Synthesis and enzymatic evaluation of modified acceptors of recombinant blood group A and B glycosyltransferases

www.elsevier.nl/locate/carres

Carbohydrate Research 326 (2000) 1–21

Synthesis and enzymatic evaluation of modiﬁed

acceptors of recombinant blood group A and B

glycosyltransferases

Ali Mukherjee, Monica M. Palcic, Ole Hindsgaul *

Department of Chemistry, Uni6ersity of Alberta, Edmonton, AB, Canada T6G 2G2

Received 26 August 1999; accepted 16 December 1999

Abstract

The disaccharide a-

L

-Fucp-(12)-b- -Galp-(1O)-Octyl (1) is an acceptor for the human blood group A and B

D

glycosyltransferases. Seven analogues of 1, containing deoxy, methoxy and arabino modiﬁcations of the Fuc residue,

were chemically synthesized and kinetically evaluated in radioactive enzymatic assays. Both the enzymes tolerate

modiﬁcation of the 3%-OH on the fucose residue. The 2%-OH was found to be key to the recognition of the acceptors

by these enzymes. The arabino derivative was recognized as an acceptor by the A transferase (K_mof 200 mM), but

not the B transferase and is the ﬁrst synthetic acceptor capable of distinguishing between the two enzyme activities.

Keywords: Glycosyltransferase; Human blood group; Synthesis; Enzyme; Acceptor, enzyme

1. Introduction

to take a closer look at some of the glycosyl-

transferases that synthesize these structures in

vivo, viz., the blood group A and B glycosyl-

transferases, respectively, the a-(13)-N-

acetylgalactosaminyl and the a-(13)-

galactosyltransferase. In recent years, the

genes coding for these enzymes have been

cloned using cDNA libraries. These studies

have shown that the number of amino acids is

the same in both the enzymes, and they differ

in their amino acid sequences at only four

positions in the catalytic domain of the

proteins [5–7]. In vivo, the B transferase

transfers Gal from UDP-Gal, and the A trans-

ferase uses UDP-GalNAc as the donor to

form the B and the A antigens, respectively.

These carbohydrate structures are found on

the surfaces of erythrocytes and other cells

and in soluble form in the cytoplasm [8]. The

The blood group A and B glycosyltrans-

ferases are two enzymes responsible for the

biosynthetic formation of the human blood

group A and B antigens from the common

acceptor, the blood group O(H) antigen. They

are morphologically typical of the glycosyl-

transferase family of enzymes as they possess

a cytoplasmic tail consisting of 16 amino

acids, a trans-membrane region with 21 amino

acids, a stem region and a large C-terminal

catalytic domain consisting of 316 amino acids

located inside the Golgi apparatus [1–4]. The

great interest generated in understanding the

involvement of blood group antigens in the

invasion and metastasis of tumor cells led us

disaccharide, a-

L

-Fucp-(12)-b-

D

-Galp-OR,

* Corresponding author. Tel.: +1-780-4923254; fax: +1-

780-4928231.

serves as the common acceptor for both the

PII: S0008-6215(00)00003-3

2

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

thesized modiﬁed acceptors to probe the

enzyme active site, an approach that has been

used frequently in our group [14–18]. The

systematic modiﬁcation on the galactose moi-

ety had already been described as part of the

initial work on these enzymes [10,11]. For the

present project it was decided that the hy-

droxyl groups on the Fuc residue would be

replaced with H and O-methyl groups, and

the C-6% methyl on the Fuc residue would be

replaced by H.

enzymes, R being a glycoprotein or a glyco-

lipid in vivo. Various alkyl groups can be used

as the aglycon without loss of acceptor activ-

ity [9]. Prior to this study, it was not known

whether these two enzymes differed in their

acceptor speciﬁcity, although it had been

shown that the B transferase is in general less

tolerant of changes in the acceptor structure

[10,11].

The blood group A and B glycosyltrans-

ferases represent an ideal system for the study

of a glycosyltransferase active site and the

design and synthesis of speciﬁc glycosyltrans-

ferase inhibitors since they differ in their se-

quence by only four amino acids and because

they have been cloned and expressed in E. coli

[12,13], giving us access to substantial

amounts of these enzymes. To identify the

sites of molecular recognition on the protein

in the active site, the hydroxyl groups on the

acceptor and/or the donor structure can be

modiﬁed to see which part of these com-

pounds is required in the recognition process.

In order to gain a detailed understanding of

the interactions between the acceptor sub-

strate and the enzyme, we designed and syn-

This report describes the synthesis of some

analogues of the minimum acceptor structure

recognized by these two enzymes, a-

L

-Fucp-

(12)-b- -Galp-O-Octyl (1) and their enzy-

D

matic evaluation. Several acceptor analogues

were synthesized by modifying the hydroxyl

groups on the fucose residue (Fig. 1) and

assayed with these enzymes in order to deter-

mine the functional group speciﬁcities, i.e., the

steric and hydrogen-bonding requirements in

the active site of the enzymes. Deoxy ana-

logues of compound 1 were prepared to assess

the H-bonding interactions between the hy-

droxyl groups on the Fuc moiety and polar

groups in the active site. Moreover, a hydro-

gen is non-polar and the smallest group that

can replace a hydroxyl [19,20]. Methoxy

groups can be H-bond acceptors but not

donors and they also probe the steric require-

ments of the active site. Additionally, they are

more hydrophobic than the native hydroxyl

group [21]. The arabino analogue was pre-

pared to determine the role of the 6%-methyl

group on the Fuc residue. The octyl aglycon

facilitates the puriﬁcation of these compounds

by reverse-phase C₁₈cartridges. It also permit-

ted the use of radioactive Sep-Pak assays [22]

of these synthetic analogues with the glycosyl-

transferases being studied. The biochemical

evaluations of these compounds are discussed

and an analysis of the results obtained is

presented.

Fig. 1. Some modiﬁed disaccharides synthesized for this

study.

1

2

3

4

5

6

7

8

R¹=R²=R³=OH, R⁴=CH₃(native)

R¹=R²=OH, R³=OMe, R⁴=CH₃(4%-methoxy)

R¹=R²=OH, R³=H, R⁴=CH₃(4%-deoxy)

R¹=R³=OH, R²=OMe, R⁴=CH₃(3%-methoxy)

R¹=R³=OH, R²=H, R⁴=CH₃(3%-deoxy)

R²=R³=OH, R¹=OMe, R⁴=CH₃(2%-methoxy)

R²=R³=OH, R¹=H, R⁴=CH₃(2%-deoxy)

R¹=R²=R³=OH, R⁴=H (arabino)

2. Results and discussion

Chemical synthesis of the disaccharide ana-

logues.—The monosaccharide building blocks

were synthesized using established protection–

deprotection strategies. In order to obtain the

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

3

Compound 20 was deacetylated with sodium

methoxide in methanol and benzylated with

benzyl bromide to give compound 22 in an

overall yield of 72%. As shown in Scheme 2,

the orthoester was converted to 2-O-acetyl-

required disaccharides modiﬁed at the 2-, 3-,

and 4-positions of the Fuc residue, an octyl

b- -galactopyranoside (25) protected at the

D

3-, 4-, and 6-positions was coupled with suit-

ably protected Fuc and Ara derivatives using

halide ion-catalyzed glycosylation or thiogly-

coside activation with dimethylmethylthiosul-

fonium triﬂate (DMTST) [23,24]. Under these

conditions, formation of b-fucosides was not

observed. Instead of using individually O-

methylated and deoxygenated fucose donors,

temporary protecting groups that can be re-

moved after glycosylation were used. Methyla-

tion and deoxygenation were performed on

the disaccharides, followed by deprotection to

obtain the target compounds. Retrosyntheti-

cally, fucose with the 3-OH or the 4-OH free

can be obtained regioselectively from a com-

mon precursor, in a single step, and this route

was chosen instead of preparing them individ-

ually by two separate chemical manipulations.

Phase-transfer catalysis was utilized in this

step, and the precursor was obtained by the

hydrolysis of the isopropylidene group on the

2-O-benzyl derivative of fucose. The free hy-

droxyl groups were then protected prior to

glycosylation.

3,4,6-tri-O-benzyl-a- -galactopyranosyl bro-

D

mide (23) with tetrabutylammonium bromide

and acetyl bromide [27–31]. Compound 23

was glycosylated with octanol under Helferich

glycosylation conditions to give compound 24

(70% over two steps), followed by deacetyla-

tion with sodium methoxide in methanol to

give 25 (97%).

Retrosynthetic analysis of the 2%-hydroxyl-

modiﬁed disaccharides leads to a fucose pre-

cursor with a non-participating protecting

group at the 2-OH that would survive glycosyl-

ation conditions, and can be removed in the

presence of a glycosidic bond and the other

protecting groups. The p-methoxybenzyl

group was considered suitable for this pur-

pose. Ethyl 3,4-di-O-isopropylidene-1-thio-b-

D-galactopyranoside (10) was alkylated with

p-methoxybenzyl chloride [34] to give com-

pound 16 (89%), which was then hydrolyzed

to remove the isopropylidene group and ben-

zylated to obtain compound 18 in 55%

(Scheme 1).

To prepare the donors, ethyl 1-thio-fucopyr-

anoside was treated with 2,2-dimethoxy

propane and a catalytic amount of camphor-

sulfonic acid to give compound 10 (88%). It

was alkylated with benzyl bromide (93%) and

hydrolyzed [32] to give compound 11 (84%).

To prepare both the analogues in one step,

compound 11 was mono-benzylated under

phase-transfer catalysis using a quaternary

ammonium salt [33], which gave both the

3-O-benzyl (12) and 4-O-benzyl (13) com-

pounds (88%).

The two compounds were acetylated (in

90% yield) to produce the donors and to

ascertain the identity of these compounds

(Scheme 1). Based on the downﬁeld shift of

H-3% in compound 15 and H-4% in compound

14 in their proton NMR spectra, their struc-

tures were unambiguously assigned.

Alcohol 25 was coupled with compound 14

to give compound 26 (71%). Compound 27

was obtained by deacetylation of 26 in sodium

methoxide in methanol (95%). Compound 27

was methylated with iodomethane to give

compound 28 (76%). Compound 28 was hy-

drogenolyzed to give compound 2 in 91%

yield (Scheme 3).

Barton’s radical deoxygenation method

[35,36] was used to obtain all the deoxy

derivatives. The precusor alcohols were ﬁrst

converted to the thiocarbonyl derivatives us-

ing pentaﬂuorophenyl chlorothionoformate

with pyridine and dimethyl aminopyridine in

dichloromethane. The thiocarbonates were

then treated with tributyltin hydride and azo-

bisisobutyronitrile (AIBN) to obtain the de-

oxygenated compounds. Compound 27 was

converted into the pentaﬂuorophenylchloro-

thiono derivative (29) in 98% yield and deoxy-

genated with tributyltin hydride [37,38] to give

compound 30 (66%). Debenzylation of this

compound gave the disaccharide 3 in 90%

(Scheme 3).

The synthesis of the acceptor 25 began with

galactose pentaacetate, which was ﬁrst con-

verted to galactosyl bromide 19 with hydrogen

bromide in acetic acid and then to the or-

thoester 20 with methanol and lutidine [25,26].

4

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

Scheme 1. Synthesis of the modiﬁed fucosyl donors. Reagents: (a) NaOMe, MeOH. (b) 2,2-Dimethoxypropane (88%). (c) BnBr,

NaH, DMF (93%). (d) 70% AcOH, 70 °C (84%). (e) 10% NaOH solution, BnBr, Bu₄NBr (88% over two steps). (f) Ac₂O, pyridine

(89%). )g) p-MeOC₆H₄CH₂Cl, NaH, DMF (89%). (h) 70% AcOH, 70 °C. (i) BnBr, NaH, DMF (55% over two steps).

Disaccharide 31 was similarly obtained by

coupling alcohol 25 with donor 15 with

DMTST (71%). Compound 32 was obtained

by Zemple´n deacetylation of 31 in sodium

methoxide in methanol (92%). Compound 32

was methylated with iodomethane to give

compound 33 (86%). It was debenzylated with

hydrogen and palladium-on-charcoal to give

the disaccharide 4 in 89% yield (Scheme 4).

Disaccharide 35 was obtained by converting

compound 32 to 34 with pentaﬂurophenyl

chlorothionoformate and reducing it with tri-

butyltin hydride and AIBN (76%). This was

followed by hydrogenation to give compound

5 in 94% yield (Scheme 4).

Compounds 25 and 18 were coupled by

activating the thioglycoside donor (18) with

DMTST to yield the disaccharide 36 (67%).

Compound 37 was obtained from compound

36 by removing the p-methoxybenzyl group

with ceric ammonium nitrate (70%) [39]. The

hydroxyl group was methylated using

iodomethane and sodium hydride to give the

disaccharide 38 (86%). Compound 38 was hy-

drogenated to give the target compound 6 in

89% yield (Scheme 5).

Compound 37 was acylated with penta-

ﬂuorophenyl chlorothionoformate to give

compound 39. Barton deoxygenation yielded

the protected disaccharide 40 in 61% over two

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

5

steps, which was then debenzylated by hy-

drogenolysis to give compound 7 in 84%

(Scheme 5).

quenching the reaction, the mixtures were

then loaded onto a C₁₈Sep-Pak cartridge that

had been washed with methanol and water.

The unreacted donor was washed away with

water, and the products were eluted with

methanol. The radioactivity of the eluant was

determined in a liquid scintillation counter.

Percentage activity was calculated as the num-

ber of decays per minute (dpm) observed for

each compound relative to the native accep-

tor. The potential inhibitor was similarly

treated after incubating with the enzyme, the

corresponding donor and the native acceptor.

Percentage inhibition was determined as the

relative loss of activity of the native acceptor

in the presence of the inhibitor.

E6aluation of the analogues as potential sub-

strates.—Preliminary screening, the results of

which are shown in Table 1, shows that the A

transferase can recognize all the disaccharides

as acceptors to various extents except the 2%-

methoxy derivative 6. All the analogues tested

were less active than the native disaccharide

except the 3%-methoxy derivative. For the B

transferase, all the modiﬁcations caused loss

of activity to some extent. The 2%-hydroxyl

group is crucial to the recognition of the

acceptor by the A enzyme. When it is replaced

2,3,4-Tri-O-benzyl- -arabinopyranose (41)

D

[40] was converted into the corresponding ara-

binopyranosyl bromide 42 with oxalyl bro-

mide and DMF, and this compound was

directly coupled with the alcohol 25 under

halide ion catalysis [41,42] to give compound

43 in 78% yield. Only b-arabinoside was ob-

served to be formed in this reaction. Com-

pound 43 was hydrogenolyzed to give

compound 8 in 91% yield (Scheme 6).

Enzymatic assays of the disaccharide ana-

logues.—The modiﬁed disaccharides (2–8)

were screened in radioactive Sep-Pak assays to

determine their activities as potential accep-

tors relative to the natural acceptor 1 with the

recombinant blood group A and B glycosyl-

transferases. Compounds that were not accep-

tors were assayed as inhibitors. The radio-

active assay technique used for this purpose

quantitates the rate of transfer of radiolabeled

Gal or GalNAc from the corresponding sugar

nucleotides to the octyl glycosides. The poten-

tial acceptors were incubated with the A or

the B enzyme and UDP-GalNAc or UDP-

Gal, respectively, in a suitable buffer. After

Scheme 2. Synthesis of the acceptor alcohol. Reagents: (a) MeOH, 2,6-lutidine, Bu₄NBr. (b) NaOMe, MeOH. (c) BnBr, NaH,

DMF (72% over three steps). (d) Et₄NBr,CH₂Cl₂, CH₃COBr. (e) n-Octanol, Hg(CN)₂, CH₃CN, (70% over two steps). (f) NaOMe,

MeOH (97%).

6

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

Scheme 3. Synthesis the 4%-hydroxyl-modiﬁed analogues of 1. Reagents: (a) DMTST, DTBMP (71%). b) NaOMe, MeOH (95%).

(c) MeI, NaH, DMF (76%). (d) H₂/Pd(OH)₂, MeOH (91%). (e) Pentaﬂurophenyl chlorothionoformate, DMAP, CH₂Cl₂(98%). (f)

AIBN, Bu₃SnH, reﬂuxing toluene (66%). (g) H₂/Pd(OH)₂, MeOH (90%).

by the methoxy group, recognition is lost.

This could be in part due to the steric bulk of

the methoxy group at the 2-position, which is

close to the active site of the enzyme and the

position of transfer of the donor. The lack of

reactivity can also be explained in terms of

hydrogen bonding, since replacing the OH

with an OMe renders it a H-bond acceptor

but not a donor.

of about two-thirds of that of the natural

acceptor, since it is no longer a H-bond

donor, but it does not hinder the transfer of

the donor, probably due to its minimal steric

demand. The A transferase also tolerates

modiﬁcations on the 3- and the 4-hydroxyl

groups of fucose. The 3%-methoxy, 3%-deoxy,

and 4%-methoxy compounds have relative rates

that are quite close to that of the native

acceptor. This probably indicates that the 3-

The 2%-deoxy compound has a relative rate

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

7

and 4-hydroxyl groups of fucose are not close

enough to the active site to be able to perturb

it in any signiﬁcant way.

relative rate of half that of compound 1,

which is lower than that of the methoxy

derivative. This could indicate that 4%-OH is

also a hydrogen-bond acceptor. The arabino

analogue is an acceptor for the A transferase

with a high relative rate; thus, the methyl

group on the fucose is not critical for recogni-

tion by the A transferase.

The 3%-OH is probably not a key polar

group since both the methoxy and deoxy ana-

logues are good acceptors. It might be a

hydrogen-bond acceptor, since the deoxy com-

pound loses some activity compared with the

methoxy, but its contribution to the stability

of the enzyme substrate complex is unlikely to

be signiﬁcant. The 4%-deoxy compound has a

As observed before for the galactose modiﬁ-

cations, the B transferase is less amenable to

modiﬁcations of the hydroxyl groups on fu-

Scheme 4. Synthesis of the 3%-hydroxyl-modiﬁed analogues of 1. Reagents: (a) DMTST, DTBMP (71%). (b) NaOMe, MeOH

(92%). (c) MeI, NaH, DMF (86%). (d) H₂/Pd(OH)₂, MeOH (89%). (e) Pentaﬂurophenyl chlorothionoformate, DMAP, CH₂Cl₂

(98%). (f) AIBN, Bu₃SnH, reﬂuxing toluene (76%). (g) H₂/Pd(OH)₂, MeOH (94%).

8

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

Scheme 5. Synthesis of the 2%-hydroxyl-modiﬁed analogues of 1. Reagents: (a) DMTST, DTBMP (67%). (b) Ceric ammoniumni-

trate, 9:1 CH₃CN–H₂O (70%). (c) MeI, NaH, DMF (86%). (d) H₂/Pd(OH)₂, MeOH (89%). (e) Pentaﬂurophenyl chlorothionofor-

mate, DMAP, CH₂Cl₂(93%). (f) AIBN, Bu₃SnH, reﬂuxing toluene (61%). (g) H₂/Pd(OH)₂, MeOH (84%).

cose as well. The 2%-OH is again crucial for

recognition, and recognition of the acceptor is

lost when either modiﬁcation is made. Thus, it

seems likely that apart from strict steric re-

quirements for the groups that are tolerated at

this position, the 2%-OH is likely to be both a

hydrogen-bond acceptor and donor. The 3%-

OH is again the least signiﬁcantly involved in

steric interactions in the active site, and since

the 3%-methoxy is a good acceptor for the B

enzyme, the 3%-OH is probably a hydrogen-

bond acceptor in the active site. No other

analogues are acceptors at all or marginally

so. The 4%-OH is also probably involved in

hydrogen-bonding interactions in the active

site. The 6%-methyl group is required for the

acceptor to bind to the B transferase. Thus,

although the arabino derivative is an acceptor

for the A enzyme, it is not recognized by the B

enzyme as an acceptor.

The binding constants of the synthetic ana-

logues most often parallel the percentage ac-

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

9

Scheme 6. Synthesis of 8, the arabino analogue of 1. Reagents: (a) oxalyl bromide, DMF, CH₂Cl₂. (b) Et₄NBr, CH₂Cl₂, DMF,

,

4 A molecular sieves (76%). (c) H₂/Pd(OH)₂, MeOH (91%).

tivities of these compounds with the exception

of the 4%-methoxy and the 4%-deoxy analogues,

which have K_mvalues of about 1140 and 700

mM, respectively, with the A transferase (Table

2). The K_mvalues of the other analogues range

from 20 to 200 mM, which is about 2–20 fold

higher than the native acceptor. Not surpris-

ingly, the 3%-methoxy analogue has the lowest

K_mvalue of all the modiﬁed acceptors. It is

not clear, however, why the 4%-methoxy com-

pound is required at such a high concentration

for it to bind to the enzyme.

The K_mvalue for the arabino compound 8 is

about 208 mM, with a relative rate that is

about 2.5 times higher than the native disac-

charide. The rates of the reactions in all cases

are comparable with that of the natural accep-

tor. The 4%-methoxy compound has the highest

reaction velocity among all the synthesized

analogues, while the 3%-deoxy compound re-

acts at the slowest rate, about 2.5 and 0.6

times of compound 1, respectively.

other compounds could not be determined

since they were insoluble at the high concen-

trations that were required for the assays.

E6aluation of the analogues as potential in-

hibitors.—Since the 2%-methoxy derivative was

concluded not to be an acceptor for either the

A or the B transferase, it was assayed as an

inhibitor for the two enzymes. The B enzyme

failed to recognize it as an inhibitor. The

compound showed a calculated K_ivalue of

about 1 mM for the A transferase.

Table 1

Relative acceptor activities of disaccharides 1–8 towards

cloned blood group A and B glycosyltransferases ^a,b

Substrate

A transferase

(% activity)

B transferase

(% activity)

2%-Methoxy (6)

2%-Deoxy (7)

3%-Methoxy (4)

3%-Deoxy (5)

4%-Methoxy (2)

4%-Deoxy (3)

Arabino- (8)

4

66

108

84

86

57

1

6

78

27

11

5

The 3%-methoxy and the 3%-deoxy derivatives

were the only acceptors for the B transferase

with K_mvalues of about 200 and 400 mM,

respectively. The relative rates of the three

modiﬁed acceptors on which kinetic studies

could be performed were higher than that of

the native disaccharide. The K_mvalues of the

75

5

Native disaccharide (1) 100

100

^aUDP-N-acetylgalactosamine and UDP-galactose were

used as donors for the A and the B enzymes, respectively.

^bBoth donors were used at a ﬁxed (saturation) concentra-

tions.

10

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

Table 2

Calculated kinetic constants for the modiﬁed acceptors with blood group A and B glycosyltransferases

Substrate

A transferase

B transferase

K_m(mM)

V_max(nmol/min/mg)

K_m(mM)

V_max(nmol/min/mg)

2%-Deoxy (7)

3%-Methoxy (4)

3%-Deoxy (5)

4%-Methoxy (2)

4%-Deoxy (3)

Arabino (8)

170923

2093

84913

11409182

7229115

20896

3.590.17

3.990.4

200925

400952

40009640

8.190.9

1.790.06

2.2990.16

1.890.09

7.790.07

2.690.13

7.490.11

2.990.08

Native disaccharide (1)

12.991.2

110912

0.2490.02

3. Experimental

None of the modiﬁcations made on the

disaccharide resulted in the loss of the accep-

tor property, that is, the 3-OH remained ac-

tive. Therefore, the loss of or modiﬁcation in

the activities of the analogues was a result of

the perturbation of the interactions around

the site of transfer in the active site. Molecular

modeling of the native disaccharide shows

that the 2%-OH is in close proximity to the

3-OH group [9]; thus, it has the most signiﬁ-

cant effect on the transfer reaction.

To summarize the results, the modiﬁcations

on the Fuc residue show that an intact 2%-OH

is necessary for recognition by the enzymes,

particularly by the B enzyme. Modiﬁcations

on the 3%-OH are tolerated by both enzymes,

more so by the A enzyme. 4%-OH and C-6% are

crucial for recognition by B, but modiﬁcations

are tolerated well by the A enzyme. Thus, a

fair amount of ﬂexibility was observed in

recognition of the Fuc residue by the two

enzymes. Results obtained from previous

work on the acceptor speciﬁcity of these en-

zymes [10,11] showed that the 4-hydroxyl

group is essential for binding, while modiﬁca-

tions on the 3- and 6-hydroxyl groups are

tolerated by both enzymes without loss of

recognition. It is likely, therefore, that the

galactosyl 4-hydroxyl and the fucosyl 2-hy-

droxyl are the key polar groups involved in

the recognition of the acceptor by these en-

zymes (Fig. 2). In addition, the two enzymes

recognized the arabino and the 4%-deoxy com-

pounds differentially as acceptors. Therefore,

the acceptor speciﬁcity of the enzymes was

shown to be determined by their amino acid

sequence.

General methods.—Analytical thin-layer

chromatography (TLC) was performed on Sil-

ica Gel 60-F₂₅₄(E. Merck, Darmstadt) with

detection by quenching of ﬂuorescence (aro-

matic compounds), charring with 5% H₂SO₄

or ninhydrin. Unless otherwise noted, column

chromatography was performed on Silica Gel

60 (E. Merck). Iatrobeads were from Iatron

Laboratories Inc. (Japan). Millex-GV (0.22

mm) ﬁlters were from Millipore (Missisauga,

ON), C₁₈Sep-Pak (reversed-phase) cartridges

were from Waters Associates (Missisauga,

ON). All commercial reagents were used as

supplied, and all chromatography solvents

were distilled prior to use. Ecolite scintillation

cocktail was from ICN Radiochemicals (St.

Louis, MO) UDP-[6-³H]-Gal (speciﬁc activity

1 Ci/mmol) and UDP-[6-³H]-GalNAc (speciﬁc

Fig. 2. Recognition of the acceptor a-

L

-Fucp-(12)-b-D-

Galp-1-O-Octyl (1) by the blood group A and B glycosyl-

transferases, key polar groups shown. (Squares indicate

hydroxyl groups essential for recognition by both the en-

zymes. Circles indicate key polar groups for the B trans-

ferase.)

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

11

characterization to give the required 14 and

15, respectively. Data for compound 14: [h]_D

activity 1 Ci/mmol) were from American Ra-

diolabelled Chemicals (St. Louis, MO). UDP-

Gal and UDP-GalNac were from Sigma

Chemical Co. Optical rotation measurements

were done on a Perkin–Elmer 241 polarimeter

1

−28.8° (c 0.4 CHCl₃). H NMR (CDCl₃): l

7.2–7.4 (m, 10 H, ArCH), 5.38 (d, 1 H, J_3,4

2.5 Hz, H-4) 4.81, 4.75, 4.72, 4.50 (d, 1 H, J_gem

11.5 Hz, PhCH₂), 4.42 (d, 1 H, J_1,29.0 Hz,

H-1), 3.64 (q, 1 H, J_5,66.5 Hz, H-5), 3.53–3.61

(m, 2 H, H-2, H-3), 2.7–2.8 (m, 2 H, SCH₂),

2.19 (s, 3 H, CH₃CO), 1.30 (t, 3 H, J_vic7.5 Hz,

1

at 2292 °C. H NMR spectra were recorded

at 360 megahertz (MHz) on a Bruker WM-

360, 500 MHz (Varian unity 500) or 300 MHz

(Varian i300) in solutions of CDCl₃(proton

chemical shifts referenced to residual proton

signal of chloroform at l 7.26), CD₃OD

(residual proton signal of CD₂HOD at l 3.30)

or D₂O (DOH at l 4.85 at 22 °C or referenced

to external acetone at l 2.225). ¹³C NMR

spectra were recorded at 75 MHz (Bruker AM

300), or 125 MHz (Varian unity 500) in

CDCl₃(l 77.07), CD₃OD (l 49.0) or D₂O

(external acetone at l 31.07). Fast-atom bom-

bardment (FAB) mass spectra were recorded

on a Kratos AEIMS9 instrument with Xe as

the bombarding gas and glycerol as the ma-

trix. Electrospray-ionization mass spectra

(performed either in positive- or negative-ion

mode) were recorded on a Zabspec Hybrid

Sector–Time of Flight instrument from

Micromass (Manchester, UK). For exact mass

measurements, the spectra were obtained by

voltage scan over a narrow mass range. Data

acquisition and processing was done using the

OPUS software package on a Digital Alpha

station with VMS operating system. Elemental

analyses were performed on a Carlo Erba

EA1108 instrument.

13

SCH₂CH₃), 1.20 (d, 3 H, J_5,66.5 Hz, H-6); C

NMR (CDCl₃): l 170.9 (COCH₃), 138.3,

137.8 (ArC), 128.4, 128.3, 128.1, 128.0

(ArCH), 85.0 (C-1), 81.5, 75.8, 73.0, 71.8, 69.9

(C-3, C-4, C-5, CH₂O), 24.9, 21.0 (CH₃CO),

16.7 (C-6), 15.06 (SCH₂CH₃). Anal. Calcd for

C₂₄H₃₀O₅S (430.56): C, 66.95; H, 7.02. Found:

C, 67.33; H, 6.92. ESIMS for C₂₄H₃₀O₅SNa:

Calcd 453.1711. Found 453.1718.

Data for compound 15: [h]_D−42.4° (c 0.4

1

CHCl₃). H NMR (CDCl₃): l 7.20–7.38 (m,

10 H, ArCH), 4.89 (dd, 1 H, J_2,39.8, J_3,43.0

Hz, H-3) 4.87 (d, 1 H, J_gem11.5 Hz, PhCH₂),

4.57–4.67 (m, 3 H, PhCH₂×3), 4.43 (d, 1 H,

J_1,29.8 Hz, H-1), 3.79 (t, 1 H, J 9.8 Hz, H-2),

4.89 (dd, 1 H, J_3,43.0, J_4,50.8 Hz, H-3), 3.60

(dq, 1 H, J_5,66.5, J_4,50.8 Hz, H-5), 2.7–2.8

(m, 2 H, SCH₂), 1.89 (s, 3 H, CH₃CO), 1.38 (t,

3 H, J_vic7.5 Hz, SCH₂CH₃), 1.22 (d, 3 H, J_5,6

6.5 Hz, H-6); ¹³C NMR (CDCl₃): l 170.3

(COCH₃), 138.1, 138.0 (ArC), 128.1, 128.1,

128.1, 129.1, 128.0, 127.9, 127.9, 127.8, 127.8,

127.67 (ArCH), 84.8 (C-1), 77.4, 76.4, 76.2,

75.2, 74.1, (C-3, C-4, C-5, CH₂O), 24.7, 21.7

(CH₃CO), 16.7 (C-6), 14.8 (SCH₂CH₃). Anal.

Calcd for C₂₄H₃₀O₅S (430.56): C, 66.95;

H, 7.02. Found: C, 66.99; H, 7.02. ESIMS

for C₂₄H₃₀O₅SNa: Calcd 453.171166. Found

453.171697.

Ethyl 4-O-acetyl-2,3-di-O-benzyl-1-thio-h-

fucopyranoside (14) and ethyl 3-O-acetyl-2,3-

di-O-benzyl-1-thio-h- -fucopyranoside (15).—

Ethyl 2-O-benzyl-1-thio-a- -fucopyranoside

L

-

L

(1.1 g, 3.35 mmol) was dissolved in 60 mL of

CH₂Cl₂. A 5% soln of aq NaOH (5 mL), BnBr

(0.63 mL, 50 mmol) and Bu₄NHSO₄(0.23 g,

0.67 mmol) were added and the mixture was

stirred at room temperature (rt) for 1 day.

Additional BnBr (0.45 mL) was added, and

stirring was continued for another 24 h. After

extraction with CH₂Cl₂and washing the ex-

tract with water, drying and evaporation of

the solvent yielded a syrup that was chro-

matographed (15:1, 10:1, 5:1, 2:1 pentane–

EtOAc) to yield compounds 12 (500 mg) and

13 (600 mg) in a combined yield of 88%. Each

of the two compounds was acetylated without

Ethyl 3,4-O-isopropylidene-2-O-p-methoxy-

benzyl-1-thio-i-

3,4 - O - isopropylidene - 1 - thio - b -

L

-fucopyranoside (16).—Ethyl

- fucopyr-

L

anoside (300 mg, 1.2 mmol) was dissolved in

DMF (4 mL), 0.115 g of 50% NaH was

added, and the mixture was stirred in a cold

water bath for 10 min. p-Methoxybenzyl chlo-

ride (0.325 mL) was added slowly and stirred

for another 3 h. After completion of the reac-

tion, excess NaH was destroyed with MeOH.

The reaction mixture was extracted with

CH₂Cl₂and washed with water and dried

(Na₂SO₄), and concentrated in vacuo. The

12

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

was added slowly, and stirring was continued

for another 3 h at rt. The reaction mixture

was then cooled in an ice-bath and MeOH

was added. The mixture was diluted with

CH₂Cl₂and washed with water. The organic

layer was dried (Na₂SO₄) and evaporated in

vacuo to obtain a syrup that was chro-

matographed with (15:1 pentane–ethylacetate)

to obtain compound 18 (200 mg, 55% from

residue was chromatographed (10:1, 7:1, 5:1

pentane–EtOAc) and 16 was obtained (387

1

mg, 89%). [h]_D−42.4° (c 0.4 CHCl₃). H

NMR (CDCl₃): l 7.35 (d, 2 H, J_ortho9.0 Hz,

ArCH), 6.85 (d, 2 H, J_ortho9.0 Hz, ArCH),

4.76 (d, 1 H, J_gem11.0 Hz, PhCH₂), 4.67 (d, 1

H, J_gem11.0 Hz, PhCH₂), 4.34 (d, 1 H, J_1,29.5

Hz, H-1), 4.15 (dd, 1 H, J_2,36.5, J_3,45.5 Hz,

H-3), 4.01 (dd, 1 H, J_3,45.5, J_4,52.0 Hz, H-4),

3.78 (dq, 1 H, J_5,66.5, J_4,52.0 Hz, H-5), 3.76

(s, 3 H, CH₃O), 3.39 (dd, 1 H, J_1,29.5, J_2,36.5

Hz, H-2), 2.60–2.78 (m, 2 H, SCH₂), 1.38,

1.35 (s, 3 H, CH₃C), 1.34 (d, 3 H, J_5,66.5 Hz,

1

16). [h]_D−1.5° (c 0.4 CHCl₃). H NMR

(CDCl₃): l 7.25–7.45 (m, 12 H, ArCH), 6.86

(d, 2 H, ArCH), 5.00, 4.82, 4.79, 4.47, 4.73,

4.70 (d, 1 H, J_gem11.5 Hz, PhCH₂), 4.38 (d, 1

H, J_1,29.5 Hz, H-1), 3.81 (t, 1 H, J 9.5 Hz,

H-2), 3.80 (s, 3 H, CH₃O), 3.61 (dd, 1 H, J_3,4

2.5, J_4,50.9 Hz, H-4), 3.57 (dd, 1 H, J_2,39.5,

J_3,42.5 Hz, H-3), 3.47 (dq, 1 H, J_5,66.5, J_4,5

0.9 Hz, H-5), 2.82–2.65 (m, 2 H, SCH₂), 1.35

(t, 3 H, J_vic7.5 Hz, SCH₂CH₃), 1.20 (d, 3 H,

13

H-6), 1.27 (t, 3 H, J_vic7.5 Hz, SCH₂CH₃); C

NMR (CDCl₃): l 159.2 (ArCOCH₃), 130.1

(ArC), 129.9, 113.6 (ArCH), 109.5 (Me₂C)

83.4 (C-1), 79.8, 78.6, 76.5, 73.1, 72.4, (C-3,

C-4, C-5, CH₂O), 55.2 (OCH₃) 28.0, 26.4,

24.4, 16.8 (C-6), 14.8 (SCH₂CH₃). ESIMS for

C₁₉H₂₈O₅SNa: Calcd 391.1555. Found

391.1555.

13

J_5,66.5 Hz, H-6); C NMR (CDCl₃): l 158.9

(ArCOCH₃), 138.4, 138.2, 132.9, 130.3 (ArC),

129.6, 129.1, 128.0, 127.9, 127.8, 127.2, 127.1,

126.5 (ArCH), 84.6 (C-1), 84.1, 77.7, 76.3,

74.9, 74.2, 74.1, 72.4, (C-2, C-3, C-4, C-5,

CH₂O), 54.8 (OCH₃), 24.2, 16.9 (C-6), 14.7

(SCH₂CH₃). Anal. Calcd for C₃₀H₃₆O₅S

(508.65): C, 70.84; H, 7.13. Found: C, 70.51;

H, 7.31. ESIMS for C₃₀H₃₆O₅SNa: Calcd

531.2181. Found 531.2179.

Ethyl 2-O-p-methoxybenzyl-1-thio-i- -fu-

L

copyranoside (17).—Compound 16 (331 mg;

0.89 mmol) was dissolved in 50% aq AcOH

(20 mL) and heated with stirring at 60 °C for

2 h. After completion of reaction, the solvent

was evaporated and subsequently co-evapo-

rated with toluene to dryness to give a syrup.

It was chromatographed with 2:1 pentane–

EtOAc to give compound 17. [h]_D−11.2° (c

0.4 CHCl₃). ¹H NMR (CDCl₃): l 7.32 (d, 2 H,

J_ortho9.0 Hz, ArCH), 6.88 (d, 2 H, J_ortho9.0

Hz, ArCH), 4.88, 4.60 (d, 1 H, J_gem10.5 Hz,

PhCH₂), 4.36 (d, 1 H, J_1,29.5 Hz, H-1), 3.79

(s, 3 H, CH₃O), 4.71 (bs, 1 H, H-4), 3.58 (m,

2 H, H-3, H-5), 3.41 (t, 1 H, J 9.5 Hz, H-2),

2.67–2.82 (m, 2 H, SCH₂), 2.55, 2.28 (bs, 1 H,

OH), 1.31 (t, 3 H, J_vic7.5 Hz, SCH₂CH₃), 1.30

Octyl

galactopyranoside

tri-O-benzyl-b- -galactopyranosyl

2-O-acetyl-3,4,6,-tri-O-benzyl-i-

(24).—2-O-Acetyl-3,4,6,-

bromide

D

-

D

(13 g, 23.4 mmol) was dissolved in 100 mL

,

acetonitrile, and 3 A molecular sieves (pow-

dered, 12 g) and octanol (11 mL, 70.2 mmol)

were added and the mixture stirred under

argon for 20 min. Hg(CN)₂(8.3 g, 32.8 mmol)

and HgBr₂(catalytic amount) were added and

the reaction mixture was stirred overnight.

The mixture was diluted with three times its

volume of CH₂Cl₂and ﬁltered through Celite.

The ﬁltrate was collected and washed succes-

sively with water, 8% aq KI and aq NaHCO₃.

Drying (Na₂SO₄) and concentration yielded a

syrup that was chromatographed (15:1, 10:1,

8:1, 5:1 pentane–EtOAc) to yield 24 (8.8 mg,

74%) as a white solid. [h]_D+6.1° (c 3.3

CHCl₃); R_f0.45 (3:1 hexane–EtOAc). ¹H

NMR (CDCl₃): l 7.2–7.4 (m, 15 H, ArCH),

5.35 (dd, 1 H, J_2,310.0, J_1,28.0 Hz, H-2), 4.94,

4.67, 4.59, 4.51, 4.46, 4.41 (d, 1 H, J_gem11.5

13

(d, 3 H, J_5,66.5 Hz, H-6); C NMR (CDCl₃):

l 159.5 (ArCOCH₃), 130.2 (ArC), 130.0,

114.0 (ArCH), 84.7 (C-1), 78.4, 75.1, 74.8,

71.7 (C-3, C-4, C-5, CH₂O), 55.2 (OCH₃),

24.9, 16.5 (C-6), 14.9 (SCH₂CH₃). ESIMS for

C₁₆H₂₄O₅SNa: Calcd 351.1242. Found

351.1247.

Ethyl 3,4-di-O-benzyl-2-O-p-methoxyben-

zyl-1-thio-i-

L

-fucopyranoside

(18).—Com-

pound 17 (250 mg, 0.89 mmol) was dissolved

in DMF (5 mL), 0.2 g NaH (50% suspension

in oil) was added, and the mixture was stirred

for 10 min at 0 °C. Benzyl bromide (0.5 mL)

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

13

mL), and the mixture was stirred for 1 h with

400 mg 4 A molecular sieves. DMTST (356

Hz, PhCH₂), 4.32 (d, 1 H, J_1,28.0 Hz, H-1),

3.94 (d, 1 H, J_3,42.8, J_4,50.8 Hz, H-4), 3.83

(dt, 1 H, J_gem9.8, J_vic6.9 Hz, CH₂O), 3.53–

3.65 (m, 3 H, H-5, H-6b, H-6b), 3.50 (dd, 1 H,

J_2,310.0, J_3,42.8 Hz, H-3), 3.39 (dt, 1 H, J_gem

9.8, J_vic6.9 Hz, CH₂O), 2.02 (s, 3 H, CH₃CO),

1.52 (m, 2 H, CH₂CH₂O), 1.25 (bs, 10 H, CH₂

octyl), 0.87 (t, 3 H, CH₃octyl); ¹³C NMR

(CDCl₃): l 169.5 (COCH₃), 138.5, 138.0,

137.9, (ArC), 128.4, 128.3, 128.2, 127.9, 127.8,

127.7, 127.5, 127.4, (ArCH), 101.4 (C-1), 80.4

(C-2), 74.7, 74.4, 73.7, 73.6, 72.6, 71.9, 71.5,

69.4, 68.7, (C-3, C-4, C-5, C-6, CH₂O×5),

31.8, 29.5, 29.4, 29.3, 25.9, 22.6 (CH₂octyl),

21.0 (CH₃CO), 14.4 (CH₃octyl). Anal. Calcd

for C₃₇H₄₈O₇(604.75): C, 73.53; H, 8.00.

Found: C, 73.53; H, 8.15.

,

mg, 1.38 mmol) and DTBMP (190 mg, 0.92

mmol) were then added and stirring continued

for another 3 h. Upon completion, the reac-

tion mixture was ﬁltered through Celite,

washed with (Na₂SO₄), and evaporated.

Column chromatography (10:1, 6:1, 4:1 pen-

tane–EtOAc) gave compound 26 (170 mg,

1

71%). [h]_D−42.2° (c 0.76 CHCl₃). H NMR

(CDCl₃): l 7.20–7.40 (m, 25 H, ArH), 5.69 (d,

1 H, J_1%,2%3.5 Hz, H-1%), 5.42 (bd, 1 H, J_3%,4%2.5

Hz, H-4%), 4.93, 4.85, 4.82, 4.78, 4.75, 4.67,

4.65 (d, 1 H, J_gem11.5 Hz, PhCH₂), 4.44–4.58

(m, 3 H, PhCH₂), 4.27 (d, 1 H, J_1,27.8 Hz,

H-1), 4.1 (q, 1 H, J_5%,6%6.5 Hz, H-5%), 4.00 (dd,

1 H, J_2,310.5, J_3,43.0 Hz, H-3), 3.85–3.95 (m,

3 H, H-2, H-4, H-6a), 3.77 (dd, 1 H, J_2%,3%10.0

Hz, J_1%,2%3.5 Hz, H-2%), 3.72 (dd, 1 H, J_2%,3%

10.0, J_3%,4%2.5 Hz, H-3%), 3.51–3.64 (m, 3 H,

H-5, H-6b, CH₂O), 3.40 (dt, 1 H, J_gem10.0,

J_vic7.0 Hz, CH₂O), 2.10 (s, 3 H, CH₃CO), 1.60

(m, 2 H, CH₂CH₂O), 1.30 (bs, 10 H, CH₂

octyl), 1.12 (d, 2 H, J_5%6%6.5 Hz, H-6%), 0.88 (t,

3 H, J_vic6.9 Hz, CH₃octyl); ¹³C NMR

(CDCl₃): l 159.7 (CH₃CO), 129.3, 139.2,

138.8, 138.1, 138.0, 128.4, 128.3, 128.1, 128.0,

127.9×2, 127.8, 127.7, 127.6, 127.4, 127.3,

127.2×2, (ArC), 103.5 (C-1), 99.3 (C-1%),

81.1, 79.5, 79.4, 76.8, 75.3, 74.9, 74.2, 74.0,

74.0, 73.3, 72.7, 71.3, 71.0, 70.0, 69.7 (C-2,

C-3, C-4, C-5, C-6, C-3%, C-4%, C-5%, CH₂O),

32.3, 30.3, 30.2, 30.0, 29.2, 27.2 (CH₂octyl),

21.7 (CH₃CO), 16.7 (C-6%), 14.6 (CH₃octyl).

ESIMS for C₅₇H₇₀O₁₁Na: Calcd 953.4815.

Found 953.4815.

Octyl 3,4,6,-tri-O-benzyl-i- -galactopyran-

D

oside (25).—Compound 24 (8.8 g, 14.5 mmol)

was deacetylated with NaOMe (0.02 M) in dry

MeOH (Zemple´n deacetylation) to yield com-

pound 25 as a white solid (8.8 g, 97%). [h]_D

1

−0.8° (c 1.6 CHCl₃). H NMR (CDCl₃): l

7.2–7.4 (m, 15 H, ArCH), 4.89, 4.73, 4.68,

4.61, 4.49, 4.43 (d, 1 H, J_gem11.5 Hz, PhCH₂),

4.22 (d, 1 H, J_1,27.5 Hz, H-1), 3.95 (dd, 1 H,

J_1,27.5, J_2,310.0 Hz, H-2), 3.92 (d, 1 H, J_3,4

2.8 Hz, H-4), 3.86 (dt, 1 H, J_gem9.8, J_vic6.9

Hz, CH₂O), 3.55–3.63 (m, 3 H, H-5, H-6a,

H-6b), 3.48 (m, 1 H, CH₂O), 3.44 (dd, 1 H,

J_2,310.0, J_3,42.8 Hz, H-3), 2.25 (d, 1 H, J_H-2,OH

1.8 Hz, OH), 1.61 (m, 2 H, CH₂CH₂O), 1.28

(bs, 10 H, CH₂octyl), 0.89 (t, 3 H, CH₃octyl);

¹³C NMR (CDCl₃): l 169.7 (COCH₃), 138.2,

137.8, 137.6 (ArC), 128.4, 128.5, 128.2, 128.0,

127.9, 127.8, 127.7, 127.5, 127.4, 127.3

(ArCH), 102.0 (C-1), 80.2 (C-2), 74.6, 74.4,

73.6, 72.7, 72.1, 69.1, 68.0, 62.3, 56.6 (C-3,

C-4, C-5, C-6, CH₂O x5), 21.05 (CH₃CO),

33.0, 31.4, 30.6, 30.4, 27.3, 23.7 (CH₂octyl),

16.8 (C-6%), 14.4 (CH₃octyl). ESIMS for

C₃₇H₅₂O₁₃N₂Na: Calcd 585.336710. Found

585.336560. Anal. Calcd for C₃₇H₄₈O₇

(604.75): C, 73.53; H, 8.00. Found: C, 73.53;

H, 8.15. ESIMS for C₃₅H₄₆O₆Na: Calcd

585.3192. Found 585.3185.

Octyl 2,3-di-O-benzyl-4-O-methyl-h-

copyranosyl-(12)-3,4,6-tri-O-benzyl-i-

L

-fu-

D

-

galactopyranoside (28).—Compound 26 (140

mg, 0.15 mmol) was dissolved in dry MeOH

(10 mL), 2 mL of a 0.5 M soln of NaOMe in

MeOH was added, and the mixture was

stirred at rt for 4 h. The reaction mixture was

neutralized with Dowex 50 (H⁺) resin and

ﬁltered. The solvent was evaporated to obtain

a syrup (compound 27) that was methylated

directly with iodomethane and NaH in DMF

to obtain compound 28 (102 mg, 73% from

Octyl

4-O-acetyl-2,3-di-O-benzyl-h- -fu-

L

copyranosyl-(12)-3,4,6-tri-O-benzyl-i-

D-

galactopyranoside (26).—Compounds 25 (145

mg, 0.26 mmol) and 14 (200 mg, 0.46 mmol,

1.8 equiv) were dissolved in dry CH₂Cl₂(10

1

26). [h]_D−47.2° (c 1.1 CHCl₃). H NMR

(CDCl₃): l 7.20–7.40 (m, 25 H, ArH), 5.71 (d,

14

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

C-4, C-5, C-6, C-3%, C-4%, C-5%, CH₂O), 62.38

(OCH₃), 32.9, 30.9, 30.5, 30.3, 27.2, 23.6 (CH₂

octyl), 16.8 (C-6%), 14.4 (CH₃octyl). ESIMS

for C₂₁H₄₀O₁₀Na: Calcd 475.2519. Found

475.2512.

1 H, J_1%,2%2.4 Hz, H-1%), 4.84, 4.83, 4.78, 4.76,

4.72, 4.61, 5.59, 4.55 (d, 1 H, J_gem11.5 Hz,

PhCH₂), 4.52 (s, 2 H, PhCH₂), 4.47 (d, 1 H,

J_1,27.8 Hz, H-1), 4.43 (q, 1 H, J_5%,6%6.5 Hz,

H-5%), 4.25 (dd, 1 H, J_2,310.0, J_1,27.8 Hz,

H-2), 3.97 (bd, 1 H, J_3,42.5 Hz, H-4), 3.94 (m,

1 H, H-6a), 3.91 (dd, 1 H, J_2%,3%9.8, J_1%,2%2.4

Hz, H-2%), 3.73 (dd, 1 H, J_2%,3%9.8, J_3%,4%2.5 Hz,

H-3%), 3.55–3.65 (m, 5 H, H-2, H-3, H-5,

H-6a, CH₂O), 3.61 (s, 3 H, OCH₃), 3.40 (dt, 1

H, J_gem10.0, J_vic7.0 Hz, CH₂O), 1.55 (m, 2 H,

CH₂CH₂O), 1.32 (bs, 10 H, CH₂octyl), 1.21

(d, 2 H, J_5%6%6.5 Hz, H-6%), 0.90 (t, 3 H, J_vic6.9

Hz, CH₃octyl); ¹³C NMR (CDCl₃): l 138.9,

138.4, 138.3, 138.0, 137.9, 128.4, 128.3, 128.2,

128.1, 128.0, 127.9, 127.8, 127.6, 127.4, 127.3,

127.2, 127.2, 127.0, 126.35 (ArC), 103.6 (C-1),

97.0 (C-1%), 84.5, 80.9, 79.2, 76.0, 74.3, 73.6,

73.3, 72.9, 72.8, 72.6, 72.1, 71.2, 70.3, 70.0,

69.7 (C-2, C-3, C-4, C-5, C-6, C-3%, C-4%, C-5%,

CH₂O), 61.6 (OCH₃), 31.8, 29.7, 29.5, 29.3,

26.2, 22.6 (CH₂octyl), 16.2 (C-6%), 14.0 (CH₃

octyl). Anal. Calcd for C₅₆H₇₀O₁₀(903.18): C,

74.47; H, 7.81. Found: C, 74.54; H, 7.84.

Octyl

benzyl-2-O-(2,3-di-O-benzyl-h-

L-

xylo-hexopyranosyl-(12)-i-

D

-galactopyr-

anoside (30).—Compound 26 (483 mg, 0.52

mmol) was deacetylated with NaOMe in

MeOH as before to give 27. After evaporation

and drying of the mixture, it was acylated

with pentaﬂuorophenylchlorothionoformate

(175 mL, 1.08 mmol) and DMAP (250 mg,

0.05 mmol) in CH₂Cl₂(29). Compound 28 was

deoxygenated with AIBN (88 mg, 0.52 mmol)

and Bu₃SnH (700 mL, 2.5 mmol) in reﬂuxing

toluene to give 30 (207 mg; 65% from alcohol)

after column chromatography (10:1, 6:1 pen-

1

tane–EtOAc). [h]_D−15.2° (c 0.9). H NMR

(CDCl₃): l 7.20–7.40 (m, 25 H, ArH), 5.69 (d,

1 H, J_1%,2%3.6 Hz, H-1%), 4.85, 4.79, 4.74, 4.66,

4.64, 4.60, 4.56, 4.49 (d, 1 H, J_gem11.5 Hz,

PhCH₂), 4.47 (d, 1 H, J_1,27.8 Hz, H-1), 4.45

(s, 2 H, PhCH₂), 4.25 (dd, 1 H, J_2,39.8 Hz, J_1,2

7.8 Hz, H-2), 3.89 (m, 3 H, H-4, H-6a, CH₂O),

3.76 (dd, 1 H, J_2,39.8, J_3,42.7 Hz, H-3)

3.57–3.65 (m, 4 H, H-5, H-6b, H-2%, H-3%),

3.40–3.45 (m, 2 H, H-5%, CH₂O), 2.10 (m, 1 H,

H-4%a), 1.05 (m, 1 H, H-4%b), 1.59 (m, 2 H,

CH₂CH₂O), 1.30 (bs, 10 H, CH₂octyl), 1.16

(d, 2 H, J_5%6%6.5 Hz, H-6%), 0.90 (t, 3 H, J_vic6.9

Hz, CH₃octyl); ¹³C NMR (CDCl₃): l 139.0,

138.4, 138.2, 138.1, 137.9, 128.4, 128.2, 128.1,

128.0, 127.9, 127.8, 127.7, 127.5, 127.3, 127.2,

126.4 (ArC), 102.2 (C-1), 97.3 (C-1%), 84.5,

80.1, 75.5, 74.3, 73.6, 73.3, 72.3×2, 72.2×2,

72.1, 71.3, 69.8, 68.9, 63.3 (C-2, C-3, C-4, C-5,

C-6, C-2%, C-3% C-5%, CH₂O), 39.1 (C-4%), 31.8,

29.7, 29.5, 29.3, 26.2, 22.6 (CH₂octyl), 20.8

(C-6%), 14.0 (CH₃octyl). Anal. Calcd for

C₅₅H₆₉O₉(873.54): C, 75.66; H, 7.85. Found:

C, 75.67; H, 7.69.

Octyl 4-O-methyl-h-

L

-fucopyranosyl-(12)-

i- -galactopyranoside (2).—Compound 28

D

(80 mg, 0.088 mmol) was dissolved in MeOH

and hydrogenated in a ﬂow of hydrogen

overnight in presence of palladium hydroxide-

on-charcoal. After completion of the reaction,

the catalyst was ﬁltered off on a 0.22-mm

Millipore ﬁlter, the solvent was evaporated,

the residue was dissolved in water and passed

through Waters C₁₈Sep-Pak cartridges and

eluted with 60:40% MeOH–water. The solu-

tion was evaporated, the residue was redis-

solved in water, and the solution was ﬁltered

on a 0.22-mm Millipore ﬁlter, and lyophilized

1

to give compound 2 (36 mg, 91%). H NMR

(CD₃OD): l 5.17 (d, 1 H, J_1%,2%3.8 Hz, H-1%),

4.40 (d, 1 H, J_1,27.6 Hz, H-1), 4.28 (q, 1 H,

J_5%,6%6.5 Hz, H-5%), 3.85–3.93 (m, 2 H, H-4,

CH₂O), 3.78–3.83 (m, 2 H, H-4%, H-6a), 3.69–

3.74 (m, 3 H, H-6b, H-2%, H-3%), 3.61–3.67 (m,

2 H, H-2, H-3), 3.56 (s, 3 H, OCH₃), 3.45–

3.51 (m, 2 H, H-5, CH₂O), 1.55–1.64 (m, 2 H,

CH₂CH₂O), 1.26–1.36 (m, 10 H, CH₂octyl),

1.19 (d, 2 H, J_5%6%6.5 Hz, H-6%), 0.0.87–0.93

(m, 3 H, CH₃octyl); ¹³C NMR (CD₃OD): l

104.4 (C-1), 101.5 (C-1%), 84.5, 83.4, 79.0, 76.5,

75.7, 72.2, 70.9, 70.8, 70.4, 67.9 (C-2, C-3,

Octyl h-

L

-xylo-hexopyranosyl-(12)-i- -

D

galactopyranoside (3).—Compound 30 (60

mg, 0.068 mmol) was hydrogenated and

puriﬁed as described for compound 35 to give

compound 3 (26.6 mg, 90%). ¹H NMR

(CD₃OD): l 5.16 (d, 1 H, J_1%,2%3.8 Hz, H-1%),

4.31 (d, 1 H, J_1,27.1 Hz, H-1), 4.24 (m, 1 H,

H-5%), 3.89 (dt, 1 H, J_gem10.0, J_vic7.0 Hz,

CH₂O), 3.77–3.84 (m, 2 H, H-3%, H-4), 3.70–

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

15

(CH₂octyl), 20.9 (CH₃CO), 16.6 (C-6%), 14.1

(CH₃octyl). ESIMS for C₅₇H₇₀O₁₁Na: Calcd

953.4815. Found 953.4817.

3.74 (m, 2 H, H-6a, H-2%), 3.61–3.67 (m, 2 H,

H-2, H-3), 3.46–3.55 (m, 3 H, H-5, H-6b,

CH₂O), 1.91 (ddd, 1 H, J_gem12.7, J_vic4.7, 2.3

Hz, H-4%a), 1.54–1.64 (m, 2 H, CH₂CH₂O),

1.22–1.38 (m, 11 H, H-4%b, CH₂octyl), 1.13

(d, 1 H, J_5%6%6.3 Hz, H-6%), 0.86–0.94 (m, 3 H,

CH₃octyl); ¹³C NMR (CD₃OD): l 104.1 (C-

1), 97.3 (C-1%), 80.6, 77.0, 76.3, 75.4, 73.7,

71.9, 71.1, 69.5, 62.4 (C-2, C-3, C-4, C-5, C-6,

C-2%, C-3%, C-5%, CH₂O), 41.9 (C-4%), 33.0, 30.8,

30.5, 30.4, 27.2, 23.7 (CH₂octyl), 21.1 (C-6%),

14.4 (CH₃octyl). ESIMS for C₂₀H₃₈O₉Na:

Calcd 445.2413. Found 445.2419.

Octyl

2,4-di-O-benzyl-h-

L

-fucopyranosyl-

-galactopyr-

(1 2)-3,4,6-tri-O-benzyl-i-

D

anoside (32).—Compound 31 (160 mg, 0.172

mmol) was deacetylated by the Zemple´n

method and after the usual workup, evapora-

tion of the solvent and column chromatogra-

phy of the residue (10:1, 4:1 pentane–EtOAc)

gave compound 32 (140 mg, 92% from 31).

[h]_D−19.0° (c 2.4 CHCl₃). ¹H NMR

(CDCl₃): l 7.18–7.43 (m, 25 H, ArH), 4.97,

4.96, 4.89, 4.86, 4.69, 4.68, 4.64, 4.57, 4.47,

4.42 (d, 1 H, J_gem11.67 Hz, PhCH₂), 4.83 (d,

1 H, J_1%,2%4.03 Hz, H-1%), 4.29 (d, 1 H, J_1,27.8

Hz, H-1), 4.22 (dd, 1 H, J_2,39.1, J_1,27.8 Hz,

H-2), 3.88 (bd, 1 H, J_3,42.8 Hz, H-4), 3.82 (m,

1 H, CH₂O), 3.38–3.62 (m, 7 H, H-3, H-5,

H-6a, H-6b, H-2%, H-3%, H-4%), 3.44 (q, 1 H,

J_5%,6%6.5 Hz, H-5%), 3.34 (m, 1 H, CH₂O), 2.32

(d, 1 H, J_H-2%,OH4.3 Hz, OH), 1.55 (m, 2 H,

CH₂CH₂O), 1.25 (bs, 10 H, CH₂octyl), 1.22

(d, 2 H, J_5%,6%6.4 Hz, H-6%), 0.89 (t, 3 H, J_vic6.9

Hz, CH₃octyl); ¹³C NMR (CDCl₃): l 138.6,

138.3, 137.9, 137.8, 128.4, 128.2, 128.1, 128.1,

128.0, 127.9, 127.8, 127.8, 127.7, 127.6, 127.5,

127.5, 127.3, 127.3, 127.1 (ArC), 103.21 (C-1),

97.1 (C-1%), 81.9, 79.9, 76.0, 75.3, 74.4, 73.7,

73.6, 73.3, 73.0, 72.4, 72.3, 71.8, 71.5, 71.0,

70.4, 69.8, 68.7 (C-2, C-3, C-4, C-5, C-6, C-3%,

C-4%, C-5%, CH₂O), 31.8, 29.6, 29.4, 29.2, 26.2,

22.6 (CH₂octyl), 16.5 (C-6%), 14.0 (CH₃octyl).

ESIMS for C₅₅H₆₈O₁₀Na: Calcd 911.4710.

Found 911.4707.

Octyl 3-O-acetyl-2,4-di-O-benzyl-h-

L

-fuco-

pyranosyl - (12) - 3,4,6 - tri - O - benzyl - i -

D-

galactopyranoside (31).—Compound 25 (146

mg, 0.26 mmol) and 15 (200 mg, 0.46 mmol,

18 equiv) were dissolved in dry CH₂Cl₂(10

mL), and the mixture was stirred for 1 h with

,

500 mg 4 A molecular sieves. DMTST (356

mg, 1.38 mmol) and DTBMP (380 mg, 1.84

mmol) were then added and stirring continued

for another 3 h. Upon completion, the reac-

tion mixture was ﬁltered through Celite,

washed with (Na₂SO₄), and the solvent was

evaporated. Column chromatography (10:1,

6:1, 4:1 pentane–EtOAc) gave compound 31

(200 mg, 71% based on alcohol). [h]_D−36.8°

1

(c 1.9 CHCl₃). H NMR (CDCl₃): l 7.20–7.40

(m, 25 H, ArH), 5.74 (d, 1 H, J_1%,2%3.72 Hz,

H-1%), 5.31 (dd, 1 H, J_2%,3%10.6, J_3%,4%2.8 Hz,

H-3%), 4.85, 4.77 (d, 1 H, J_gem11.6 Hz,

PhCH₂), 4.40–4.60 (m, 8 H, PhCH₂), 4.22 (d,

1 H, J_1,27.7 Hz, H-1), 4.14 (q, 1 H, J_5%,6%6.5

Hz, H-5%), 4.09 (m, 1 H, H-6a), 4.00 (dd, 1 H,

J_2%,3%9.5, J_1%,2%3.7 Hz, H-2%), 3.97 (bs, 1 H,

H-4), 3.92 (m, 1 H, CH₂O), 3.76 (bs, 1 H,

H-4%), 3.73 (dd, 1 H, J_2,39.0 Hz, J_3,42.6 Hz,

H-3), 3.68–3.58 (m, 3 H, H-5, H-6b, H-2),

3.52–3.45 (m, 1 H, CH₂O), 2.05 (s, 3 H,

CH₃CO), 1.60 (m, 2 H, CH₂CH₂O), 1.35 (bs,

10 H, CH₂octyl), 1.20 (d, 2 H, J_5%,6%6.5 Hz,

Octyl 2,4-di-O-benzyl-3-O-methyl-h-

L

-fu-

copyranosyl-(12)-3,4,6-tri-O-benzyl-i-

D

-

galactopyranoside (33).—Compound 32 (160

mg, 0.172 mmol) was methylated with MeI

(25 mL, 0.4 mmol) and NaH (13 mg, 0.45

mmol) in DMF. Workup and column chro-

matography (10:1, 4:1 pentane–EtOAc ) gave

compound 33 (140 mg, 86% from 32). [h]_D

13

H-6%), 0.95 (t, 3 H, J_vic6.9 Hz, CH₃octyl); C

1

NMR (CDCl₃): l 170.5 (CH₃CO), 139.2,

139.2, 139.1, 138.8, 138.4, 138.0, 133.3, 128.5,

128.4, 128.3, 128.2, 128.2, 128.1, 128.0, 128.0,

127.9, 127.8, 127.7×2, 127.5, 127.4, 127.3,

127.0 (ArC), 103.5 (C-1), 99.1 (C-1%), 81.5,

76.6, 75.9, 75.3, 74.5, 74.4, 73.1, 72.9, 70.4,

69.9, 68.0 (C-2, C-3, C-4, C-5, C-6, C-3%, C-4%,

C-5%, CH₂O), 31.8, 29.5, 29.3, 29.2, 26.0, 22.6

−8.5° (c 2.8 CHCl₃). H NMR (CDCl₃): l

7.15–7.47 (m, 25 H, ArH), 5.69 (d, 1 H, J_1%,2%

3.7 Hz, H-1%), 4.95, 4.82, 4.76, 4.64, 4.61, 4.57

(d, 1 H, J_gem11.5 Hz, PhCH₂), 4.54, 4.46 (s, 2

H, PhCH₂), 4.45 (d, 1 H, J_1,27.8 Hz, H-1),

4.44 (q, 1 H, J_5%,6%6.5 Hz, H-5%), 4.24 (dd, 1 H,

J_2,310.0, J_1,27.8 Hz, H-2), 3.98 (dd, 1 H, J_2%,3%

9.5, J_1%,2%3.7 Hz, H-2%), 3.96 (bs, 1 H, H-4),

16

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

amino pyridine (100 mg, 0.84 mmol) in dry

CH₂Cl₂(10 mL). After 14 h the reaction mix-

ture was extracted with CH₂Cl₂, and the ex-

tract was washed with 0.5% aq HCl, satd

NaHCO₃, and water, dried, and concentrated

to a syrup. This was directly deoxygenated as

follows. To the syrup (109 mg) dissolved in

toluene (10 mL), tributyltin hydride (130 mL,

0.48 mmol) and azobisisobutyronitrile (40 mg,

0.24 mmol) were added, and the mixture was

reﬂuxed at 130 °C for 4 h. Evaporation of the

solvent left a syrup, which was dissolved in

acetonitrile and washed with hexane. The ace-

tonitrile layer was concentrated in vacuo and

chromatographed (6:1, 4:1 pentane–EtOAc)

to obtain 35 as a white solid (225 mg; 75%).

3.92 (dt, 1 H, J_gem10.0, J_vic7.0 Hz, CH₂O),

3.81 (s, 3 H, OCH₃), 3.52–3.75 (m, 6 H, H-3,

H-5, H-6a, H-6b, H-3%, H-4%), 3.41 (dt, 1 H,

J_gem10.0, J_vic7.0 Hz, CH₂O), 1.58 (m, 2 H,

CH₂CH₂O), 1.30 (bs, 10 H, CH₂octyl), 1.15

(d, 2 H, J_5%,6%6.5 Hz, H-6%), 0.91 (t, 3 H, J_vic6.9

Hz, CH₃octyl); ¹³C NMR (CDCl₃): l 138.9,

138.5, 138.4, 138.1, 137.9, 128.4, 128.3, 128.2,

128.1×2, 128.0, 127.9, 127.8, 127.7, 127.5,

127.4, 127.3, 127.2, 126.3 (ArC), 102.0 (C-1),

97.2 (C-1%), 84.4, 81.5, 75.4, 74.6, 74.3, 73.6,

73.3, 69.6, 68.9, 66.1 (C-2, C-3, C-4, C-5, C-6,

C-3%, C-4%, C-5%, CH₂O), 58.2 (OCH₃), 31.8,

29.7, 29.5, 29.3, 26.3, 22.6 (CH₂octyl), 16.5

(C-6%), 14.0 (CH₃octyl). Anal. Calcd for

C₅₆H₇₀O₁₀(903.16): C, 74.47; H, 7.81. Found:

C, 74.32; H, 7.84.

1

[h]_D−2.9° (c 0.8 CHCl₃). H NMR (CDCl₃):

l 7.12–7.40 (m, 25 H, ArH), 5.12 (d, 1 H,

J_1%,2a%3.3 Hz, H-1%), 4.86, 4.80, 4.64, 4.56, 4.30

(d, 1 H, J_gem11.6 Hz, PhCH₂), 4.36–4.50 (m,

5 H, PhCH₂), 4.25 (d, 1 H, J_1,27.97 Hz, H-1),

3.95 (bd, 1 H, J_3,42.0 Hz, H-4), 3.88 (q, 1 H,

J_5%,6%6.5 Hz, H-5%), 3.77 (dd, 1 H, J_2%,3%10.6,

J_1%,2%3.3 Hz, H-2%), 3.72 (dd, 1 H, J_2,39.8, J_3,4

2.0 Hz, H-3), 3.55–3.65 (m, 5 H, H-2, H-5,

H-6a, H-4%, CH₂O), 3.35–3.43 (m, 2 H, H-6b,

CH₂O), 2.05 (m, 1 H, H-3%a), 1.85 (t, 1 H, J

10.5 Hz, H-3%b), 1.50 (m, 2 H, CH₂CH₂O),

1.25 (bs, 10 H, CH₂octyl), 1.15 (d, 2 H, J_5%,6%

6.5 Hz, H-6%), 0.85 (t, 3 H, J_vic6.9 Hz, CH₃

octyl); ¹³C NMR (CDCl₃): l 138.5, 138.3,

137.9, 130.6, 128.9, 128.5, 128.4, 128.3, 128.2,

128.2, 128.1, 127.9, 127.8, 127.7, 127.5, 127.4,

127.3, 126.4, 124.6 (ArC), 102.2 (C-1), 96.4

(C-1%), 76.0, 75.7, 74.4, 73.6, 73.4, 72.4, 72.3,

71.5, 71.0, 70.6, 70.3, 69.7, 69.5, 65.9 (C-2,

C-3, C-4, C-5, C-6, C-2%, C-4%, C-5%, CH₂O),

31.8 (C-3%), 29.7, 29.5, 29.2, 27.5, 26.2, 22.6

(CH₂octyl), 16.3 (C-6%), 14.1 (CH₃octyl).

ESIMS for C₅₅H₆₈O₉Na: Calcd 895.4761.

Found 895.4757.

Octyl 3-O-methyl-h-

L

-fucopyranosyl-(12)-

i- -galactopyranoside (4).—Compound 33

D

(21.7 mg, 0.024 mmol) was dissolved in 12 mL

of EtOH, and 10 mg Pd(OH)₂-on-charcoal

was added. The mixture was hydrogenated at

rt and atmospheric pressure overnight. After

completion of the reaction, the catalyst was

ﬁltered off on a 0.22 mm Millipore ﬁlter. The

usual workup and chromatography on C₁₈

Sep-Pak cartridges gave compound 4 (10 mg,

1

89%). H NMR (CD₃OD): l 5.19 (dd, 1 H,

J_1%,2%3.9 Hz, H-1%), 4.33 (d, 1 H, J_1,27.0 Hz,

H-1), 4.27 (q, 1 H, J_5%,6%6.6 Hz, H-5%), 3.90 (dt,

1 H, J_gem10.0, J_vic7.0 Hz, CH₂O), 3.80–3.85

(m, 3 H, H-3, H-4%, H-6a), 3.62–3.76 (m, 4 H,

H-2, H-4, H-5, H-2%), 3.46–3.54 (m, 2 H,

H-6b, CH₂O), 3.45 (s, 3 H, OCH₃), 3.41 (dd, 1

H, J_2%,3%10.0, J_3%,4%3.2 Hz, H-3%), 1.56–1.64 (m,

2 H, CH₂CH₂O), 1.28–1.38 (m, 10 H, CH₂

octyl), 1.20 (d, 2 H, J_5%6%6.6 Hz, H-6%), 0.90 (t,

3 H, J_vic6.7 Hz, CH₃octyl); ¹³C NMR

(CD₃OD): l 104.3 (C-1), 101.2 (C-1%), 84.3,

79.7, 76.4, 73.8, 71.9, 71.5, 71.1, 69.5, 68.6,

62.4 (C-2, C-3, C-4, C-5, C-6, C-3%, C-4%, C-5%,

CH₂O), 57.2 (OCH₃), 33.0, 30.8, 30.6, 30.4,

27.3, 23.7 (CH₂octyl), 16.7 (C-6%), 14.4 (CH₃

octyl). ESIMS for C₂₁H₄₀O₁₀Na: Calcd

475.2519. Found 475.2513.

Octyl h-

L

-xylo-hexopyranosyl-(12)-i- -

D

galactopyranoside (5).—Compound 35 (15

mg, 0.017 mmol) was dissolved in 10 mL of

EtOH, and 10 mg of Pd(OH)₂-on-charcoal

was added. Compound 35 was hydrogenated

at rt and atmospheric pressure overnight. Af-

ter completion of the reaction, the catalyst

was ﬁltered off on a 0.22 mm Millipore ﬁlter,

the solvent was evaporated, and the residue

was dissolved in water and passed through

Octyl 2,4-di-O-benzyl-h-

osyl-(12)-3,4,6-tri-O-benzyl-i-

pyranoside (35).—Compound 32 was acetyl-

ated with pentaﬂuorophenylchlorothionofor-

mate (126 mL, 0.78 mmol) and 4-dimethyl-

L

-xylo-hexopyran-

-galacto-

D

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

17

H-3), 3.72 (s, 3 H, OCH₃), 3.55–3.67 (m, 5 H,

H-4, H-5, H-6b, H-3%, H-4%), 3.35–3.43 (m, 1

H, CH₂O), 1.53 (m, 2 H, CH₂CH₂O), 1.25 (bs,

10 H, CH₂octyl), 1.14 (d, 2 H, J_5%6%6.5 Hz,

Waters C₁₈Sep-Pak cartridges and eluted with

1:1 MeOH–water. The solvent was evapo-

rated, and the residue was redissolved in wa-

ter, ﬁltered on a 0.22-mm Millipore ﬁlter, and

lyophilized to give compound 5 (6.8 mg; 94%).

¹H NMR (CD₃OD): l 5.14 (dd, 1 H, J_1%,2%3.5

Hz, H-1%), 4.33 (d, 1 H, J_1,27.5 Hz, H-1), 4.22

(q, 1 H, J_5%,6%6.6 Hz, H-5%), 3.98 (ddd, 1 H,

J_2%,3a%17.0, J_1%,2%3.5, J_2%3%b2.2 Hz, H-2%), 3.88

(dt, 1 H, J_gem10.0, J_vic7.0 Hz, CH₂O), 3.81

(m, 1 H, H-6a), 3.62–3.76 (m, 5 H, H-2, H-3,

H-4, H-5, H-4%), 3.44–3.58 (m, 2 H, H-6b,

CH₂O), 1.85–2.00 (m, 2 H, H-3a, H-3%b), 1.60

(m, 2 H, CH₂CH₂O), 1.25–1.40 (m, 10 H,

CH₂octyl), 1.12 (d, 2 H, J_5%6%6.6 Hz, H-6%),

13

H-6%), 0.90 (t, 3 H, J_vic7.0 Hz, CH₃octyl); C

NMR (CDCl₃): l 158.9 (ArC-OMe), 139.0,

138.8, 138.4, 138.1, 137.9, 130.3, 129.4×2,

128.3, 128.2, 128.1, 127.9, 127.8, 127.5, 127.3,

126.4, 113.4 (ArC), 102.1 (C-1), 97.2 (C-1%),

84.5, 79.7, 78.1, 75.1, 74.7, 74.3, 73.6, 73.3,

72.9, 72.3, 72.1, 72.0, 71.3, 69.7, 68.9, 66.1

(C-2, C-3, C-4, C-5, C-6, C-2%, C-3%, C-4%, C-5%,

CH₂O), 55.1 (CH₃O), 31.8, 29.7, 29.5, 29.3,

26.3, 22.6 (CH₂octyl), 16.5 (C-6%), 14.1 (CH₃

octyl). ESIMS for C₆₃H₇₆O₁₁Na: Calcd

1031.5285. Found 1031.5280.

13

0.90 (t, 3 H, J_vic6.8 Hz, CH₃octyl); C NMR

(CD₃OD): l 104.9 (C-1), 101.4 (C-1%), 77.7,

75.5, 75.1, 72.5, 71.4, 70.4, 69.0, 66.2, 61.5

(C-2, C-3, C-4, C-5, C-6, C-2%, C-4%, C-5%,

CH₂O), 37.8 (C-3%), 32.1, 29.9, 29.6, 29.5, 26.4,

23.0 (CH₂octyl), 16.5 (C-6%), 14.3 (CH₃octyl).

ESIMS for C₂₀H₃₈O₉Na: Calcd 445.2413.

Found 445.2417.

Octyl

3,4-di-O-benzyl-h-

L

-fucopyranosyl-

-galactopyr-

(1 2)-3,4,6-tri-O-benzyl-i-

D

anoside (37).—Compound 36 (150 mg, 0.148

mmol) and ceric ammonium nitrate (154 mg,

0.28 mmol) were dissolved in 9:1 CH₃CN–wa-

ter (10 mL) and the mixture stirred for 30 min

at rt. The reaction mixture was diluted with

CH₂Cl₂, washed with satd NaHCO₃and wa-

ter, dried (Na₂SO₄), ﬁltered and evaporated in

vacuo to yield a syrup that was chro-

matographed (6:1 pentane–EtOAc) to give

compound 37 (100 mg, 70%). [h]_D−11.9° (c

Octyl 3,4-di-O-benzyl-2-O-p-methoxyben-

zyl - h -

L

- fucopyranosyl - (12) - 3,4,6, - tri - O-

-galactopyranoside (36).—Com-

benzyl-i-

D

pound 25 (140 mg, 0.25 mmol) and compound

18 (230 mg, 0.45 mmol, 1.8 equiv) were dis-

solved in dry CH₂Cl₂(10 mL) and stirred for

1

1.2 CHCl₃). H NMR (CDCl₃): l 7.2–7.4 (m,

,

1 h with 500 mg 4 A molecular sieves.

25 H, ArH), 5.49 (d, 1 H, J_1%,2%3.9 Hz, H-1%),

4.93, 4.85, 4.59, 4.56, 4.47, 4.43 (d, 1 H, J_gem

11.44 Hz, PhCH₂), 4.69 (s, 2 H, PhCH₂), 4.65,

4.62 (d, 1 H, J_gem11.0 Hz, PhCH₂), 4.33 (d, 1

H, J_1,27.77 Hz, H-1), 4.27 (q, 1 H, J_5%,6%6.5

Hz, H-5%), 4.17 (m, 1 H, H-6a), 4.10 (dd, 1 H,

J_2,39.76, J_1,27.77 Hz, H-2), 3.95 (bd, 1 H, J_3,4

2.4 Hz, H-4), 3.86 (dd, 1 H, J_gem10.0, J_vic7.0

Hz, CH₂O), 3.70 (dd, 1 H, J_2%,3%9.9, J_3%,4%2.7

Hz, H-3%), 3.53–3.63 (m, 5 H, H-2%, H-3, H-5,

H-6b, H-4%), 3.40 (dd, 1 H, J_gem10.0, J_vic7.0

Hz, CH₂O), 2.44 (d, 1 H, J_OH,H-2%7.4 Hz, OH),

1.56 (m, 2 H, CH₂CH₂O), 1.27 (bs, 10 H, CH₂

octyl), 1.14 (d, 2 H, J_5%6%6.5 Hz, H-6%), 0.89 (t,

3 H, J_vic7.0 Hz, CH₃octyl); ¹³C NMR

(CDCl₃): l 138.8, 138.7, 138.6, 137.6, 137.0,

128.4×2, 128.3, 128.2, 127.9, 127.8, 127.5

(ArC), 102.3 (C-1), 99.2 (C-1%), 83.7, 80.4,

77.0, 74.7, 74.3, 73.6, 73.4, 72.5, 72.4, 72.2,

69.9, 69.2, 68.2, 66.8 (C-2, C-3, C-4, C-5, C-6,

C-2%, C-3%, C-4%, C-5%, CH₂O), 31.8, 29.6, 29.5,

29.3, 26.2, 22.7 (CH₂octyl), 16.7 (C-6%), 14.1

DMTST (356 mg, 1.38 mmol) and DTBMP

(380 mg, 1.84 mmol) were then added, and the

mixture was stirred for another 3 h. Upon

completion of the reaction as determined from

the TLC of the reaction mixture, the reaction

was quenched with Et₃N, ﬁltered through

Celite, and the solvent was evaporated.

Column chromatography of the syrup thus

obtained (10:1, 6:1, 4:1 pentane–EtOAc) gave

35 (185 mg, 67% isolated yield). [h]_D−28.8°

1

(c 0.26 CHCl₃). H NMR (CDCl₃): l 7.2–7.4

(m, 25 H, ArH), 6.95, 6.61 (d, 2 H, J_ortho8.6

Hz, ArH), 5.69 (d, 1 H, J_1%,2%3.56 Hz, H-1%),

4.96, 4.85, 4.82, 4.77, 4.73, 4.66 (d, 1 H, J_gem

11.6 Hz, PhCH₂), 4.64, 4.54, 4.53 (d, 1 H, J_gem

11.5 Hz, PhCH₂), 4.47 (d, 1 H, J_1,27.8 Hz,

H-1), 4.46 (s, 2 H, PhCH₂), 4.25 (dd, 1 H, J_2,3

9.6, J_1,27.8 Hz, H-2), 4.04 (dd, 1 H, J_2%,3%10.2,

J_1%,2%3.6 Hz, H-2%), 3.95–3.99 (m, 2 H, H-5%,

H-6a), 3.90 (dd, 1 H, J_gem10.0, J_vic7.0 Hz,

CH₂O), 3.76 (dd, 1 H, J_2,39.6, J_3,42.68 Hz,

18

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

C₁₈column. The column was washed with

water and eluted with MeOH. The fractions

containing the product were pooled, and the

solvent was again evaporated. The residue was

re-dissolved in water, passed though a 0.22 mm

Millipore ﬁlter and lyophilized to give com-

(CH₃octyl). Anal. Calcd for C₅₅H₆₈O₁₀

(889.09): C, 74.30; H, 7.71. Found: C, 74.06;

H, 7.84.

Octyl 3,4-di-O-benzyl-2-O-methyl-h-

L

-fu-

copyranosyl -(12)-3,4,6,-tri-O-benzyl-i-

D-

galactopyranoside (38).—Compound 37 (200

mg, 0.225 mmol) was dissolved in DMF (10

mL) and iodomethane (0.028 mL, 0.45 mmol)

and NaH (50%, 25 mg, 0.9 mmol) were added

at 0 °C, the mixture was then stirred for 4 h at

rt. The reaction was quenched with MeOH in

the cold, and the mixture diluted with CH₂Cl₂.

It was washed with water, dried (Na₂SO₄),

and evaporated. Column chromatography

(10:1, 6:1 pentane–EtOAc) gave 38 (176 mg,

1

pound 6 (7 mg, 89%). H NMR (CD₃OD): l

5.49 (d, 1 H, J_1%,2%3.8 Hz, H-1%), 4.41 (d, 1 H,

J_1,27.8 Hz, H-1), 4.35 (q, 1 H, J_5%,6%6.5 Hz,

H-5%), 3.86–3.94 (m, 2 H, H-2, H-4), 3.68–

3.80 (m, 5 H, H-3, H-6a, H-2%, H-3%, H-4%),

3.52–3.64 (m, 3 H, H-6b, CH₂O), 3.50 (s, 3 H,

OCH₃), 3.45 (dd, 1 H, J_gem10.0, J_vic7.0 Hz,

CH₂O), 1.57 (m, 2 H, CH₂CH₂O), 1.28 (bs, 10

H, CH₂octyl), 1.17 (d, 2 H, J_5%6%6.5 Hz, H-6%),

1

13

86%). [h]_D−71.8° (c 3.0 CHCl₃). H NMR

0.86 (t, 3 H, J_vic7.0 Hz, CH₃octyl); C NMR

(CDCl₃): l 7.20–7.45 (m, 25 H, ArH), 5.70 (d,

1 H, J_1%,2%3.8 Hz, H-1%), 4.94, 4.93, 4.81, 4.78,

4.73, 4.67, 4.61, 4.59, 4.53 (d, 1 H, J_gem11.5

Hz, PhCH₂), 4.44, 4.40 (d, 1 H, J_gem11.8 Hz,

PhCH₂), 4.39 (d, 1 H, J_1,27.8 Hz, H-1), 4.19

(q, 1 H, J_5%,6%6.5 Hz, H-5%), 4.17 (dd, 1 H, J_2,3

9.8, J_1,27.8 Hz, H-2), 3.92 (bd, 1 H, J_3,42.5

Hz, H-4), 3.82–3.85 (m, 2 H, H-3, H-6a), 3.80

(dd, 1 H, J_2%,3%10.0, J_1%,2%3.8 Hz, H-2%), 3.72

(bd, 1 H, J_2%,3%10.0 Hz, H-3%), 3.67 (bs, 1 H,

H-4%), 3.55–3.61 (m, 2 H, H-5, H-6b), 3.52

(dd, 1 H, J_gem10.0, J_vic7.0 Hz, CH₂O), 3.43 (s,

3 H, OCH₃), 3.35 (dd, 1 H, J_gem10.0, J_vic7.0

Hz, CH₂O), 1.49 (m, 2 H, CH₂CH₂O), 1.22

(bs, 10 H, CH₂octyl), 1.12 (d, 2 H, J_5%6%6.5

Hz, H-6%), 0.85 (t, 3 H, J_vic7.0 Hz, CH₃octyl);

¹³C NMR (CDCl₃): l 139.0, 138.8, 138.6,

138.5, 138.0, 128.4, 128.3, 128.2, 128.1, 127.9,

127.8, 127.5, 127.4, 127.3×2, 126.7 (ArC),

102.0 (C-1), 97.0 (C-1%), 84.5, 78.5, 78.2, 78.0,

77.6, 74.9, 74.3, 73.6, 73.4, 73.0, 72.9, 72.4,

71.8, 69.6, 66.6 (C-2, C-3, C-4, C-5, C-6, C-2%,

C-3%, C-4%, C-5%, CH₂O), 58.1 (OCH₃), 31.8,

29.6, 29.5, 29.3, 26.2, 22.6 (CH₂octyl), 16.7

(C-6%), 14.0 (CH₃octyl). Anal. Calcd for

C₅₆H₇₀O₁₀(903.08): C, 74.47; H, 7.81. Found:

C, 74.57; H, 7.86.

(CD₃OD): l 104.6 (C-1), 100.1 (C-1%), 82.3,

80.3, 76.4, 74.5, 73.9, 72.8, 71.8, 71.0, 69.5,

62.4 (C-2, C-3, C-4, C-5, C-6, C-2%, C-3%, C-4%,

C-5%, CH₂O), 61.0 (OCH₃), 38.0, 30.8, 30.6,

30.4, 27.3, 23.7 (CH₂octyl), 16.6 (C-6%), 14.4

(CH₃octyl). ESIMS for C₂₁H₄₀O₁₀Na: Calcd

475.2519. Found 475.2514.

Octyl 3,4-di-O-benzyl-h-

L

-xylo-hexopyran-

-galacto-

osyl-(12)-3,4,6-tri-O-benzyl-i-

D

pyranoside (40).—Compound 37 (50 mg, 0.09

mmol) was dissolved in CH₂Cl₂(10 mL) and

4-dimethylaminopyridine (55 mg, 0.45 mmol)

and

pentaﬂuorophenylchlorothionoformate

(42 mL, 0.26 mmol) were added in the cold.

The mixture was stirred overnight at rt. After

completion of the reaction, the reaction mix-

ture was washed successively with 5% aq HCl,

satd aq NaHCO₃and water and then dried.

The solvent was evaporated in vacuo, and this

was taken to the next step as follows. Tri-

butyltin hydride (39 mL, 0.52 mmol) and

AIBN (42.3 mg, 0.25 mmol) were added to it

in toluene and reﬂuxed at 130 °C bath temper-

ature for 4 h. The solvent was evaporated and

chromatographed (6:1 pentane–EtOAc) to

give compound 40 (50 mg, 57%). [h]_D−10.4°

1

(c 0.74 CHCl₃). H NMR (CDCl₃): l 7.12–

Octyl 2-O-methyl-h-

L

-fucopyranosyl-(12)-

7.40 (m, 25 H, ArH), 5.50 (d, 1 H, J_1%,2a%3.7

Hz, H-1%), 4.95, 4.84, 4.68, 4.66, 4.57, 4.49 (d,

1 H, J_gem11.6 Hz, PhCH₂), 4.58, 4.42 (s, 2 H,

PhCH₂), 4.24 (d, 1 H, J_1,27.8 Hz, H-1), 4.22

(q, 1 H, J_5%,6%6.5 Hz, H-5%), 4.06 (dd, 1 H, J_2,3

9.8, J_1,27.8 Hz, H-2), 3.92 (bd, 1 H, J_3,42.6

Hz, H-4),), 3.84 (dd, 1 H, J_gem10.0, J_vic7.0

Hz, CH₂O), 3.59–3.64 (m, 2 H, H-3%, H-6a),

i- -galactopyranoside (6).—Compound 38

D

(16 mg; 0.017 mmol) was hydrogenated with

hydrogen and palladium-on-charcoal in

MeOH and puriﬁed as follows. The solution

was ﬁltered to remove the catalyst, and the

solvent was evaporated. The crude product

was dissolved in water and passed though a

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

19

the solvent was partially evaporated and then

3.58 (bs, 1 H, H-4%), 3.53–3.56 (m, 2 H, H-5,

H-6b), 3.36 (dd, 1 H, J_gem10.0 Hz, J_vic7.0 Hz,

CH₂O), 2.09–2.18 (ddd, 1 H, J_gem16.3 Hz,

J_2%a,3%12.2 Hz, J_2%a,1%3.7 Hz, H-2%a), 1.89 (m, 1

H, H-2%b), 1.60 (m, 2 H, CH₂CH₂O), 1.25 (bs,

10 H, CH₂octyl), 1.12 (d, 2 H, J_5%6%6.5 Hz,

,

dried over 4 A molecular sieves. The resulting

syrup was transferred to the reaction vessel

containing compound 25 (68 mg, 0.12 mmol),

,

Bu₄NBr (72 mg, 0.22 mmol) and 1 g 4 A

molecular sieves in 2:1 CH₂Cl₂–DMF (15 mL)

and the mixture was stirred for 2 days. The

mixture was ﬁltered through Celite, and the

solvents were evaporated. Column chromatog-

raphy (10:1 pentane–EtOAc) gave 43 (100

mg, 76% based on alcohol). [h]_D−53.8° (c

13

H-6%), 0.89 (t, 3 H, J_vic6.9 Hz, CH₃octyl); C

NMR (CDCl₃): l 139.5, 1349.4, 139.3, 139.2,

138.9, 128.4, 128.3, 128.1, 127.9, 127.8, 127.7,

127.5, 127.2 (ArC), 102.5 (C-1), 98.1 (C-1%),

84.2, 82.7, 77.2, 76.5, 74.4, 74.1, 73.6, 73.5,

72.9, 72.6, 72.1, 70.3, 69.4, 68.9 (C-2, C-3,

C-4, C-5, C-6, C-3%, C-4%, C-5%, CH₂O), 33.8

(C-2%), 29.5, 29.4, 29.3, 27.3, 26.2, 22.6 (CH₂

octyl), 17.2 (C-6%), 14.1 (CH₃octyl). Anal.

Calcd for C₅₅H₆₉O₉(873.05): C, 75.66; H,

7.85. Found: C, 74.39; H, 7.88.

1

1.6 CHCl₃). H NMR (CDCl₃): l 7.20–7.40

(m, 30 H, ArH), 5.78 (d, 1 H, J_1%,2%3.8 Hz,

H-1%), 4.85, 4.77, 4.75, 4.72, 4.63, 4.55, 4.49,

4.45 (d, 1 H, J_gem11.5 Hz, PhCH₂), 4.74, 4.61

(s, 2 H, PhCH₂), 4.46 (d, 1 H, J_1,27.8 Hz,

H-1), 4.25 (dd, 1 H, J_2,310.0 Hz, J_1,27.8 Hz,

H-2), 4.08 (dd, 1 H, J_2%,3%10.0, J_1%,2%3.8 Hz,

H-2%), 3.98 (dd, 1 H, J_2,310.0, J_3,43.0 Hz,

H-3), 3.94 (m, 1 H, H-4, H-6a), 3.89 (dt, 1 H,

J_gem10.0, J_vic7.0 Hz, CH₂O), 3.80 m, 1 H,

H-5%a), 3.74 (dd, 1 H, J_2%,3%9.8, J_3%,4%2.8 Hz,

H-3%), 3.56–3.69 (m, 4 H, H-4, H-6b, H-4%,

H-5%b), 3.42 (m, 1 H, CH₂O), 1.56 (m, 2 H,

CH₂CH₂O), 1.26 (bs, 10 H, CH₂octyl), 0.91

(t, 3 H, J_vic6.9 Hz, CH₃octyl); ¹³C NMR

(CDCl₃): l 138.8, 138.6, 138.5, 138.4, 138.0,

137.9, 128.5, 128.4, 128.3, 128.2, 128.1×3,

128.0, 127.9, 127.8, 127.7×2, 127.6, 127.5,

127.4, 127.3, 127.2, 126.5 (ArC), 102.1 (C-1),

97.5 (C-1%), 84.4, 77.6, 76.0, 74.4×2, 73.6,

73.3, 73.0, 72.4, 72.4, 72.1, 71.5, 71.5, 69.6,

68.8 (C-2, C-3, C-4, C-5, C-6, C-2%, C-3% C-4%,

CH₂O), 60.3 (C-5%), 31.8, 29.7, 29.4, 29.2, 26.2,

22.6 (CH₂octyl), 14.1 (CH₃octyl). ESIMS for

Octyl h-

L

-xylo-hexopyranosyl-(12)-i- -

D

galactopyranoside (7).—Compound 40 (25

mg, 0.028 mmol) was hydrogenated as de-

scribed for 35. After the usual workup, it was

chromatographed (10:1 CH₂Cl₂–MeOH) and

then puriﬁed on reversed-phase C₁₈cartridges,

as described for compound 6, to give com-

1

pound 7 (10 mg; 84%). H NMR (CD₃OD): l

5.38 (dd, 1 H, J_vic2.2, 2.1 Hz, H-1%), 4.31 (q,

1 H, J_5%,6%6.6 Hz, H-5%), 4.25 (d, 1 H, J_1,27.5

Hz, H-1), 3.86–3.96 (m, 2 H, H-3%, CH₂O),

3.62–3.78 (m, 4 H, H-2, H-4, H-5, H-6a), 3.58

(dd, 1 H, J_2,39.5, J_3,43.4 Hz, H-3), 3.44–3.53

(m, 3 H, H-4%, H-6b, CH₂O), 1.80–1.88 (m, 2

H, H-2%), 1.52–1.60 (m, 2 H, CH₂CH₂O),

1.26–1.36 (m, 10 H, CH₂octyl), 1.17 (d, 2 H,

J_5%6%6.6 Hz, H-6%), 0.88–0.92 (m, 3 H, CH₃

octyl); ¹³C NMR (CD₃OD): l 104.1 (C-1),

101.8 (C-1%), 80.6, 76.4, 73.8, 72.1, 71.2, 70.8,

69.9, 69.5, 62.4 (C-2, C-3, C-4, C-5, C-6, C-3%,

C-4%, C-5%, CH₂O), 35.2 (C-2%), 31.6, 30.8, 30.5,

30.4, 27.3, 23.7 (CH₂octyl), 17.0 (C-6%), 14.4

(CH₃octyl). ESIMS for C₂₀H₃₈O₉Na: Calcd

445.2413. Found 445.2414.

C₆₁H₇₂O₁₀Na:

Calcd

987.5502.

Found

987.5502.

Octyl i -

D

- arabinopyranosyl - (12) - i - -

D

galactopyranoside (8).—Compound 43 (50

mg, 0.052 mmol) was hydrogenated as de-

scribed before and puriﬁed in the usual way to

1

Octyl 2,3,4-tri-O-benzyl-i-

osyl-(12)-3,4,6-tri-O-benzyl-i-

pyranoside (43).—2,3,4-Tri-O-benzyl-

D

-arabinopyran-

-galacto-

-ara-

give compound 8 (20 mg, 91%). H NMR

D

(CD₃OD): l 5.21 (d, 1 H, J_1%,2%2.75 Hz, H-1%),

4.32 (d, 1 H, J_1,27.23 Hz, H-1), 4.15 (dd, 1 H,

J_gem11.72, J_4%,5%a1.5 Hz, H-5%a), 3.89 (dt, 1 H,

J_gem10.0, J_vic7.0 Hz, CH₂O), 3.80–3.84 (m, 2

H, H-3%, H-3), 3.76–3.79 (m, 2 H, H-4, H-2%),

3.70–3.74 (m, 2 H, H-4%, H-5%b), 3.45–3.58

(m, 3 H, H-2, H-5, CH₂O), 1.59 (m, 2 H,

CH₂CH₂O), 1.30 (m, 10 H, CH₂octyl), 0.90

(m, 3 H, CH₃octyl); ¹³C NMR (CD₃OD): l

D

binopyranose (41, 238 mg, 0.566 mmol) was

dissolved in CH₂Cl₂(2 mL), and DMF (22.3

mL) was added with stirring. Oxalyl bromide

(0.184 mL, 1.3 mmol) was added dropwise,

and stirring was continued for 20 min after

evolution of gas ceased. The reaction mixture

was washed with water, the extract was dried,

20

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

0.12, 0.06, 0.03, 0.015, 0.0075, 0.00375 mM

for kinetic studies, and under the conditions

stated above, K_mwas determined to be 1.00,

0.750, 0.500, 0.250, 0.125, 0.0625 and

0.03125 mM. K_mdetermination for com-

pound 3 was carried out at concentrations

of 5.00, 2.50, 1.250, 0.625, 0.3125, 0.15625,

0.07812, 0.03906 and 0.01953 mM. To deter-

mine the K_mvalue of compound 4, it was

used in concentrations of 1.00, 0.500, 0.250,

0.125, 0.0625, 0.03125, 0.0156, 0.0078, 0.0039

mM. Compounds 2 and 5 were assayed in

concentrations of 1.00, 0.500, 0.250, 0.125,

0.0625, 0.03125, 0.0156 and 0.0078 mM and

compound 7 at concentrations of 0.250,

0.125, 0.0625, 0.03125, 0.0156, 0.0078 and

0.0039 mM for K_mvalue determination.

103.5 (C-1), 98.5 (C-1%), 77.6, 75.6, 73.0,

72.4, 71.8, 71.6, 70.4, 69.1, 68.9 (C-2, C-3,

C-4, C-5, C-6, C-2%, C-3%, CH₂O), 61.5 (C-

5%), 32.0, 29.8, 29.5, 29.4, 26.3, 22.9 (CH₂

octyl), 14.3 (CH₃octyl). ESIMS for

C₁₉H₃₆O₁₀Na:

447.2202.

Enzyme kinetics.—The enzymatic assays

performed were based on a method devel-

oped that uses the rate of transfer of sugar

donors incorporated with a radioisotope

onto an acceptor. All the compounds synthe-

sized had a hydrophobic aglycon and there-

fore C₁₈(reversed-phase) cartridges were

used to separate the products from unre-

acted donors. The recombinant enzymes

were expressed in E. coli and the isolation

and puriﬁcation was performed according to

the established protocol [12,13]. They were

stored at −70 °C with 1 mg/mL bovine

serum albumin (BSA) until used.

Calcd

447.2206.

Found

UDP-Gal (0.6 mM) and UDP-[6-³H]Gal

(0.2 mCi) were used for assays with the B

transferase. The acceptors were used in con-

centrations of 1 mM. 0.1 mL of the enzyme

(0.997 mg/mL) was used with 0.15 mM of

UDP-Gal. To test the inhibitory activities of

potential inhibitors, the analogues were

added in concentrations of 0.5–0.1 mM of

the native acceptor 1. To determine K_mval-

ues for compound 4, it was used in concen-

trations of 0.4125, 0.2062, 0.0625, 0.03125,

0.0156, 0.0078 and 0.0039 mM. K_mdetermi-

nation for compound 5 was carried out at

concentrations of 4.00, 2.00, 1.00, 0.500,

0.250, 0.125, 0.0625, 0.03125 and 0.0156

mM. The data obtained were then ﬁt to the

Michaelis–Menten equation using un-

weighted non-linear regression with the

Sigma Plot 4.0 Program (Jandel Scientiﬁc) to

estimate the kinetic parameters.

All reactions were carried out in 450 mL

microfuge tubes in a total volume of 33 mL.

The modiﬁed derivatives were screened at 1

mM concentrations with the A and B en-

zymes, all activity being measured relative to

the natural acceptor [octyl a-

L

-fucopyra-

nosyl-(12)-b- -galactopyranoside (1)]. All

D

assays were carried out in 35 mM sodium

cacodylate buffer containing 0.02 M MnCl₂

and 1 mg/mL BSA at 37 °C. The reactions

were carried out for 30 min before being

quenched with water. The reaction mixture

was transferred to a C₁₈Sep-Pak cartridge

pre-equilibrated with MeOH and water. The

cartridge was washed with water to remove

unreacted donor until background counts

were obtained. The products were then

eluted with MeOH (3.5 mL), and the ra-

dioactivity in dpm (decays per minute) was

measured in a liquid scintillation counter.

UDP-GalNAc (0.152 mM) and UDP-[6-

³H]GalNAc (0.2 mCi) were used as donors

for the assay with the A transferase. The

acceptors were used in concentrations of 1

mM. 0.25 mL of the enzyme (0.165 mg/mL)

was used with 0.15 mM of UDP-GalNAc.

When tested as inhibitors, the analogues

were used in concentrations of 1 mM with

0.01 mM of the native acceptor 1. Com-

pound 1 was tested at concentrations of

Acknowledgements

We thank Dr N.O.L. Seto for the cloned

enzymes, Ms C.A. Compston for assistance

in performing the enzyme kinetic experi-

ments and Ms L. Lechelt for assistance in

preparing the manuscript. This work was

supported by a NSCERC/NRC partner-

ship grant with Synsorb Biotech Inc. A.M. is

the recipient of a studentship from the Al-

berta Heritage Foundation for Medical Re-

search.

A. Mukherjee et al. / Carbohydrate Research 326 (2000) 1–21

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Article Doi

Synthesis and enzymatic evaluation of modified acceptors of recombinant blood group A and B glycosyltransferases

DOI: 10.1016/S0008-6215(00)00003-3

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Full text of DOI:10.1016/S0008-6215(00)00003-3

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