Synthetic studies on glycosphingolipids from protostomia phyla: Synthesis of glycosphingolipids and related carbohydrate moieties from the parasite Schistosoma mansoni

Carbohydrate Research 361 (2012) 55–72

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Carbohydrate Research

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Synthetic studies on glycosphingolipids from protostomia phyla: synthesis

of glycosphingolipids and related carbohydrate moieties from the parasite

Schistosoma mansoni

Takayuki Kanaya ^a, Frank Schweizer ^b,c, Tadahiro Takeda ^a, Fumiyuki Kiuchi ^a, Noriyasu Hada ^a,

⇑

^aFaculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan

^bDepartment of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2

^cDepartment of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 8 June 2012

Received in revised form 9 August 2012

Accepted 14 August 2012

Available online 21 August 2012

Stereocontrolled syntheses of three neutral glycosphingolipids and six oligosaccharide derivatives found

from the parasite Schistosoma mansoni have been accomplished. A pentasaccharide glycosphingolipid b-

D

-Galp-(1?4)-[

hexasaccharide glycosphingolipids

(1?3)-b- -GalpNAc-(1?4)-b- -Glcp-(1M1)-Cer (2) and b-

NAc-(1?3)-b- -GlcpNAc-(1?3)-b- -GalpNAc-(1?4)-b- -Glcp-(1M1)-Cer (3), together with their

non-reducing end tri- and tetrasaccharides, b- -Galp-(1?4)-[ -Fucp-(1?3)]-b- -GlcpNAcOR (4) and

-Fucp-(1?3)-b- -Galp-(1?4)-[ -Fucp-(1?3)]-b- -GlcpNAcOR (5), were synthesized by block

synthesis. Moreover, non-reducing end oligosaccharides of schistosomal glycosphingolipids, b- -Galp-

NAc-(1?4)-[ -Fucp-(1?3)]-b- -GlcpNAcOR (6), -Fucp-(1?3)-b- -GalpNAc-(1?4)-[ -Fucp-(1?3)]

-b- -GlcpNAcOR (7), -Fucp-(1?3)-b- -GalpNAc-(1?4)-[ -Fucp-(1?2)- -Fucp-(1?3)]-b- -GlcpNA-

cOR (8) and -Fucp-(1?3)-b- -GalpNAc-(1?4)-[ -Fucp-(1?2)- -Fucp-(1?2)- -Fucp-(1?3)]-b-

-GlcpNAcOR (9) [R = 2-(trimethylsilyl)ethyl], were synthesized as probes to explore their diagnostic

potential to detect schistosomiasis from patients’ sera.

a

-

L

-Fucp-(1?3)]-b-

D

-GlcpNAc-(1?3)-b-

D

-GalpNAc-(1?4)-b-

-Galp-(1?4)-[

D

-Glcp-(1M1)-Cer (1), two

-Fucp-(1?3)]-b- -GlcpNAc-

-Fucp-(1?3)]-b- -Glcp-

a-

L

-Fucp-(1?3)-b-

D

a

-

L

D

a

-

L

D

Keywords:

D

a

-L

D

Glycosphingolipid

Schistosoma mansoni

Schisto-core

a

-

L

D

a

-

L

D

a-

L

D

a-

L

D

a-L

Stereocontrolled synthesis

D

a-

L

D

a-L

D

a

-

L

D

a

-

L

a-

L

a-L

D

1. Introduction

(1?3)]-b-

core types have been identiﬁed along with the conventional type

1 [b- -Galp-(1?3)-b- -GalpNAc] and type 2 [b- -Galp-(1?3)-

[b- -GlcpNAc-(1?6)]-b- -GalpNAc] in the O-linked glycans of

schistosoma. Several unusually long and complex O-glycan chains

were found which are multifucosylated.⁷Furthermore, some of the

fucosylated schistosome glycosphingolipids in cercariae are domi-

nated not only by terminal Le^Xgroups, but also abundantly express

D

-GlcpNAc (Lewis X, Le^X).⁶On the other hand, novel

Schistosomiasis is a human parasitic disease caused by the

digenetic blood ﬂukes of the genus Schistosoma that affect more

than 200 million people in the tropics.¹There is no vaccine to pre-

vent the disease, and although drug therapy is effective in most

cases, reinfection is common in endemic areas.²Schistosomes pro-

duce a variety of remarkable and complex glycoconjugates such as

glycoproteins and glycosphingolipids.³It has been known for more

than a quarter century that glycosylated antigens play a major role

in the development of the characteristic innate humoral immune

response in infected hosts.⁴In the past decade, many studies have

investigated global structural features of glycans derived from

schistosomal glycoprotein and glycolipid preparations in particular

from Schistosoma mansoni and Schistosoma japonicum.⁵S. mansoni

adult worm- and egg-derived glycoproteins contain common

N-linked oligomannose structures that occur in most eukaryotic

organisms. Typical elements of the complex antennary N-linked

D

pseudo-Lewis Y (Le^Y) variants of

-Fucp-(1?3)]-b-

shown that S. mansoni glycolipids from adult worms consisting of

a b- -GalpNAc-(1?4)-b- -Glcp-(1M1)-Cer sequence called ‘schis-

to-core’ instead of the more common mammalian b- -Galp-

(1?4)-b- -Glcp-(1M1)-Cer core structure, and this core structure

a

-

L

-Fucp-(1?3)-b-

D-Galp-(1?4)-

[a

-L

D

-GlcpNAc-structures⁸Makaaru et al.⁹have

D

is also found in other unique glycan sequences from eggs and

cercariae (Fig. 1). Thus, S. mansoni synthesizes a multitude of com-

plex carbohydrates, including both parasite- and stage-speciﬁc gly-

cans. These glycoconjugates are interesting from two points of

view. One is their antigenicity against sera of schistosomiasis pa-

tients and the other is their function in immune responses against

host mammals. Therefore, schistosome glycans and glycoconju-

gates are good targets not only for immunodiagnostic purposes

but also for vaccine developments. Chemical synthesis of glycocon-

jugates provides not only access to large amounts of structurally

glycans from adult worm are b-

(LacdiNAc, LDN), b- -GalpNAc-(1?4)-[

(fucosylated LacdiNAc, LDN-F), and b-

D

-GalpNAc-(1?4)-b- -GlcpNAc

-Fucp-(1?3)]-b- -GlcpNAc

-GalpNAc-(1?4)-[a-L-Fucp-

D

a

D

-L

D

⇑

Corresponding author. Tel.: +81 3 5400 2666; fax: +81 3 5400 2556.

E-mail address: hada-nr@pha.keio.ac.jp (N. Hada).

http://dx.doi.org/10.1016/j.carres.2012.08.008

56

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

β

-

D

-Gal

p

NAc-(1

→

4)-

β

-

D

-Glc

p

-(1

↔1)-Cer

(

A)

β-

D

-Glc

p

NAc-(1

→3)-

β

-

D

p

NAc-(1

β

-

D

p

-(1

B)

β

-

D

-Gal

p

-(1

→

4)-

-Glc

-

C

D

)

-Galp

→

-Glc

3

↑

1

α

-

L

-Fuc

p

α

-

L

-Fuc

p

-(1

→

3)-

β

-

D

-Gal

p

-(1

→

4)-

β

-

D

-Glc

3

p

NAc-(1

→

3)-

β

-

D

-Gal

p

NAc-(1

→

4)-

β

-

D

-Glc

p

-(1

↔

1)-Cer

(

E)

↑

1

α

-

L

-Fuc

p

β

-

D

-Gal

p

-(1→

4)-

β-

D

-Glc

p

NAc-(1

→

3)-

β

-

D

-Glc

pNAc-(1

→

β

-

D

-Gal

p

NAc-(1

→

4)-

β

-

D

-Glc

p

-(1

↔

1)-Cer

F)

3

↑

1

α-

L-Fuc

p

β

-

D

-Gal

p

NAc-(1

→4)-

-

D

-Glc

3

pNAc-

β

-

D

-Glc

pNAc-(1

→

3)-

β

-

D

-Gal

p

NAc-(1

→

4)-

β

-

D

-Glc

p

-(1

↔

G

)

↑

1

α

β

-L

-Fuc

p

α

-

L

-Fuc

p

-(1

→

3)-

β

-

D

-Gal

p

NAc-(1

→4)-

-

D

-Glc

p

NAc-

β

-

D

-Glc

p

NAc-(1

→

3)-

β

-

D

-Gal

p

NAc-(1

→

4)-

β

-

D

-Glc

p

-(1

↔

1)-Cer

(

H

3

↑

1

α

β

-L

-Fuc

p

α-

-Fucp

→

β

-

p

→4)-

-

D

-Glc

p

NAc-

β

-

-Glc

→

3)-

D

-Gal

→

-

-Glc

p

↔

I)

3

↑

1

α

-

L

-Fuc

p

-(1

→

2)-

α

-

L

-Fuc

p

α-L

-Fucp

-(1→3)-

β

-D

-Gal NAc-(1

p

→

4)-

β

-

D

-Glc

NAc-

β

-D

-Glcp

NAc-(1

→3)-β-

D

-Gal

p

NAc-(1

→4)-

β

-D

-Glc

p

-(1

↔1)-Cer

(J)

3

↑

1

α-L

-Fuc

p

-(1

→

2)-

α

-

L

-Fuc

p

-(1

→

2)-

α

-

L

-Fuc

p

Figure 1. Structures of glycosphingolipid (A–J) from Schistosoma mansoni.

well-deﬁned natural compounds, but also to a variety of structur-

ally related non-natural compounds.

acceptor 14 and trisaccharide donor 13 (Scheme 1). Initially, we at-

tempted to synthesize the trisaccharide donor via glycosylation of

In the course of our studies on natural oligosaccharides, we are

interested in the unique glycolipids and glycoproteins found in

various parasites. We initiated a program to study biological func-

tions of various parasitic glycoconjugates and have (a) devised syn-

thetic strategies for their preparation and (b) examined their

antigenicity against patient sera.¹⁰In our previous paper,¹¹we re-

ported the total syntheses of di-, tri-, and tetrasaccharide glycolip-

ids of schistosoma (A, B, and C, Fig. 1) and examined their

antigenicity by an enzyme linked immunosorbent assay (ELISA).

However, these glycolipids did not react with sera of patients suf-

fering from schistosomiasis (data not shown), most likely because

of insufﬁcient size of the presented carbohydrate epitope. In this

study, we describe total syntheses of glycosphingolipids 1–3 (D–

F) together with the non-reducing end fragments of D and E, that

is Le^X(4) and pseudo Le^Y(5). In addition, we describe syntheses

of the non-reducing oligosaccharide fragments 6–9 that are pre-

cursors of glycosphingolipids G–J (Fig. 2). These compounds are ex-

pected to be good probes to clarify the structural requirements for

antigenicity against sera of schistosomiasis patients.

LacNAc acceptor 10¹¹with phenyl 2,3,4-tri-O-benzyl-1-thio-b-

D-

fucopyranoside¹²using N-iodosuccinimide (NIS)/triﬂuoromethane-

sulfonic acid (TfOH) as promoter.¹³However, this resulted in poor

yield of corresponding trisaccharide because of a side reaction lead-

ing to a formation of an NIS–fucose complex. In contrast, signiﬁ-

cantly improved yield of the trisaccharide was obtained with

fucosyl imidate donor 11¹⁴bearing benzoyl protecting groups at

3- and 4-positions. Glycosylation of 10 with 11 in the presence of

trimethylsilyl triﬂuoromethanesulfonate (TMSOTf)¹⁵and 4 Å

molecular sieves (MS 4 Å) in CH₂Cl₂afforded desired trisaccharide

(12) in 79% yield. The nature of the new glycosidic linkage was

determined by the coupling constant of anomeric proton (H-1 of

Fuc, d = 5.33 ppm, J = 4.9 Hz). Removal of the 2,2,2-trichloroethoxy-

carbonyl (Troc) group and N-acetylation of 12 with Zn–Ac₂O–

AcOH,¹⁶followed by debenzylation with catalytic hydrogenolysis

over 10% Pd/C in THF and acetylation afforded O-acetylated inter-

mediate. Zemplén-based deacetylation of the intermediate and

puriﬁcation by Sephadex LH-20 column chromatography furnished

Le^Xtrisaccharide 4. Once we had an access to the trisaccharide

derivative 12, it was used as a precursor for the synthesis of the tar-

get glycosphingolipid 1. Selective removal of 2-(trimethylsilyl)ethyl

(TMS-ethyl) group in 12 with triﬂuoroacetic acid (TFA) in CH₂Cl₂,

followed by treatment with CCl₃CN in the presence of 1,8-diazabi-

2. Results and discussion

2.1. Total synthesis of glycosphingolipid 1 and its non-reducing

end trisaccharide 4

cyclo[5,4,0]-7-undecene (DBU)¹⁷provided corresponding

a-tri-

chloroacetimidate 13 in 73% yield. Glycosylation of 13 with

schisto core derivative 14¹¹in the presence of TMSOTf and MS

4 Å in CH₂Cl₂produced desired pentasaccharide 15 in 78% yield.

The anomeric proton of newly established anomeric center

Glycosphingolipid 1 which contains the Le^Xtrisaccharide and

schisto-core disaccharide [b-

D-GalpNAc-(1?4)-b-D-Glcp] sequence

was prepared by block synthesis of corresponding disaccharide

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

57

β

-

D

-Gal

p

-(1

→

4)-

β

-

D

-Glc

p

NAc-(1

→

3)-

β

-

D

-Gal

p

NAc-(1

→

4)-

β

-

D

-Glc

p

-(1

↔

1)-Cer

(1)

(2)

3

↑

1

α

4)-

-

L

-Fuc

p

α

-

L

-Fuc

p

-(1

→

3)-

β-

D

-Gal

p

-(1

β

-

D

-Glc

3

p

3)-

β

-

D

4)-

β

-

↑

1

α

3)-

-

L

-Fuc

p

β-D

-Gal

p

-(1

→

4)-

β-

D

-Glc

p

NAc-(1

β

-

D

-Glc

p

3)-

β

-

D

4)-

β

-

D

(3)

(4)

3

↑

1

β

-

D

-Gal

p

-(1

→

4)-

β

-D

-Glc

p

NAcOR

α-

L

-Fuc

3

↑

1

α

-

L

-Fuc

p

O

α

-

L

-Fuc

p

-(1

→

3)-

β

-

D

-Gal

p

-(1

→

4)-β

-

D

-Glc

3

NAcOR

(5)

(6)

(7)

(8)

(9)

C₁₃H₂₇

HN

OH

Cer =

↑

O

1

α

-

L

-Fuc

p

OH

β

-

D

-Gal

p

NAc-(1

4)-

β

-

D

-Glc

3

p

↑

R = CH₂CH₂Si(CH₃)₃

1

α-

L

-Fuc

p

α

-

L

-Fuc

p

-(1

→

3)-

-

D

4)-

β

-

D

-Glc

3

p

↑

1

α-

L

-Fuc

α-

p

→

4)-

β

-

D

-Glc

3

p

↑

1

α

-

L

-Fuc

p

-(1

→

2)-

α-

L

-Fuc

p

α

-

L

-Fuc

p

-(1

→

3)-

β-

D

-Gal NAc-(1

p

→

4)-

β

-

D

-Glc NAcOR

p

3

↑

1

α-

L

-Fuc

p

-(1

→

2)-

α

-

L

-Fuc

p

-(1

→

2)-

α

-

L

-Fuc

p

Figure 2. Structures of target compounds (1–9).

appeared as a doublet at d 4.72 (J = 8.0 Hz) indicating exclusive for-

mation of b-anomeric linkage. Removal of the Troc-protecting

group of 15, followed by catalytic hydrogenation over 10% Pd-C in

MeOH–AcOH and acetylation gave protected pentasaccharide ana-

log 16. Compound 16 was exposed to TFA and resulted hemiacetal

was converted into glycosyl imidate 17 using standard conditions.

In a synthesis of glycosyl ceramide, a phytoceramide acceptor gives

lower yield compared to, for example, an azidosphingosine. How-

ever, we employed a ceramide type acceptor because of the easy

conversion to the ﬁnal product. TMSOTf-promoted glycosylation

(1?4)-[a-L-Fucp-(1?3)]-b-D-GlcpNAc-}, at the non-reducing end.

Prior to the synthesis of glycosphingolipid 2, pseudo Le^Ytetrasac-

charide derivative 5 was synthesized (Scheme 2). Galactopyranosyl

donor 22 was obtained from phenyl 4,6-O-benzylidene-1-thio-b-D-

galactopyranoside (20),¹⁹by a two-step procedure. Regioselective

chloroacetylation of the in situ prepared stannylidene derivative

of 20 with chloroacetyl chloride produced 21 in 75% yield. This

chloroacetylation was found to be highly regio-speciﬁc in this case.

Benzoylation of 21 in pyridine resulted in partial replacement of

the chloroacetyl group with a benzoyl group. Thus, this reaction

had to be carried out under a milder condition to prevent cleavage

of the chloroacetyl group and treatment with benzoyl chloride in

CH₂Cl₂/pyridine (5:3) mixture successfully gave glycosyl donor

22 in 85% yield. Glycosylation of the donor 22 with acceptor 23¹³

in the presence of NIS, TfOH, and MS 4 Å in CH₂Cl₂afforded desired

disaccharide 24 in 84% yield. The anomeric proton of the galactose

unit appeared as a doublet at d 4.63 (J = 8.3 Hz), indicating a b-link-

age formation. The two chloroacetyl groups in 24 were cleaved by

treatment with pyridine/H₂O yielding diol 25 in 70% yield. Glyco-

sylation of the diol acceptor 25 with glycosyl donor 11 in the pres-

ence of TMSOTf produced tetrasaccharide 26 in 82% yield. The

of phytoceramide

acceptor, (2S,3R,4R)-3,4-di-O-benzoyl-2-

hexadecanamido-octadecane-1,3,4-triol 18¹⁸with glycosyl imidate

donor 17 afforded desired protected glycosphingolipid-based b-

glycoside 19 in 23% yield. Finally, standard deacetylation of 19

and puriﬁcation by column chromatography on Sephadex LH-20

furnished glycolipid 1 (Scheme 1). The structure and purity of 1

were demonstrated by its ¹H NMR and HR-FABMS data.

2.2. Total synthesis of glycosphingolipid 2 and its non-reducing

end tetrasaccharide 5

Glycosphingolipid 2 contains pseudo Le^Ytetrasaccharide {

Fucp-(1?3)]-b- -Galp-(1?4)-[ -Fucp-(1?3)]-b- -GlcpNAc-}, in

which the terminal Fuc-residue is bound to 3-position of galactose

instead of 2-position in Le^Ystructure {

-Fucp-(1?2)-b- -Galp-

a-L

-

presence of two a-inter fucosidic linkages in 26 was indicated by

D

a-

L

D

two doublets at d 5.36 and 5.04 ppm both showing the same small

homonuclear coupling constant of 3.6 Hz in the ¹H NMR spectrum.

Removal of the Troc-protecting group of 26 was achieved with Zn

L

D

58

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

Scheme 1. Reagents and conditions: (a) TMSOTf, MS 4 Å, CH₂Cl₂, 12 79%, 15 78%, 19 23%; (b) (1) Zn, Ac₂O, AcOH; (2) Pd-C/H₂, THF-MeOH; (3) Ac₂O/pyridine, 58% (three

steps); (4) MeONa, MeOH, 86%; (c) (1) TFA, CH₂Cl₂; (2) CCl₃CN, DBU, CH₂Cl₂, 73% (two steps); (d) (1) Zn, Ac₂O, AcOH; (2) Pd-C/H₂, MeOH/AcOH; (3) Ac₂O/pyridine, 37% (three

steps); (e) (1) TFA, CH₂Cl₂; (2) CCl₃CN, DBU, CH₂Cl₂, quant. (two steps); (f) MeONa, 1,4-dioxane/MeOH, 65%.

Scheme 2. Reagents and conditions: (a) (1) Bu₂SnO, toluene; (2) Bu₄NBr, chloroacetyl chloride, DMF, 75%; (b) benzoyl chloride, CH₂Cl₂/pyridine, 85%; (c) NIS, TfOH, MS 4 Å,

CH₂Cl₂, 84%; (d) pyridine, H₂O, 70%; (e) TMSOTf, MS 4 Å, CH₂Cl₂, 82%; (f) (1) Zn, Ac₂O, AcOH; (2) Pd-C/H₂, AcOH/MeOH; (3) Ac₂O/pyridine; (4) NaOMe, MeOH, 23% (four steps).

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

59

Scheme 3. Reagents and conditions: (a) (1) CF₃CO₂H/CH₂Cl₂, MS 4 Å; (2) DBU, CCl₃CN, CH₂Cl₂, 27 64% (two steps), 32 82% (two steps); (b) TMSOTf, MS 4 Å, CH₂Cl₂, 28 65%, 30

72%, 33 28%; (c) pyridine, H₂O, quant.; (d) 1) Zn, Ac₂O, AcOH; (2) Pd-C/H₂, MeOH/AcOH; (3) Ac₂O/pyridine, 43% (three steps); (e) MeONa, dioxane/MeOH, quant.

Ac₂O and AcOH mixture, followed by catalytic hydrogenation over

10% Pd-C in MeOH–AcOH, acetylation and deacetylation provided

tetrasaccharide 5 which was puriﬁed by column chromatography

on Sephadex LH-20 (Scheme 2).

of the Troc groups of 30 with Zn–AcOH followed by debenzylidena-

tion and debenzylation with catalytic hydrogenolysis over 10% Pd/

C in MeOH/AcOH and acetylation afforded 31 in 43% yield over

three steps. Selective removal of the TMS-ethyl group in 31 with

TFA, followed by exposure of resulted hemiacetal to CCl₃CN and

Hexasaccharide-based core of glycosphingolipid 2 containing

pseudo Le^Ytetrasaccharide moiety was planned to be synthesized

by double glycosylation with fucosyl imidate 11 of tetrasaccharide

acceptor 29 which can be obtained by glycosylation of donor 27

with schisto core disaccharide acceptor 14 (Scheme 3). Disaccha-

ride donor 27 was prepared from protected disaccharide 24 by

cleavage of the anomeric TMS-ethyl protecting group and conver-

sion of resulted hemiacetal into imidate. During the deprotection

of TMS-ethyl group, presence of MS 4 Å was necessary to prevent

hydrolysis of the benzylidene acetal in the molecule. Linear tetra-

saccharide acceptor 29 was synthesized by TMSOTf promoted gly-

cosylation of acceptor 14 with the donor 27 followed by removal of

the chloroacetyl group with aqueous pyridine in 65% yield over

two steps. Having obtained the tetrasaccharide acceptor 29, we

performed TMSOTf-promoted glycosylation of 29 with fucopyr-

anosyl donor 11 in dichloromethane at ꢀ60 °C, which produced

desired hexasaccharide 30 (72%), as evidenced by ¹H NMR spec-

troscopy (H-1 of Fuc a,b, d 5.30 and 5.03 ppm, J = 3.6 Hz). Reduction

DBU afforded corresponding

a-trichloroacetimidate 32. TMSOTf-

promoted glycosylation of phytosphingosine-based lipid 18 with

the glycosyl donor 32 was carried out in the presence of TMSOTf

and MS 4 Å to afford desired b-glycoside 33 in 23% yield. Finally,

removal of the acyl groups in 33 under Zemplén conditions and

column chromatography on Sephadex LH-20 furnished desired gly-

colipid 2 (Scheme 3). The structure and purity of 2 were demon-

strated by its ¹H NMR and HR-FABMS data.

2.3. Total synthesis of glycosphingolipid 3

Glycosphingolipid 3 is a hexasaccharide containing an additional

GlcpNAc moiety between the Le^Xtrisaccharide and the schisto core

disaccharide. The hexasaccharide portion of 3 can be synthesized by

glycosylation of a linear trisaccharide-based acceptor 37 with the

previously prepared trisaccharide imidate donor 13. The synthesis

of acceptor 37 started from known phenyl 4,6-O-benzylidene-2-

60

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

Scheme 4. Reagents and conditions: (a) chloroacetyl chloride, CH₂Cl₂/pyridine, 99%; (b) NIS, TfOH, MS 4 Å, CH₂Cl₂, 73%; (c) pyridine, H₂O, 86%; (d) TMSOTf, MS 4 Å, CH₂Cl₂, 38

63%, 41 20%; (e) (1) Zn, Ac₂O, AcOH; (2) Pd-C/H₂, MeOH/AcOH; (3) Ac₂O/pyridine, 40% (three steps); (f) (1) CF₃CO₂H/CH₂Cl₂, MS 4 Å; (2) DBU, CCl₃CN, CH₂Cl₂, 77% (two steps);

(g) MeONa, dioxane/MeOH, 79%.

Scheme 5. Reagents and conditions: (a) TMSOTf, MS 4 Å, CH₂Cl₂, 43 79%, 45 80%; (b) pyridine, H₂O, 70%; (c) (1) Zn, Ac₂O, AcOH; (2) Pd-C/H₂, MeOH/AcOH; (3) Ac₂O/pyridine,

44% (three steps); (d) MeONa, MeOH, 94%.

deoxy-2-(2,2,2-trichloroethoxy-carbonylamino)-1-thio-b-

D

-gluco-

2.4. Synthesis of oligosaccharide fragments 6–9

pyranoside (34).²⁰At ﬁrst chloroacetylation of the free hydroxyl

group of 34 using standard conditions provided acetate 35. The thio-

glycoside donor 35 was then condensed with the schisto core accep-

tor 14 using NIS/TfOH promoter to furnish trisaccharide 36 in 73%

yield. Hydrolysis of the chloroacetyl group in 36 with aqueous pyr-

idine produced trisaccharide acceptor 37 in 86% yield. Glycosylation

of 37 with 13 produced hexasaccharide 38 which was further con-

verted into glycosphingolipid 3 by a series of reactions as described

previously. The structure and purity of 3 were demonstrated by its

¹H NMR and HR-FABMS data (Scheme 4).

Glycosphingolipids F–J contain a common b-

D-GlcpNAc-(1?3)-

b-

D

-GalpNAc-(1?4)-b- -Glcp-sequence. The synthesis of glyco-

D

sphingolipids G–J can be envisaged by coupling of the common

trisaccharide sequence to oligosaccharide fragments 6–9 which

corresponded to the non-reducing ends of G–J, respectively.

Oligosaccharide fragments 6–9 are not only important intermedi-

ates toward the synthesis of glycosphingolipids G–J but also impor-

tant molecular probes to explore their potential use as diagnostic

tools for serum-based-antigen recognition in patients and

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

61

Scheme 6. Reagents and conditions: (a) (1) guanidinium nitrate, MeONa, MeOH/CHCl₃; (2) benzaldehyde dimethyl acetal, NaHSO₄ꢁSiO₂, CH₃CN 58% (two steps); (b) TMSOTf,

MS 4 Å, CH₂Cl₂; (c) (1) Zn, Ac₂O, AcOH; (2) Pd-C/H₂, MeOH/AcOH; (3) Ac₂O/pyridine; 4) MeONa, MeOH, 23% (four steps).

Scheme 7. Reagents and conditions: (a) (1) 2,2,2-trichloroethanol, NaHSO₄ꢁSiO₂; (2) 2,2-dimethyl propane, NaHSO₄ꢁSiO₂, acetone 59% (two steps); (b) TMSOTf, MS 4 Å,

CH₂Cl₂, 52 75%, 55 80%; (c) (1) 80% AcOH; 2) Ac₂O/pyridine, 53 86%, 56 63%; (d) (1) Zn, AcOH; (2), CF₃CO₂H/CH₂Cl₂; (3) DBU, CCl₃CN, CH₂Cl₂, 54 77%, 57 76% (three steps).

62

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

Scheme 8. Reagents and conditions: (a) TMSOTf, MS 4 Å, CH₂Cl₂, 58 67%, 59 43%; (b) (1) Zn, Ac₂O, AcOH; (2) Pd-C/H₂, MeOH/AcOH; (3) Ac₂O/pyridine; (4) MeONa, MeOH, 8

32%, 9 35% (four steps).

immunomodulator. The desired oligosaccharide fragments 6–9

were prepared by a systematic and integrated approach (Schemes

tive fucosylation of acceptor 47 was achieved by using a reduced

amount of 11 (3 equiv). In this case desired 49, which is the inter-

mediate to prepare 8 and 9, was obtained in 66% yield together

with recovered acceptor 47 (11%).

Difucosyl donor 54 and trifucosyl donor 57, which are the build-

ing blocks of 8 and 9, respectively, were obtained from fucosyl

derivative 51 which was prepared by trichloroethylation and iso-

5–8). Trisaccharide fragment 6 is the LDN-F antigen {b-

(1?4)-[ -Fucp-(1?3)]-b- -GlcpNAc} while tetrasaccharide frag-

ment 7 contains an additional 1?3 fucosyl GalpNAc sequence at

D-GalpNAc-

a-L

D

a

the non-reducing end. Oligosaccharide fragments 8 and 9, precur-

sors of glycosphingolipids I and J, can be derived from fragment 7

to which di and tri

attached at the 3-position of GlcpNAc.

a

1?2 fucopyranosyl repeating units are to be

propylidenation of

of 51 with the common fucosyl donor 11 provided disaccharide

52 in 75% yield. The -glycosidic linkage between the fucose units

L-fucose 50. TMSOTf-promoted glycosylation

Trisaccharide 6 was synthesized by a coupling of the fucopyr-

anosyl donor 11 with a disaccharide acceptor 44. The acceptor 44

was obtained by a condensation of acceptor 23 with a known glyco-

syl imidate 42²¹to afford disaccharide 43 followed by hydrolysis of

chloroacetyl group. TMSOTf-promoted glycosylation of fucosyl

imidate 11 with acceptor 44 produced trisaccharide 45 which was

subjected to standard deblocking conditions as described for the

preparation of 1 to afford unprotected trisaccharide 6 (Scheme 5).

Compound 47 was chosen as a common acceptor for the syn-

thesis of oligosaccharide fragments 7–9. It was obtained from

disaccharide intermediate 43 by ester hydrolysis followed by ben-

zylidenation²²of 4⁰,6⁰-diol using benzaldehyde dimethyl acetal in

58% yield. Three glycosylation conditions of acceptor 47 with the

fucosyl donor 11 were tested (Table 1). Tetrasaccharide 48, which

can be converted into 7, was obtained by condensation of 47 with

large excess of 11 (6 equiv) in the presence of TMSOTf. Deprotec-

tion of 48 as previously described for oligosaccharide 6 afforded

target tetrasaccharide 7 (Scheme 6). On the other hand, regioselec-

a

in 52 was established from the homonuclear coupling constant (d,

J = 3.3 Hz) of the newly introduced anomeric proton signal at

d = 5.58. Compound 52 was converted into 53 by deisopropylide-

nation followed by acetylation. The anomeric trichloroethyl

protecting group in 53 was selectively cleaved by exposure to

Zn/AcOH and resulted hemiacetal was converted into imidate 54

using standard conditions. Coupling of acceptor 51 to disaccharide

donor 54 afforded trisaccharide 55 which was deprotected and

activated to provide trisaccharide imidate 57 (Scheme 7). Similarly,

TMSOTf-promoted glycosylation of trisaccharide acceptor 49 with

glycosyl donors 54 and 57 afforded desired a-glycosides 58 (67%)

and 59 (43%), respectively. Finally, standard deprotection of 58

and 59, followed by puriﬁcation by column chromatography on

Sephadex LH-20 furnished penta- and hexasaccharides 8 and 9,

respectively (Scheme 8). All the intermediates and the unprotected

target oligosaccharides were fully characterized by NMR spectros-

copy and MS spectrometry.

3. Conclusions

Table 1

In summary, a systematic and integrated approach for the syn-

thesis of three glycosphingolipids 1–3 found in the parasite, Schis-

tosoma mansoni and six non-reducing end oligosaccharide

fragments 4–9 has been accomplished. Fragments 6–9 are ex-

pected to be probes to clarify structural requirements for antigen

recognition in glycosphingolipids G, H, I, and J. Although, S. man-

soni possesses unique carbohydrate antigens, so far it has not been

possible to exploit them for diagnostic and therapeutic purposes.

As isolation of carbohydrate antigens from natural sources requires

time consuming procedures and skill, the demand for homoge-

neous synthetic carbohydrate antigens is growing. Effective

Fucosylation under various conditions

Ent. Donor 11 Acceptor 47 Time Promoter Solvent Temp Yield %

No. (equiv)

(

l

mol)

(h)

(equiv)

(°C)

1

2

3

6

62.7

94.9

114

18

TMSOTf

(0.15)

TMSOTf

(0.1)

TMSOTf

(0.15)

CH₂Cl₂ꢀ40

48: 78

49: 28^a

49: 66^b

1.5

3

CH₂Cl₂ꢀ40

a

With 52% recovery of 47.

With 11% recovery of 47.

b

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

63

synthetic methods are expected to contribute toward development

of rapid, cost-effective, and reliable diagnostic tools in the near

future.

74.4, 74.1, 73.7, 73.6, 73.2, 72.8, 70.94, 70.88, 70.8, 69.2, 67.6,

67.2, 67.0, 64.8, 61.1, 60.3, 59.4, 21.0, 20.8, 20.7, 20.6, 18.0, 16.0,

14.2, ꢀ1.4. HR-FABMS: calcd for

C₆₂H₇₄Cl₃NO₂₂SiNa: m/z

1340.3435; found: m/z 1340.3441 [M+Na]⁺.

4. Experimental

4.1. General

4.3. 2-(Trimethylsilyl)ethyl b-D-galactopyranosyl-(1?4)-[a-L-

fucopyranosyl-(1?3)]-2-acetamido-2-deoxy-b-D-

glucopyranoside (4)

Optical rotations were measured with a Jasco P-1020 digital polar-

imeter. ¹H and ¹³C NMR spectra were recorded with a Varian 500 FT

NMR spectrometer. Me₄Si and acetone were used as internal stan-

dards for CDCl₃and CD₃OD, respectively. MALDI-TOFMS was re-

corded on an AB SCIEX Voyager RP mass spectrometer. High-

resolution mass spectra were recorded on a JEOL JMS-700 under

FAB conditions. TLC was performed on Silica Gel 60 F254 (E. Merck)

with detection by quenching of UV ﬂuorescence and by charring with

10% H₂SO₄. Column chromatography was carried out on Silica Gel 60

(E. Merck) and Iatrobeads 6RS-8060 (Iatron Lab., Tokyo). 2-(Trimeth-

A mixture of 12 (111 mg, 76.4 lmol) and zinc powder (1.2 g) in

AcOH–Ac₂O (1:1, 12 mL) was stirred for 18 h at 55 °C. After com-

pletion of the reaction, the mixture was ﬁltered and the ﬁltrate

was diluted with water and extracted with CHCl₃. The extract

was washed with water, dried (MgSO₄), and concentrated. The res-

idue was dissolved in THF–MeOH (1:1, 3 mL) and hydrogenolysed

under hydrogen (0.4 MPa) in the presence of 10% Pd/C (300 mg) for

18 h at room temperature. The mixture was then ﬁltered and con-

centrated. The residue was acetylated with acetic anhydride (2 mL)

in pyridine (3 mL). The reaction mixture was poured into ice H₂O

and extracted with CHCl₃. The extract was washed sequentially

with 5% HCl, aq NaHCO₃, and brine, dried (MgSO₄), and concen-

trated. The residue was puriﬁed by silica gel column chromatogra-

phy using 2:1 CHCl₃–acetone as eluent to give acylated

ylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-

benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-b-

pyranoside (10),¹²2-(trimethylsilyl)ethyl 4,6-O-benzylidene-2-

deoxy-2-(2,2,2-trichloroethoxy carbonylamino)-b- -galactopyrano-

syl-(1?4)-2,3-di-O-benzoyl-6-O-benzyl-b-

-glucopyranoside (14),¹¹

phenyl 4,6-O-benzylidene-1-thio-b-

-galactopyranoside (20),¹⁹2-

(trimethylsilyl)ethyl 6-O-benzyl-3-O-chloroacetyl-2-deoxy-2-(2,2,

2-trichloro-ethoxycarbonyl amino)-b-

-glucopyranoside (23),¹¹

D-galactopyranosyl-(1?4)-6-O-

D-gluco-

D

trisaccharide intermediate (48.3 mg, 58%). [

a

]

D

ꢀ51.4 (c 0.3,

D

MeOH); ¹H NMR (600 MHz, CD₃OD): d 5.76 (d, 1H, J_3,4= 3.3 Hz,

H-4 of Fuc), 5.62 (d, 1H, NH), 5.60 (dd, 1H, J_2,3= 10.7 Hz, H-3 of

Fuc), 5.54 (d, 1H, J_1,2= 4.1 Hz, H-1 of Fuc), 5.47 (d, 1H,

J_3,4= 3.8 Hz, H-4 of Gal), 5.34 (dd, 1H, H-2 of Fuc), 5.16 (dd, 1H,

J_{1,2 =}7.3 Hz, J_2,3= 10.2 Hz, H-2 of Gal), 5.04–4.99 (m, 2H, H-3 of

Gal, H-5 of Fuc), 4.69 (d, 1H, J_1,2= 6.9 Hz, H-1 of GlcN), 4.62 (dd,

1H, J_5,6a= 2.8 Hz, J_6a,6b= 11.8 Hz, H-6a of GlcN), 4.58 (dd, 1H,

J_5,6a= 6.5 Hz, J_6a,6b= 10.6 Hz, H-6a of Gal), 4.52 (d, 1H, H-1 of

Gal), 4.41 (dd, 1H, J_5,6a= 7.4 Hz, H-6b of Gal), 4.22–4.18 (m, 2H,

H-3 of GlcN, H-6b of Gal), 3.93 (dd, 1H, H-5 of Gal), 3.91–3.86

(m, 2H, H-4 of GlcN, CH₂CH₂Si(CH₃)₃), 3.70 (br d, H-2 of GlcN),

3.63–3.60 (m, 1H, H-5 of GlcN), 3.54–3.49 (m, 1H, CH₂CH₂Si(CH₃)₃),

2.27, 2.17, 2.15, 2.10, 2.08, 2.01, 1.99 (each s, 21H, 7 Ac), 1.30 (d,

3H, J_5,6= 6.6 Hz, H-6 of Fuc), 0.96–0.85 (m, 2H, CH₂CH₂Si(CH₃)₃),

0.001 (s, 9H, Si(CH₃)₃). ¹³C NMR (CDCl₃):d 171.2, 170.6, 170.5,

170.3, 170.0, 169.3, 166.0, 165.3, 133.3, 133.0, 129.6, 129.51,

129.47, 128.5, 128.2, 100.5 (C-1 of Gal), 99.3 (C-1 of GlcN), 95.4

(C-1 of Fuc), 74.6, 73.1, 72.8, 72.00, 72.19, 70.9, 69.5, 69.0, 68.8,

68.6, 66.9, 66.8, 64.9, 64.8, 62.3, 61.0, 53.8, 31.7, 30.9, 29.7, 23.5,

20.97, 20.95, 20.8, 20.68, 20.65, 20.6, 20.5, 18.0, 16.0, ꢀ1.5. MAL-

DI-TOFMS: calcd for C₅₁H₆₇NO₂₃SiNa ([M+Na]⁺), m/z 1112; found

1112. To a solution of the intermediate (17.3 mg) in MeOH

(1.5 mL) was added NaOMe (15.0 mg) at 40 °C. The mixture was

stirred for 5 h and then neutralized with Amberlite IR 120 [H⁺].

The mixture was ﬁltered and concentrated. The product was

puriﬁed by Sephadex LH-20 column chromatography with

MeOH to give 4 (5.3 mg, 86%). ¹H NMR (500 MHz, CD₃OD): d 4.42

(d, 1H, J_1,2= 4.5 Hz, H-1 of Fuc), 3.91 (d, 1H, J_1,2= 7.5 Hz, H-1 of

Gal), 3.84 (d, 1H, J_1,2= 7.5 Hz, H-1 of GlcN). HR-FABMS: calcd

D

were prepared as reported. Benzoylceramide 18 was prepared

from phytosphingosine, which was purchased from Degussa (The

Netherlands) by the conventional four-step procedure.¹⁸

4.2. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-D-

galactopyranosyl-(1?4)-[3,4-di-O-benzoyl-2-O-benzyl-

a-L-

fucopyranosyl-(1?3)]-6-O-benzyl-2-deoxy-2-(2,2,2-

trichloroethoxycarbonylamino)-b-D-glucopyranoside (12)

A mixture of 10 (335 mg, 0.38 mmol), 12 (581 mg, 0.96 mmol,

2.5 equiv), and MS 4 Å (500 mg) in dry CH₂Cl₂(4 mL) was stirred

for 2 h at room temperature, then cooled to ꢀ30 °C. TMSOTf

(34.8 lL, 0.19 mmol) was added, and the mixture was stirred for

18 h at ꢀ30 °C, then neutralized with Et₃N. The precipitates were

ﬁltrated off and washed with CHCl₃. The combined ﬁltrate and

washings were washed with water, dried (MgSO₄), and concen-

trated. The product was puriﬁed by silica gel column chromatogra-

phy (2:1 hexane–EtOAc) to give 12 (399 mg, 79%) as an amorphous

powder. [

a]

D

ꢀ61.7 (c 3.2); ¹H NMR (600 MHz, CDCl₃) d 7.95–7.21

(m, 20H, 4 Ph), 5.68–5.64 (m, 2H, H-3, 4 of Fuc), 5.37 (d, 1H, NH),

5.33 (d, 1H, J_1,2= 4.9 Hz, H-1 of Fuc), 5.32 (d, 1H, J_3,4= 3.8 Hz, H-4

of Gal), 5.13 (br dd, 1H, H-5 of Fuc), 5.05 (dd, 1H, J_1,2= 8.2 Hz,

J_2,3= 10.2 Hz, H-2 of Gal), 4.92 (d, 1H, J_1,2= 8.3 Hz, H-1 of GlcN),

4.82–4.76 (m, 4H, H-3 of Gal, benzylmethylene, NHCOCH₂CCl₃),

4.71–4.66 (m, 2H, 2 benzylmethylene), 4.65 (d, 1H, H-1 of Gal),

4.49–4.45 (m, 2H, H-6a of Gal, benzylmethylene), 4.37 (dd, 1H,

J

_5,6b= 7.3 Hz, J _6b,6a= 11.6 Hz, H-6b of Gal), 4.33 (t, 1H, J

=

2,3

for

C₆₂H₇₄Cl₃NO₂₂SiNa: m/z 652.2613; found: m/z 652.2642

[M+Na]⁺.

J_3,4= 9.3 Hz, H-3 of GlcN), 4.15 (dd, 1H, J_1,2= 3.9 Hz, J_2,3= 10.2 Hz,

H-2 of Fuc), 4.06 (t, 1H, J_4,5= 9.1 Hz, H-4 of GlcN), 3.96–3.92 (m,

1H, CH₂CH₂Si(CH₃)₃), 3.81 (dd, 1H, J _5,6a= 3.0 Hz, J _6a,6b= 10.7 Hz,

H-6a of GlcN), 3.77 (dd, 1H, J_5,6b= 1.6 Hz, H-6b of GlcN), 3.55 (t,

4.4. 2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl-(1?4)-[3,4-di-O-

benzoyl-2-O-benzyl-a-L-fucopyranosyl-(1?3)]-6-O-benzyl-2-

deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-b-D-

1H,

J _5,6a= J _5,6b= 6.9 Hz, H-5 of Gal), 3.53–3.48 (m, 1H,

CH₂CH₂Si(CH₃)₃), 3.42 (br d, 1H, H-5 of GlcN), 3.16 (br d, 1H, H-2

of GlcN), 2.22, 2.06, 1.99, 1.96 (each s, 12H, 4 Ac), 1.25 (d, 3H, H-

6 of Fuc), 0.95–0.88 (m, 2H, CH₂CH₂Si(CH₃)₃), ꢀ0.01 (s, 9H,

Si(CH₃)₃). ¹³C NMR (CDCl₃):d 170.6, 170.3, 170.0, 168.9, 165.9,

165.1, 153.6, 137.63, 137.60, 133.1, 132.7, 129.9, 129.8, 129.7,

129.5, 128.6, 128.43, 128.38, 128.13, 128.10, 128.07, 128.0, 127.8,

99.6 (C-1 of Gal), 98.7 (C-1of GlcN), 97.2 (C-1 of Fuc), 95.5, 74.8,

glucopyranosyl trichloroacetimidate (13)

To a solution of 12 (247 mg, 0.19 mmol) in CH₂Cl₂(3.0 mL)

cooled to 0 °C was added CF₃CO₂H (1.5 mL), and the mixture was

stirred for 3 h at room temperature and concentrated. EtOAc and

toluene (1:2) were added and evaporated to give the reducing su-

gar. To a solution of the residue in CH₂Cl₂(3.0 mL) cooled at 0 °C

64

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

were added DBU (42.0

l

L, 0.28 mmol) and CCl₃CN (191

l

L,

99.5 (C-1 of GalN), 99.4 (C-1 of GlcN), 95.6 (C-1 of Fuc). MALDI-

TOFMS: calcd for C₈₅H₁₀₄N₂O₃₈SiNa: m/z 1812; found: m/z 1812

[M+Na]⁺.

1.87 mmol). The reaction mixture was stirred for 6 h at room tem-

perature. After completion of the reaction, the mixture was con-

centrated. The residue was puriﬁed by silica gel column

chromatography using 100:1 chloroform–acetone as eluent to give

4.7. 2,3,4,6-Tetra-O-acetyl-b-

acetyl-3,4-di-O-benzoyl- -fucopyranosyl-(1?3)]-2-

acetamido-6-O-acetyl-2-deoxy-b- -glucopyranosyl-(1?3)-2-

acetamido-4,6-di-O-acetyl-2-deoxy-b- -galactopyranosyl-

-glucopyranosyl

D-galactopyranosyl-(1?4)-[2-O-

13 (187 mg, 73%). [

a]

D

ꢀ85.6 (c 1.3); ¹H NMR (600 MHz, CDCl₃) d

a-L

6.46 (d, 1H, J_1,2= 3.3 Hz, H-1 of GlcN), 5.49 (d, 1H, J_1,2= 3.3 Hz, H-

1 of Fuc), 4.61 (d, 1H, J_1,2= 8.3 Hz, H-1 of Gal). ¹³C NMR (CDCl₃):

d 170.7, 170.4, 170.0, 169.3, 166.0, 165.2, 154.6, 137.5, 137.4,

137.3, 133.1, 132.7, 129.8, 128.74, 128.66, 128.6, 128.5, 128.4,

128.3, 128.22, 128.19, 128.1, 127.7, 99.8 (C-1 of Gal), 97.5 (C-1 of

Fuc), 95.3, 91.7 (C-1of GlcN), 74.7, 74.2, 73.7, 73.1, 72.8, 72.6,

72.1, 70.9, 70.8, 70.4, 69.4, 67.5, 66.9, 65.0, 61.1, 61.0, 59.3, 20.8,

20.7, 20.64, 20.57, 16.0.

D

(1?4)-6-O-acetyl-2,3-di-O-benzoyl-a-D

trichloroacetimidate (17)

Compound 17 was prepared from 16 (34.0 mg, 18.8 lmol) as

described for preparation of 13. The product was puriﬁed by silica

gel column chromatography (5:1 chloroform–methanol) to give 17

25

(34.4 mg, quant.). [

a]

ꢀ40.5 (c 0.85, CHCl₃); ¹H NMR (600 MHz,

D

4.5. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-D-

galactopyranosyl-(1?4)-[3,4-di-O-benzoyl-2-O-benzyl-

fucopyranosyl-(1?3)]-6-O-benzyl-2-deoxy-2-(2,2,2-

trichloroethoxycarbonylamino)-b-

O-benzylidene-2-deoxy-2-(2,2,2-

trichloroethoxycarbonylamino)-b-

CDCl₃): d 4.70 (d, 1H, J_1,2= 3.5 Hz, H-1 of Glc), 5.44 (d, 1H,

J _1,2= 4.1 Hz, H-1 of Fuc), 4.82 (d, 1H, J _1,2= 8.5 Hz,H-1 of GalN),

5.09 (d, 1H, J_1,2= 7.1 Hz, H-1 of GlcN), 4.88 (d, 1H, J_1,2= 8.3 Hz,

H-1 of Gal).

a-L-

D-glucopyranosyl-(1?3)-4,6-

D-galactopyranosyl-(1?4)-

4.8. 2,3,4,6-Tetra-O-acetyl-b-

acetyl-3,4-di-O-benzoyl- -fucopyranosyl-(1?3)]-2-

acetamido-6-O-acetyl-2-deoxy-b- -glucopyranosyl-(1?3)-2-

acetamido-4,6-di-O-acetyl-2-deoxy-b- -galactopyranosyl-

(1?4)-6-O-acetyl-2,3-di-O-benzoy-b- -glucopyranosyl-(1?1)-

D-galactopyranosyl-(1?4)-[2-O-

2,3-di-O-benzoyl-6-O-benzyl-b-D-glucopyranoside (15)

a-L

D

A mixture of 14 (65.7 mg, 65.5

and MS 4 Å (120 mg) in dry CH₂Cl₂(1 mL) was stirred for 5 h at

room temperature, then cooled to ꢀ40 °C. TMSOTf (1.3 L,

7.21 mol) was added, and the mixture was stirred for 12 h at

lmol), 13 (98.3 mg, 72.1 lmol),

D

l

(2S,3S,4R)-3,4-di-O-benzoyl-2-hexadecanamido-octadecane-

1,3,4-triol (19)

l

ꢀ40 °C, then neutralized with Et₃N. The precipitates were ﬁltrated

off and washed with CHCl₃. The combined ﬁltrate and washings

were washed with water, dried (MgSO₄), and concentrated. The

product was puriﬁed by silica gel column chromatography (2:1

hexane–EtOAc) to give 15 (113 mg, 78%). [

A mixture of 17 (54.4 mg, 36.5

zoyl-2-hexadecanamido-octadecane-1,3,4-triol

72.8 mol), and activated MS 4 Å (200 mg) in dry CH₂Cl₂

(0.4 mL) was stirred under an atmosphere of argon for 18 h at

room temperature, then cooled to 0 °C. TMSOTf (5.3 L,

29.2 mol) was added, and the mixture was stirred for 3 h at

l

mol), (2S,3S,4R)-3,4-di-O-ben-

18 (55.7 mg,

25

l

a]

D

ꢀ37.0 (c 1.5,

CHCl₃); ¹H NMR (600 MHz, CDCl₃): d 5.95 (d, 1H, NH of GlcN),

5.67 (d, 1H, NH of GalN), 5.30 (d, 1H, J_1,2= 3.3 Hz, H-1 of Fuc),

4.99 (br d, 1H, H-1 of GalN), 4.72 (br d, 1H, H-1 of GlcN), 4.66 (d,

1H, J_1,2= 8.0 Hz, H-1 of Glc), 4.64 (d, 1H, J_1,2= 7.0 Hz, H-1 of Gal).

¹³C NMR (150 MHz, CDCl₃):d 100.4 (C-1 of Glc), 99.8 (C-1 of GalN),

99.3 (C-1 of Gal), 98.7 (C-1 of GlcN), 97.1 (C-1 of Fuc). MALDI-TOF-

MS: calcd for C₁₀₅H₁₁₄Cl₆N₂O₃₅SiNa: m/z 2224; found: m/z 2224c.

l

room temperature, then neutralized with Et₃N. The solids were ﬁl-

trated off and washed with CHCl₃. The combined ﬁltrate and

washings were washed with brine, dried (MgSO₄), and concen-

trated. The product was puriﬁed by ﬂash silica gel column chro-

matography using 30:1 chloroform–methanol as eluent to give

25

19 (25 mg, 23%). [

a]

ꢀ13.6 (c 0.2, CHCl₃). ¹H NMR (600 MHz,

D

4.6. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-

galactopyranosyl-(1?4)-[2-O-acetyl-3,4-di-O-benzoyl-

fucopyranosyl-(1?3)]-2-acetamido-6-O-acetyl-2-deoxy-b-

glucopyranosyl-(1?3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-b-

-galactopyranosyl-(1?4)-6-acetyl-2,3-di-O-benzoyl-b-

D-

CDCl₃): d 8.12–7.26 (m, 30H, 6 Ph), 6.10 (d, 1H, NH of ceramide),

5.81 (d, 1H, NH of GlcN), 5.43 (d, 1H, J_1,2= 4.1 Hz, H-1 of Fuc),

4.66 (d, 1H, J_1,2= 8.3 Hz, H-1 of GlcN), 4.62 (d, 1H, J_1,2= 7.6 Hz,

H-1 of Glc), 4.60 (d, 1H, J_1,2= 8.3 Hz, H-1 of GalN), 4.55 (d, 1H,

J_1,2= 7.4 Hz, H-1 of Gal). ¹³C NMR (150 MHz, CDCl₃): d 100.5 (C-1

of Glc, Gal), 99.3 (C-1 of GalN, GlcN), 95.6 (C-1 of Fuc). MALDI-

TOFMS: Calcd for C₁₂₈H₁₆₇N₃O₄₃Na: m/z 2457.1; found: m/z 2458.4

[M+Na]⁺.

a-L-

D

-

D

D-

glucopyranoside (16)

A mixture of 15 (113 mg, 51.3 lmol) and zinc powder (1.0 g) in

AcOH–Ac₂O (1:1, 10 mL) was stirred for 18 h at 55 °C. After com-

pletion of the reaction, the mixture was ﬁltered and the ﬁltrate

was diluted with water and extracted with CHCl₃.The extract was

washed with water, dried (MgSO₄), and concentrated. The residue

in MeOH–AcOH (1:1, 4 mL) was hydrogenolysed (0.4 MPa) under

hydrogen in the presence of 10% Pd/C (300 mg) for 18 h at room

temperature, then ﬁltered and concentrated. The residue was acet-

ylated with acetic anhydride (2 mL) in pyridine (3 mL). The reac-

tion mixture was poured into ice H₂O and extracted with CHCl₃.

The extract was washed sequentially with 5% HCl, aq NaHCO₃,

and brine, dried (MgSO₄), and concentrated. The residue was puri-

ﬁed by silica gel column chromatography using 5:1 CHCl₃–acetone

as eluent to give 16 (34.0 mg, 37%). [a]

NMR (600 MHz, CDCl₃): d 5.95 (d, 1H, NH of GlcN), 5.67 (d, 1H, NH

of GalN), 5.43 (d, 1H, J_1,2= 3.9 Hz, H-1 of Fuc), 4.82 (br d, 1H, H-1 of

GalN), 4.72 (d, 1H, J_1,2= 8.2 Hz, H-1 of GlcN), 4.68 (d, 1H,

J_1,2= 8.0 Hz, H-1 of Glc), 4.60 (d, 1H, J_1,2= 8.4 Hz, H-1 of Gal). ¹³C

NMR (150 MHz, CDCl₃):d 100.5 (C-1 of Glc), 100.5 (C-1 of Gal),

4.9. b-

acetamido-2-deoxy-b-

deoxy- -galactopyranosyl-(1?4)-b-

(2S,3S,4R)-2-hexadecanamido-octadecane-1,3,4-triol (1)

D

-Galactopyranosyl-(1?4)-[

-glucopyranosyl-(1?3)-2-acetamido-2-

-glucopyranosyl-(1?1)-

a-L-fucopyranosyl-(1?3)]-2-

D

To a solution of 19 (25 mg, 10.3 lmol) in MeOH (1.0 mL) was

added dioxane (1.0 mL) and NaOMe (20 mg) at 40 °C. The mixture

was stirred for 18 h and then neutralized with Amberlite IR 120

[H⁺]. The mixture was ﬁltered and concentrated. The product was

puriﬁed by Sephadex LH-20 column chromatography with 1:1

ꢀ26.4 (c 0.61, CHCl₃); ¹H

25

CHCl₃–MeOH to give 1 (9.5 mg, 65%). [

a

]

ꢀ7.0 (c 0.08, CHCl₃/

D

MeOH = 1:1). ¹H NMR (600 MHz, CDCl₃/CD₃OD = 1:1): d 4.90 (d,

1H, J_1,2= 4.0 Hz, H-1 of Fuc), 4.52 (d, 1H, J_1,2= 8.3 Hz, H-1 of GlcN),

4.45 (d, 1H, J_1,2= 8.5 Hz, H-1 of GalN), 4.36 (d, 1H, J_1,2= 7.6 Hz, H-1

of Gal), 4.30 (d, 1H, J_1,2= 7.5 Hz, H-1 of Glc). HR-FABMS: calcd for

C

₆₈H₁₂₅N₃NaO₂₈: m/z 1454.8347; found: m/z 1454.8300 [M+Na]⁺.

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

65

4.10. Phenyl 4,6-O-benzylidene-3-O-chloroacetyl-1-thio-b-

galactopyranoside (21)

D

-

Gal), 4.32 (br d, 1H, H-6a of Gal), 4.25, 4.15 (each d, 2H, COCH₂Cl

of GlcN), 4.07–4.01 (m, 2H, H-4 of GlcN, H-6b of Gal), 3.97 (d, 1H,

COCH₂Cl of Gal), 3.93–3.87 (m, 2H, COCH₂Cl of Gal,

CH₂CH₂Si(CH₃)₃), 3.73 (dd, 1H, H-2 of GlcN), 3.59 (dd, 1H, J

_5,6a= 2.8 Hz, J_6a,6b= 11.0 Hz, H-6ª of GlcN), 3.51–3.45 (m, 2H, H-6b

of GlcN, CH₂CH₂Si(CH₃)₃), 3.34 (br s, 1H, H-5 of Gal), 3.27 (br d,

1H, H-5of GlcN), 0.91–0.88 (m, 2H, CH₂CH₂Si(CH₃)₃), ꢀ0.06 (s, 9H,

Si(CH₃)₃). ¹³C NMR (150 MHz, CDCl₃):d 167.7, 167.1, 164.3, 154.2,

138.0, 137.0, 133.4, 129.6, 129.3, 129.0, 128.6, 128.5, 128.4, 128.2,

128.1, 126.3, 101.3, 100.5 (C-1 of GlcN), 100.0 (C-1 of Gal), 95.5,

74.5, 74.29, 74.26, 74.0, 73.5, 73.0, 69.1, 68.7, 67.3, 67.2, 66.1,

55.9, 40.8, 40.5, 18.0, ꢀ1.4. HR-FABMS: calcd for C₄₅H₅₂Cl₅NO₁₅Si-

Na: m/z 1072.1447; found 1072.1423: m/z [M+Na]⁺.

A solution of 20 (2.17 g, 6.02 mmol) and dibutyltin oxide (2.3 g,

9.03 mmol) in 60 mL of dry toluene was stirred under reﬂux for

5 h. Toluene was distilled off, the stannylidene derivative was dis-

solved in DMF (15 mL) and Bu₄NBr (2.4 g, 7.23 mmol) and chloro-

acetyl chloride (0.72 mL, 9.03 mmol) were added at room

temperature. After being stirred for 5 h, the solution was concen-

trated. The residue was dissolved with EtOAc, washed with aq

NaCl, dried (MgSO₄), and concentrated. The residue was puriﬁed

by silica gel column chromatography using 20:1 toluene–ethyl

acetate to give 21 (1.97 g, 75%). [

a]

ꢀ22.0 (c 1.9, CHCl₃). ¹H

D

NMR (600 MHz, CDCl₃): d 7.68–7.25 (m, 10H, 2Ph), 5.59 (s, 1H,

PhCH), 5.09 (dd, 1H, J_2,3= 9.6 Hz, J_3,4= 3.6 Hz, H-3), 4.81 (d, 1H, J

_1,2= 9.6, H-1), 4.64 (br s, 1H, OH), 4.46 (d, 1H, H-4), 4.25–4.22 (m,

3H, H-6a, ClCH₂CO), 4.14 (dd, 1H, J _5,6b= 1.7 Hz, J _6a,6b= 12.6 Hz,

H-6b), 3.93 (t, 1H, H-2), 3.83 (dd, 1H, H-5). ¹³C NMR (150 MHz,

CDCl₃):d 167.6, 139.5, 133.5, 133.2, 129.5, 128.7, 128.0, 127.3,

101.3, 87.7 (C-1), 77.6, 74.6, 70.2, 69.7, 66.5, 41.6.

4.13. 2-(Trimethylsilyl)ethyl 2-O-benzoyl-4,6-O-benzylidene-b-

D-galactopyranosyl-(1?4)-6-O-benzyl-3-O-chloroacetyl-2-

deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-b-D-

glucopyranoside (25)

A solution of 24 (137 mg, 0.130 mmol) in pyridine (4.0 mL) and

H₂O (2.0 mL) was stirred for 18 h at 80 °C. The mixture was diluted

with CHCl₃, washed with aq 5%HCl, aq NaHCO₃and brine, dried

(MgSO₄), and concentrated. The product was puriﬁed by silica gel

column chromatography using 10:1 chloroform–acetone to give

HR-FABMS: calcd for C₂₁H₂₂ClO₆S: m/z 437.0826; found: m/z

437.0759 [M]⁺.

4.11. Phenyl 2-O-benzoyl 4,6-O-benzylidene-3-O-chloroacetyl-

thio-b-

D-galactopyranoside (22)

25 (81.7 mg, 70%).

[

a]

ꢀ2.5 (c 3.0, CH₃COCH₃). ¹H NMR

D

(600 MHz, CD₃COCD₃): d 8.09–7.21 (m, 15H, 3 Ph), 6.83 (d, 1H,

NH), 5.70 (s, 1H, PhCH), 5.43 (dd, 1H, J _{1,2 =}7.8 Hz, J_2,3= 10.2 Hz,

H-2 of Gal), 4.88 (d, 1H, H-1 of Gal), 4.79, 4.70 (each d, 2H,

NHCOCH₂CCl₃), 4.46 (d, 1H, J _1,2= 8.5 Hz, H-1 of GlcN), 4.43 (d,

1H, J_3,4= 3.0 Hz, H-4 of Gal), 4.40–4.37 (m, 2H, 2 OH), 4.25–4.23

(m, 2H, H-6 of Gal, benzylmethylene), 4.17 (d 1H, benzylmethyl-

ene), 4.09 (dt, 1H, H-3 of GlcN), 3.92–3.88 (m, 2H, H-3 of Gal,

CH₂CH₂Si(CH₃)₃), 3.70 (d, 1H, J_{2, 3}= J _3,4= 9.6 Hz, H-3 of GlcN),

3.59 (d, 1H, H-4 of Gal), 3.55–3.46 (m, 3H, H-2, 6 of GlcN,

CH₂CH₂Si(CH₃)₃), 3.39–3.36 (m, 1H, H-5 of GlcN), 0.94–0.82 (m,

2H, CH₂CH₂Si(CH₃)₃), ꢀ0.02 (s, 9H, Si(CH₃)₃). ¹³C NMR (150 MHz,

CD₃COCD₃):d 165.8, 155.2, 139.7, 139.4, 134.1, 131.1, 130.5,

129.5, 129.4, 129.0, 128.7, 128.1, 127.2, 102.3 (C-1 of Gal), 101.6

(C-1 of GlcN), 101.5, 97.1, 82.1, 76.8, 74.8, 74.7, 73.64, 73.55,

73.3, 71.6, 69.41, 69.39, 68.0, 67.0, 58.3, 18.6, ꢀ1.2. HR-FABMS:

calcd for C₄₁H₅₀Cl₃NO₁₃SiNa: m/z 920.2015; found: m/z 920.2032

[M+Na]⁺.

To a solution of 21 (1.18 g, 2.70 mmol) in CH₂Cl₂(8.0 mL) was

added pyridine (4.8 mL) and benzoyl chloride (0.47 mL,

4.05 mmol), and the reaction mixture was stirred for 5 h at room

temperature. Toluene was added and evaporated, then the residue

was dissolved in CHCl₃, washed with 5% HCl, aq NaHCO₃and water,

dried (MgSO₄), and concentrated. The product was puriﬁed by sil-

ica gel column chromatography using 3:1 hexane–EtOAc as eluent

to give 22 (3.2 g, 85.1%). [a]

_D+5.3 (c 3.0, CHCl₃). ¹H NMR (600 MHz,

CDCl₃): d 8.02–7.23 (m, 15H, 3Ph), 5.60 (t, 1H, J_1,2= J_2,3= 9.9 Hz, H-

2), 5.50 (s, 1H, PhCH), 5.24 (dd, 1H, J_3,4= 3.6 Hz, H-3), 4.88 (d, 1H,

H-1), 4.46 (d, 1H, H-4), 4.41 (dd, 1H, J_5,6a= 1.4 Hz, J_6a,6b= 12.4 Hz,

H-6a), 4.06 (dd, 1H, J _5,6b= 1.4 Hz, H-6b), 3.67 (d, 1H, H-5). ¹³C

NMR (150 MHz, CDCl₃):d 167.1, 164.8, 137.3, 133.9, 133.4, 130.9,

129.8, 129.4, 129.2, 128.8, 128.5, 128.3, 128.2, 126.5, 101.1, 85.2

(C-1), 74.9, 73.3, 69.7, 69.0, 67.1, 40.6.

HR-FABMS: calcd for C₂₈H₂₅ClO₇S: m/z 541.1088; found: m/z

541.1055 [M]⁺.

4.14. 2-(Trimethylsilyl)ethyl 3,4-di-O-benzoyl-2-O-benzyl-

a-L-

4.12. 2-(Trimethylsilyl)ethyl 2-O-benzoyl-4,6-O-benzylidene-3-

fucopyranosyl-(1?3)-2-O-benzoyl-4,6-O-benzylidene-b-

D-

O-chloroacetyl-b-

D

-galactopyranosyl-(1?4)-6-O-benzyl-3-O-

galactopyranosyl-(1?4)-[3,4-di-O-benzoyl-2-O-benzyl-

a-

L-

chloroacetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-

fucopyranosyl-(1?3)]-6-O-benzyl-2-deoxy-2-(2,2,2-

b-

D-glucopyranoside (24)

trichloroethoxycarbonylamino)-b-

D-glucopyranoside (26)

A mixture of 22 (190 mg, 0.35 mmol), 23 (169 mg, 0.27 mmol),

Compound 26 was prepared from 25 (81.6 mg, 90.7

lmol) and

and powdered MS 4 Å (350 mg) in dry CH₂Cl₂(3 mL) was stirred un-

der Ar atmosphere for 2 h at room temperature, then cooled to

11 (110 mg, 0.18 mmol) as described for preparation of 12. The

product was puriﬁed by silica gel column chromatography (2:1

ꢀ40 °C. NIS (119 mg, 0.53 mmol) and TfOH (3.1

l

L, 35.2

l

mol)

hexane–ethyl acetate) to give 26 (132 mg, 82%). [

a

]

ꢀ79.5 (c

D

were added and the mixture was stirred for 1 h at ꢀ40 °C, then neu-

tralized with Et₃N. The precipitates were ﬁltered off and washed

with CHCl₃. The combined ﬁltrate and washings were successively

washed with saturated aqueous Na₂S₂O₃and water, dried (MgSO₄),

and concentrated. The product was puriﬁed by silica gel column

chromatography (8:1 toluene–acetone) to give 24 (238 mg, 84%).

2.04): ¹H NMR (600 MHz, CDCl₃): d 8.01–6.86 (m, 45H, 9 Ph),

5.82 (dd, 1H, J_2,3= 10.7 Hz, J_3,4= 3.0 Hz, H-3 of Fuc a), 5.70 (dd,

1H, J_2,3= 10.5 Hz, J_3,4= 3.3 Hz, H-3 of Fuc b), 5.65 (d,1H, H-4 of

Fuc a), 5.63 (d,1H, H-4 of Fuc b), 5.60–5.57 (m, 2H, H-2 of Gal,

PhCH), 5.52 (br d, 1H, NH), 5.36 (d, 1H, J_1,2= 3.6 Hz, H-1 of Fuc a),

5.22 (br dd, 1H, H-5 of Fuc a), 5.04 (d, 1H, J_1,2= 3.6 Hz, H-1 of Fuc

b), 4.89 (d, 1H, benzylmethylene), 4.83–4.79 (m, 4H, H-1 of GlcN,

H-1, 6a of Gal, benzylmethylene), 4.83–4.79 (H-5 of Fuc b,

NHCOCH₂CCl₃, benzylmethylene), 4.47 (d, 1H, benzylmethylene),

4.27 (d, 1H, J_3,4= 3.6 Hz, H-4 of Gal), 4.20 (t, 1H, J_2,3= J_3,4= 9.1 Hz,

H-3of GlcN), 4.16 (br d, H-6 of Gal), 4.12–4.09 (m, 2H, H-2 of Fuc

a, benzylmethylene), 4.05 (t, 1H, J_4,5= 8.8 Hz, H-4 of GlcN), 3.99–

3.96 (m, 3H, H-6a of GlcN, H-2 of Fuc b, benzylmethylene),

[a]

+17.6 (c 3.4, CHCl₃). ¹H NMR (600 MHz, CDCl₃): d 7.86–7.37

D

(m, 15H, 3 Ph), 5.49 (s, 1H, PhCH), 5.44 (dd, 1H, J _{1,2 =}8.3 Hz, J

_2,3= 9.9 Hz, H-2 of Gal), 5.25 (d, 1H, NH), 5.17 (t, 1H, J _2,3= J

_3,4= 10.5 Hz, H-3 of GlcN), 4.99 (dd, 1H, J_3,4= 3.9 Hz, H-3 of Gal),

4.75–4.71 (m, 2H, benzylmethylene, NHCOCH₂CCl₃), 4.65 (d, 1H,

NHCOCH₂CCl₃), 4.63 (d, 1H, H-1 of Gal), 4.41 (d, 1H, J_1,2= 8.2 Hz,

H-1 of GlcN), 4.38 (d 1H, benzylmethylene), 4.36 (d, 1H, H-4 of

66

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

3.86–3.82 (m, 1H, CH₂CH₂Si(CH₃)₃), 3.68 (br d, 1H, H-3 of Gal), 3.62

(br d, 1H, H-6b of GlcN), 3.39–3.35 (m, 1H, CH₂CH₂Si(CH₃)₃), 3.34

(s, 1H, H-5 of Gal), 3.27 (br d, 1H, H-5 of GlcN), 3.13 (br. dd, H-2

of GlcN), 1.07 (d, 3H, H-6 of Fuc b), 0.89–0.82 (m, 2H,

CH₂CH₂Si(CH₃)₃), 0.79 (d, 3H, H-6 of Fuc a), ꢀ0.06 (s, 9H, Si(CH₃)₃).

¹³C NMR (CDCl₃):d 166.0, 165.6, 165.2, 165.0, 164.3, 153.4, 138.5,

137.7, 137.5, 137.1, 133.1, 132.8, 132.69, 132.65, 132.6, 139.4,

130.3, 130.1, 129.9, 129.71, 129.66, 129.6, 129.4, 128.5, 128.44,

128.39, 128.3, 128.2, 128.2, 128.11, 128.08, 128.0, 127.9, 127.8,

127.5, 100.3, 100.20 (C-1 of Fuc a), 100.16 (C-1 of Gal), 98.7 (C-

1of GlcN), 97.5 (C-1 of Fuc b), 95.6, 79.8, 75.5, 75.1, 74.7, 74.5,

74.4, 73.8, 73.6, 73.1, 72.3, 71.8, 71.7, 71.5, 70.8, 70.3, 69.2, 68.0,

67.0, 66.9, 65.7, 65.5, 59.3, 29.6, 17.9, 16.2, 15.1, ꢀ1.4. MALDI-TOF-

product was puriﬁed by silica gel column chromatography (5:1

chloroform–acetone) to give 28 (118 mg, 65%). [a] +21.4 (c 2.8),

D

¹H NMR (600 MHz, CDCl₃) d 7.96–7.23 (m, 35H, 7 Ph), 4.84 (br d,

1H, H-1 of GlcN), 4.67 (d, 1H, J_1,2= 8.0 Hz, H-1 of Glc), 4.62 (d,

1H, J _1,2= 7.8 Hz, H-1 of Gal), 4.58 (br d, 1H, H-1 of GalN). ¹³C

NMR (150 MHz, CDCl₃) d 101.6 (C-1 of GalN), 100.3 (C-1 of Glc),

100.2 (C-1 of Gal), 99.2 (C-1 of GlcN). MALDI-TOFMS: Calcd for

C

₈₈H₉₂Cl₈N₂O₂₈SiNa: m/z 1955.3; found: m/z 1955.1[M+Na]⁺.

4.18. 2-(Trimethylsilyl)ethyl 2-O-benzoyl-4,6-O-benzylidene-b-

D-

galactopyranosyl-(1?4)-6-O-benzyl-2-deoxy-2-(2,2,2-

trichloroethoxycarbonylamino)-b-

O-benzylidene-2-deoxy-2-(2,2,2-

trichloroethoxycarbonylamino)-b-

D

-glucopyranosyl-(1?3)-4,6-

-galactopyranosyl-(1?4)-

MS: Calcd for C₉₅H₉₈Cl₃NO₂₅SiNa: m/z 1808.5; found: 1807.7 m/z

D

+

[M+Na]

.

2,3-di-O-benzoyl-6-O-benzyl-b-D-glucopyranoside (29)

4.15. 2-(Trimethylsilyl)ethyl

galactopyranosyl-(1?4)-[

acetamido-2-deoxy-b- -glucopyranoside (5)

a

-

L

-fucopyranosyl-(1?3)-b-

D-

Compound 29 was prepared from 28 (118 mg, 63.4

scribed for preparation of 25. The product was puriﬁed by silica gel

l

mol) as de-

a

-L-fucopyranosyl-(1?3)]-2-

D

column chromatography (5:1 chloroform–methanol) to give 29

(34.4 mg, quant.). [a]

+12.9 (c 2.6), ¹H NMR (600 MHz, CDCl₃) d

D

A mixture of 26 (52.0 mg, 67.7 lmol) and zinc powder (600 mg)

8.07–7.20 (m, 35H, 7 Ph), 4.83 (br d, 1H, H-1 of GlcN), 4.62 (d, 2H,

J_1,2= 7.4 Hz, H-1 of Glc, Gal), 4.54 (d, 1H, J_1,2= 8.3 Hz, H-1 of GalN).

¹³C NMR (150 MHz, CDCl₃) d 101.4 (C-1 of Glc), 100.7 (C-1 of GalN),

100.3 (C-1 of Gal), 99.3 (C-1 of GlcN). MALDI-TOFMS: Calcd for

in AcOH–Ac₂O (2:1, 6 mL) was stirred for 18 h at 55 °C. After com-

pletion of the reaction, the mixture was ﬁltered and the ﬁltrate was

diluted with water and extracted with CHCl₃. The extract was

washed with water, dried (MgSO₄), and concentrated. A solution

of the residue in MeOH–AcOH (2:1, 3 mL) was hydrogenolysed

(0.4 MPa) under hydrogen in the presence of 10% Pd/C (300 mg)

for 18 h at room temperature, then ﬁltered and concentrated. The

residue was de-acylated with NaOMe (20 mg) in MeOH (2 mL) at

40 °C. The mixture was stirred for 5 h and then neutralized with

Amberlite IR 120 [H⁺]. The mixture was ﬁltered and concentrated.

The product was puriﬁed by Sephadex LH-20 column chromatog-

C

₈₄H₉₀Cl₆N₂O₂₆SiNa: m/z 1803.4; found: m/z 1803.9 [M+Na]⁺.

4.19. 2-(Trimethylsilyl)ethyl 3,4-di-O-benzoyl-2-O-benzyl-

fucopyranosyl-(1?3)-2-O-benzoyl-4,6-O-benzylidene-b-

galactopyranosyl-(1?4)-[3,4-di-O-benzoyl-2-O-benzyl-

fucopyranosyl-(1?3)]-6-O-benzyl-2-deoxy-2-(2,2,2-

a-L-

D

-

a-

L-

trichloroethoxycarbonylamino)-b-

O-benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonyl

amino)-b- -galactopyranosyl-(1?4)-2,3-di-O-benzoyl-6-O-

benzyl-b- -glucopyranoside (30)

D-glucopyranosyl-(1?3)-4,6-

raphy with MeOH to give 5 (7.2 mg, 23%). [a]_D+88.0 (c 0.3, MeOH),

D

¹H NMR (600 MHz, CD₃OD) d 4.90 (d, 1H, J_1,2= 3.8 Hz, H-1 of Fuc b),

4.78 (d, 1H, J_1,2= 3.8 Hz, H-1 of Fuc a), 4.29 (d, 1H, J_1,2= 8.0 Hz, H-1

of Gal), 4.25 (d, 1H, J_1,2= 8.0 Hz, H-1 of GlcN). ¹³C NMR (125 MHz,

CDCl₃) d 101.7 (C-1 of Gal), 100.8 (C-1 of Fuc b), 99.7 (C-1 of GlcN),

98.2 (C-1 of Fuc a).

D

Compound 30 was prepared from 11 (178 mg, 293

29 (104.3 mg, 58.5 mol) as described for preparation of 12. The

product was puriﬁed by silica gel column chromatography (2:1

hexane–ethyl acetate) to give 30 (110 mg, 72%). [

lmol) and

l

HR-FABMS: Calcd for C₃₁H₅₇NO₁₉SiNa: m/z 798.3192; found: m/z

a

]

D

ꢀ23.5 (c 2.8,

798.3213 [M+Na]⁺.

CHCl₃), ¹H NMR (600 MHz, CDCl₃) d 8.02–6.85 (m, 60H, 12 Ph),

5.30 (br s, H-1 of Fuc a), 5.03 (d, J_1,2= 3.6 Hz, H-1 of Fuc b), 4.96

(br, 1H, H-1 of GlcN), 4.80 (d, 1H, J_1,2= 8.0 Hz, H-1 of Glc), 4.64

(d, 1H, J _1,2= 7.4 Hz, H-1 of GalN), 4.63 (d, J _1,2= 8.0 Hz, H-1 of

Gal), 1.05 (d, 1H, H-6 of Fuc a), 0.75 (d, 1H, H-6 of Fuc b). ¹³C

NMR (150 MHz, CDCl₃) d 100.5 (C-1 of Glc), 100.3 (C-1 of Fuc b),

100.2 (C-1 of Gal), 99.3 (C-1 of GalN, GlcN). MALDI-TOFMS: Calcd

for C₁₃₈H₁₃₈Cl₆N₂O₃₈SiNa: m/z 2691.7; found: m/z 2692.1 [M+Na]⁺.

4.16. 2-O-Benzoyl-4,6-O-benzylidene-3-O-chloroacetyl-b-

galactopyranosyl-(1?4)-6-O-benzyl-3-O-chloroacetyl-2-deoxy-

D-

2-(2,2,2-trichloroethoxycarbonylamino)-

trichloroacetimidate (27)

D-glucopyranosyl

To a mixture of 24 (100.3 mg, 0.10 mmol) and MS 4 Å (150 mg)

in CH₂Cl₂(20 mL) cooled to 0 °C was added CF₃CO₂H (1.5 mL), and

the mixture was stirred for 3 h at room temperature. The solids

were ﬁltrated off and concentrated. EtOAc and toluene (1:2) were

added and evaporated to give the reducing sugar. To a solution of

the residue in CH₂Cl₂(2.0 mL) cooled at 0 °C were added DBU

4.20. 2-(Trimethylsilyl)ethyl 2-O-acetyl-3,4-di-O-benzoyl-

fucopyranosyl-(1?3)-4,6-di-O-acetyl-2-O-benzoyl-b-

galactopyranosyl-(1?4)-[2-O-acetyl-3,4-di-O-benzoyl-

fucopyranosyl-(1?3)]-2-acetramide-6-O-acetyl-2-deoxy-b-

glucopyranosyl-(1?3)-2-acetamide-4,6-di-O-acetyl-2-deoxy-b-

-galactopyranosyl-(1?4)-6-acetyl-2,3-di-O-benzoyl-

a-L-

D

-

a

-L

-

D-

(14.7 lL, 98.6 lmol) and CCl₃CN (52.7 lL, 0.53 lmol). The reaction

mixture was stirred for 6 h at room temperature. After completion

of the reaction, the mixture was concentrated. The residue was

puriﬁed by silica gel column chromatography using chloroform

to give 27 (65.7 mg, 64%).

D

D-

glucopyranoside (31)

Compound 31 was prepared from 30 (110 mg, 41.2 lmol) as de-

scribed for preparation of 16. The product was puriﬁed by silica gel

4.17. 2-(Trimethylsilyl)ethyl 2-O-benzoyl-4,6-O-benzylidene-3-

O-chloroacetyl-b-

chloroacetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-

b- -glucopyranosyl-(1?3)-4,6-O-benzylidene-2-deoxy-2-(2,2,2-

trichloroethoxycarbonyl-amino)-b- -galactopyranosyl-(1?4)-

2,3-di-O-benzoyl-6-O-benzyl-b- -glucopyranoside (28)

D-galactopyranosyl-(1?4)-6-O-benzyl-3-O-

column chromatography (15:1 chloroform–methanol) to give 31

ꢀ26.4 (c 0.61, CHCl₃); ¹H NMR (600 MHz,

25

(39.1 mg, 43%). [a]

D

CDCl₃): d 7.98–6.90 (m, 35H, 7 Ph), 5.40 (d, 1H, J_1,2= 3.6 Hz, H-1

of Fuc a), 5.20 (d, 1H, J_1,2= 3.9 Hz, H-1 of Fuc b), 4.84 (br d, 1H,

H-1 of GalN), 4.75 (d, 1H, J_1,2= 8.0 Hz, H-1 of GlcN), 4.66 (d, 1H, J

_1,2= 7.4 Hz, H-1 of Glc), 4.60 (d, 1H, J_1,2= 8.0 Hz, H-1 of Gal), 0.98

(d, 1H, H-6 of Fuc a), 0.80 (d, 1H, H-6 of Fuc b). ¹³C NMR

(150 MHz, CDCl₃):d101.3 (C-1 of Glc), 100.5 (C-1 of Gal), 100.0

D

Compound 28 was prepared from 24 (123.2 mg, 112

lmol) and

14 (93.6 mg, 93.3 mol) as described for preparation of 12. The

l

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

67

(C-1 of Fuc b), 99.5 (C-1 of GalN), 99.2 (C-1 of GlcN), 95.4 (C-1 of

Fuc a). MALDI-TOFMS: calcd for C₁₁₀H₁₂₄N₂O₄₄SiNa: m/z 2228.7;

found: m/z 2227.9 [M+Na]⁺.

column chromatography (20:1 chloroform–methanol) to give 35

(2.18 g, 99%). [

a

]

D

ꢀ22.0 (c 1.9, CHCl₃). ¹H NMR (600 MHz,

CD₃COCD₃): d 7.54–7.31 (m, 10H, 2Ph), 7.29 (d, 1H, NH), 5.65 (s,

1H, PhCH), 5.46 (t, 1H, J_2,3= J_3,4= 9.6 Hz, H-3), 5.25 (d, 1H, J_1,2

=

4.21. 2-O-Acetyl-3,4-di-O-benzoyl-

di-O-acetyl-2-O-benzoyl-b- -galactopyranosyl-(1?4)-[2-O-

acetyl-3,4-di-O-benzoyl- -fucopyranosyl-(1?3)]-2-

acetramide-6-O-acetyl-2-deoxy-b- -glucopyranosyl-(1?3)-2-

acetamide-4,6-di-O-acetyl-2-deoxy-b- -galactopyranosyl-

-glucopyranosyl

a

-

L

-fucopyranosyl-(1?3)-4,6-

10.4 Hz, H-1), 4.82 (q, 2H, NHCOOCH₂CCl₃), 4.33 (dd, 1H,

J_5,6a= 4.9 Hz, J_6a,6b= 10.2 Hz, H-6a), 4.22 (q, 2H, ClCH₂CO), 3.88–

3.82 (m, 3H, H-2, 4, 6b), 3.69 (dt, 1H, H-5). ¹³C NMR (150 MHz,

CD₃ODCD₃):d 167.4, 155.2, 138.4, 133.8, 132.7, 129.8, 129.6,

128.8, 128.5, 127.0, 101.8, 96.8, 87.6 (C-1), 79.1, 75.3, 74.8, 71.1,

D

a

-L

D

(1?4)-6-acetyl-2,3-di-O-benzoyl-b-

D

68.8, 56.1, 41.4. HR-FABMS: calcd for C₂₄H₂₃Cl₄NO₇S: m/z

trichloroacetimidate (32)

610.0028; found: m/z 610.0039 [M]⁺.

Compound 32 was prepared from 31 (39.1 mg, 17.7 lmol) as

described for preparation of 17. The product was puriﬁed by silica

4.25. 2-(Trimethylsilyl)ethyl 4,6-O-benzylidene-3-O-

chloroacetyl-2-deoxy-2-(2,2,2-trichloroethoxy-

gel column chromatography (20:1 chloroform–methanol) to give

ꢀ1.5 (c 0.3, CHCl₃). ¹H NMR (600 MHz,

carbonylamino)-b-D-glucopyranosyl-(1?3)-4,6-O-benzylidene-

32 (32.6 mg, 82%). [a]

D

2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-b-

D

-

CDCl₃): d 5.70 (d, 1H, J_1,2= 3.5 Hz, H-1 of Glc), 5.44 (d, 1H, J_1,2

= 3.6 Hz, H-1 of Fuc a), 5.30 (d, 1H, J_1,2= 4.0 Hz, H-1 of Fuc b),

4.91 (br d, 1H, H-1 of GalN), 4.82 (d, 1H, J_1,2= 8.0 Hz, H-1 of GlcN),

4.68 (d, 1H, J_1,2= 8.5 Hz, H-1 of Gal).

galactopyranosyl-(1?4)-2,3-di-O-benzoyl-6-O-benzyl-b-D-

glucopyranoside (36)

Compound 36 was prepared from 35 (150 mg, 0.150 mmol) and

14 (183 mg, 0.300 mmol) as described for preparation of 24. The

product was puriﬁed by silica gel column chromatography (6:1

4.22. 2-O-Acetyl-3,4-di-O-benzoyl-

di-O-acetyl-2-O-benzoyl-2-deoxy-b-

[2-O-acetyl-3,4-di-O-benzoyl- -fucopyranosyl-(1?3)]-2-

acetramide-6-O-acetyl-2-deoxy-b- -glucopyranosyl-(1?3)-2-

acetamide-4,6-di-O-acetyl-2-deoxy-b-

(1?4)-6-O-acetyl-2,3-di-O-benzoyl-b-

a

-

D

L

-fucopyranosyl-(1?3)-4,6-

-galactopyranosyl-(1?4)-

chloroform–acetone) to give 36 (165 mg, 73%). [

a]

+4.6 (c 3.6,

D

a-L

CHCl₃). ¹H NMR (600 MHz, CD₃COCD₃): d 6.87 (d, 1H, NH of GlcN),

6.66 (d, 1H, NH of GalN), 5.04 (1H, d, J_1,2= 8.2 Hz, H-1 of GalN), 4.92

(1H, d, J_1,2= 8.0 Hz, H-1 of Glc), 4.87 (1H, d, J_1,2= 8.2 Hz, H-1 of

GlcN). ¹³C NMR (125 MHz, CDCl₃): d178.8, 167.3, 165.7, 155.0,

139.9, 139.6, 138.4, 133.9, 133.6, 131.0, 130.9, 130.3, 129.6,

129.5, 129.24, 129.15, 129.1, 128.8, 128.7, 128.5, 128.4, 128.2,

127.3, 127.0, 103.0 (C-1 of GalN), 101.8, 101.6 (C-1 of GlcN),

101.2, 100.7 (C-1 of Glc), 97.0, 96.8, 79.3, 79.1, 78.0, 76.6, 76.0,

75.6, 75.3, 74.9, 74.8, 74.3, 73.7, 69.7, 69.0, 67.4, 67.2, 66.8, 57.4,

57.3, 54.5, 41.4, 18.4, –1.32. MALDI-TOFMS: calcd for

D

-galactopyranosyl-

D-glucopyranosyl-(1?1)-

(2S,3S,4R)-3,4-di-O-benzoyl-2-hexadecanamido-octadecane-

1,3,4-triol (33)

Compound 33 was prepared from 32 (32.6 mg, 14.5

lmol) and

18 (16.7 mg, 21.8 mol) as described for preparation of 19. The

l

product was puriﬁed by silica gel column chromatography (20:1

hexane–ethyl acetate) to give 33 (11.6 mg, 28%) as an amorphous

ꢀ23.4 (c 0.2, CHCl₃). ¹H NMR (600 MHz, CDCl₃):

C

₆₆H₇₁Cl₇N₂O₂₁SiNa: m/z 1523; found: m/z 1523 [M+Na]⁺.

powder. [a]

D

d 8.10–7.14 (m, 45H, 9 ꢂ Ph), 5.45 (d, 1H, J_1,2= 4.1 Hz, H-1 of Fuc

4.26. 2-(Trimethylsilyl)ethyl 4,6-O-benzylidene-2-deoxy-2-

(2,2,2-trichloroethoxy-carbonylamino)-b- -glucopyranosyl-

(1?3)-4,6-O-benzylidene-2-deoxy-2-(2,2,2-

trichloroethoxycarbonyl-amino)-b- -galactopyranosyl-(1?4)-

2,3-di-O-benzoyl-6-O-benzyl-b- -glucopyranoside (37)

a), 5.32 (d, 1H,

J _1,2= 3.6 Hz, H-1 of Fuc b), 4.70 (d, 1H,

D

J_1,2= 8.3 Hz, H-1 of GlcN), 4.65 (d, 1H, J_1,2= 7.6 Hz, H-1 of Glc),

4.60 (br d, 1H, H-1 of GalN), 4.56 (d, 1H, J_1,2= 8.5 Hz, H-1 of Gal).

¹³C NMR (150 MHz, CDCl₃):d 100.8 (C-1 of Glc, Gal), 99.5 (C-1 of

GalN, GlcN), 96.2 (C-1 of Fuc). MALDI-TOFMS: calcd for

D

C

₁₅₃H₁₈₇N₃O₄₉Na: m/z 2873.2; found 2873.4: m/z [M+Na]⁺.

Compound 37 was prepared from 36 (120 mg, 79.8 lmol) as

described for preparation of 25. The product was puriﬁed by silica

4.23.

-fucopyranosyl-(1?3)]-2-acetramide-2-deoxy-b-

glucopyranosyl-(1?3)-2-acetamide-2-deoxy-b-

galactopyranosyl-(1?4)-b- -glucopyranosyl-(1?1)-(2S,3S,4R)-

a

-

L

-Fucopyranosyl-(1?3)-b-

D

-galactopyranosyl-(1?4)-[

a

-

gel column chromatography (6:1 chloroform–acetone) to give 37

L

D-

(97.5 mg, 86%). [

a

]

D

+36.1 (c 0.9, CHCl₃). ¹H NMR (600 MHz,

D

-

CD₃COCD₃): d 7.95–7.28 (m, 25H, 5 Ph), 6.74 (br d, 1H, NH of

GalN), 6.64 (br d, 1H, NH of GlcN), 5.67 (1H, d, J_2,3= J_3,4= 9.1 Hz,

H-3 of GlcN), 5.56 (s, 1H, PhCH), 5.40 (s, 1H, PhCH), 5.21 (1H, t,

J_1,2= 9.1 Hz, H-2 of Glc), 4.92 (1H, br. d, H-1 of Glc), 4.91–4.81

(m, 4H, H-1 of GalN, H-1 of GlcN, NHCOCH₂CCl₃, benzylmethyl-

ene), 4.72–4.63 (m, 3H, NHCOCH₂CCl₃, benzylmethylene, OH),

4.28 (1H, d, J_3,4= 2.8 Hz, H-4 of GalN), 4.24–4.19 (m, 3H, H-4 of

Glc, H-3 of GalN, H-4 of GlcN), 4.04–4.01 (m, 1H, CH₂CH₂Si(CH₃)₃),

4.00–4.93 (m, 2H, H-6 of Glc), 3.90–3.86 (m, 2H, H-5 of Glc, H-3 of

D

2-hexadecanamido-octadecane-1,3,4-triol (2)

Compound 2 was prepared from 33 (11.6 mg, 4.1

l

mol) as de-

scribed for preparation of 1. The product was puriﬁed by Sephadex

LH-20 column chromatography in 1:1 CHCl₃–MeOH to give 2

(6.8 mg, quant.). [

(600 MHz, CDCl₃/CD₃OD = 1:1): d 4.93 (d, 1H, J_1,2= 4.1 Hz, H-1 of

a

]

ꢀ7.0 (c 0.08, CHCl₃/CH₃OH = 1:1). ¹H NMR

D

Fuc), 4.79 (d, 1H, J_1,2= 3.9 Hz, H-1 of Fuc), 4.48 (d, 1H, J _1,2

=

GlcN), 3.75–3.63 (m, 3H, H-2,

6 of GalN, H-6a of GlcN,

8.3 Hz, H-1 of GlcN), 4.30 (d, 1H, J_1,2= 8.0 Hz, H-1 of GalN), 4.28

(d, 1H, J_1,2= 8.5 Hz, H-1 of Gal), 4.05 (d, 1H, J_1,2= 7.8 Hz, H-1 of

Glc). 0.99 (d, 2H, H-6 of Fuc). MALDI-TOFMS: calcd for

CH₂CH₂Si(CH₃)₃), 3.51 (t, 1H, H-6b of GlcN), 3.44–3.37 (m, 2H,

H-3, 5 of GalN), 0.93–0.79 (m, 2H, CH₂CH₂Si(CH₃)₃), ꢀ0.08 (m,

9H, Si(CH₃)₃). ¹³C NMR (125 MHz, CD₃COCD₃): d 165.7, 155.2,

155.0, 139.9, 139.0, 133.8, 133.6, 131.0, 130.9, 130.3, 129.5,

129.21, 129.15, 129.1, 128.7, 128.5, 128.4, 128.2, 127.3, 127.2,

103.4 (C-1 of GalN), 102.0, 101.7 (C-1 of GlcN), 101.1, 100.7 (C-1

of Glc), 97.1, 82.5, 79.1, 77.3, 76.7, 76.2, 75.6, 75.4, 74.9, 73.7,

71.6, 69.8, 69.1, 69.0, 67.4, 67.3, 67.1, 59.6, 54.5, 18.4, –1.32. MAL-

DI-TOFMS: calcd for C₆₄H₇₀Cl₆N₂O₂₀SiNa: m/z 1447; found: m/z

1447 [M+Na]⁺.

C

₇₄H₁₃₅N₃O₃₂Na: m/z 1600.9; found: m/z 1600.5 [M+Na]⁺.

4.24. Phenyl 4,6-O-benzylidene-3-O-chloroacetyl-2-deoxy-2-

(2,2,2-trichloro-ethoxycarbonyl amino)-thio-b-D-

gulcopyranoside (35)

Compound 35 was prepared from 34 (1.93 g, 3.61 mmol) as de-

scribed for preparation of 22. The product was puriﬁed by silica gel

68

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

4.27. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-

galactopyranosyl-(1?4)-[3,4-di-O-benzoyl-2-O-benzyl-

fucopyranosyl-(1?3)]-6-O-benzyl-2-deoxy-2-(2,2,2-

D

-

product was puriﬁed by silica gel column chromatography (20:1

hexane–ethyl acetate) to give 41 (8.6 mg, 20%). [

a-L-

a

]

D

ꢀ23.4 (c 0.2,

CHCl₃). ¹H NMR (600 MHz, CDCl₃): d 8.08–7.22 (m, 30H, 6 ꢂ Ph),

5.70 (d, 1H, J_1,2= 3.6 Hz, H-1 of Fuc), 4.79 (br d, 1H, H-1 of GalN),

4.70 (br d, 1H, H-1 of GlcN a), 4.65 (d, 1H, J_1,2= 8.5 Hz, H-1 of GlcN

b), 4.59 (d, 1H, J_1,2= 7.9 Hz, H-1 of Glc), 4.56 (d, 1H, J_1,2= 8.5 Hz, H-

1 of Gal). ¹³C NMR (150 MHz, CDCl₃):d100.8 (C-1 of Glc, Gal), 99.5

(C-1 of GalN, GlcN), 96.2 (C-1 of Fuc). MALDI-TOFMS: calcd for

trichloroethoxycarbonylamino)-b-

benzylidene-2-deoxy-2-(2,2,2-trichloroethoxy-carbonylamino)-b-

-glucopyranosyl-(1?3)-4,6-O-benzylidene-2-deoxy-2-(2,2,2-

trichloro-ethoxycarbonylamino)-b- -galactopyranosyl-(1?4)-2,3-

di-O-benzoyl-6-O-benzyl-b- -glucopyranoside (38)

D-glucopyranosyl-(1?3)-4,6-O-

D

C

₁₄₀H₁₈₄N₄O₅₀Na: m/z 2744.1; found: m/z 2744.5 [M+Na]⁺.

Compound 38 was prepared from 37 (97.5 mg, 68.4

13 (112.2 mg, 82.1 mol) as described for preparation of 15. The

product was puriﬁed by silica gel column chromatography (3:1 hex-

ane–ethyl acetate) to give 38 (114 mg, 63%). [ +13.6 (c 1.5,

lmol) and

l

4.31. b-

acetamido-2-deoxy-b-

deoxy-b- -glucopyranosyl-(1?3)-2-acetamido-2-deoxy-b-

galactopyranosyl-(1?4)-b- -glucopyranosyl-(1?1)-(2S,3S,4R)-

D

-Galactopyranosyl-(1?4)-[

a-L-fucopyranosyl-(1?3)]-2-

D-glucopyranosyl-(1?3)-2-acetamido-2-

a]

D

D-

D

CHCl₃). ¹H NMR (600 MHz, CDCl₃): d 8.03–7.24 (m, 45H, 9 Ph),

5.62 (br s, 1H, H-1 of Fuc), 4.95 (br d, 1H, H-1 of GalN), 4.91 (d,

1H, J_1,2= 8.2 Hz, H-1 of GlcN a), 4.87 (br d, 1H, H-1 of GlcN b), 4.75

(d, 1H, J_1,2= 8.2 Hz, H-1 of Glc), 4.69 (d, 1H, J_1,2= 8.5 Hz, H-1 of

Gal), 1.25 (d, 1H, H-6 of Fuc). ¹³C NMR (150 MHz, CDCl₃):d101.0

(C-1 of Glc), 100.8 (C-1 of GalN), 100.7 (C-1 of Gal, GlcN a, GlcN b),

96.7 (C-1 of Fuc). MALDI-TOFMS: calcd for C₁₂₁H₁₃₀Cl₉N₃O₄₁SiNa:

m/z 2646.5; found: m/z 2647.5 [M+Na]⁺.

D

2-hexadecanamido-octadecane-1,3,4-triol (3)

Compound 3 was prepared from 41 (8.6 mg, 2.9 lmol) as de-

scribed for preparation of 1. The product was puriﬁed by Sephadex

LH-20 column chromatography with 1:1 CHCl₃–MeOH to give 3

(3.4 mg, 79%). [

a

]

ꢀ35.6 (c 0.09, CHCl₃/CH₃OH = 1:1). ¹H NMR

D

(600 MHz, CDCl₃/CD₃OD = 1:1): d 4.78 (d, 1H, J_1,2= 3.6 Hz, H-1 of

Fuc), 4.48 (d, 1H, J _1,2= 8.3 Hz, H-1 of GlcN), 4.37 (d, 1H, J _1,2

= 8.5 Hz, H-1 of GlcN), 4.35 (d, 1H, J_1,2= 8.0 Hz, H-1 of GalN), 4.20

(d, 1H, J_1,2= 8.5 Hz, H-1 of Gal), 4.05 (d, 1H, J_1,2= 7.8 Hz, H-1 of

Glc). 0.92 (d, 1H, H-6 of Fuc). MALDI-TOFMS: calcd for

4.28. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-

galactopyranosyl-(1?4)-[2-O-acetyl-3,4-di-O-benzoyl-

fucopyranosyl-(1?3)]-2-acetamido-6-O-acetyl-2-deoxy-b-

D-

a-L-

D-

glucopyranosyl-(1?3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-b-

D

-

C

₇₆H₁₃₈N₄O₃₃Na: m/z 1657.9; found: m/z 1658.3 [M+Na]⁺.

glucopyranosyl-(1?3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-b-

D

galactopyranosyl-(1?4)-6-O-acetyl-2,3-di-O-benzoyl-b-D-

glucopyranoside (39)

4.32. 2-(Trimethylsilyl)ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-

trichloroethoxy-carbonylamino)-b- -galactopyranosyl-(1?4)-

6-O-benzyl-3-O-chloroacetyl-2-deoxy-2-(2,2,2-trichloroethoxy-

carbonylamino)-b- -glucopyranoside (43)

D

Compound 39 was prepared from 38 (114 mg, 51.7 lmol) as de-

scribed for preparation of 16. The product was puriﬁed by silica gel

D

column chromatography (10:1 chloroform–methanol) to give 39

ꢀ23.4 (c 0.2, CHCl₃). ¹H NMR (600 MHz,

Compound 43 was prepared from 23 (221 mg, 0.36 mmol) and

42 (266 mg, 0.43 mmol) as described for preparation of 15. The

product was puriﬁed by silica gel column chromatography (4:1

(43.0 mg, 40%). [

a]

D

CDCl₃): d 5.60 (d, 1H, J_1,2= 3.9 Hz, H-1 of Fuc), 4.89 (d, 1H, J_1,2

=

8.5 Hz, H-1 of Glc), 4.85 (d, 1H, J_1,2= 8.5 Hz, H-1 of GalN), 4.79 (d,

1H, J_1,2= 7.9 Hz, H-1 of GlcN a), 4.70 (d, 1H, J_1,2= 8.0 Hz, H-1 of GlcN

b), 4.56 (d, 1H, H-1 of Gal), ¹³C NMR (CDCl₃):d 100.6 (C-1 of Glc,

Gal), 99.9 (C-1 of GlcN a), 99.3 (C-1 of GalN, GlcN b), 96.1 (C-1 of

Fuc). MALDI-TOFMS: calcd for C₉₇H₁₂₁N₃O₄₅SiNa: m/z 2098.6;

found: m/z 2098.2 [M+Na]⁺.

hexane–ethyl acetate) to give 43 (313 mg, 79%). [

a]

ꢀ15.2 (c 7.8,

D

CHCl₃). ¹H NMR (600 MHz, CDCl₃): d 7.45–7.33 (m, 5H, Ph), 5.27

(d, 1H, NH of GlcN), 5.21 (d, 1H, J_3,4= 3.3 Hz, H-4 of GalN), 5.16

(t, 1H, J_2,3= J_3,4= 8.6 Hz, H-3 of GlcN), 4.79, 4.35 (each d, 2H, 2 ben-

zylmethylene), 4.69–4.60 (m, 5H, H-3 of GalN, 2 NHCOCH₂CCl₃),

4.48 (d, 1H, J_1,2= 8.0 Hz, H-1 of GlcN), 4.26 (br d, 1H, H-1 of GalN),

4.10–4.01 (m, 5H, H-6 of GalN, 2 OCOCH₂Cl, NH of GalN), 3.96–3.89

(m, 2H, H-4 of GlcN, CH₂CH₂Si(CH₃)₃), 3.69 (br d, 1H, H-6a of GlcN),

3.62–3.56 (m, 3H, H-2, 6b of GlcN, H-5 of GalN), 3.53–3.48 (m, 2H,

H-2 of GalN, CH₂CH₂Si(CH₃)₃), 3.39 (br d, 1H, H-5 of GlcN), 3.53–

3.48 (m, 1H, CH₂CH₂Si(CH₃)₃), 3.42 (br d, 1H, H-5 of GlcN), 2.08,

2.05, 1.91 (each s, 9H, 3 Ac), 0.91–0.86 (m, 2H, CH₂CH₂Si(CH₃)₃),

ꢀ0.03 (s, 9H, Si(CH₃)₃). ¹³C NMR (150 MHz, CDCl₃):d 170.3, 170.2,

170.0, 167.1, 154.1, 153.7, 137.4, 129.1, 129.0, 128.6, 100.4 (C-1of

GlcN), 100.1 (C-1 of GalN), 95.5, 95.4, 74.4, 74.3, 73.8, 73.5, 70.4,

70.3, 69.8, 67.4, 67.0, 66.21, 66.15, 61.2, 60.0, 56.0, 52.4, 40.8,

20.7, 20.64, 20.58, 20.48, 20.45, 18.0, ꢀ1.4, ꢀ1.5. HR-FABMS: calcd

for C₃₈H₅₁Cl₇N₂O₁₇SiNa: m/z 1103.0674; found: m/z 1103.0670

[M+Na]⁺.

4.29. 2,3,4,6-Tetra-O-acetyl-b-

acetyl-3,4-di-O-benzoyl- -fucopyranosyl-(1?3)]-2-acetamido-

6-O-acetyl-2-deoxy-b- -glucopyranosyl-(1?3)-2-acetamido-4,6-

di-O-acetyl-2-deoxy-b- -glucopyranosyl-(1?3)-2-acetamido-

4,6-di-O-acetyl-2-deoxy-b- -galactopyranosyl-(1?4)-6-O-acetyl-

2,3-di-O-benzoyl- -glucopyranosyl trichloroacetimidate (40)

D-galactopyranosyl-(1?4)-[2-O-

a-L

D

Compound 40 was prepared from 39 (43.0 mg, 20.7 lmol) as

described for preparation of 17. The product was puriﬁed by silica

gel column chromatography (20:1 chloroform–methanol) to give

40 (33.8 mg, 77%). [

CDCl₃): d 5.68 (d, 1H, J_1,2= 3.3 Hz, H-1 of Glc), 5.45 (d, 1H, J_1,2

a

]

ꢀ1.5 (c 0.8, CHCl₃). ¹H NMR (600 MHz,

D

=

3.6 Hz, H-1 of Fuc), 4.86 (br d, 1H, H-1 of GalN), 4.71 (d, 1H,

J_1,2= 7.8 Hz, H-1 of GlcN a), 4.70 (d, 1H, J_1,2= 8.0 Hz, H-1 of GlcN

b), 4.60 (d, 1H, J_1,2= 8.3 Hz, H-1 of Gal).

4.33. 2-(Trimethylsilyl)ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-

4.30. 2,3,4,6-Tetra-O-acetyl-b-

acetyl-3,4-di-O-benzoyl- -fucopyranosyl-(1?3)]-2-acetamido-

6-O-acetyl-2-deoxy-b- -glucopyranosyl-(1?3)-2-acetamido-4,6-

D-galactopyranosyl-(1?4)-[2-O-

trichloroethoxycar bonylamino)-b-

6-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-b-

D-galactopyranosyl-(1?4)-

a-L

D

D-glucopyranoside (44)

di-O-acetyl-2-deoxy-b-D-glucopyranosyl-(1?3)-2-acetamido-4,6-

di-O-acetyl-2-deoxy-b-D-galactopyranosyl-(1?4)-6-acetyl-2,3-di-

Compound 44 was prepared from 43 (0.64 g, 0.59 mmol) as de-

O-benzoyl-b-D-glucopyranosyl-(1?1)-(2S,3S,4R)-3,4-di-O-benzoyl-

2-hexadecanamido-octadecane-1,3,4-triol (41)

scribed for preparation of 25. The product was puriﬁed by silica gel

column chromatography (4:1 toluene–ethyl acetate) to give 44

(0.43 g, 70%). [

a

]

_Dꢀ23.5 (c 1.0, CHCl₃). ¹H NMR (600 MHz, CDCl₃):

Compound 41 was prepared from 40 (32.6 mg, 14.5 lmol) and

d 7.45–7.12 (m, 5H, Ph), 5.25 (d, 1H, J_3,4= 3.3 Hz, H-4 of GalN), 5.16

18 (16.7 mg, 21.8 lmol) as described for preparation of 19. The

(br s, 1H, NH of GlcN), 4.83, 4.32 (each d, 2H, 2 benzylmethylene),

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

69

4.73–4.70 (m, 2H, NHCOCH₂CCl₃), 4.65–4.58 (m, 4H, H-1 of GlcN,

H-3 of GalN, NHCOCH₂CCl₃), 4.11 (d, 1H, J_1,2= 8.5 Hz, H-1 of GalN),

4.09–4.06 (m, 2H, H-6 of GalN), 4.02 (br d, NH of GalN), 3.97–3.93

(m, 1H, CH₂CH₂Si(CH₃)₃), 3.86–3.80 (m, 3H, H-3 of GlcN, H-2,5 of

GalN), 3.71 (dd, 1H, J_6a,5= 3.8 Hz, J _6a,6b= 10.7 Hz), 3.63 (t, 1H, J

_3,4= J_{4, 5}= 9.3 Hz, H-4 of GlcN), 3.59 (br d, 1H, H-6b of GlcN),

3.52–3.47 (m, 1H, CH₂CH₂Si(CH₃)₃), 3.38 (br d, 1H, H-5 of GlcN),

3.28 (br dd, 1H, H-2 of GalN), 2.10, 2.04, 1.94 (each s, 9H, 3 Ac),

0.96–0.85 (m, 2H, CH₂CH₂Si(CH₃)₃), ꢀ0.03 (s, 9H, Si(CH₃)₃). ¹³C

NMR (CDCl₃):d 170.5, 170.0, 154.1, 154.0, 138.1, 129.1, 128.9,

128.6, 128.2, 101.9 (C-1of GalN), 99.9 (C-1 of GlcN), 95.5, 95.4,

81.4, 74.6, 74.4, 73.4, 71.5, 71.0, 70.0, 67.2, 66.2, 66.1, 61.3, 58.0,

51.9, 20.6, 18.0, ꢀ1.4. HR-FABMS: calcd for C₃₆H₅₀Cl₆N₂O₁₆SiNa:

m/z 1027.0958; found: m/z 1027.0980 [M+Na]⁺.

(3 mL). The reaction mixture was poured into ice H₂O and ex-

tracted with CHCl₃. The extract was washed sequentially with 5%

HCl, aq NaHCO₃, and brine, dried (MgSO₄), and concentrated. The

residue was puriﬁed by silica gel column chromatography using

3:1 CHCl₃–acetone to give 46 (36.5 mg, 44%). [

a]

ꢀ86.3 (c 0.6,

D

CHCl₃): ¹H NMR (600 MHz, CDCl₃): d 5.54 (d, 1H, J _1,2= 3.9 Hz,

H-1 of Fuc), 4.61 (d, 1H, J_1,2= 6.4 Hz, H-1 of GlcN), 4.61 (d, 1H, J

_1,2= 6.4 Hz, H-1 of GalN). ¹³C NMR (150 MHz, CDCl₃):d 171.2,

171.0, 170.8, 170.6, 166.0, 165.3, 133.3, 133.0, 129.8, 129.6,

129.48, 129.45, 128.5, 128.2, 101.2 (C-1 of GalN), 99.5 (C-1 of

GlcN), 95.4 (C-1 of Fuc), 73.8, 73.6, 73.0, 72.0, 71.0, 70.3, 69.5,

68.9, 68.2, 66.8, 66.2, 65.1, 63.2, 61.2, 53.8, 50.8, 31.7, 29.7, 29.3,

23.3, 21.0, 20.9, 20.8, 20.7, 20.6, 18.0, 16.1, ꢀ0.04, ꢀ1.5. HR-FABMS:

calcd for C₅₁H₆₈N₂O₂₂SiNa: m/z 1111.3931; found: m/z 1111.3956

[M+Na]⁺.

4.34. 2-(Trimethylsilyl)ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-

trichloroethoxy-carbonylamino)-b-

D

-galactopyranosyl-(1?4)-

4.36. 2-(Trimethylsilyl)ethyl 2-acetamido-2-deoxy-b-

galactopyranosyl-(1?4)-[ -fucopyranosyl- (1?3)]-2-

acetamido-2-deoxy-b- -glucopyranoside (6)

D-

[3,4-di-O-benzoyl-2-O-benzyl- -fucopyranosyl-(1?3)]-6-O-

a

-L

a-L

benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-b-

D-

D

glucopyranoside (45)

Compound 6 was prepared from 46 (36.5 mg, 33.5 lmol) as de-

Compound 45 was prepared from 44 (122 mg, 121

11 (147 mg, 242 mol) as described for preparation of 15. The

product was puriﬁed by silica gel column chromatography (3:1

hexane–ethyl acetate) to give 45 (141 mg, 80%). [ +29.7 (c

3.2, CHCl₃). ¹H NMR (600 MHz, CDCl₃): d 7.94–7.21 (m, 20H, 4

l

mol) and

scribed for preparation of 1. The product was puriﬁed by Sephadex

l

LH-20 column chromatography with MeOH to give 6 (21.1 mg,

94%). [

a

]

ꢀ28.1 (c 0.4, MeOH). ¹H NMR (600 MHz, CD₃OD): d

D

a]

4.99 (d, 1H, J_1,2= 3.8 Hz, H-1 of Fuc), 4.44 (d, 1H, J_1,2= 8.5 Hz, H-1

of GalN), 4.41 (d, 1H, J_1,2= 7.7 Hz, H-1 of GlcN). HR-FABMS: calcd

D

Ph), 5.68 (d, 1H, J_3,4= 2.5 Hz, H-4 of Fuc), 5.62 (dd, 1H, J _2,3

10.4 Hz, H-3 of Fuc), 5.40 (d, 1H, NH of GlcN), 5.34 (d, 1H, J_3,4

=

for C₂₇H₅₀N₂O₁₅SiNa: m/z 693.2878; found: m/z 693.2898

[M+Na]⁺.

3.3 Hz, H-4 of GalN), 5.32 (d 1H, J_1,2= 3.6 Hz, H-1 of Fuc), 5.09

(br. dd, 1H, H-5 of Fuc), 4.91 (d, 1H, J_1,2= 7.4 Hz, H-1 of GlcN),

4.87 (d, 1H, benzylmethylene), 4.79–4.66 (m, 7H, NH of Gal, 2

NHCOCH₂CCl₃, 2 benzylmethylene), 4.57 (br d, 1H, H-3 of GalN),

4.50 (dd, 1H, J_6a,5= 6.3 Hz, J_6a,6b= 11.3 Hz, H-6a of GalN), 4.42–

4.39 (m, 3H, H-1, 6b of GalN, benzylmethylene), 4.30 (br t, 1H,

H-3 of GalN), 4.14 (dd, 1H, H-2 of Fuc), 3.97–3.92 (m, 3H, H-4,5

4.37. 2-(Trimethylsilyl)ethyl 4,6-O-benzylidene-2-deoxy-2-

(2,2,2-trichloroethoxy-carbonylamino)-b-

(1?4)-6-O-benzyl-2-deoxy-2-(2,2,2-

D-galactopyranosyl-

trichloroethoxycarbonylamino)-b-D-glucopyranoside (47)

To a solution of 43 (311 mg, 0.287 mmol) in MeOH/CHCl₃

(25 mL, 9:1) was added guanidinium nitrate (311 mg) and MeONa

(27.1 mg). The reaction mixture was stirred for 3 h at room tem-

perature then neutralized with Amberlite IR 120 [H⁺]. The mixture

was ﬁltered and the ﬁltrate was washed with brine, dried (MgSO₄),

and concentrated. A mixture of the residue (756 mg, 0.83 mmol) in

CH₃CN (5 mL), NaHSO₄ꢁSiO₂(300 mg), and benzaldehyde dimethyl

of GlcN, CH₂CH₂Si(CH₃)₃), 3.77 (dd, 1H, J_6a,5= 2.2 Hz, J

=

6a,6b

10.7 Hz, H-6a of GlcN), 3.72–3.67 (m, 2H, H-2, 5 of GalN), 3.61

(br d, 1H, H-6b of GlcN), 3.52–3.47 (m, 1H, CH₂CH₂Si(CH₃)₃),

3.40 (br d, 1H, H-5 of GlcN), 3.14 (br d, 1H, H-2 of GlcN), 2.21,

2.08, 1.96 (each s, 9H, 3 Ac), 1.26 (d, 3H, H-6 of Fuc), 0.95–0.86

(m, 2H, CH₂CH₂Si(CH₃)₃), ꢀ0.01 (s, 9H, Si(CH₃)₃). ¹³C NMR

(150 MHz, CDCl₃):d 170.7, 170.5, 170.1, 166.0, 165.9, 165.3,

154.0, 153.7, 137.6, 137.1, 133.1, 132.8, 129.9, 129.8, 129.7,

129.6, 129.4, 129.3, 128.8, 128.5, 128.4, 128.2, 128.1, 127.9,

100.4 (C-1 of GalN), 98.8 (C-1 of GlcN), 97.3 (C-1 of Fuc), 95.6,

74.7, 74.5, 74.4, 74.1, 73.8, 73.3, 72.8, 71.0, 70.7, 70.4, 67.8, 67.3,

66.1, 64.8, 61.2, 59.4, 52.4, 29.3, 20.9, 20.6, 18.1, 16.0, ꢀ0.02,

ꢀ1.4. HR-FABMS: calcd for C₆₃H₇₄Cl₆N₂O₂₂SiNa: m/z 1471.2531;

found: m/z 1471.2535 [M+Na]⁺.

acetal (BDA) (87.6 lL, 574 lmol, 2.0 equiv) was stirred for 3 h at

room temperature, then neutralized with Et₃N. The solids were ﬁl-

trated off and the ﬁltrated was concentrated. The product was

puriﬁed by silica gel column chromatography using 5:1 toluene–

acetone to give 47 (161 mg, 58%). [

a]

ꢀ13.0 (c 2.0, CHCl₃). ¹H

D

NMR (600 MHz, CD₃COCD₃): d 7.54–7.12 (m, 10H, Ph), 6.82 (d,

1H, NH of GlcN), 6.68 (d, 1H, NH of GalN), 5.68 (s, 1H, PhCH),

4.80–4.78 (m, 2H, NHCOCH₂CCl₃), 4.72–4.69 (m, 2H,

NHCOCH₂CCl₃), 4.67 (d, 1H, J_1,2= 8.0 Hz, H-1 of GalN), 4.63 (s, 2H,

2 benzylmethylene), 4.51 (d, 1H, J_1,2= 8.5 Hz, H-1 of GlcN), 4.46

(s, 1H, OH of GlcN), 4.33 (d, 1H, J_3,4= 3.0 Hz, H-4 of GalN), 4.20

(br d, 2H, H-6 of GalN), 4.14 (br d, OH of GalN), 3.97–3.93 (m,

1H, CH₂CH₂Si(CH₃)₃), 3.91–3.86 (m, 2H, H-2, 5 of GalN), 3.80–

3.78 (m, 2H, H-6 of GlcN, H-3 of GalN), 3.71 (br t, 1H, H-3 of GlcN),

3.63 (br t, 1H, H-4 of GlcN), 3.59–3.55 (m, 1H, CH₂CH₂Si(CH₃)₃),

4.35. 2-(Trimethylsilyl)ethyl 2-acetamido-3,4,6-tri-O-acetyl-

deoxyb-

benzoyl-a-L

deoxy-b-

D

-galacto-pyranosyl-(1?4)-[2-O-acetyl-3,4-di-O-

-fucopyranosyl-(1?3)]-2-acetamido-6-O-acetyl-2-

D-glucopyranoside (46)

A mixture of 45 (106 mg, 73.2 lmol) and zinc powder (900 mg)

in AcOH–Ac₂O (2:1, 9 mL) was stirred for 18 h at 55 °C. After com-

pletion of the reaction, the mixture was ﬁltered and the ﬁltrate was

diluted with water and extracted with CHCl₃. The extract was

washed with water, dried (MgSO₄), and concentrated. A solution

of the residue in MeOH–THF (1:1, 2 mL) was hydrogenolysed

(0.4 MPa) under hydrogen in the presence of 10% Pd/C (150 mg)

for 18 h at room temperature, then ﬁltered and concentrated. The

residue was acetylated with acetic anhydride (2 mL) in pyridine

3.51–3.47 (m, 2H, H-2,

5

of GlcN), 0.95–0.86 (m, 2H,

CH₂CH₂Si(CH₃)₃), 0.00 (s, 9H, Si(CH₃)₃). ¹³C NMR (150 MHz,

CD₃COCD₃): d 155.5, 155.3, 154.1, 140.0, 139.5, 129.4, 129.1,

129.0, 128.7, 128.4, 128.1, 127.2, 103.1 (C-1of GalN), 101.7 (C-1

of GlcN), 101.4, 97.1, 96.9, 81.8, 76.2, 75.1, 75.0, 74.9, 73.7, 73.6,

70.8, 69.6, 67.84, 67.81, 67.1, 58.4, 56.0, 18.6, ꢀ1.2. HR-FABMS:

calcd for

C₃₇H₄₈Cl₆N₂O₁₃SiNa: m/z 989.0955; found: m/z

989.0976 [M+Na]⁺.

70

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

4.38. 2-(Trimethylsilyl)ethyl 3,4-di-O-benzoyl-2-O-benzyl-

fucopyranosyl- (1?3)-4,6-O-benzylidene-2-deoxy-2-(2,2,2-

trichloroethoxycarbonylamino)-b- -galactopyranosyl-(1?4)-

[3,4-di-O-benzoyl-2-O-benzyl- -fucopyranosyl-(1?3)]-6-O-

benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-b-

glucopyranoside (48) 2-(trimethylsilyl)ethyl 3,4-di-O-benzoyl-

2-O-benzyl- -fucopyranosyl-(1?3)-4,6-O-benzylidene-2-

deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-b-

galactopyranosyl-(1?4)-6-O-benzyl-2-deoxy-2-(2,2,2-

trichloroethoxycarbonylamino)-b- -glucopyranoside (49)

a

-

L

–

L

-

4.39. 2-(Trimethylsilyl)ethyl

a-L-fucopyranosyl-(1?3)-2-

acetamido-2-deoxy-b- -galactopyranosyl-(1?4)-[a-L-

D

fucopyranosyl-(1?3)]-2-acetamido-2-deoxy-b-D-

glucopyranoside (7)

a

-L

D

-

Compound 7 was prepared from 48 70.5 mg, 38.0 lmol) as de-

scribed for preparation of 4. The product was puriﬁed by Sephadex

a

-L

D-

LH-20 column chromatography in MeOH to give 7 (7.2 mg, 23%).

[

a

]

ꢀ88.0 (c 0.3, MeOH). ¹H NMR (600 MHz, CD₃OD): d 5.00 (d,

D

1H, J_1,2= 4.1 Hz, H-1 of Fuc a), 4.87 (d, 1H, J_1,2= 4.1 Hz, H-1 of

Fuc b), 4.51 (d, 1H, J _1,2= 8.5 Hz, H-1 of GalN), 4.40 (d, 1H, J

_1,2= 7.7 Hz, H-1 of GlcN). ¹³C NMR (150 MHz, CDCl₃):d 103.0 (C-1

of Fuc b), 101.9 (C-1 of GalN), 101.7 (C-1 of GlcN), 100.0 (C-1 of

(Ent. 1) A mixture of 47 (60.8 mg, 62.7

376 mol, 6.0 equiv), and MS 4 Å (250 mg) in dry CH₂Cl₂

(1.8 mL) was stirred for 3 h at room temperature, then cooled to

L, 47.1 mol, 0.15 equiv) was added, and

lmol), 11 (228 mg,

l

Fuc a). HR-FABMS:: calcd for

C₁₀₁H₁₁₀Cl₆N₂O₃₁SiNa: m/z

ꢀ40 °C. TMSOTf (8.5

l

839.3457; found: m/z 839.3489 [M+Na]⁺.

the mixture was stirred for 18 h at ꢀ40 °C, then neutralized with

Et₃N. The precipitates were ﬁltrated off and washed with CHCl₃.

The combined ﬁltrate and washings were washed with water,

dried (MgSO₄), and concentrated. The product was puriﬁed by sil-

ica gel column chromatography (2:1 hexane–EtOAc) to give 48

(91.0 mg, 78%).

4.40. 2,2,2-Trichloroethyl 3,4-O-isopropylidene-a-L-

fucopyranoside (51)

A mixture of L-fucose (50) (200 mg, 1.22 mmol) and NaH-

SO₄ꢁSiO₂(500 mg) in 2,2,2-trichloroethanol (5 mL) was stirred for

8 h at 50 °C, then neutralized with Et₃N. The solids were ﬁltrated

off and the ﬁltrated was concentrated. A mixture of the residue

in acetone (4.0 mL), NaHSO₄ꢁSiO₂(500 mg), and 2,2-dimethoxypro-

pane (2 mL) was stirred for 3 h at room temperature, then neutral-

ized with Et₃N. The solids were ﬁltrated off and the ﬁltrated was

concentrated. The product was puriﬁed by silica gel column chro-

matography using 10:1 toluene–acetone to give 51 (240 mg, 59%).

¹H NMR (600 MHz, CD₃COCD₃): d 5.03 (d, J_1,2= 3.6 Hz, H-1), 4.35,

4.26 (each d, 2H, CH₂CCl₃), 4.32–4.30 (m, 1H, H-5), 4.23 (t, 1H, J

_2,3= J_3,4= 6.1 Hz, H-3), 4.15 (dd, 1H, H-4), 3.98 (d, 1H, OH), 3.79–

3.76 (m, 1H, H-2), 1.41, 1.29 (each s, 6H, O(CH₃)₂), 1.26 (d, 3H,

H-6). ¹³C NMR (150 MHz, CD₃COCD₃): d 109.2, 100.2 (C-1), 97.8,

80.2, 76.8, 76.4, 70.1, 65.5, 28.2, 26.2, 16.6.

(Ent. 2) A mixture of 47 (92.0 mg, 94.9

142 mol, 1.5 equiv), and MS 4 Å (150 mg) in dry CH₂Cl₂(1.2 mL)

was stirred for 3 h at room temperature, then cooled to ꢀ40 °C.

TMSOTf (2.6 L, 14.2 mol, 0.10 equiv) was added, and the mixture

lmol), 11 (86.4 mg,

l

was stirred for 3 h at ꢀ40 °C, then neutralized with Et₃N. The pre-

cipitates were ﬁltrated off and washed with CHCl₃. The combined

ﬁltrate and washings were washed with water, dried (MgSO₄),

and concentrated. The product was puriﬁed by silica gel column

chromatography (5:2 hexane–EtOAc) to give 49 (91.0 mg, 28%) as

an amorphous powder and was recovered compound 47 (52%).

(Ent. 3) A mixture of 47 (110.5 mg, 114

342 mol, 3.0 equiv), and MS 4 Å (200 mg) in dry CH₂Cl₂(2.0 mL)

was stirred for 3 h at room temperature, then cooled to ꢀ40 °C.

TMSOTf (9.3 L, 51.3 mol, 0.15 equiv) was added, and the mixture

lmol), 11 (208 mg,

l

was stirred for 3 h at ꢀ40 °C, then neutralized with Et₃N. The pre-

cipitates were ﬁltrated off and washed with CHCl₃. The combined

ﬁltrate and washings were washed with water, dried (MgSO₄),

and concentrated. The product was puriﬁed by silica gel column

chromatography (5:2 hexane–EtOAc) to give 49 (106 mg, 66%) as

an amorphous powder and was recovered compound 47 (11%).

4.41. 2,2,2-Trichloroethyl 3,4-di-O-benzoyl-2-O-benzyl-a-L-

fucopyranosyl-(1?2)-3,4-O-isopropylidene-a-L-fucopyranoside

(52)

Compound 52 was prepared from 51 (175 mg, 0.522 mmol) and

11 (475 mg, 0.783 mmol, 5.0 eq.) as described for preparation of

15. The product was puriﬁed by silica gel column chromatography

48: [a

]

D

ꢀ109.9 (c 1.6, CHCl₃). ¹H NMR (600 MHz, CDCl₃): d 5.36

(d, 1H, J_1,2= 3.0 Hz, H-1 of Fuc a), 4.79 (br s, 1H, H-1 of Fuc b),

4.85 (br d, 1H, H-1 of GlcN), 4.65 (d, 1H, J_1,2= 8.5 Hz, H-1 of GalN).

¹³C NMR (150 MHz, CDCl₃): d 165.9, 165.8, 165.5, 165.3, 165.2,

154.4, 153.6, 138.5, 137.8, 137.4, 137.2, 133.2, 132.9, 132.8,

130.3,3 130.26, 129.7, 129.64, 129.56, 128.6, 128.49, 128.45,

128.4, 128.23, 128.19, 128.0, 127.92, 127.86, 127.7, 125.6, 100.1,

99.7 (C-1 of GalN; Fuc b), 98.9 (C-1 of GlcN), 97.6 (C-1 of Fuc a),

95.7, 95.5, 76.1, 74.8, 74.6, 74.3, 74.1, 74.0, 73.3, 73.0, 72.9, 72.3,

71.5, 70.8, 69.4, 68.8, 66.9, 66.6, 66.0, 65.4, 60.4, 58.8, 53.6, 29.7,

18.0, 16.2,15.3, 14.2, –1.5. MALDI-TOFMS: calcd for

(3:1 hexane–ethyl acetate) to give 52 (326 mg, 75%). [

a

]

_Dꢀ114.0 (c

1.3, CHCl₃). ¹H NMR (600 MHz, CD₃COCD₃): d 7.97–7.22 (m, 15H, 3

Ph), 5.74 (dd, 1H, J_{2 ,3}= 10.5 Hz, J_{3 ,4}= 3.3 Hz, H-3⁰), 5.64 (d, 1H,

0

H-4⁰), 5.58 (d, 1H, J_{1 ,2}= 3.3 Hz, H-1⁰), 5.30 (d, 1H, J_1,2= 3.3 Hz, H-1),

4.76 (q, 2H, benzylmethelene), 5.70–5.69 (m, 1H, H-5⁰), 4.45–4.42

(m, 2H, H-3, 5), 4.33–4.26 (m, 4H, H-4, 2⁰, OCH₂CCl₃), 3.96 (dd,

1H, J_2,3= 7.7 Hz, H-2), 1.52, 1.36 (each s, 6H, C(CH₃)₂), 1.31 (d, 1H,

H-6), 1.19 (d, 1H, H-6⁰). ¹³C NMR (150 MHz, CD₃COCD₃): d 166.5,

165.9, 139.3, 134.2, 133.9, 130.8, 130.3, 130.2, 129.5, 129.2,

129.0, 128.6, 128.3, 109.4, 98.3 (C-1), 97.5, 96.5 (C-1⁰), 81.0, 76.7,

75.3, 75.2, 74.2, 73.3, 72.7, 71.4, 65.6, 16.6, 16.4.

0

C

₉₁H₉₆Cl₆N₂O₂₅SiNa: m/z 1877; found: m/z 1877 [M+Na]⁺. 49:

[a

]

D

ꢀ61.7 (c 0.95, CHCl₃). ¹H NMR (600 MHz, CDCl₃): d 5.47 (d,

1H, J_1,2= 3.0 Hz, H-1 of Fuc), 4.79 (d, 1H, J_1,2= 7.6 Hz, H-1 of GalN),

4.52 (d, 1H, J_1,2= 8.5 Hz, H-1 of GlcN). ¹³C NMR (150 MHz, CDCl₃):d

166.4, 165.8, 154.3, 140.0, 139.4, 139.3, 134.1, 133.9, 130.8, 130.7,

130.2, 129.6, 129.5, 129.13, 129.07, 129.0, 128.9, 128.5, 128.3,

128.1, 127.1, 102.5 (C-1 of GalN), 101.8, 101.5 (C-1 of GlcN),

100.2 (C-1 of Fuc), 97.1, 96.8, 81.7, 79.7, 75.6, 75.2, 74.89, 74.85,

73.9, 73.8, 73.7, 73.2, 72.0, 71.2, 69.6, 69.5, 67.5, 67.1, 66.2, 58.4,

54.2, 18.6, 16.6, ꢀ1.2. HR-FABMS: calcd for C₆₄H₇₂Cl₆N₂O₁₉SiNa:

m/z 1433.2527; found: m/z 1433.2567 [M+Na]⁺.

4.42. 2,2,2-Trichloroethyl 3,4-di-O-benzoyl-2-O-benzyl-

a-L-

fucopyranosyl-(1?2)-3,4-di-O-acetyl- -fucopyranoside (53)

a-L-L

A solution of 52 (131 mg, 0.168 mmol) in 80% AcOH (5 mL) was

stirred at 50 °C for 5 h, then diluted with toluene and concentrated.

The residue was acetylated with acetic anhydride (2.0 mL) in pyr-

idine (3.0 mL). After the reaction was quenched with MeOH, tolu-

ene was added and co-evaporated several times. The product was

puriﬁed by silica gel column chromatography (4:1 hexane–ethyl

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

71

acetate) to give 53 (118 mg, 86%). [

a

]

ꢀ154.1 (c 1.4, CHCl₃). ¹H

128.16, 125.2, 99.5 (C-1⁰), 98.8 (C-1⁰⁰), 98.7 (C-1), 96.4, 80.1, 76.0,

74.6, 73.2, 72.5, 72.4, 71.63, 71.56, 70.6, 69.8, 69.0, 65.7, 65.6,

65.2, 29.7, 21.4, 21.0, 20.8, 20.6, 20.5, 16.0, 15.8, 15.7.

D

NMR (600 MHz, CD₃COCD₃): d 8.00–7.23 (m, 15H, 3 Ph), 5.72 (dd,

1H, J_{2 ,3}= 9.4 Hz, J_{3 ,4}= 3.3 Hz, H-3⁰), 5.63 (d, 1H, H-4⁰), 5.43 (d,

0

1H, J _{1 ,2}= 3.6 Hz, H-1⁰), 5.37 (dd, 1H, J _2,3= 9.5 Hz, J _3,4= 3.3 Hz,

H-3), 5.32–5.31 (d, 2H, J_1,2= 3.6 Hz, H-1, 4), 4.77 (q, 2H, benzylm-

ethelene), 4.59–4.56 (m, 1H, H-5⁰), 4.26 (dd, 1H, H-2⁰), 4.20 (q,

2H, OCH₂CCl₃), 4.18 (dd, 1H, H-2), 2.15, 2.03 (each s, 6H, 2 Ac),

1.18 (d, 1H, H-6⁰), 1.14 (d, 1H, H-6). ¹³C NMR (150 MHz,

CD₃COCD₃):d 170.9, 170.3, 170.0, 166.5, 165.9, 139.1, 134.2,

133.9, 130.8, 130.3, 130.2, 129.5, 129.2, 129.1, 128.9, 128.5, 99.0

(C-1), 98.9 (C-1⁰), 97.3, 81.0, 74.8, 74.4, 73.5, 73.3, 72.0, 71.6,

70.0, 66.4, 66.2, 20.9, 20.5, 16.3, 16.1.

0

4.46. 3,4-Di-O-benzoyl-2-O-benzyl-

3,4-di-O-acetyl- -fucopyranosyl-(1?2)-3,4-di-O-acetyl-

fucopyranosyl trichloroacetimidate (57)

a-L-fucopyranosyl-(1?2)-

a-

L

a-L-

Compound 57 was prepared from 56 (151 mg, 0.18 mmol) as

described for preparation of 54. The product was puriﬁed by silica

gel column chromatography (chloroform) to give 57 (75.8 mg,

76%). [

a

]

ꢀ156.3 (c 1.9, CHCl₃). ¹H NMR (600 MHz, CDCl₃): d

D

8.60 (s, 1H, C(NH)CCl₃), 6.61 (d, 1H, J _1,2= 3.6 Hz, H-1), 5.06 (d,

1H, J_{1 ,2}= 3.5 Hz, H-1⁰). 4.96 (d, 1H, J_{1 ,2}= 3.6 Hz, H-1⁰⁰).

0

00 00

4.43. 3,4-Di-O-benzoyl-2-O-benzyl-a-L-fucopyranosyl-(1?2)-

3,4-di-O-acetyl- -fucopyranosyl trichloroacetimidate (54)

a-L

4.47. 2-(Trimethylsilyl)ethyl 3,4-di-O-benzoyl-2-O-benzyl-

a-L-

A mixture of 53 (1.75 g, 0.13 mmol) and zinc powder (1.50 g) in

AcOH (15.0 mL) was stirred for 18 h at 55 °C. After completion of

the reaction, the mixture was ﬁltered and the ﬁltrate was diluted

with toluene and concentrated to give the intermediate. To a solu-

tion of the residue in CH₂Cl₂(5.0 mL) cooled at –20 °C were added

fucopyranosyl-(1?3)-4,6-O-benzylidene-2-deoxy-2-(2,2,2-

trichloroethoxycarbonylamino)-b- -galactopyranosyl-(1?4)-

[3,4-di-O-benzoyl-2-O-benzyl- -fucopyranosyl-(1?2)-3,4-di-

O-acetyl- -fucopyranosyl-(1?3)]-6-O-benzyl-2-deoxy-2-

D

a-L

(2,2,2-trichloro-ethoxycarbonylamino)-b-D-glucopyranoside

DBU (120

lL, 0.822 mmol) and CCl₃CN (280

lL, 2.74 mmol). The

(58)

reaction mixture was stirred for 5 h at ꢀ20 °C. After completion

of the reaction, the mixture was concentrated. The residue was

puriﬁed by silica gel column chromatography using chloroform

Compound 58 was prepared from 49 (83.3 mg, 59.1

54 (59.4 mg, 70.9 mol) as described for preparation of 15. The

product was puriﬁed by silica gel column chromatography (5:2

hexane–ethyl acetate) to give 58 (82.7 mg, 67%). [

lmol) and

l

to give 54 (350 mg, 77%). [

a

]

D

ꢀ117.2 (c 3.0, CHCl₃). ¹H NMR

(600 MHz, CDCl₃): d 8.52 (s, 1H, C(NH)CCl₃), 7.94–7.22 (m, 15H, 3

a]

ꢀ101.4 (c

D

Ph), 6.56 (d, 1H, J_1,2= 3.6 Hz, H-1), 5.63 (dd, 1H, J_{2 ,3}= 10.4 Hz,

2.0, CHCl₃). ¹H NMR (600 MHz, CDCl₃): d 5.37 (d, 1H, J_1,2= 3.5 Hz,

H-1 of Fuc a), 5.16 (d, 1H, J_1,2= 3.6 Hz, H-1 of Fuc c), 5.12 (d, 1H,

J_1,2= 3.0 Hz, H-1 of Fuc b), 4.65 (d, 1H, J_1,2= 7.5 Hz, H-1 of GlcN).

¹³C NMR (150 MHz, CDCl₃):d 99.9 (C-1 of GlcN), 99.7 (C-1 of Fuc

b), 98.5 (C-1 of GalN), 98.2 (C-1 of Fuc c), 96.7 (C-1 of Fuc a). MAL-

DI-TOFMS: calcd for C₁₀₁H₁₁₀Cl₆N₂O₃₁SiNa: m/z 2108; found: m/z

2108 [M+Na]⁺.

0

J_{3 ,4}= 3.3 Hz, H-3⁰), 5.59 (d, 1H, H-4⁰), 5.45–5.43 (m, 2H, H-3, 4),

0

5.06 (d, 1H, J _{1 ,2}= 3.5 Hz, H-1⁰), 4.58 (d, 2H, benzylmethelene),

4.42 (br dd, 1H, H-5⁰), 4.37 (br dd, 1H, H-5), 4.22 (dd, 1H,

J _2,3= 9.9 Hz, H-2), 4.07 (dd, 1H, H-2⁰), 2.19, 2.07 (each s, 6H, 2

Ac), 1.19 (d, 1H, H-6⁰), 1.17 (d, 1H, H-6). ¹³C NMR (150 MHz,

CDCl₃):d 170.3, 169.8, 165.8, 165.3, 161.0, 137.8, 133.2, 132.9,

129.7, 129.6, 129.5, 128.4, 128.3, 128.2, 128.0, 127.8, 98.6 (C-1⁰),

94.3 (C-1), 73.1, 73.0, 72.8, 72.2, 70.9, 70.8, 70.6, 69.4, 67.3, 65.6,

60.3, 30.9, 21.0, 20.8, 20.6, 15.99, 15.95, ꢀ0.04.

0

4.48. 2-(Trimethylsilyl)ethyl 3,4-di-O-benzoyl-2-O-benzyl-

fucopyranosyl-(1?3)-4,6-O-benzylidene-2-deoxy-2-(2,2,2-

trichloroethoxycarbonylamino)-b- -galactopyranosyl-(1?4)-

[3,4-di-O-benzoyl-2-O-benzyl- -fucopyranosyl-(1?2)-3,4-di-

O-acetyl- -fucopyranosyl-(1?2)-3,4-di-O-acetyl-

fucopyranosyl-(1?3)]-6-O-benzyl-2-deoxy-2-(2,2,2-

trichloroethoxycarbonylamino)-b- -glucopyranoside (59)

a-L-

D

4.44. 2,2,2-Trichloroethyl 3,4-di-O-benzoyl-2-O-benzyl-

fucopyranosyl-(1?2)-3,4-di-O-acetyl- -fucopyranosyl-(1?2)-

3,4-O-isopropylidene- -fucopyranoside (55)

a-

L-

a-L

a-

L

a-

L

a-L-

a-L

D

Compound 55 was prepared from 51 (151 mg, 0.18 mmol) and

54 (122 mg, 0.36 mmol) as described for preparation of 15. The

product was puriﬁed by silica gel column chromatography (2:1

Compound 59 was prepared from 49 (46.5 mg, 33.0

57 (82.7 mg, 39.6 mol) as described for preparation of 15. The

product was puriﬁed by silica gel column chromatography (5:2

hexane–ethyl acetate) to give 59 (32.9 mg, 43%). [

lmol) and

l

hexane–ethyl acetate) to give 55 (141 mg, 80%). [

a

]

D

ꢀ157.2 (c

1.8, CHCl₃). ¹H NMR (600 MHz, CDCl₃): d 5.21 (d, 1H, J _{1 ,2}

=

a]

ꢀ101.4 (c

0

D

3.3 Hz, H-1⁰), 5.16 (d, 1H, J_1,2= 3.0 Hz, H-1), 5.05 (d, 1H, J_{1 ,2}

2.0, CHCl₃). ¹H NMR (600 MHz, CDCl₃): d 5.37 (d, 1H, J_1,2= 3.5 Hz,

H-1 of Fuc a), 5.20 (d, 1H, J_1,2= 3.6 Hz, H-1 of Fuc d), 5.15 (d, 1H,

J_1,2= 3.0 Hz, H-1 of Fuc b), 5.12 (d, 1H, J_1,2= 3.0 Hz, H-1 of Fuc c),

4.65 (d, 1H, J_1,2= 7.5 Hz, H-1 of GlcN). ¹³C NMR (150 MHz, CDCl₃):d

99.9 (C-1 of GlcN), 99.7 (C-1 of Fuc b), 98.6 (C-1 of GalN), 98.2 (C-1

of Fuc c, d), 96.5 (C-1 of Fuc a). MALDI-TOFMS: calcd for

00 00

3.6 Hz). ¹³C NMR (150 MHz, CDCl₃):d 170.4, 169.9, 165.7, 165.2,

137.4, 133.2, 132.9, 129.7, 129.6, 129.5, 128.5, 128.4, 128.2,

128.1, 128.04, 127.99, 108.7, 98.7 (C-1⁰⁰), 98.0 (C-1), 97.8 (C-1⁰),

96.5, 80.4, 76.6, 76.0, 74.6, 74.5, 73.1, 72.3, 71.9, 70.6, 69.2, 65.5,

64.9, 64.6, 28.3, 26.3, 20.9, 20.6, 16.2, 15.98, 15.95, 15.7.

C

₁₁₁H₁₂₄Cl₆N₂O₃₇SiNa: m/z 2337.6; found: m/z 2337.1 [M+Na]⁺.

4.45. 2,2,2-Trichloroethyl 3,4-di-O-benzoyl-2-O-benzyl-

fucopyranosyl-(1?2)-3,4-di-O-acetyl- -fucopyranosyl-(1?2)-

3,4-di-O-acetyl- -fucopyranoside (56)

a-L-

a-

L

4.49. 2-(Trimethylsilyl)ethyl

acetamido-2-deoxy-b- -galactopyranosyl-(1?4)-[

fucopyranosyl-(1?2)- -fucopyranosyl-(1?3)]-2-acetamido-

2-deoxy-b- -gluco-pyranoside (8)

a-L-fucopyranosyl-(1?3)-2-

a-

L

D

a-L-

a

-L

Compound 56 was prepared from 55 (131 mg, 0.17 mmol) as

described for preparation of 53. The product was puriﬁed by silica

gel column chromatography (3:1 hexane–ethyl acetate) to give 56

D

Compound 8 was prepared from 58 (57.6 mg, 27.6 lmol) as de-

(98.4 mg, 63%). [

a

]

ꢀ138.2 (c 2.5, CHCl₃). ¹H NMR (600 MHz,

scribed for preparation of 4. The product was puriﬁed by silica gel

column chromatography on Iatrobeads (2:1 chloroform–methanol)

to give 8 (8.5 mg, 32%). ¹H NMR (600 MHz, CD₃OD) d 5.29 (d, 1H,

J_1,2= 3.6 Hz, H-1 of Fuc a), 4.91 (d, 1H, J_1,2= 3.8 Hz, H-1 of Fuc c),

4.90 (d, 1H, J_1,2= 3.8 Hz, H-1 of Fuc b), 4.60 (d, 1H, J_1,2= 8.5 Hz,

D

0

CDCl₃): d 5.50 (d, 1H, J_1,2= 3.8 Hz, H-1), 5.05 (d, 1H, J_{1 ,2}= 3.3 Hz,

H-1⁰), 4.89 (d, 1H, J_{1 ,2}= 3.6 Hz, H-1⁰⁰). ¹³C NMR (150 MHz, CDCl₃):d

00 00

170.4, 170.3, 170.0, 169.6, 165.7, 165.2, 137.8, 137.4, 133.3, 133.0,

129.71, 129.6, 129.6, 129.0, 128.6, 128.5, 128.3, 128.23, 128.19,

72

T. Kanaya et al. / Carbohydrate Research 361 (2012) 55–72

3. Kamerling, J. K. In Comprehensive Glycoscience; Elsevier: New York, 2007; Vol. 4,

473–492.

H-1 of GalN), 4.48 (d, 1H, J_1,2= 7.7 Hz, H-1 of GlcN). HR-FABMS:

calcd for C₃₉H₇₀N₂O₂₃SiNa: m/z 985.4036; found: m/z 985.4062

[M+Na]⁺.

4. Nash, T. E.; Lunnde, M. N.; Cheever, A. W. J. Immunol. 1981, 126, 805–810.

5. (a) Hokke, C. H.; Yazdnbakhsh, M. Parasite Immunol. 2005, 27, 257–264; (b)

Harnett, W. Parasite Immunol. 2005, 27, 357–359; (a) Hokke, C. H.; Deelder, A.

M. Glycoconjugate J. 2001, 18, 573–587; (b) Dell, A.; Haslam, S. M.; Morris, H. R.;

Khoo, K. H. Biochim. Biophys. Acta 1999, 1455, 353–362; (c) Cummings, R. D.;

Nyame, A. K. Biochim. Biophys. Acta 1999, 1455, 363–374; (d) Khoo, K. H. Trends

Glycosci. Glycotech. 2001, 13, 493–506.

6. (a) Nyame, A. K.; Cummings, R. D.; Damian, R. T. Mol. Biochem. Parasitol. 1988,

28, 265–274; (b) Nyame, A. K.; Smith, D. F.; Damian, R. T.; Cummings, R. D. J.

Biol. Chem. 1989, 264, 3235–3243; (c) Srivatsan, J.; Smith, D. F.; Cummings, R. D.

Glycobiology 1992, 2, 445–452.

7. Hung, H. H.; Tsai, P. L.; Khoo, K. H. Glycobiology 2001, 11, 395–406.

8. (a) Wuhrer, M.; Kantelhardt, S. R.; Dennis, R. D.; Doenhoff, M. J.; Lochnit, G.;

Geyer, R. Glycobiology 2000, 10, 89–101; (b) Wuhrer, M.; Kantelhardt, S. R.;

Dennis, R. D.; Doenhoff, M. J.; Lochnit, G.; Geyer, R. Eur. J. Biochem. 2000, 269,

481–493.

4.50. 2-(Trimethylsilyl)ethyl

acetamido-b- -galacto-pyranosyl-(1?4)-[

(1?2)- -fucopyranosyl-(1?2)- -fucopyranosyl-(1?3)]-2-

acetamido-2-deoxy-b- -glucopyranoside (9)

a-

L

-fucopyranosyl-(1?3)-2-

D

a-L-fucopyranosyl-

a-L

D

Compound 9 was prepared from 59 (32.9 mg, 14.2 lmol) as de-

scribed for preparation of 4. The product was puriﬁed by silica gel

column chromatography on Iatrobeads (2:1 chloroform–methanol)

to give 9 (5.0 mg, 35%). [

a

]

ꢀ76.2 (c 0.06, MeOH). ¹H NMR

D

(600 MHz, CD₃OD) d 5.28 (d, 1H, J_1,2= 3.6 Hz, H-1 of Fuc a), 4.92

(d, 1H, J_1,2= 3.8 Hz, H-1 of Fuc d), 4.90 (d, 1H, J_1,2= 3.8 Hz, H-1

of Fuc c), 4.88 (d, 1H, J _1,2= 3.8 Hz, H-1 of Fuc b), 4.60 (d, 1H,

J_1,2= 8.3 Hz, H-1 of GalN), 4.50 (d, 1H, J_1,2= 7.9 Hz, H-1 of GlcN).

HR-FABMS: calcd for C₄₅H₈₀N₂O₂₇SiNa: m/z 1131.4615; found: m/z

1131.4631 [M+Na]⁺.

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Acknowledgements

This work was supported by a Grant-in-Aid for Scientiﬁc Re-

search (No. 22590014) from the Ministry of Education, Culture,

Sports, Science and Technology of Japan (MEXT), and the Strategic

Research Foundation at Private Universities of the MEXT. We grate-

fully acknowledge ﬁnancial support in the form of the Uehara

Memorial Foundation. The authors are grateful to Ms. J. Hada for

providing HR-FABMS measurement data.

Supplementary data

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.carres.2012.08.008.

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Synthetic studies on glycosphingolipids from protostomia phyla: Synthesis of glycosphingolipids and related carbohydrate moieties from the parasite Schistosoma mansoni

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