopyranosyl-disaccharides by transglycosylation

with b-

D

-galactosidases

Xiaoxiong Zeng ^a, Rika Yoshino ^a, Takeomi Murata ^a, Katsumi Ajisaka ^b,

Taichi Usui ^a,*

^aDepartment of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka Uni6ersity, Ohya 836,

Shizuoka 422-8529, Japan

^bMeiji Institute of Health Science, Meiji Milk Products Co., Ltd., 540 Naruda, Odawara 250-0862, Japan

Received 1 November 1999; accepted 5 November 1999

Abstract

The b-

D

-galactosidase from porcine liver induced regiospeciﬁc transglycosylation of b-

D

-galactose from b-D-Gal-

OC₆H₄NO₂-o to OH-6 of, respectively, p-nitrophenyl glycoside acceptors of Gal, GlcNAc and GalNAc to afford

b-Gal-(16)-a-Gal-OC₆H₄NO₂-p, b-Gal-(16)-b-Gal-OC₆H₄NO₂-p, b-Gal-(16)-a-GalNAc-OC₆H₄NO₂-p, b-Gal-

(16)-b-GalNAc-OC₆H₄NO₂-p, b-Gal-(16)-a-GlcNAc-OC₆H₄NO₂-p, and b-Gal-(16)-b-GlcNAc-OC₆H₄NO₂-p.

The enzyme showed much higher transglycosylation activity for the a-glycoside acceptors than the corresponding

b-glycoside acceptors. The regioselectivity of the b-

D

-galactosidase from Bacillus circulans ATCC 31382 greatly

-GlcNAc-OC₆H₄NO₂-p were used

depended on the nature of the acceptor. When a- -GalNAc-OC₆H₄NO₂-p and a-

D

as acceptors, the enzyme showed high potency for regioselective synthesis of b-Gal-(13)-a-GalNAc-OC₆H₄NO₂-p

and b-Gal-(13)-a-GlcNAc-OC₆H₄NO₂-p in high respective yields of 75.9 and 79.3% based on the acceptors added.

However, replacement of b-

D

-Gal-OC₆H₄NO₂-p by b-D-GalNAc-OC₆H₄NO₂-p did change the direction of galactosyl-

ation. The enzyme formed regioselectively b-Gal-(16)-b-Gal-OC₆H₄NO₂-p with (b-Gal-1(6-b-Gal-1)_n6-b-Gal-

OC₆H₄NO₂-p, n=1–4). No b-(13)-linked product was detected during the reaction. Use of the two readily

available b-

D

-galactosidases facilitates the preparation of (13)- and (16)-linked disaccharide glycosides of

b- -Gal-GalNAc and b-

D

Keywords: b-

D

-Galactosidase; p-Nitrophenyl glycoside of b-

D

-galactosyl-disaccharides; p-Nitrophenyl b-glycoside of (16)-galac-

tosyl-oligosaccharides; Regioselectivity; Transglycosylation

1. Introduction

eties of asialoglycoproteins, and the usual

linkages are (14) to GlcNAc, (13) to

GlcNAc, and (13) to GalNAc in sugar-

chain components of glycoproteins [1–4].

There is consequently much interest in the

chemical or enzymatic synthesis of oligosac-

charides containing these linkages. From a

practical viewpoint, the use of glycosidases is

attractive for oligosaccharide synthesis [5–12],

because glycosidases do have some regioselec-

D

-Galactose is an important constituent of

the carbohydrate chains of glycoconjugates

involved in a variety of biological recognition

events. The

D

-galactose residue is generally

found at terminal positions in the sugar moi-

* Corresponding author. Tel./fax: +81-54-238-4873.

E-mail address: actusui@agr.shizuoka.ac.jp (T. Usui)

PII: S0008-6215(99)00303-1

X. Zeng et al. / Carbohydrate Research 325 (2000) 120–131

121

tivity for the hydroxyl linkage to the acceptor,

and their selectivity may vary with different

enzymes [13]. For example, the b- -galactosi-

dase from Bacillus circulans mediated the

-galactosyl from lactose to the

D

transfer of b-

D

secondary hydroxyl group (OH-4) over the

primary hydroxyl group of GlcNAc, whereas

the b-

D

-galactosidase from Kluy6eromyces lac-

-Gal-(16)- -GlcNAc as the

tis affords b-

D

main transglycosylation product from lactose

and GlcNAc [14]. Our interest is aimed at

Fig. 1. Toyopearl HW-40S chromatographic separation of

transglycosylation products with b-

D

-Gal-OC₆H₄NO₂-o and

-galac-

b- -galactosidase-mediated synthesis of p-ni-

D

a-

D

-GalNAc-OC₆H₄NO₂-p as substrates by use of b-

D

trophenyl glycosides of galactosyl-disaccha-

rides with different linkages as the units

mimicking the carbohydrate chains of glyco-

tosidase from porcine liver. (ꢀ) Absorbance at 210 nm, (ꢁ)

absorbance at 300 nm, and ( ) absorbance at 485 nm.

conjugates [15–17]. In our recent work, a-

fucosidase and b- -galactosidase from porcine

liver have been separated from each other.

L-

D

tion. The enzyme was used for the regioselec-

tive synthesis of p-nitrophenyl galactosyl-

disaccharides without further puriﬁcation. The

galactosidase from porcine liver had an opti-

The former catalyzes the formation of an a- -

L

(12)-linked product with its isomers fucosyl-

ated at OH-3 or OH-6 of the galactosyl

mum pH of 5.5 with b- -Gal-OC₆H₄NO₂-o as

D

residue of LacNAc or p-nitrophenyl b- -lac-

D

substrate. The enzyme showed an optimum

temperature of 50 °C, while the enzyme was

stable only below 40 °C. The enzyme hy-

toside [18,19]. In the present report, we de-

scribe the transglycosylation by b- -gal-

D

actosidases from porcine liver and B. circulans

ATCC 31382 for the synthesis of p-nitro-

phenyl glycosides of galactosyl-disaccharides

and the regioselectivity of the two enzymes

toward various of p-nitrophenyl glycoside ac-

ceptors.

drolyzed b-

D

-Gal-OC₆H₄NO₂-o and b- -Gal-

D

OC₆H₄NO₂-p as good substrates, but not

lactose and N-acetyllactosamine (data not

shown).

When a-

D

-GalNAc-OC₆H₄NO₂-p and b-

D

-

Gal-OC₆H₄NO₂-o were incubated with b-

D

galactosidase from porcine liver, the transfer

product was monitored by high-performance

liquid chromatography (HPLC). HPLC

showed that one transfer product was formed

during the incubation. The mixture was read-

ily separated on a column of Toyopearl HW-

40S to afford compound 1 in a yield of 78.7%

based on the acceptor added (Fig. 1). Simi-

2. Results and discussion

Transglycosylation of i- -galactosidase

D

from porcine li6er.—The fresh extract from

porcine liver was ﬁrst dialyzed against 50 mM

NaOAc buffer (pH 5.2) overnight. The super-

natant from the dialyzate was loaded directly

onto an ion-exchange column pre-equilibrated

with 50 mM NaOAc buffer (pH 5.2). The

larly with b-

GlcNAc-OC₆H₄NO₂-p, b-

NO₂-p, b- -Gal-OC₆H₄NO₂-p, and a-

D

-GalNAc-OC₆H₄NO₂-p, a-

-GlcNAc-OC₆H₄-

-Gal-

D

-

D

fractions containing b- -galactosidase activity

D

OC₆H₄NO₂-p as acceptors, the b-(16)-

linked transfer products were obtained in

yields of 28.1, 83.8, 25.7, 21.2, and 63.9%,

respectively (Table 1). No b-(14)- or b-(1

3)-linked product was detected during the re-

action. The use of a-glycoside acceptors gave

much higher yields (63–84%) than those of

their corresponding b-glycoside acceptors

(21–28%). The enzyme transferred the Gal

were pooled, concentrated to low volume, and

loaded onto the same column treated as al-

ready described, but the pH of the buffer was

changed to 4.5. The partially puriﬁed b- -

D

galactosidase, completely devoid of N-acetyl-

hexosaminidases [which degrades the acceptor

substrates a- and b-HexNAc-OC₆H₄NO₂-p

(see later)], was obtained in a speciﬁc activity

of 1.9 U/mg protein with a 51-fold puriﬁca-

122

X. Zeng et al. / Carbohydrate Research 325 (2000) 120–131

Table 1

The yields of transfer products mediated by b-D-galactosidases from porcine liver and Bacillus circulans ATCC 31382

Sources of b-

D

-galactosidase

Acceptor

Product

Yield ^a(%)

Porcine li6er

GalNAca-pNP

GalNAcb-pNP

GlcNAca-pNP

GlcNAcb-pNP

Gala-pNP

Galb16GalNAca-pNP (1)

Galb16GalNAcb-pNP (3)

Galb16GlcNAca-pNP (7)

Galb16GlcNAcb-pNP (5)

Galb16Gala-pNP (14)

Galb16Galb-pNP (9)

78.7

28.1

83.8

25.7

63.9

21.2

Galb-pNP

Bacillus circulans ATCC 31382

GalNAca-pNP

GalNAcb-pNP

GlcNAca-pNP

GlcNAcb-pNP

Gala-pNP

Galb13GalNAca-pNP (2)

Galb16GalNAca-pNP (1)

Galb13GalNAcb-pNP (4)

Galb16GalNAcb-pNP (3)

Galb13GlcNAca-pNP (8)

Galb16GlcNAca-pNP (7)

Galb13GlcNAcb-pNP (6)

Galb16GlcNAcb-pNP (5)

Galb13Gala-pNP (15)

75.9

3.2

20.9

18.6

79.3

0.3

29.5

6.0

9.3

22.8

4.3

2.6

11.7

6.8

4.3

Galb16Gala-pNP (14)

Galb16Galb16Gala-pNP (16)

Galb1(6Galb1)₂6Gala-pNP (17)

Galb16Galb-pNP (9)

Galb16Galb16Galb-pNP (10)

Galb1(6Galb1)₂6Galb-pNP (11)

Galb1(6Galb1)₃6Galb-pNP (12)

Galb1(6Galb1)₄6Galb-pNP (13)

Galb-pNP

1.7

0.5

^aThe yield is calculated based on the acceptor added.

group much more extensively to OH-6 of the

a-glycoside acceptor than that of the corre-

sponding b-glycoside. This shows that the ori-

entation of the hydrophobic p-nitrophenyl

group in the acceptor greatly inﬂuences the

efﬁciency of transglycosylation. The efﬁciency

of the transglycosylation was strongly depen-

dent on the molar ratio of the donor to accep-

tor substrates. The reaction employing the

molar ratio of 3.0 was suitable for the effective

syntheses of the corresponding b-(16)-

linked disaccharide glycosides (Fig. 2). Once

formation of compound 7 reached its maxi-

mum, no decomposition of product could be

observed. This was also the same trend for

other acceptors (data not shown).

saccharides closely corresponds to that of

transglycosylation activity. In this case, how-

ever, the enzyme produced only (16)-linked

transfer product, and no (13) transfer

product was detected during the reaction.

Transglycosylation of i-

D-galactosidase

from B. circulans ATCC 31382.—b-

D

-Galac-

In a separate experiment, the relative hy-

drolytic rates of p-nitrophenyl galactosyl-N-

acetylglycosaminides toward porcine liver

b- -galactosidase were measured and are

D

Fig. 2. Effects of the molar ratio of donor/acceptor on the

formation of b-Gal-(16)-a-GlcNAc-OC₆H₄NO₂-p mediated

shown in Table 2. The initial rate of reaction

was (16)\(13). The enzyme did not hy-

drolyze the (14) linkage. In general, the

order of hydrolytic activity toward those di-

by b-D-galactosidase from porcine liver. (ꢀ), ( ), and (ꢁ)

represent molar ratios of 1.5, 3.0, and 4.0 for donor/acceptor,

respectively.

X. Zeng et al. / Carbohydrate Research 325 (2000) 120–131

123

Table 2

The hydrolytic rates of b-

D

-galactosidase acting on p-nitrophenyl galactosyl-N-acetylglucosaminides

Compounds

Enzyme

b-

D

-Galactosidase from

b-D-Galactosidase

from B. cirulans ATCC 31382

porcine liver

b-Gal-(13)-b-GlcNAc-OC₆H₄NO₂-p

b-Gal-(14)-b-GlcNAc-OC₆H₄NO₂-p

b-Gal-(16)-b-GlcNAc-OC₆H₄NO₂-p

18

0

100 ^a

5

14

100 ^b

^aThe hydrolytic rate on b-Gal-(13)-b-GlcNAc-OC₆H₄NO₂-p by b-

D

-galactosidasc from B. cirulans ATCC 31382 was

arbitrarily set at 100.

^bThe hydrolytic rate on b-Gal-(16)-b-GlcNAc-OC₆H₄NO₂-p by b-

D-galactosidase from porcine liver was arbitrarily set at

100.

enzyme can be used to synthesize b-glycosides

-galactopyranosyl-oligosaccha-

rides (degree of polymerization 2–5) in one

step. Much work has been done on the chem-

tosidase from B. circulans ATCC 31382 pre-

dominated the formation of the (13)-linked

compound 2 over the (16)-linked isomer

of (16)-b-

D

(1) through Gal transfer from b-

D-Gal-

ical synthesis of O-b-(16)-

D

-galactopyr-

OC₆H₄NO₂-o to a- -GalNAc-OC₆H₄NO₂-p.

D

anosyl-oligosaccharides that act as ligands

The transfer products 2 and 1 were obtained

in 79.1% overall yield based on the acceptor

added and in a ratio of 96:4. This was also the

case for the formation of compound 8 with

of anti-(16)-b- -galactopyranan antibodies

D

[20–24]. For example, Ekborg et al. have re-

ported the synthesis of compounds 9 and 10

from b- -Gal-OC₆H₄NO₂-p using a chemical

D

a- -GlcNAc-OC₆H₄NO₂-p as acceptor (Table

D

method [20]. The present process for obtaining

-galactosyl-oligo-

1). These transfer products were readily sepa-

rated on Chromatorex-ODS and Toyopearl

HW-40S columns. The unreacted acceptors

could be recovered by straightforward chro-

matography and reutilized. The enzymatic

process for obtaining the disaccharides 2 and

8 was simple and the yield is sufﬁciently high

to make the method practical. The enzyme

formed regioselectively the b-(16)-linked

disaccharide 9 along with a series of b-(16)-

linked glycosides of galactopyranosyl-oli-

gosaccharides (10–13). No other linkage in

the transfer products was detected during the

b-glycosides of (16)-b-

D

saccharides is simple and the yield is sufﬁ-

ciently high to make the method practical.

Recently, Fransen et al. have reported the

enzymatic synthesis of [(b-

Glcp] and [(b- -Galp-(16))_n-b-

4)-

b-

D

-Galp-(14))_n-

D-

D

-Galp-(1

D

-Glcp] (n=0–5) from lactose using

D

-galactosidase [25].

Regioselecti6ity of formation of p-nitro-

phenyl glycosides of i-

saccharides by i-

culans ATCC 31382.—The positions of galac-

tosylation mediated by b- -galactosidase from

B. circulans ATCC 31382 with a- -GalNAc-

-GalNAc-OC₆H₄NO₂-p, a-

-GlcNAc-OC₆-

D

-galactopyranosyl-di-

D

-galactosidase from B. cir-

D

reaction. In the case of the a- -Gal-

D

OC₆H₄NO₂-p acceptor, the b-(16)-linked

disaccharide (14) was formed along with the

b-(13)-linked one (15) in a molar ratio of

19:6, and together with the b-(16)-linked

galactosyl-trisaccharide (16) and galactosyl-te-

trasaccharide (17). In both reactions, b-(1

6)-linked galactosyl-oligosaccharides larger

than the dimer were formed as transfer prod-

ucts. It is apparent that these oligomers are

produced by successive regioselective galacto-

sylation from the disaccharides 9 and 14

formed initially. The results indicate that the

OC₆H₄NO₂-p, b-

D

-GlcNAc-OC₆H₄NO₂-p, b-

D

H₄NO₂-p, a-

D-Gal-OC₆H₄NO₂-p, and b- -

D

Gal-OC₆H₄NO₂-p acceptors are depicted by

arrows in Fig. 3. It may be seen that the

regioselectivity of the b- -galactosidase from

D

B. circulans ATCC 31382 is markedly inﬂu-

enced by the presence of the 2-acetamido

group and the anomeric conﬁguration of the

aglycon in the acceptor. In the case of a-

GalNAc-OC₆H₄NO₂-p and a- -GlcNAc-OC₆-

H₄NO₂-p acceptors, galactosylation occurs

D-

D

124

X. Zeng et al. / Carbohydrate Research 325 (2000) 120–131

only the regioselectivity at OH-3, but also the

efﬁciency of transglycosylation. The decrease

in activity may be attributed to the unsuitable

conﬁguration of the p-nitrophenyl group to

the hydrophobic binding locus in the active

site. On the other hand, if this concept is

overwhelmingly at OH-3 and is accompanied

by a high efﬁciency of transglycosylation. This

can be tentatively explained by comparing the

stereochemical environment between acceptor

and the active site of the enzyme with regard

to reaction sites. Thus, two hydrophobic

groups, the 2-acetamido and p-nitrophenyl

groups in the a-glycoside acceptor, may bind

to the enzyme and position the substrate

molecular properly on the active site so that

they are located in relation to the hydrophobic

site of the enzyme, as shown in Fig. 4(A). This

applied to the b- -Gal-OC₆H₄NO₂-p acceptor,

D

it would not adopt a favorable orientation

along C-1 to C-2, because of an unsuitable

conﬁguration of the 1-p-nitrophenyl group

and a repulsion of the hydrophilic 2-hydroxyl

group to the hydrophobic binding locus in the

active site, as shown in Fig. 4(B-a). In an

alternative plan (shown in Fig. 4(B-b)), when

shows that the regioselectivity of b- -galacto-

D

syl transfer to the acceptor catalyzed by the

enzyme is strongly determined by the 1,2-cis

conﬁguration of 2-acetamido and 1-p-nitro-

phenyl groups, which adopt an orientation

favorable to the enzyme. Replacement of b-

glycosides of GalNAc and GlcNAc by their

corresponding a-glycosides diminishes not

the structural form of b- -Gal-OC₆H₄NO₂-p

D

to the binding locus is reversed along C-1 to

C-4, the acceptor will be properly located in

relation to the hydrophobic site so that it

adopts a favorable orientation along C-1 to

the ring oxygen atoms. This enables us to

assume that only OH-6 of the b- -Gal-

D

OC₆H₄NO₂-p acceptor is galactosylated to

form the b-(16)-galactosyl-disaccharide gly-

coside. It has already been reported that the

regioselectivity of glycosidase-catalyzed for-

mation of disaccharides is greatly inﬂuenced

by the nature of the acceptor, such as its

anomeric conﬁguration, presence of a 2-ac-

etamido group, and the substitution mode at

the anomeric carbon atoms [15,26–28].

Table 2 shows the relative hydrolytic rates

of p-nitrophenyl galactosyl-N-acetylglucos-

aminide toward b- -galactosidase from B. cir-

D

culans ATCC 31382. The relative rates of

hydrolysis for compound 5 and b-Gal-

(14)-b-GlcNAc-OC₆H₄NO₂-p as compared

with compound 6 (set at 100) were 5 and 14,

respectively, namely, 7–14-fold differences.

Compound 6 should be a much better sub-

strate than compound 5. Thus, the order of

the hydrolytic rates of compounds 6 and 5

corresponds to that of their transglyco-

sylation.

In conclusion, the regiospeciﬁc synthesis

of b-(16)-galactosyl-disaccharide glycosides

was achieved by using transglycosylation by

Fig. 3. Structures of acceptors for b-

D-galactosidase-mediated

galactosylation (b- -galactosidase from B. circulans ATCC

D

31382). Arrows indicate the positions of galactosylation. Per-

centages above and below the arrows are of the formation of

a given transglycosylation as compared with the total. The

percentages of galactosylation are based on the time at which

the (13)-linked disaccharide derivative production reaches

its maximum. R¹: N-acetyl group, and R²: p-nitrophenyl

group.

the readily available b- -galactosidase from

D

porcine liver. We also developed a synthetic

method for compound 2, which is a mimic

unit of mucin type I and its analogs, by trans-

glycosylation mediated by the b- -galactosi-

D

X. Zeng et al. / Carbohydrate Research 325 (2000) 120–131

125

Fig. 4. (A) The postulated hydrophobic–acceptor binding-site of b-

active site. Arrows indicate the positions of galactosylation. (A) a-

b- -Gal-OC₆H₄NO₂-p.

D

-galactosidase, which positions the acceptor properly on the

D

-GalNAc-OC₆H₄NO₂-p and a- -GlcNAc-OC₆H₄NO₂-p, (B)

D

dase from B. circulans ATCC 31382. The en-

zymatic process for obtaining the desired com-

pounds 2 and 8 is simple and the yields

(75–80%) are remarkably high. The synthetic

oligosaccharide glycosides are useful as start-

ing substances for glycopolymers, which are

valuable for investigating biological recogni-

tion phenomena using lectins [29–31].

1.0 M Na₂CO₃soln, and the liberated o-nitro-

phenol was determined spectrophotometri-

cally at 420 nm. One unit of enzyme activity

was deﬁned as the amount of enzyme hy-

drolyzing 1 mmol of b- -Gal-OC₆H₄NO₂-o per

D

minute.

Analytical methods.—HPLC was performed

with a Hitachi 6000 liquid chromatograph

with a column of Shodex Asahipak NH2P-50

4E (4.6×250 mm, Showa Denko Corp.,

Tokyo, Japan) developed with 80% of MeCN

in aq soln at a ﬂow rate of 0.8 mL/min, and a

column of TSKgel Super ODS (4.6×100 mm,

Tosoh Corp., Tokyo, Japan) eluted with 10%

MeOH at a ﬂow rate of 1.0 mL/min, both

3. Experimental

Materials.—a -

was purchased from Yaizu Suisan Kagaku

Co., Ltd. (Yaizu, Japan). a- -Gal-OC₆H₄-

NO₂-p, b- -Gal-OC₆H₄NO₂-p, b- -GalNAc-

OC₆H₄NO₂-p, and -GlcNAc-OC₆H₄-

D

- GalNAc - OC₆H₄NO₂- p

D

1

with an L-4000 UV detector. H and ¹³C

D

NMR spectra were recorded with a Jeol JNM-

EX 270 Fourier transform NMR spectrometer

at 25 °C. Chemical shifts are expressed in l

relative to sodium 4,4-dimethy-4-silapen-

tanoate (TPS) as the external standard in

D₂O. The assignment of signals in the transfer

a-D

NO₂-p were obtained from Sigma Chemical

Co. (St Louis, MO, USA). CM-Sepharose

Fast Flow was a product of Pharmacia. b- -

D

Galactosidase from B. circulans ATCC 31382

was kindly supplied from Meiji Institute of

Health Science (Odawara, Japan).

13

products was obtained by C–¹H COSY and

also by analogy with the chemical shifts of

related compounds based on earlier data

[7,16,17,20]. FABMS was performed in the

positive mode using a Jeol JMS DX-303 HF

mass spectrometer coupled to a Jeol DA-800

mass data system. An accelerating voltage of

10 kV and a mass soln of 1000 were em-

Assay of i-

activity was assayed as follows. A mixture

-Gal-OC₆H₄NO₂-o in

D

-galactosidase acti6ity.—The

containing 2.0 mM b-

D

0.9 mL NaOAc buffer (pH 5.5) and an appro-

priate amount of enzyme to a total volume of

1.0 mL was incubated for 10 min at 37 °C.

The reaction was stopped by adding 0.5 mL of

126

X. Zeng et al. / Carbohydrate Research 325 (2000) 120–131

ATCC 31382 (0.4 U). The soln was incubated

at 37 °C, samples (100 mL) were taken out at

appropriate time intervals (0, 5, 10, 15, 20

min) during the incubation, and were analyzed

by HPLC with a column of TSKgel G-Oligo-

PW (4.6×100 mm, Tosoh Corp., Tokyo,

Japan). The following p-nitrophenyl galacto-

syl-glycosides were used: b-Gal-(13)-b-Glc-

NAc-OC₆H₄NO₂-p, b-Gal-(14)-b-GlcNAc-

OC₆H₄NO₂-p, and b-Gal-(16)-b-GlcNAc-

OC₆H₄NO₂-p.

ployed. Mass calibration was achieved using

Ultramark. Speciﬁc rotation was determined

with a Digital Automatic Polarimeter DIP-

1000 apparatus (Jasco, Japan).

Preparation of i- -galactosidase from

D

porcine li6er.—A crude enzyme soln from

fresh porcine liver was prepared by the

method reported [19]. The extract was dia-

lyzed overnight against 50 mM NaOAc buffer

(pH 5.2). After centrifugation (24,100g, 20

min), the supernatant (20 mL) was applied to

a column (3.2×26 cm) of CM-Sepharose Fast

Flow pre-equilibrated with 50 mM NaOAc

buffer (pH 5.2). The column was washed with

400 mL of the same buffer. Next the column

was eluted with a linear gradient of NaCl

concentration from 0 (400 mL) to 1.0 M (400

Preparation of i-Gal-(16)-h-GalNAc-

OC₆H₄NO₂-p (1) and i-Gal-(13)-h-Gal-

NAc-OC₆H₄NO₂-p (2)

(A) Using i-

li6er. To a soln of b-

mg) and a- -GalNAc-OC₆H₄NO₂-p (44 mg)

in 8 mL of 40 mM NaOAc buffer (pH 5.5)

was added partially puriﬁed b- -galactosidase

D

-galactosidase from porcine

D

-Gal-OC₆H₄NO₂-o (120

D

mL). The fractions containing b- -galactosi-

D

dase activity (20 mL/tube, tubes 9–13) were

combined, concentrated to a low volume with

an Amicon Diaﬂo unit equipped with a PM10

membrane operating at 2 kg/cm², and loaded

to the same column pre-equilibrated with 50

mM NaOAc buffer (pH 4.5). Then the column

was treated as already described. The fractions

from porcine liver (0.8 U). The reaction was

terminated by heating in a boiling-water bath

for 5 min after 8.5 h of incubation at 40 °C.

The supernatant from the centrifugation was

extracted with diethyl ether to remove the

o-nitrophenol liberated during the reaction,

concentrated, and loaded onto a Toyopearl

HW-40S column (4.5×90 cm) equilibrated

with 25% MeOH in water. The column was

eluted with the same soln. The elution was

monitored by measuring the absorbances at

300 nm (the p-nitrophenyl group) and 485 nm

(carbohydrate content, determined by the phe-

nol–sulfuric acid method). The fractions

(tubes 57–64) containing transfer product as

shown in Fig. 1 were collected, concentrated,

and lyophilized to afford compound 1 (51.0

mg). The physical and NMR data for com-

pound 1 were identical to those of b-Gal-(1

6)-a-GalNAc-OC₆H₄NO₂-p reported previous-

ly [17].

containing b- -galactosidase activity (tubes

D

31–35) were combined and concentrated to a

low volume, giving 1.9 U/mg protein of spe-

ciﬁc activity.

Effects of pH and temperature on the acti6ity

of i-

the determination of optimum pH, the activity

-galactosidase from porcine liver was

D-galactosidase from porcine li6er.—For

of b-

D

measured at 37 °C for 10 min at various pH

values using sodium citrate buffer (pH 2.0–

3.0), NaOAc buffer (pH 3.0–6.0), and sodium

phosphate buffer (pH 6.0–9.0). To determine

optimum temperature, the b- -galactosidase

D

was incubated for 10 min at various tempera-

tures in 0.1 M NaOAc buffer (pH 5.5). The

thermal stability of the enzyme was examined

by incubating the enzyme at various tempera-

tures for 30 min, and the activity was mea-

sured as already described.

(B) Using i-

lans ATCC 31382. To a soln of b-

OC₆H₄NO₂-o (600 mg) and a- -GalNAc-

OC₆H₄NO₂-p (220 mg) in 40 mM NaOAc

buffer (40 mL, pH 5.5) was added b- -galac-

D

-galactosidase from B. circu-

D

-Gal-

D

Hydrolysis reaction by i- -galactosidases

D

tosidase from B. circulans ATCC 31382 (2.0

U). After 3.5 h of incubation at 50 °C, the

reaction was terminated by heating in a boil-

ing-water bath for 5 min. The supernatant

from the centrifugation was applied to a

column (3.6×52 cm) of Chromatorex-

on p-nitrophenyl galactosyl-N-acetylglucos-

aminides.—To a soln containing p-nitro-

phenyl galactosyl-N-acetylglucosaminide (250

mg) in 50 mM NaOAc buffer (pH 5.5, 1.0 mL)

was added b- -galactosidase from porcine

D

liver (partially puriﬁed) or from B. circulans

X. Zeng et al. / Carbohydrate Research 325 (2000) 120–131

127

Preparation of i-Gal-(16)-i-GlcNAc-

OC₆H₄NO₂-p (5) and i-Gal-(13)-i-Glc-

NAc-OC₆H₄NO₂-p (6)

ODS DM 1020T equilibrated with 10%

MeOH in water. The column was eluted with

the same soln. The fractions (60 mL/tube,

tubes 58–101) were combined, concentrated,

and loaded onto a Toyopearl HW-40S column

treated as already described. The F-1 (30 mL/

tube, tubes 59–63) and F-2 (tubes 67–75)

fractions were combined, concentrated, and

lyophilized to afford compounds 1 (10.5 mg)

and 2 (245.7 mg), respectively. The physical

and NMR data for compound 2 were identical

to those of b-Gal-(13)-a-GalNAc-OC₆H₄-

NO₂-p, reported previously [17].

(A) Using i-

li6er. To a soln of b-

mg) and b- -GlcNAc-OC₆H₄NO₂-p (330 mg)

in 40 mM NaOAc buffer (120 mL) was added

-galactosidase (5 U) from

D

-galactosidase from porcine

D

-Gal-OC₆H₄NO₂-o (900

D

partially puriﬁed b-

D

porcine liver. The reaction was terminated by

heating in a boiling-water bath for 5 min after

8 h of incubation at 40 °C. The supernatant

from centrifugation was treated as described

for the preparation of compound 1 using b- -

D

Preparation of i-Gal-(16)-i-GalNAc-

OC₆H₄NO₂-p (3) and i-Gal-(13)-h-Gal-

NAc-OC₆H₄NO₂-p (4)

galactosidase from porcine liver. The fractions

(tubes 60–71) were combined, concentrated,

and lyophilized to afford compound 5 (98.4

mg). The physical and NMR data for com-

pound 5 were identical to those of b-Gal-(1

6)-b-GlcNAc-OC₆H₄NO₂-p, reported previous-

ly [16].

(A) Using i-

li6er. To a soln of b-

mg) and b- -GalNAc-OC₆H₄NO₂-p (330 mg)

in 40 mM NaOAc buffer (120 mL) was added

-galactosidase (5 U) from

D

-galactosidase from porcine

D-Gal-OC₆H₄NO₂-o (900

D

(B) Using i-

lans ATCC 31382. To a soln of b-

OC₆H₄NO₂-o (900 mg) and b- -GlcNAc-

OC₆H₄NO₂-p (330 mg) in 40 mM NaOAc

buffer (120 mL) was added b- -galactosidase

D

-galactosidase from B. circu-

partially puriﬁed b-

D

-Gal-

porcine liver. The reaction was terminated by

heating in a boiling-water bath for 5 min after

8 h of incubation at 40 °C. The reaction mix-

ture was treated as described for the prepara-

D

(5.0 U) from B. circulans ATCC 31382. After

9 h of incubation at 50 °C, the reaction mix-

ture was terminated by heating in a boiling-

water bath for 5 min. The supernatant from

centrifugation was treated as described for the

tion of compound 1 using b- -galactosidase

D

from porcine liver. The fractions (tubes 58–

66) were combined, concentrated, and lyo-

philized to afford compound 3 (107.6 mg).

The physical and NMR data for compound 3

were identical to those of b-Gal-(16)-b-Gal-

NAc-OC₆H₄NO₂-p, reported previously [17].

preparation of compounds 1 and 2 using b- -

D

galactosidase from B. circulans ATCC 31382.

The F-1 (tubes 72–83) and F-2 (tubes 98–115)

fractions were concentrated, and lyophilized

to afford compounds 5 (29.2 mg) and 6 (143.9

mg), respectively. The physical and NMR

data for compound 6 were identical to those

of b-Gal-(13)-b-GlcNAc-OC₆H₄NO₂-p, re-

ported previously [7].

(B) Using i-

lans ATCC 31382. To a soln of b-

OC₆H₄NO₂-o (900 mg) and b- -GalNAc-

OC₆H₄NO₂-p (330 mg) in 40 mM NaOAc

buffer (120 mL) was added b- -galactosidase

D

-galactosidase from B. circu-

D

-Gal-

D

(5.0 U) from B. circulans ATCC 31382. After

6 h of incubation at 50 °C, the reaction was

terminated by heating in a boiling-water bath

for 5 min. The supernatant from centrifuga-

tion was treated as described for the prepara-

Preparation of i-Gal-(16)-h-GlcNAc-

OC₆H₄NO₂-p (7) and i-Gal-(13)-h-Glc-

NAc-OC₆H₄NO₂-p (8)

(A) Using i-

li6er. To a soln of b-

mg) and a- -GlcNAc-OC₆H₄NO₂-p (66 mg) in

40 mM NaOAc buffer (15 mL) was added

-galactosidase (1.8 U)

D

-galactosidase from porcine

tion of compounds 1 and 2 using b- -

D

-Gal-OC₆H₄NO₂-o (200

galactosidase from B. circulans ATCC 31382.

F-1 (20 mL/tube, tubes 84–97) and F-2 (tubes

104–117) fractions were concentrated and

lyophilized to afford compounds 3 (90.1 mg)

and 4 (101.5 mg), respectively. The physical

and NMR data for compound 4 were identical

to those of b-Gal-(13)-b-GalNAc-OC₆H₄-

NO₂-p, reported previously [17].

D

partially puriﬁed b-

D

from porcine liver. The reaction was termi-

nated by heating in a boiling-water bath for 5

min after 6 h of incubation at 40 °C. The

reaction mixture was treated as described for

128

X. Zeng et al. / Carbohydrate Research 325 (2000) 120–131

(A) Using i-

li6er. To a soln of b-

mg) and b- -Gal-OC₆H₄NO₂-p (200 mg) in 40

mM NaOAc buffer (50 mL) was added par-

-galactosidase (5.7 U) from

D

-galactosidase from porcine

the preparation of compound 1 using b- -

D

-Gal-OC₆H₄NO₂-o (600

galactosidase from porcine liver. The fractions

(tubes 53–61) were collected, concentrated,

and lyophilized to afford compound 7 (81.5

mg).

D

tially puriﬁed b-

D

Compound 7 had [h]²_D⁵+181.9° (c 0.6, wa-

porcine liver. The reaction was terminated by

heating in a boiling-water bath for 5 min after

12 h of incubation at 40 °C. The reaction

mixture was treated as described for the

1

ter) and m/z 505 (M+H)⁺; H NMR: l 8.26

(d, 2 H, J 8.9 Hz, m-Ph), 7.30 (d, 2 H, J 8.9

Hz, o-Ph), 5.81 (d, 1 H, J_1,23.5 Hz, H-1), 4.42

(d, 1 H, J_1%,2%7.6 Hz, H-1%), and 2.09 (s, 3 H,

preparation of compound 1 using b- -galac-

D

13

tosidase from porcine liver. The fractions

(tubes 63–72) were combined, concentrated,

and lyophilized to afford compound 9 (65.3

mg). The NMR data for compound 9 were

identical to those of b-Gal-(16)-b-Gal-

OC₆H₄NO₂-p, reported respectively [20].

Me of Ac); C NMR: l 177.50 (CꢀO of Ac),

164.22 (Ph carbon attached to the phenolic

oxygen), 145.35 (p-Ph), 128.97 (m-Ph carbon),

119.66 (o-Ph carbon), 106.25 (C-1%), 98.58 (C-

1), 78.04 (C-5%), 75.67 (C-5), 75.09 (C-3%),

73.64 (C-2%, C-3), 72.31 (C-6), 71.57 (C-4),

71.03 (C-4%), 63.86 (C-6%), 56.14 (C-2), and

24.76 (Me of Ac).

(B) Using i-

lans ATCC 31382. To a soln of b-

OC₆H₄NO₂-p (280 mg) in 40 mM NaOAc

-galactosidase

D

-galactosidase from B. circu-

D

-Gal-

(B) Using i-

lans ATCC 31382. To a soln of b-

OC₆H₄NO₂-o (120 mg) and a- -GlcNAc-

OC₆H₄NO₂-p (44 mg) in 40 mM NaOAc

buffer (8 mL) was added b- -galactosidase

D-galactosidase from B. circu-

buffer (20 mL) was added b-

D

D-Gal-

(4.0 U) from B. circulans ATCC 31382. After

20 h of incubation at 50 °C, the reaction mix-

ture was treated as described for the prep-

D

aration of compounds 1 and 2 using b- -

D

(0.4 U) from B. circulans ATCC 31382. After

3.5 h of incubation at 50 °C, the reaction

mixture was terminated by heating in a boil-

ing-water bath for 5 min. The supernatant

from centrifugation was treated as described

for the preparation of compounds 1 and 2

galactosidase from B. circulans ATCC 31382.

For the separation on a Chromatorex-ODS

DM 1020T column (Fig. 5(A)), F-2 (tubes

18–24) was concentrated and lyophilized to

afford compound 9 (50.5 mg). The F-1 (tubes

9–17) fraction was concentrated and loaded

onto a Toyopearl HW-40S column treated as

already described (Fig. 5(B)). The F-1-1 (tubes

56–58), F-1-2 (tubes 60–64), F-1-3 (tubes 67–

71), and F-1-4 (tubes 77–82) fractions were

combined, concentrated, and lyophilized to

afford compounds 13 (b-Gal-1(6-b-Gal-1)₄-

6-b-Gal-OC₆H₄NO₂-p, 5.1 mg), 12 (b-Gal-

1(6-b-Gal-1)₃6-b-Gal-OC₆H₄NO₂-p, 15.1

mg), 11 (b-Gal-1(6-b-Gal-1)₂6-b-Gal-

OC₆H₄NO₂-p, 31.8 mg), and 10 (b-Gal-

(16)-b-Gal-(16)-b-Gal-OC₆H₄NO₂-p,

42.7 mg), respectively. The NMR data for

compound 10 were identical to those of b-Gal-

(16)-b-Gal-(16)-b-Gal-OC₆H₄NO₂-p, re-

ported respectively [20].

using b- -galactosidase from B. circulans

D

ATCC 31382. The F-1 (tubes 54–58) and F-2

(tubes 67–77) fractions were concentrated,

and lyophilized to afford compounds 7 (0.2

mg) and 8 (51.4 mg), respectively.

Compound 8 had [h]²_D⁵+178.7° (c 0.6, wa-

1

ter) and m/z 505 (M+H)⁺; H NMR: l 8.26

(d, 2 H, J 8.9 Hz, m-Ph), 7.30 (d, 2 H, J 8.9

Hz, o-Ph), 5.81 (d, 1 H, J_1,23.5 Hz, H-1), 4.42

(d, 1 H, J_1%,2%7.6 Hz, H-1%), and 2.09 (s, 3 H,

13

Me of Ac); C NMR: l 177.41 (CꢀO of Ac),

163.92 (Ph carbon attached to the phenolic

oxygen), 145.21 (p-Ph), 128.86 (m-Ph carbon),

119.35 (o-Ph carbon), 106.38 (C-1%), 98.49 (C-

1), 82.48 (C-3), 78.13 (C-5%), 75.58 (C-5), 75.36

(C-3%), 73.51 (C-2%), 71.43 (C-4), 71.07 (C-4%),

63.89 (C-6%), 63.05 (C-6), 54.97 (C-2), and

24.74 (Me of Ac).

Compound 11 had [h]²_D⁵−35.7° (c 0.5, wa-

1

ter) and m/z 788 (M+H)⁺; H NMR l 8.34

(d, 2 H, J 9.2 Hz, m-Ph), 7.38 (d, 2 H, J 9.2

Hz, o-Ph), 5.28 (d, 1 H, J_1,27.0 Hz, H-1), 4.51

(d, 1 H, J_1§,2§8.2 Hz, H-1§), and 4.48 (2 H, J

7.9 Hz, H-1%, H-1¦); ¹³C NMR l 164.83 (Ph

Preparation of i-Gal-(16)-i-Gal-OC₆-

H₄NO₂-p (9) and i-Gal-1(6-i-Gal-1)_n-

6-i-Gal-OC₆H₄NO₂-p (10–13)

X. Zeng et al. / Carbohydrate Research 325 (2000) 120–131

129

Fig. 5. Chromatorex-ODS DM 1020T (A) and Toyopearl HW-40S (B) chromatographic separation of transglycosylation product

with b- -Gal-OC₆H₄NO₂-p as substrate by use of b- -galactosidase from B. circulans ATCC 31382. Absorbance at 300 nm (ꢀ)

and absorbance at 485 nm ( ).

D

78.07, 77.39 and 76.67 (C-5, C-5%, C-5¦, C-5§,

C-5¨, C-5¨%), 75.70, 75.54, 75.51 and 75.24

(C-3, C-3%, C-3¦, C-3§, C-3¨, C-3¨%), 73.69,

73.64 and 73.30 (C-2, C-2%, C-2¦, C-2§, C-2¨,

C-2¨%), 72.33 (C-6, C-6%, C-6¦, C-6§, C-6¨),

71.57 (C-4, C-4%, C-4¦, C-4§, C-4¨, C-4¨%),

and 63.95 (C-6¨%).

carbon attached to the phenolic oxygen),

145.53 (p-Ph), 129.11 (m-Ph), 119.64 (o-Ph),

106.32 (C-1%, C-1¦, C-1§), 102.93 (C-1), 78.08,

77.38 and 76.66 (C-5, C-5%, C-5¦, C-5§), 75.69,

75.56, 75.51 and 75.24 (C-3, C-3%, C-3¦, C-3§),

73.67, 73.58 and 73.30 (C-2, C-2%, C-2¦, C-2§),

72.29 and 72.09 (C-6, C-6%, C-6¦), 71.54 and

71.46 (C-4, C-4%, C-4¦, C-4§), and 63.93 (C-

6§).

Preparation of i-Gal-(16)-h-Gal-OC₆H₄-

NO₂-p (14), i-Gal-(13)-h-Gal-OC₆H₄NO₂-

Compound 12 had [h]²_D⁵−25.8° (c 0.5, wa-

ter) and m/z 972 (M+Na)⁺; ¹H NMR: l 8.34

(d, 2 H, J 9.2 Hz, m-Ph), 7.38 (d, 2 H, J 9.2

Hz, o-Ph), 5.28 (d, 1 H, J_1,27.0 Hz, H-1), 4.52

(d, 1 H, J_1¨,2¨6.8 Hz, H-1¨), and 4.49 (3 H,

J 6.5 Hz, H-1%, H-1¦, H-1§); ¹³C NMR: l

164.85 (Ph carbon attached to the phenolic

oxygen), 145.55 (p-Ph), 129.11 (m-Ph), 119.66

(o-Ph), 106.32 (C-1%, C-1¦, C-1§, C-1¨),

102.93 (C-1), 78.07, 77.39 and 76.64 (C-5,

C-5%, C-5¦, C-5§, C-5¨), 75.70, 75.54, 75.51

and 75.24 (C-3, C-3%, C-3¦, C-3§, C-3¨),

73.69, 73.62 and 73.30 (C-2, C-2%, C-2¦, C-2§,

C-2¨), 72.31 and 72.13 (C-6, C-6%, C-6¦, C-

6§), 71.55 (C-4, C-4%, C-4¦, C-4§,C-4¨), and

63.93 (C-6¨).

p

(15), i-Gal-(16)-i-Gal-(16)-h-Gal-

OC₆H₄NO₂-p (16), i-Gal-(16)-i-Gal-

(16)-i-Gal-(16)-h-Gal-OC₆H₄NO₂-p (17)

(A) Using i- -galactosidase from porcine

li6er. To a soln of b- -Gal-OC₆H₄NO₂-o (180

mg) and a- -Gal-OC₆H₄NO₂-p (60 mg) in 40

mM NaOAc buffer (14 mL) was added par-

tially puriﬁed b- -galactosidase (2.0 U) from

D

porcine liver. The reaction was terminated by

heating in a boiling-water bath for 5 min after

12 h of incubation at 40 °C. The reaction

mixture was treated as described for the

preparation of compound 1 using b- -galac-

D

tosidase from porcine liver. The fractions

(tubes 64–74) were combined, concentrated,

and lyophilized to afford compound 14 (59

mg).

Compound 13 had m/z 1134 (M+Na)⁺;

¹H NMR: l 8.34 (d, 2 H, J 9.2 Hz, m-Ph),

7.38 (d, 2 H, J 9.2 Hz, o-Ph), 5.28 (d, 1 H, J_1,2

7.0 Hz, H-1), 4.50 (d, 1 H, J_1¨%,2¨%7.6 Hz,

H-1¨%), and 4.49 (4 H, J 7.6 Hz, H-1%, H-1¦,

1

Compound 14 had m/z 464 (M+H)⁺; H

NMR: l 8.31 (d, 2 H, J 9.2 Hz, m-Ph), 7.34

(d, 2 H, J 9.2 Hz, o-Ph), 5.93 (d, 1 H, J_1,23.0

13

Hz, H-1), 4.35 (d, 1 H, J_1%,2%7.8 Hz, H-1%); C

13

NMR: l 164.66 (Ph carbon attached to the

phenolic oxygen), 145.35 (p-Ph), 129.00 (m-

Ph), 119.98 (o-Ph), 105.89 (C-1%), 99.99 (C-1),

78.00 (C-5%), 75.65 (C-3%), 73.85 (C-2), 73.60

H-1§, H-1¨); C NMR: l 164.85 (Ph carbon

attached to the phenolic oxygen), 145.55 (p-

Ph), 129.13 (m-Ph), 119.66 (o-Ph), 106.34 (C-

1%, C-1¦, C-1§, C-1¨, C-5¨%), 102.95 (C-1),

130

X. Zeng et al. / Carbohydrate Research 325 (2000) 120–131

4.45 (d, 1 H, J_1¦,2¦7.2 Hz, H-1¦), and 4.40 (d,

(C-2%), 72.09 (C-3), 72.00 (C-6), 71.52 (C-5),

71.46 (C-4%), 70.71 (C-4), and 63.91 (C-6%).

13

1 H, J 6.8 Hz, H-1%); C NMR: l 164.71 (Ph

carbon attached to the phenolic oxygen),

145.37 (p-Ph), 129.04 (m-Ph), 120.07 (o-Ph),

106.29 and 106.02 (C-1%, C-1¦, C-1§), 100.11

(C-1), 78.08, 76.67 and 76.59 (C-5%, C-5¦, C-

5§), 75.70, 75.54 and 75.49 (C-3%, C-3¦, C-3§),

73.89 (C-2), 73.67 and 73.58 (C-2%, C-2¦, C-

2§), 72.06 (C-3), 71.99 (C-6, C-6%, C-6¦), 71.88

(C-5), 71.55 and 71.45 (C-4%, C-4¦, C-4§),

70.73 (C-4), and 63.93 (C-6§).

(B) Using i- -galactosidase from B. circu-

D

lans ATCC 31382. To a soln of b-

D

-Gal-

OC₆H₄NO₂-o (240 mg) and a-

D

OC₆H₄NO₂-p (80 mg) in 40 mM NaOAc

buffer (16 mL) was added b- -galactosidase

D

(2.2 U) from B. circulans ATCC 31382. After

5.5 h of incubation at 50 °C, the reaction

mixture was terminated by heating in a boil-

ing-water bath for 5 min. The reaction mix-

ture was treated as described for the

preparation of compound 1 using b- -galac-

D

tosidase from porcine liver. The F-1 (tubes

68–71), F-2 (tubes 72–77), F-3 (tubes 78–82),

F-4 (tubes 89–95), F-5 (tubes 98–103), and

F-6 (tubes 121–127) fractions were combined,

concentrated, and lyophilized to afford com-

pounds 17 (5.5 mg), b-Gal-(16)-b-Gal-(1

6)-b-Gal-OC₆H₄NO₂-p (18.7 mg, NMR data

not shown), 16 (7.1 mg), b-Gal-(16)-b-Gal-

OC₆H₄NO₂-p (84.7 mg, NMR data not

shown), 14 (28.1 mg) and 15 (11.5 mg),

respectively.

Acknowledgements

This work was supported by Grants-in-Aid

from the Ministry of Education, Science,

Sports, and Culture, Japan, by a research

grant for the Leading Research Utilizing Po-

tential of Regional Science and Technology

from the Science and Technology Agency,

Japan, and by a research grant from the Min-

istry of Agriculture, Forestry, and Fisheries,

Japan.

Compound 15 m/z 464 (M+H)⁺; ¹H

NMR: l 8.30 (d, 2 H, J 9.0 Hz, m-Ph), 7.34

(d, 2 H, J 9.0 Hz, o-Ph), 5.93 (d, 1 H, J_1,23.0

13

Hz, H-1), 4.35 (d, 1 H, J_1%,2%8.9 Hz, H-1%); C

References

NMR: l 164.38 (Ph carbon attached to the

phenolic oxygen), 145.26 (p-Ph), 128.95 (m-

Ph), 119.75 (o-Ph), 107.40 (C-1%), 99.71 (C-1),

82.01 (C-3), 78.04 (C-5%), 75.49 (C-3%), 74.81

(C-2), 74.05 (C-2%), 71.93 (C-5), 71.55 and

69.88 (C-4, C-4%), 63.93 and 63.82 (C-6, C-6%).

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Article Doi

Regioselective synthesis of p-nitrophenyl glycosides of β-D-galactopyranosyl-disaccharides by transglycosylation with β-D-galactosidases

DOI: 10.1016/S0008-6215(99)00303-1

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