Synthesis of galactofuranosyl-containing oligosaccharides corresponding to the glycosylinositolphospholipid of Trypanosoma cruzi.

www.elsevier.nl/locate/carres

Carbohydrate Research 325 (2000) 253–264

Synthesis of galactofuranosyl-containing oligosaccharides

corresponding to the glycosylinositolphospholipid of

Trypanosoma cruzi

Karla D. Randell, Blair D. Johnston, Paula N. Brown, B. Mario Pinto *

Department of Chemistry and Biochemistry, Simon Fraser Uni6ersity, Burnaby, B.C., Canada V5A 1S6

Received 8 December 1999; accepted 14 December 1999

Abstract

The oligosaccharide b-

D

-Galf-(13)-a-

D

-Manp-(12)-[b-

D

-Galf-(13)]-a-

D

-Manp-(12)-a-D-Manp corre-

sponds to the terminal end of the glycosylinositolphospholipid oligosaccharide of the protozoan Trypanosoma cruzi,

the causative agent of Chagas’ disease. Syntheses of methyl or ethylthio glycosides of the terminal disaccharide,

trisaccharide, tetrasaccharide, and pentasaccharide corresponding to this structure are described. These syntheses

employ the selective activation of a phenyl 1-selenogalactofuranoside or a phenyl 1-selenomannopyranoside donor

Keywords: Glycosylation; Glycosides; Galactofuranose; Trypanosoma cruzi

Manp-(12)-a-Manp-(16)-a-Manp-(14)-

1. Introduction

a-GlcNp-(16)-myoinositol backbone, sug-

gesting that this sequence is likely to be con-

served in all GPI anchors. In protozoa, GPI

anchors have been widely studied in their role

as anchors for cell-surface proteins. Several

protozoa also synthesize unique GPI deriva-

tives that are not covalently linked to protein

or modiﬁed by additional glycoconjugates.

These low-molecular-weight structures, re-

ferred to as glycosylinositolphospholipids

(GIPLs), are included as members of the GPI

family by virtue of the core sequence a-Manp-

(14)-a-GlcNp-(16)-myoinositol [6]. It is

known that Galf is part of the oligosaccharide

core of the glycosylinositolphospholipid from

the protozoan Trypanosoma cruzi, the infec-

tious agent of Chagas’ disease [8]. The GIPL

structure of T. cruzi contains the same te-

trasaccharide core sequence as the protein-

bound GPI anchors, but diverges from the

Galactofuranose (Galf ) is present as a con-

stituent of external cellular structures in pro-

tozoa [1], bacteria [2,3], and fungi [4]. These

structures do not appear to be present on

mammalian cells and elicit a strong antigenic

response during infection [5]. Many glycocon-

jugates are anchored to the plasma membrane

via glycosylphosphatidylinositol (GPI) an-

chors. The GPI anchors of plasma membrane

proteins have been detected in organisms

ranging from yeast to man, but occur with a

much higher frequency in lower eukaryotes

such as protozoa [6,7]. All of the GPI anchors

that have been characterized to date contain

an identical ethanolamine phosphate–a-

* Corresponding author. Tel.: +1-604-2914327; fax: +1-

604-2913765.

E-mail address: bpinto@sfu.ca (B.M. Pinto)

PII: S0008-6215(00)00007-0

254

K.D. Randell et al. / Carbohydrate Research 325 (2000) 253–264

contains both an internal Galf and a

branched Galf moiety [16]. The acidic glyco-

lipid, puriﬁed from Paracoccidioides brasi-

liensis, containing a terminal Galf, has been

shown to be reactive with sera of patients

infected with paracoccidioidomycosis [17].

The synthesis of oligosaccharides containing

Galf may therefore be useful for understand-

ing the role Galf plays in microorganisms

and for studying the biosynthesis of fura-

protein anchors beyond this sequence. The

GIPL contains up to two additional b-Galf

residues and there is a 2-aminoethylphospho-

nic acid group located at the C-6 position of

the glucosamine residue. The lipid moiety is

a ceramide containing sphinganine and N-

linked lignoceric (C_24:0) acid instead of the

alkylglycerol found in the protein anchors

[6,9,10]:

nosyl-containing

glycoconjugates.

Com-

pounds containing Galf may also be used as

inhibitors to probe the development of infec-

tions, to develop diagnostic methods, or as

vaccines.

This structure is the most abundant cell-sur-

face glycoconjugate present in the insect-

dwelling epimastigote stage of the T. cruzi

life cycle [1]. The glycoconjugates on the cell

surface during the infectious stage of T.

cruzi are not modiﬁed with galactofuranose;

Oligosaccharide synthesis involving furan-

osyl glycosyl donors has not been studied to

the same extent as with pyranosyl donors,

but methods have been developed that em-

ploy thioglycosides [18], n-pentenyl gly-

cosides [19,20], anomeric benzoates [21],

anomeric xanthates [22], trichloroacetimi-

dates [23,24], and selenoglycosides [25,26].

Anomeric benzoates [21] and n-pentenyl gly-

cosides [19] give stereoselective syntheses of

the b anomers, while activation of anomeric

xanthates leads to a:b mixtures. In addition,

an indirect approach to galactofuranosyl-

containing disaccharides involving acyclic

glycosyl donors has recently been reported

however, it has been shown that the b- -

D

Galf moiety is recognized by antibodies that

inhibit T. cruzi internalization into mam-

malian cells [5]. Recently, it was demon-

strated that T. cruzi GIPLs are able to block

T-lymphocyte activation [11]. Thus, interac-

tion between host cellular defense mecha-

nisms and the GIPLs of T. cruzi may play a

role in establishment and maintenance of

chronic infection [12].

Galactofuranose has also been isolated

from another class of GIPLs found in Leish-

mania [13]. This GIPL differs from T. cruzi

by having a glycerolipid instead of a ce-

[27]. The syntheses of a-

containing oligosaccharides have not been as

widely investigated as those of the

anomers, but have been achieved in high

D

-galactofuranose-

b

ramide and the b- -Galf-(13)-a-Manp

D

yields, using ethyl 2,3,5,6-tetra-O-benzyl-a- -

D

moiety as an internal unit in the oligosac-

charide core. Glycoconjugates containing

galactofuranose have also been found in the

GIPLs of Leptomonas samueli [14] and En-

dotrypanum schaudinni [15], both of which

contain a ceramide lipid. The Galf moiety is

the terminal non-reducing sugar in L. sa-

mueli, and it is internal in E. schaudinni. In

thiogalactofuranoside and N-bromosuccin-

imide [18].

A previous report from our laboratory de-

scribed the viability of phenyl 2,3,5,6-tetra-

O-acetyl-b- -selenogalactofuranoside (16) as

D

a glycosyl donor [26]. As an extension of

this and other studies of the selective activa-

tion of selenoglycoside donors in the pres-

ence of thioglycoside acceptors [28,29], we

now report the syntheses of the terminal dis-

accharide 1, trisaccharide 2, tetrasaccharide

3, and pentasaccharide 4 corresponding to

the terminus of the glycosylinositolphospho-

lipid oligosaccharide of T. cruzi, which em-

ploy 16 as a glycosyl donor.

all of the above examples, the b- -galacto-

D

furanose is linked (13) to a-Manp. This

speciﬁcity suggests that a b-(13)-galacto-

furanosyltransferase might be involved in the

biosynthesis, although a sugar donor has not

been identiﬁed [10].

The speciﬁc capsular polysaccharide pro-

duced by Streptococcus pneumoniae type 20

K.D. Randell et al. / Carbohydrate Research 325 (2000) 253–264

255

cannot be used as a reliable indicator of the a

or b conﬁguration for the galactofuranosyl

residues [31]. For the trisaccharide 2, the 2D

NOESY spectrum showed an NOE between

H-1A and H-3A (of the Galf ring A) and no

NOE between H-1A and H-4A, indicating the

presence of a b linkage between the Galf (A)

and Manp (B) rings. The J_C1H1values are 171

Hz for the Manp (B) ring and 172 Hz for the

Manp (C) ring, indicating the presence of a

conﬁgurations about C-1 for both manno-

pyranosyl residues [32].

2. Results and discussion

The required disaccharide, ethyl 2-O-

benzoyl-4,6-O-benzylidene-3-O-(2,3,4,6-tetra-

O-acetyl-b-

nopyranoside (5) [26] and the monosaccha-

rides methyl 3,4,6-tri-O-benzyl-a- -manno-

pyranoside (6) [30] and phenyl 2,3,5,6-tetra-

O-acetyl-b- -selenogalactofuranoside (16) [26]

D

-galactofuranosyl)-a-

D

-thioman-

D

were synthesized following literature methods.

The disaccharide 5 was deprotected by

methanolysis of the esters, followed by hydrol-

ysis of the benzylidene acetal to give com-

pound 1 in 70% yield.

The reaction of the glycosyl donor, disac-

charide 5, with an excess of the acceptor 6,

mediated by NIS–TfOH, resulted in the pro-

tected trisaccharide 7 as a colorless foam in

68% yield (see Scheme 1). The trisaccharide

was deprotected by aminolysis of the esters,

followed by hydrolysis of the benzylidene ac-

etal and hydrogenolysis of the benzyl ethers,

to give compound 2 in 85% yield.

Synthesis of the monosaccharide acceptor

phenyl 3-O-benzoyl-2-O-benzyl-4,6-O-benzyl-

idene-a-

carried out from phenyl 2,3,4,6-tetra-O-acetyl-

a- -selenomannopyranoside (8). Compound 8

D-selenomannopyranoside (12) was

D

was deacetylated using NaOMe–MeOH, and

the crude product 9 was selectively protected

as a 4,6-O-benzylidene acetal, in a manner

analogous to that reported by Franzyk et al.

[33], to give 10 (see Scheme 2). Phase-transfer-

catalyzed benzylation gave the 2-O-benzyl

compound 11 that was subsequently benzoy-

lated to give 12 [34]. Ethyl 3-O-benzoyl-4,6-

The stereochemical integrity of the oligosac-

charides 1 and 2 was conﬁrmed by examina-

tion of the J_C1H1values of the manno-

pyranosyl residues and NOE contacts for the

galactofuranosyl residues. The J_C1H1values

O-benzylidene-a- -thiomannopyranoside (13)

D

was synthesized in an analogous manner to

that reported by Seymour [35] for the methyl

glycoside.

Glycosylation of the acceptor 13 with the

donor 12 was again performed using NIS–

TfOH, with selective activation of the phenyl

selenomannoside over the ethyl thioman-

noside; the protected disaccharide 14 was ob-

tained as a white foam in 60% yield (see

Scheme 2). The benzoate esters at the 3-posi-

tions of both mannose residues were removed

using NaOMe–MeOH to give the disaccha-

ride 15, which was subsequently used as an

acceptor. The tetrasaccharide 17 was synthe-

sized in one pot using the disaccharide 15

and 2.4 equivalents of phenyl 2,3,5,6-tetra-

O-acetyl-b-

Again, selective activation of the phenyl se-

lenogalactofuranoside over the ethyl

D

-selenogalactofuranoside

(16).

thiomannopyranoside yielded the desired te-

trasaccharide 17 as white crystals in 85% yield.

Compound 17 was then deprotected by

aminolysis of the esters followed by hydrolysis

of the benzylidene acetals and hydrogenolysis

of the benzyl ether to give compound 3 in 71%

yield.

Scheme 1.

256

K.D. Randell et al. / Carbohydrate Research 325 (2000) 253–264

Scheme 2.

Assignment of the NMR signals of the

Manp (C) rings. The J_C1H1values are 170 Hz

for the Manp (B) ring and 167 Hz for the

Manp (C) ring, indicating the presence of a

conﬁgurations about C-1 for both mannopyr-

anosyl residues [32].

Manp (C) ring of the tetrasaccharide 3 was

based on the fact that C-1 for this residue has

a characteristic upﬁeld chemical shift due to

the ethyl thioglycoside. A C–H correlation

spectrum, together with a COSY and a

TOCSY spectrum, then permitted complete

The tetrasaccharide 17 contains the ethyl

thioglycoside at C-1 of the Manp (C) ring, and

without any manipulation, could be used as a

donor to make the pentasaccharide. Thus,

glycosylation of the acceptor 6 with the donor

tetrasaccharide 17 gave the desired compound

18 as a clear syrup. Both the donor 17 and the

product of the reaction 18 had similar R_f

1

assignment of the H and ¹³C NMR signals.

The assignment of signals for the Manp (B)

ring was based on the presence of a NOE

contact across the glycosidic linkage between

H-1B and H-2C, and COSY and TOCSY

1

transfer between H NMR signals of the B

1

ring. Assignment of signals of the Galf rings A

and D was based on NOE contacts across the

glycosidic linkages between H-1A and H-3B,

and H-1D and H-3C, respectively. The 2D

NOESY spectrum also showed an NOE be-

tween H-1A and H-3A (of the Galf ring A),

and another NOE between H-1D and H-3D

(of the Galf ring D), indicating the presence of

a b linkage between the Galf (A) and Manp

(B) rings, and also between the Galf (D) and

values: a H NMR spectrum showed the pres-

ence of 18:17 in a ratio of 3:1, with an esti-

mated 35% yield of the desired penta-

saccharide 18. Extensive chromatography

gave an analytically pure sample of 18. In

subsequent experiments, the mixture of 17 and

18 was subjected to methanolysis, hydrolysis,

and hydrogenolysis; the deprotected pentasac-

charide 4 could be isolated as a pure com-

pound (see below).

K.D. Randell et al. / Carbohydrate Research 325 (2000) 253–264

257

the protozoan, T. cruzi, the causative agent of

Chagas’ disease, have been synthesized by se-

lective activation of selenoglycoside donors in

the presence of thioglycoside acceptors. The

selenoglycoside 16 is a versatile furanosyl

donor that gives oligosaccharides with b selec-

tivity. The four target compounds 1–4 will be

tested as inhibitors against T. cruzi prolifera-

tion and also in the inhibition of proliferation

of B lymphocytes.

In a recent paper from Zhang et al., the

synthesis of oligosaccharides in one pot by

sequential addition of acceptors and pro-

moters was reported [36]. Using this ap-

proach, a second attempt at the synthesis of

18 was carried out in one pot starting with

disaccharide 15. The tetrasaccharide 17 was

ﬁrst synthesized by reacting the disaccharide

15 with 2.1 equivalents of the galactofuranosyl

donor 16 at 0 °C; then, without any workup,

the acceptor 6 was added together with an

additional 1.2 equivalents of NIS and acti-

,

vated 4 A molecular sieves. Again, a mixture

of the tetrasaccharide 17 and the product 18

was obtained, but 18 was formed in a slightly

better yield (28% for two steps). The pentasac-

charide 18 was deprotected by methanolysis of

the esters followed by hydrolysis of the ben-

zylidene acetals and hydrogenolysis of the

benzyl ethers, to give the target pentasaccha-

ride 4 in 60% yield.

The assignment of the NMR signals of the

Manp (E) ring of the pentasaccharide 4 was

1

based on the H NMR spectrum in which this

H-1 signal was the most upﬁeld. The 2D

NOESY spectrum also showed a contact be-

tween H-1E and the methyl aglycon. The as-

signment of the remaining signals of the Manp

(E) ring then followed from a COSY experi-

ment. The signals of the Manp rings B and C

were assigned based on NOE contacts across

the glycosidic linkages between H-1C and H-

2E, and H-1B and H-2C, respectively. Assign-

ment of signals of the Galf rings A and D was

based on NOE contacts across the glycosidic

linkages between H-1A and H-3B, and H-1D

and H-3C, respectively. The 2D NOESY spec-

trum also showed an NOE between H-1A and

H-3A (of the Galf ring A), and another NOE

between H-1D and H-3D (of the Galf ring D),

indicating the presence of b linkages between

the Galf (A) and Manp (B) rings, and between

the Galf (D) and Manp (C) rings. The J_C1H1

values were 171 Hz for the Manp (B) ring, 172

Hz for the Manp (C) ring, and 173 Hz for the

Manp (E) ring, indicating the presence of a

conﬁgurations about C-1 for all three

mannopyranosyl residues [32].

3. Experimental

General methods.—Melting points were de-

termined on a Fisher–Johns melting point

apparatus and are uncorrected. Optical rota-

tions were measured at 21 °C with a Rudolph

Research Autopol II automatic polarimeter.

¹H NMR and ¹³C NMR spectra were

recorded on a Bruker AMX-400 NMR spec-

trometer at 400.13 and 100.6 MHz, for proton

and carbon, respectively. Chemical shifts are

given in ppm downﬁeld from TMS for those

measured in CDCl₃or CD₂Cl₂and from 2,2-

dimethyl-2-silapentane-5-sulfonate (DSS) for

those spectra measured in D₂O. Chemical

shifts and coupling constants were obtained

from a ﬁrst-order analysis of the spectra. All

assignments were conﬁrmed with the aid of

two-dimensional H–¹H (COSYDFTP), H–

¹³C (INVBTP), ¹H (NOESYTP), and ¹H

(MLEVTP) experiments using standard

1

In summary, di- up to pentasaccharides 1–4

corresponding to the terminal end of the gly-

cosylinositolphospholipid oligosaccharide of

258

K.D. Randell et al. / Carbohydrate Research 325 (2000) 253–264

oligosaccharide was dissolved in 4:1 HOAc–

H₂O (10 mL) and stirred with Pd–C (100 mg)

under H₂(52 psi). After 20 h the reaction

mixture was ﬁltered through a pad of Celite,

which was subsequently washed with water.

The combined ﬁltrates were evaporated to

dryness, and the residue was co-evaporated

several times with distilled water to remove

any traces of HOAc. The target oligosaccha-

ride was puriﬁed by column chromatography.

Bruker pulse programs and an inverse detec-

1

tion, H–X double-resonance probe. Sugar

rings are denoted A, B, C, D, and E, respec-

tively, as shown in the diagrams for com-

pounds 1–4; intermediates are labeled

correspondingly. Analytical thin-layer chro-

matography (TLC) was performed on alu-

minum plates precoated with E. Merck Silica

Gel 60 F254 as the adsorbent. The developed

plates were air-dried, exposed to UV light

and/or sprayed with a solution containing 1%

Ce(SO₄)₂and 1.5% molybdic acid in 10% aq

H₂SO₄and heated. Compounds were puriﬁed

by ﬂash column chromatography on Kieselgel

60 (230–400 mesh). Solvents were distilled

before use and were dried as necessary, by

literature procedures. Solvents were evapo-

rated under reduced pressure and below

40 °C.

Ethyl i-

D

-galactofuranosyl-(13)-1-thio-

-mannopyranoside (1).—To a solution of

h-D

the disaccharide 5 (45 mg, 0.06 mmol) in

freshly distilled MeOH (5 mL) was added 1 M

NaOMe–MeOH (0.5 mL). The reaction mix-

ture was stirred at rt, overnight, under an N₂

atmosphere. The reaction mixture was neu-

tralized with Rexyn 101 (H⁺), the resin was

ﬁltered, and the solvent was removed in

vacuo. The crude product was then dissolved

in 80% AcOH (5 mL), and the solution was

stirred at rt overnight. The solvent was re-

moved, and the crude product was puriﬁed by

column chromatography using 7:2:1 EtOAc–

MeOH–H₂O as the eluant to yield the desired

disaccharide 1 as a clear glass (13 mg, 70%):

General procedure for glycosylation reac-

tions.—A mixture of the glycosyl donor, the

,

acceptor, and activated 4 A molecular sieves

was stirred in dry CH₂Cl₂at room tempera-

ture (rt) under an N₂atmosphere. The reac-

tion mixture was cooled in an ice bath and

NIS (1.2–1.3 equiv relative to the donor) was

added, followed by addition of TfOH (0.05

equiv). The reaction mixture was stirred at

0 °C, under an N₂atmosphere, until TLC

showed that the reaction was complete. The

mixture was quenched by addition of Et₃N,

diluted with CH₂Cl₂and ﬁltered through a

pad of Celite. The mixture was washed with

10% Na₂S₂O₃, followed by satd aq NaHCO₃.

The organic layer was dried over Na₂SO₄, the

solvent was removed in vacuo, and the residue

was puriﬁed by column chromatography.

General procedure for deprotection.—The

protected oligosaccharide was dissolved in

freshly distilled MeOH, and NH₃(g) was bub-

bled through the solution, while stirring under

an N₂atmosphere, until TLC indicated that

no further change was occurring. The reaction

mixture was concentrated by rotary evapora-

tion, then placed under high vacuum (ꢀ0.05

torr), at 50 °C overnight to remove NH₄OAc.

The residue was puriﬁed by column chro-

matography to give the desired partially de-

protected oligosaccharide. The ¹H NMR

spectrum conﬁrmed that the acyl groups had

been removed. The partially deprotected

1

[h]_D+35° (c 0.028, H₂O); H NMR (D₂O): l

5.31 (d, 1 H, J_1,21.6 Hz, H-1B), 5.09 (d, 1 H,

J_1,21.4 Hz, H-1A), 4.21 (dd, 1 H, J_2,33.1 Hz,

H-2B), 4.11 (dd, 1 H, J_2,33.2 Hz, H-2A), 4.04

(dd, 1 H, J_3,46.6 Hz, H-3A), 4.01 (m, 1 H,

H-5A), 4.00 (dd, 1 H, J_4,53.8 Hz, H-4A), 3.85

(dd, 1 H, J_5,62.3, J_6,6%12.4 Hz, H-6A), 3.81

(m, 2 H, H-3B, H-5B), 3.74 (dd, 1 H, J_5,66.0

Hz, H-6A%), 3.70 (dd, 1 H, J_3,4, J_4,59.7 Hz,

H-4B), 3.67 (dd, 1 H, J_5,64.5, J_6,6%11.7 Hz,

H-6B), 3.61 (dd, 1 H, J_5,67.4 Hz, H-6B%), 2.64

(m, 2 H, SCH₂CH₃), 1.24 (t, 3 H, J 7.4 Hz,

SCH₂CH₃), ¹³C NMR (D₂O): l 106.90 (C-

1A), 86.60 (C-1B), 85.51 (C-4A), 83.97 (C-

2A), 79.62 (C-3A), 78.31 (C-3B), 75.65

(C-5A), 73.28 (C-5B), 71.06 (C-2B), 67.99 (C-

4B), 65.41 (C-6B), 63.47 (C-6A), 27.45

(SCH₂CH₃), 16.67 (SCH₂CH₃). Anal. Calcd

for C₁₄H₂₆O₁₀S: C, 43.52; H, 6.78. Found: C,

43.85; H, 6.78.

Methyl 2,3,5,6-tetra-O-acetyl-i-

furanosyl-(13)-2-O-benzoyl-4,6-O-benzyli-

dene - h - - mannopyranosyl - (12) - 3,4,6 - tri-

O-benzyl-h- -mannopyranoside (7).—The

methyl mannopyranoside acceptor 6 (308 mg,

D-galacto-

D

K.D. Randell et al. / Carbohydrate Research 325 (2000) 253–264

259

J_1,21.6 Hz, H-1A), 5.03 (d, 1 H, J_1,21.5 Hz,

H-1B), 4.96 (d, 1 H, J_1,21.9 Hz, H-1C), 4.24

(dd, 1 H, J_2,32.9 Hz, H-2B), 4.20 (dd, 1 H, J_2,3

3.1 Hz, H-2A), 4.04 (dd, 1 H, J_3,46.5 Hz,

H-3A), 4.01 (dd, 1 H, J_4,53.8 Hz, H-4A), 3.93

(dd, 1 H, J_2,33.3 Hz, H-2C), 3.89 (dd, 1 H, J_3,4

9.4 Hz, H-3B), 3.83 (dd, 1 H, J_3,49.7 Hz,

H-3C), 3.83–3.36 (m, 11 H, H-4C, H-4B,

H-5C, H-5B, H-5A, H-6C, H-6C%, H-6B, H-

0.66 mmol) was glycosylated with the thiogly-

coside donor 5 (379 mg, 0.51 mmol) following

the general procedure. An immediate reaction

to produce a dark purple–brown color en-

sued; reaction time was 3.5 h at rt. The crude

product was puriﬁed by column chromatogra-

phy using 3:1 toluene–EtOAc as the eluant.

The desired trisaccharide 7 was obtained as a

colorless foam (393 mg, 68%): [h]_D−36° (c

1

13

0.25, CH₂Cl₂); H NMR (CD₂Cl₂): l 8.12–

6B%, H-6A, H-6A%), 3.37 (s, 3 H, ꢀOCH₃). C

7.09 (m, 25 H, aromatic), 5.70 (dd, 1 H, J_1,2

1.5, J_2,33.6 Hz, H-2B), 5.64 (s, 1 H, PhCH),

5.29 (bs, 1 H, H-1A), 5.25 (ddd, 1 H, J_4,53.4,

J_5,67.5, J_5,6%4.1 Hz, H-5A), 5.24 (d, 1 H,

H-1B), 4.91 (d, 1 H, J_2,31.5 Hz, H-2A), 4.85,

4.57 (2 d, 2 H, J_gem11.1 Hz, OCH₂Ph), 4.82

(dd, 1 H, J_3,45.6 Hz, H-3A), 4.80 (d, 1 H, J_1,2

1.9 Hz, H-1C), 4.70, 4.66 (2 d, 2 H, J_gem11.8

Hz, OCH₂Ph), 4.64, 4.58 (2 d, 2 H, J_gem12.2

Hz, OCH₂Ph), 4.41 (m, 1 H, H-3B), 4.32 (dd,

1 H, J_5,64.1, J_6,6%10.2 Hz, H-6B), 4.24 (dd, 1

H, H-4A), 4.11–4.01 (m, 4 H, H-2C, H-4B,

H-5B, H-6A), 3.96 (dd, 1 H, J_6,6%11.8 Hz,

H-6A%), 3.92–3.83 (m, 3 H, H-3C, H-6B%,

H-4C), 3.77–3.72 (m, 3 H, H-5C, H-6C, H-

6C%), 3.37 (s, 3 H, ꢀOCH₃), 2.11, 2.08, 1.89,

1.82 (4 s, 3 H each, ꢀC(O)CH₃). ¹³C NMR

(CD₂Cl₂): l 170.55, 170.28, 170.20, 169.45

(COCH₃), 165.62 (COPh), 139.13–126.46 (30

C, aromatic), 103.14 (C-1A), 102.21 (CHPh),

100.64 (C-1B), 100.24 (C-1C), 81.18 (C-2A),

81.05 (C-4A), 80.41 (C-3C), 77.69 (C-4B),

77.25 (C-3A), 75.37 (2 C, C-4C CH₂Ph), 75.05

(C-2C), 73.60 (CH₂Ph), 72.76 (CH₂Ph), 72.25

(C-5C), 70.47 (C-3B), 69.98 (C-6C), 69.64 (2

C, C-5A and C-2B), 69.17 (C-6B), 64.76 (C-

5B), 62.96 (C-6A), 55.01 (ꢀOCH₃), 20.97,

20.94, 20.78, 20.54 (COCH₃). Anal. Calcd for

C₆₂H₆₈O₂₁: C, 64.80; H, 5.96. Found: C, 64.95;

H, 5.91.

NMR (D₂O): l 107.02 (C-1A), 104.73 (C-1B),

101.93 (C-1C), 85.50 (C-4A), 83.98 (C-2A),

81.27 (C-2C), 79.63 (C-3A), 77.87 (C-3B),

75.82, 75.20, 75.35, 72.81, 69.60 (C-4B, C-4C,

C-5A, C-5B, C-5C), 69.31 (C-2B), 67.80,

65.48, 63.58 (C-6A, C-6B, C-6C), 63.76 (C-

3C), 57.46 (ꢀOCH₃). Anal. Calcd for

C₁₉H₃₄O₁₆: C, 44.02; H, 6.61. Found: C, 43.68;

H, 6.30.

Phenyl 2,3,4,6-tetra-O-acetyl-1-seleno-h- -

D

mannopyranoside (8).—To a solution of 50%

H₃PO₂(90 mL) was added diphenyl diselenide

(9.1 g, 29 mmol), and the mixture was rapidly

stirred at reﬂux, under an N₂atmosphere until

the yellow color disappeared. The reaction

mixture was cooled to 0 °C and extracted with

CH₂Cl₂and washed with cold water. The

combined extracts were washed with half-satu-

rated NaCl and dried over MgSO₄. The solu-

tion was ﬁltered into a ﬂask containing

peracetylated mannose (15 g, 39 mmol) and

BF₃·Et₂O (9.9 mL, 78 mmol) was added. The

reaction was stirred overnight under an N₂

atmosphere. The reaction mixture was cooled

in an ice bath and quenched with Et₃N (4 mL)

and satd aq NaHCO₃(50 mL), and extracted

with CH₂Cl₂. The extracts were washed with

satd aq NaHCO₃, followed by cold water and

dried over Na₂SO₄. The crude product was

puriﬁed by column chromatography using 2:1

hexanes–EtOAc as the eluant. The white solid

was recrystallized from hexanes–EtOAc to

yield white crystals of compound 8 (9.2 g,

49%): mp 89–92 °C; [h]_D+138° (c 0.33,

Methyl i-

D

-galactofuranosyl-(13)-h-

D

-

mannopyranosyl-(12)-h-

D

-mannopyranoside

(2).—The trisaccharide 7 (129 mg, 0.11 mmol)

was deprotected following the general proce-

dure. After deacylation, the partially depro-

tected trisaccharide was puriﬁed by column

chromatography using 5:1 CH₂Cl₂–MeOH as

the eluant. This product was further depro-

tected to yield the target trisaccharide 2 as a

colorless foam, (43 mg, 96%): [h]_D−54.5° (c

1

CH₂Cl₂); H NMR (CDCl₃): l 7.60–7.20 (m,

5 H, aromatic), 5.75 (d, 1 H, J_1,21.1 Hz, H-1),

5.56 (dd, 1 H, J_2,33.2 Hz, H-2), 5.34 (dd, 1 H,

H-4), 5.28 (dd, 1 H, J_3,410.0 Hz, H-3), 4.46

(ddd, 1 H, J_4.59.8, J_5,65.8, J_5,6%2.3 Hz, H-5),

4.31 (dd, 1 H, J_6,6%12.3 Hz, H-6), 4.09 (dd, 1

H, H-6%), 2.13, 2.07, 2.06, 2.01 (4 s, 3 H each,

1

0.16, H₂O); H NMR (D₂O): l 5.13 (d, 1 H,

260

K.D. Randell et al. / Carbohydrate Research 325 (2000) 253–264

ꢀC(O)CH₃). ¹³C NMR (CDCl₃): l 170.40,

169.76, 169.71, 169.61 (COCH₃), 134.37–

128.30 (6 C, aromatic), 82.51 (C-1), 71.57

(C-2), 71.31 (C-5), 69.82 (C-3), 66.41 (C-4),

62.41 (C-6), 20.76, 20.60, 20.52 (4 C, COCH₃).

Anal. Calcd for C₂₀H₂₄O₉Se: C, 49.29; H, 4.96.

Found: C, 49.38; H, 5.03.

mmol), benzyl bromide (0.83 mL, 7.0 mmol)

and 5% aq NaOH (8 mL). The reaction mix-

ture was stirred at reﬂux, under an N₂atmo-

sphere, for 40 h. The reaction mixture was

cooled to rt and diluted with CH₂Cl₂, washed

with water and dried over Na₂SO₄. The sol-

vent was removed in vacuo and the white

solid was puriﬁed by column chromatography

using 4:1 hexanes–EtOAc as the eluant. The

product 11 was recrystallized from hexanes–

EtOAc to give white needle-like crystals, (1.35

g, 68%): mp 149–150 °C; [h]_D+142° (c 0.76,

Phenyl4,6-O-benzylidene-1-seleno-h-

nopyranoside (10).—To a solution of phenyl

2,3,4,6-tetra-O-acetyl-1-seleno-a- -manno-

D-man-

D

pyranoside (8, 2.0 g, 4.1 mmol) in freshly

distilled MeOH (60 mL) was added 1 M

NaOMe–MeOH (2 mL). The reaction mix-

ture was stirred overnight, under an N₂atmo-

sphere. The solution was neutralized with

Rexyn 101 (H⁺). The resin was ﬁltered, the

ﬁltrate was concentrated in vacuo and placed

under high vacuum overnight. To a solution

of the crude product 9 in DMF (6 mL) was

added p-toluenesulfonic acid (7.6 mg, 0.04

mmol) and benzaldehyde dimethyl acetal (0.72

mL, 4.8 mmol). The reaction mixture was

heated at 45–50 °C for 1.5 h. An excess of

K₂CO₃was added, and the solvent was re-

moved by rotary evaporation under a high

vacuum. The white solid was dissolved in

EtOAc, washed with H₂O, dried over Na₂SO₄,

and the solvent was removed in vacuo. The

white solid was recrystallized from hexanes–

EtOAc to yield white powdery crystals of

compound 10 (1.20 g, 72%). This compound

was very insoluble in both EtOAc and

CH₂Cl₂, and in subsequent repetitions of this

reaction it was not puriﬁed; mp softens at

ꢀ70 °C, melts 214–216 °C; [h]_D+237° (c

1

CH₂Cl₂); H NMR (CDCl₃): l 7.56–7.25 (m,

15 H, aromatic), 5.85 (d, 1 H, J_1,21.0 Hz,

H-1), 5.59 (s, 1 H, CHPh), 4.73, 4.59 (2 d, 2

H, J_gem11.6 Hz, OCH₂Ph) 4.25–4.15 (m, 2 H,

H-5, H-6), 4.17 (dd, 1 H, J_2,33.6 Hz, H-2),

4.11, (m, 1 H, H-3), 3.99 (bt, 1 H, J_3,4, J_4,59.3

Hz, H-4), 3.84 (m, 1 H, H-6%), 2.42 (bd, 1 H,

13

OH). C NMR (CDCl₃): l 137.33–126.34 (18

C, aromatic), 102.19 (CHPh), 83.96 (C-1),

80.72 (C-2), 79.56 (C-4), 73.04 (CH₂Ph), 69.34

(C-3), 68.34 (C-6), 66.59 (C-5). Anal. Calcd

for C₂₆H₂₆O₅Se: C, 62.78; H, 5.27. Found: C,

63.01; H, 5.26.

Phenyl 3-O-benzoyl-2-O-benzyl-4,6-O-ben-

zylidene-1-seleno-h- -mannopyranoside (12).

D

—To a solution of 11 (620 mg, 1.25 mmol) in

pyridine (10 mL) at 0 °C; was added benzoyl

chloride (0.26 mL, 2.3 mmol) and DMAP

(catalytic). The reaction mixture was allowed

to warm to rt and stirred overnight. The reac-

tion was quenched with MeOH, and the

pyridine was removed by rotary evaporation

under a high vacuum. The crude product was

puriﬁed by column chromatography using 4:1

hexanes–EtOAc as the eluant. The desired

product 12 was obtained as a white foam, (650

1

0.54, DMSO); H NMR (DMSO-d₆): l 7.60–

7.30 (m, 10 H, aromatic), 5.71 (d, 1 H, J_1,21.2

Hz, H-1), 5.62 (s, 1 H, CHPh), 5.54 (d, 1 H,

J_2,OH4.0 Hz, OH-2), 5.20 (d, 1 H, J_3,OH6.2

Hz, OH-3), 4.07 (m, 2 H, H-2, H-6), 3.95 (m,

2 H, H-4, H-5), 3.79 (m, 1 H, H-6%), 3.73 (m,

1 H, H-3). ¹³C NMR (DMSO-d₆): l 137.75–

126.34 (12 C, aromatic), 101.13 (CHPh), 87.79

(C-1), 78.32 (C-4), 73.02 (C-2), 68.32 (C-3),

67.36 (C-6), 66.93 (C-5). Anal. Calcd for

C₁₉H₂₀O₅Se: C, 56.03; H, 4.95. Found: C,

56.18; H, 4.91.

1

mg, 86%): [h]_D+67° (c 0.60, CH₂Cl₂); H

NMR (CDCl₃): l 8.20–7.10 (m, 20 H, aro-

matic), 5.83 (d, 1 H, J_1,21.2 Hz, H-1), 5.64 (s,

1 H, CHPh), 5.54 (dd, 1 H, J_2,33.4, J_3,410.3

Hz, H-3), 4.63, 4.50 (2 d, 2 H, J_gem12.0 Hz,

OCH₂Ph), 4.43 (dd, 1 H, H-4), 4.41, (dd, 1 H,

H-2), 4.36 (m, 1 H, J_5,64.9, J_4,5, J_5,6%10.0 Hz,

H-5), 4.28 (dd, 1 H, J_6,6%10.2 Hz, H-6), 3.93

(dd, 1 H, H-6%). ¹³C NMR (CDCl₃): l 165.82

(COPh), 137.28–126.20 (24 C, aromatic),

101.81 (CHPh), 83.91 (C-1), 78.44 (C-2), 76.34

(C-4), 73.04 (CH₂Ph), 71.44 (C-3), 68.42 (C-

6), 67.22 (C-5). Anal. Calcd for C₃₃H₃₀O₆Se:

C, 65.89; H, 5.03. Found: C, 65.75; H, 5.01.

Phenyl 2-O-benzyl-4,6-O-benzylidene-1-se-

leno-h- -mannopyranoside (11).—To a solu-

D

tion of 10 (1.63 g, 4.0 mmol) in CH₂Cl₂(75

mL) was added (Bu)₄NHSO₄(272 mg, 0.8

K.D. Randell et al. / Carbohydrate Research 325 (2000) 253–264

261

H-1C), 4.96 (d, 1 H, J_1,21.5 Hz, H-1B), 4.46–

4.38 (m, 3 H, H-2C, H-4C, H-5C), 4.34, 4.21

(2 d, 2 H, J_gem11.6 Hz, OCH₂Ph), 4.31–4.23

(m, 3 H, H-6B, H-6C, H-4B), 4.18–4.10 (m, 2

H, H-2B, H-5B), 4.01 (dd, 1 H, J_5,6, J_6,6%10.0

Hz, H-6C%), 3.86 (dd, 1 H, J_5,6, J_6,6%10.3 Hz,

H-6B%), 2.72 (m, 2 H, SCH₂CH₃), 1.38 (t, 3 H,

J 7.5 Hz, SCH₂CH₃). ¹³C NMR (CD₂Cl₂): l

166.18, 166.04 (COPh), 137.97–126.52 (30 C

aromatic), 102.21 (CHPh), 102.12 (CHPh),

101.85 (C-1B), 84.94 (C-1C), 79.68 (C-2C),

77.56 (C-2B), 76.88 (C-4C), 76.69 (C-4B),

74.28 (CH₂Ph), 71.45 (C-3C), 71.26 (C-3B),

68.42 (2 C, C-6B, C-6C), 65.36 (C-5B), 65.04

(C-5C), 26.01 (SCH₂CH₃), 15.19 (SCH₂CH₃).

Anal. Calcd for C₄₉H₄₈O₁₂S: C, 68.36; H, 5.62.

Found: C, 68.35; H, 5.66.

Ethyl 3-O-benzoyl-4,6-O-benzylidene-1-thio-

-mannopyranoside (13).—To a solution of

ethyl 4,6-O-benzylidene-1-thio-a- -manno-

h-D

D

pyranoside [34] (770 mg, 2.47 mmol) in

pyridine (15 mL) at −25 °C; was added ben-

zoyl chloride (0.34 mL, 2.9 mmol). The reac-

tion mixture was stirred at −25 °C for 4 h.

The pyridine was removed by rotary evapora-

tion under a high vacuum. The residue was

dissolved in CHCl₃, washed with 0.1 M HCl,

followed by satd aq NaHCO₃and then water.

The solution was dried over Na₂SO₄, and the

solvent was removed in vacuo to give a white

solid. The crude product was puriﬁed by

column chromatography using 3:1 hexanes–

EtOAc as the eluant. The desired product 13

was obtained as a white foam, (711 mg, 69%):

[h]_D+80° (c 0.28, CH₂Cl₂); ¹H NMR

(CD₂Cl₂): l 8.10–7.30 (m, 10 H, aromatic),

5.60 (s, 1 H, CHPh), 5.48 (dd, 1 H, J_2,33.3,

J_3,410.1 Hz, H-3), 5.38 (d, 1 H, J_1,21.1 Hz,

H-1), 4.42 (ddd, 1 H, J_5,64.8, J_5,6%J_4,59.9 Hz,

H-5), 4.36 (m, 1 H, H-2), 4.31, (dd, 1 H, H-4),

4.28 (dd, 1 H, J_6,6%10.3 Hz, H-6), 3.91 (dd, 1

H, H-6%), 2.70 (m, 2 H, SCH₂CH₃), 2.49 (d, 1

H, J_{OH, 2}4.0 Hz, OH), 1.32 (t, 3 H, J 7.5 Hz,

SCH₂CH₃). ¹³C NMR (CD₂Cl₂): l 165.78

(COPh), 137.93–126.56 (12 C, aromatic),

102.27 (CHPh), 85.55 (C-1), 76.86 (C-4), 72.03

(C-3), 71.84 (C-2), 69.04 (C-6), 64.91 (C-5),

25.61 (SCH₂CH₃), 15.1 (SCH₂CH₃). Anal.

Calcd for C₂₂H₂₄O₆S: C, 63.45; H, 5.81.

Found: C, 63.19; H, 5.91.

Ethyl

mannopyranosyl-(12)-4,6-O-benzylidene-1-

thio-h- -mannopyranoside (15).—To a solu-

2-O-benzyl-4,6-O-benzylidene-h-

D

-

D

tion of the disaccharide 14 (110 mg, 0.128

mmol) in freshly distilled MeOH (10 mL) was

added 1 M NaOMe–MeOH (0.5 mL). The

reaction mixture was stirred at rt, overnight,

under an N₂atmosphere. The solution was

neutralized with Rexyn 101 (H⁺), the resin

was ﬁltered, and the solvent was removed in

vacuo. The partially deprotected disaccharide

was puriﬁed by column chromatography using

2:1 hexanes–EtOAc as the eluant. The desired

product 15 was obtained as a white foam (81

1

mg, 96%): [h]_D+69° (c 0.67, CH₂Cl₂); H

NMR (CDCl₃): l 7.60–7.30 (m, 15 H, aro-

matic), 5.60 (s, 1 H, CHPh), 5.59 (s, 1 H,

CHPh), 5.33 (bs, 1 H, H-1C), 5.21 (d, 1 H, J_1,2

1.3 Hz, H-1B), 4.76, 4.66 (2 d, 2 H, J_gem11.7

Hz, OCH₂Ph), 4.25–4.15 (m, 4 H, H-6B, H-

6C, H-5C, H-3B), 4.11 (m, 2 H, H-2C, H-3C),

3.98 (dd, 1 H, J_2,33.4 Hz, H-2B), 3.96–3.87

(m, 5 H, H-6B%, H-6C%, H-4B, H-4C, H-5B),

2.65 (m, 2 H, SCH₂CH₃), 1.35 (t, 3 H, J 7.4

Hz, SCH₂CH₃). ¹³C NMR (CDCl₃): l 137.61–

126.17 (18 C aromatic), 102.29 (CHPh),

102.07 (CHPh), 100.71 (C-1B), 84.73 (C-1C),

79.93 (C-2C), 79.48 (2 C, C-4B, C-4C), 78.39

(C-2B), 73.55 (CH₂Ph), 69.55 (C-3C), 68.74

(C-6C), 68.64, 68.60 (C-3B, C-6B), 64.34 (C-

5B), 64.04 (C-5C), 25.62 (SCH₂CH₃), 14.94

(SCH₂CH₃). Anal. Calcd for C₃₅H₄₀O₁₀S: C,

64.40; H, 6.18. Found: C, 64.60; H, 6.16.

Ethyl 3-O-benzoyl-2-O-benzyl-4,6-O-ben-

zylidene - h -

benzoyl-4,6-O-benzylidene-1-thio-h-

D

- mannopyranosyl - (12) - 3 - O-

-manno-

D

pyranoside (14).—The thioglycoside acceptor

13 (375 mg, 0.900 mmol) was glycosylated

with the selenoglycoside donor 12 (650 mg,

1.08 mmol) following the general procedure.

The reaction time was 1.5 h at 0 °C. The

disaccharide was puriﬁed by column chro-

matography using 4:1 hexanes–EtOAc as the

eluant. The desired product 14 was obtained

as a white foam (464 mg, 60%): [h]_D−49° (c

1

0.60, CH₂Cl₂); H NMR (CD₂Cl₂): l 8.12–

7.00 (m, 25 H, aromatic), 5.70 (s, 1 H, CHPh),

5.67 (dd, 1 H, J_2,33.4, J_3,410.4 Hz, H-3B),

5.61 (s, 1 H, CHPh), 5.51 (dd, 1 H, J_2,33.6,

J_3,49.9 Hz, H-3C), 5.50 (d, 1 H, J_1,21.2 Hz,

262

K.D. Randell et al. / Carbohydrate Research 325 (2000) 253–264

26.14 (SCH₂CH₃), 21.0, 20.84, 20.74 (8 C,

COCH₃), 15.29 (SCH₂CH₃). Anal. Calcd for

C₆₃H₇₆O₂₈S: C, 57.62; H, 5.83. Found: C,

57.80; H, 5.99.

Ethyl

furanosyl-(13)-2-O-benzyl-4,6-O-benzyli-

dene-h- -mannopyranosyl-(12)-[2,3,5,6-te-

tra-O-acetyl-i- -galactofuranosyl-(13)]-

4,6-O-benzylidene-1-thio- h- -mannopyran-

2,3,5,6-tetra-O-acetyl-i- -galacto-

D

Ethyl i-

nopyranosyl - (12) - [i -

(13)]-1-thio-h- -mannopyranoside

D

-galactofuranosyl-(13)-h-

- galactofuranosyl-

(3).—

D

-man-

oside (17).—The thioglycoside acceptor 15

(156 mg, 0.239 mmol) was glycosylated with

the selenoglycoside donor 16 (280 mg, 0.574

mmol) following the general procedure. The

reaction time was 40 min at 0 °C. Pure 17 (265

mg, 85%) was crystallized from the crude

product mixture using hexanes–EtOAc. An

analytical sample was obtained as colorless

needles by column chromatography using 2:1

hexanes–EtOAc as the eluant and recrystal-

lization from hexanes–EtOAc: mp 137–

D

The tetrasaccharide 17 (50 mg, 0.038 mmol)

was deprotected as described in the general

procedure. The intermediate deacylated

product was puriﬁed by column chromatogra-

phy using 12:1 CH₂Cl₂–MeOH as the eluant

to give a clear glass. The compound was then

subjected to hydrogenolysis and puriﬁed by

column chromatography using 5:2:1 EtOAc–

MeOH–H₂O as the eluant. The tetrasaccha-

ride 3 was obtained as a syrup (19 mg, 71%):

1

140 °C; [h]_D−5° (c 0.60, CH₂Cl₂); H NMR

(CD₂Cl₂): l 7.50–7.25 (m, 15 H, aromatic),

5.62 (s, 1 H, CHPh), 5.60 (s, 1 H, CHPh),

5.40 (bs, 1 H, H-1C), 5.34 (m, 1 H, H5A), 5.29

(d, 1 H, J_1,21.1 Hz, H-1B), 5.25 (m, 1 H, J_4,5

4.0, J_5,67.6 Hz, H-5D), 5.21 (s, 1 H, H-1A),

5.13 (s, 1 H, H-1D), 5.06 (d, 1 H, J_2,31.1 Hz,

H-2A), 4.96 (dd, 1 H, J_3,45.8 Hz, H-3D), 4.93

(d, 1 H, J_2,31.6 Hz, H-2D), 4.91 (bdd, 1 H,

J_3,45.4 Hz, H-3A), 4.79, 4.75 (2 d, 2 H, J_gem

11.1 Hz, OCH₂Ph), 4.37 (dd, 1 H, J_4,53.2 Hz,

H-4A), 4.30 (dd, 1 H, H-4D), 4.28 (dd, 1 H,

J_2,32.3, J_3,410.1 Hz, H-3B), 4.25–4.18 (m, 3

H, H-5C, H-6C, H-6B), 4.18 (m, 1 H, H-2C),

4.17 (dd, 1 H, J_2,33.2 Hz, H-3C), 4.09 (dd, 1

H, J_5,67.4 Hz, H-6A), 4.10–4.05 (m, 4 H,

H-6D, H-4B, H-4C, H-2B), 3.95 (dd, 1 H, J_5,6%

4.6, J_6,6%11.7 Hz, H-6A%), 3.93 (m, 1 H, H-5B),

3.92 (dd, 1 H, J_5,6%, J_6,6%11.6 Hz, H-6C%), 3.85

(dd, 1 H, J_5,6%4.1, J_6,6%12.0 Hz, H-6D%), 3.84

(dd, 1 H, J_5,6%, J_6,6%10.0 Hz, H-6B%), 2.65 (m, 2

H, SCH₂CH₃), 2.10, 2.08 (2 s, 3 H each,

ꢀC(O)CH₃), 2.06 (s, 6 H, ꢀC(O)CH₃), 1.93,

1.92, 1.91, 1.90 (4 s, 3 H each, C(O)CH₃), 1.30

(t, 3 H, J 7.5 Hz, SCH₂CH₃). ¹³C NMR

(CD₂Cl₂): l 170.62, 170.34 (8 C, COCH₃),

138.9–126.5 (18 C, aromatic), 103.22 (C-1D),

103.04 (C-1A), 102.13 (3 C, C-1B, 2×CHPh),

85.33 (C-1C), 82.58 (C-2D), 82.04 (C-2A),

81.16 (2 C, C-4A, C-4D), 77.81 (C-2C), 77.47

(2 C, C-4B and C-4C), 77.35 (C-3A), 76.98

(C-3D), 76.35 (C-2B), 74.53 (CH₂Ph), 72.42 (2

C, C-3B, C-3C), 69.85 (C-5A), 69.75 (C-5D),

69.14 (C-6C), 68.99 (C-6B), 65.58 (C-5B),

65.44 (C-5C), 63.08 (C-6D), 62.84 (C-6A),

1

[h]_D−10° (c 0.079, H₂O); H NMR (D₂O): l

5.55 (d, 1 H, J_1,21.4 Hz, H-1C), 5.13 (d, 1 H,

J_1,21.6 Hz, H-1A), 5.11 (d, 1 H, J_1,21.7 Hz,

H-1D), 5.08 (d, 1 H, J_1,21.7 Hz, H-1B), 4.29

(dd, 1 H, J_2,33.0 Hz, H-2C), 4.21 (dd, 1 H, J_2,3

3.1 Hz, H-2B), 4.12 (dd, 1 H, J_2,33.3 Hz,

H-2A), 4.08 (dd, 1 H, J_2,33.5 Hz, H-2D)

4.07–3.96 (m, 5 H, H-3A, H-3D, H-4A, H-

4D, H-5C), 3.92–3.74 (m, 9 H, H-3B, H-3C,

H-5A, H-5B, H-5D, H-6B, H-6C, H-6C%, H-

4C), 3.71 (dd, 1 H, J_5,6%4.2, J_6,6%10.9 Hz,

H-6B%), 3.69 (m, 2 H, H-6A, H-6D), 3.65 (dd,

1 H, J_3,4, J_4,59.9 Hz, H-4B), 3.62 (dd, 1 H, J_5,6%

7.4, J_6,6%11.6 Hz, H-6A%), 3.61 (dd, 1 H, J_5,6%

7.4, J_6,6%11.6 Hz, H-6D%), 2.65 (m, 2 H,

SCH₂CH₃), 1.30 (t, 3 H, J 7.5 Hz, SCH₂CH₃).

¹³C NMR (D₂O): l 107.30 (C-1D), 107.18

(C-1A), 104.47 (C-1B), 85.53 (2 C, C-4A, C-

4D), 85.46 (C-1C), 83.98 (2 C, C-2A, C-2D),

79.68 (C-2C), 79.60 (C-3A), 79.45 (C-3D),

78.51 (C-3B), 78.05 (C-3C), 76.06 (C-5D),

78.85 (C-5C), 73.35 (C-5A), 73.26 (C-5B),

69.56 (C-2B), 68.29 (C-4C), 67.92 (C-4B),

65.50, 65.45 (2 C, C-6A, C-6D), 63.78 (C-6B),

63.54 (C-6C), 27.84 (SCH₂CH₃), 16.91

(SCH₂CH₃). Anal. Calcd for C₂₆H₄₆O₂₀S: C,

43.94; H, 6.52. Found: C, 43.71; H, 6.39.

Methyl 2,3,5,6-tetra-O-acetyl-i-

furanosyl-(13)-2-O-benzyl-4,6-O-benzyli-

dene-h- -mannopyranosyl-(12)-[2,3,5,6-te-

tra-O-acetyl-i- -galactofuranosyl-(13)]-

4,6-O-benzylidene-h- -mannopyranosyl-(12)-

3,4,6-tri-O-benzyl-h- -mannopyranoside (18).

D-galacto-

D

K.D. Randell et al. / Carbohydrate Research 325 (2000) 253–264

263

77.16 (2 C, C-4B, C-4C), 76.96 (C-3D), 76.22

(C-2B), 75.51 (C-4E), 75.40 (C-2C), 75.32

(CH₂PhE), 75.19 (C-2E), 74.42 (CH₂PhB),

75.57, 72.42 (2 C, CH₂PhE), 72.32 (C-5E),

72.08 (C-3C), 69.92 (C-3B), 69.73 (C-5A),

69.65 (2 C, C-5D, C-6E), 68.97 (2 C, C-6B,

C-6C), 65.41 (C-5C), 65.08 (C-5B), 63.02 (C-

6D), 62.77 (C-6A), 55.02 (OCH₃), 21.00, 20.75

(8 C, COCH₃). Anal. Calcd for C₈₉H₁₀₂O₃₄: C,

62.30; H, 5.99. Found: C, 62.62; H, 5.88.

One-pot synthesis of 18.—The thioglycoside

acceptor 15 (58 mg, 0.088 mmol) was glycosyl-

ated with the selenoglycoside donor 16 (94

mg, 0.19 mmol) following the general proce-

dure. After 40 min at 0 °C, a solution of the

methyl glycoside acceptor 6 (74 mg, 0.16

—The methyl mannopyranoside acceptor 6

(30 mg, 0.064 mmol) was glycosylated with

the thioglycoside donor 17 (100 mg, 0.076

mmol) following the general procedure. The

reaction time was 4 h at rt. Puriﬁcation of the

pentasaccharide 18 was attempted by column

chromatography, but the donor and product

eluted together. The ¹H NMR spectrum

showed a 3:1 ratio of product–donor (57 mg,

35%, corrected for presence of donor). Exten-

sive column chromatography using 1.5:1

toluene–EtOAc as the eluant gave a small

amount of pure material for characterization:

[h]_D−6.4° (c 2.35, CH₂Cl₂); ¹H NMR

(CD₂Cl₂): l 7.60–7.15 (m, 30 H, aromatic),

5.64 (s, 1 H, CHPhB), 5.59 (s, 1 H, CHPhC),

5.33 (d, 1 H, H-1C), 5.32 (d, 1 H, H-1B), 5.30

(m, 1 H, H-5A), 5.26 (m, 1 H, H-5D), 5.23 (s,

1 H, H-1A), 5.17 (s, 1 H, H-1D), 5.06 (d, 1 H,

J_2,31.2 Hz, H-2A), 4.96 (m, 2 H, H-2D,

H-3D), 4.93 (m, 1 H, H-3A), 4.83, 4.54 (2 d, 2

H, J_gem10.9 Hz, OCH₂PhE), 4.80, 4.75 (2 d, 2

H, J_gem12.5 Hz, OCH₂PhB), 4.77 (d, 1 H, J_1,2

1.8 Hz, H-1E), 4.73, 4.68 (2 d, 2 H, J_gem12.3

Hz, OCH₂PhE), 4.63, 4.57 (2 d, 2 H, J_gem12.2

Hz, OCH₂PhE), 4.36 (dd, 1 H, J_3,45.3, J_4,53.2

Hz, H-4A), 4.30 (dd, 1 H, J_3,46.0, J_4,53.2 Hz,

H-4D), 4.26 (dd, 1 H, J_5,63.3, J_6,6%9.9 Hz,

H-6B), 4.26–4.22 (m, 3 H, H-2C, H-3C, H-

3B), 4.17 (dd, 1 H, J_5,64.6, J_6,6%10.0 Hz,

H-6C), 4.10 (dd, 1 H, J_5,67.6, J_6,6%11.7 Hz,

H-6A), 4.08 (dd, 1 H, J_1,2, J_2,31.7 Hz, H-2B),

4.07 (dd, 1 H, J_3,48.1, J_4,59.1 Hz, H-4B), 4.06

(dd, 1 H, J_3,4, J_4,59.5 Hz, H-4C), 4.05 (dd, 1

H, J_5,64.0, J_6,6%11.9 Hz, H-6D), 4.01 (dd, 1 H,

J_2,32.8 Hz, H-2E), 3.99–3.85 (m, 2 H, H-5B,

H-5C), 3.91 (dd, 1 H, J_5,64.4 Hz, H-6A%), 3.89

(dd, 1 H, J_5,6%9.9 Hz, H-6B%), 3.88 (dd, 1 H,

J_3,49.2 Hz, H-3E), 3.81 (dd, 1 H, J_4,59.2 Hz,

H-4E), 3.80 (dd, 1 H, J_5,6%3.8 Hz, H-6D%), 3.79

(dd, 1 H, J_5,610.3 Hz, H-6C%), 3.77–3.68 (m, 3

H, H-5E, H-6E, H-6E%), 3.35 (s, 3 H, OCH₃),

2.10, 2.08, 2.06, 2.04, 1.951, 1.950, 1.89, 1.86

(8 s, 3 H each, C(O)CH₃). ¹³C NMR

(CD₂Cl₂): l 170.57, 170.47, 170.40, 170.31,

170.27, 170.08 (8 C, COCH₃), 136.21–126.41

(36 C, aromatic), 103.26 (C-1D), 102.94 (C-

1A), 102.06 (CHPhB), 101.96 (CHPhC),

101.68 (2 C, C-1B, C-1C), 100.24 (C-1E),

82.45 (C-2D), 81.90 (C-2A), 81.05 (C-4A),

81.00 (C-4D), 79.59 (C-3E), 77.26 (C-3A),

,

mmol) in dry CH₂Cl₂(2 mL), activated 4 A

molecular sieves (200 mg), and NIS (52 mg,

0.23 mmol) were added. The reaction mixture

was stirred for 1 h at 0 °C and then warmed to

rt and stirred for an additional 2 h. The color

of the solution changed from a deep purple to

dark rose. The reaction was worked up as

1

described in the general procedure. The H

NMR spectrum showed a 3:1 ratio of

product–donor (42 mg, 28%, corrected for

presence of donor).

Methyl i-

mannopyranosyl - (12) - [i -

osyl-(13)]-h- -mannopyranosyl-(12)-h-

D

-galactofuranosyl-(13)-h-

D

-

D

- galactofuran-

D

-

mannopyranoside (4).—To a solution of the

impure pentasaccharide 18 (55 mg, 0.032

mmol) in freshly distilled MeOH (5 mL) was

added 1 M NaOMe–MeOH (0.5 mL). The

reaction mixture was stirred at rt overnight

under an N₂atmosphere. The reaction mix-

ture was neutralized with Rexyn 101 (H⁺), the

resin was ﬁltered, and the solvent was re-

moved in vacuo. The crude product was

puriﬁed by column chromatography using

10:1:1 EtOAc–MeOH–H₂O as the eluant to

give a clear glass. This compound was then

subjected to hydrogenolysis as described in the

general procedure for deprotection. The sol-

vent was removed in vacuo and the crude

product puriﬁed by column chromatography

using 5:2:1 EtOAc–MeOH–H₂O as the elu-

ant. The pentasaccharide 4 was obtained as a

clear syrup, (12 mg, 60%): [h]_D−30° (c 0.029,

1

H₂O); H NMR (D₂O): l 5.24 (d, 1 H, J_1,21.7

Hz, H-1C), 5.18 (d, 1 H, J_1,21.7 Hz, H-1D),

264

K.D. Randell et al. / Carbohydrate Research 325 (2000) 253–264

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5.14 (d, 1 H, J_1,21.5 Hz, H-1A), 5.09 (d, 1 H,

J_1,21.7 Hz, H-1B), 4.97 (d, 1 H, J_1,21.4 Hz,

H-1E), 4.31 (dd, 1 H, H-2C), 4.21 (dd, 1 H,

J_2,32.9 Hz, H-2B), 4.12 (dd, 1 H, J_2,33.3 Hz,

H-2A), 4.09 (dd, 1 H, J_2,33.4 Hz, H-2D),

4.08–4.01 (m, 4 H, H-3A, H-3D, H-4A, H-

4D), 4.00 (dd, 1 H, J_2,33.0, J_3.410.2 Hz,

H-3C), 3.93 (dd, 1 H, J_2,33.4 Hz, H-2E)

3.91–3.55 (m, 20 H, H-3B, H-3E, H-4B, H-

4C, H-4E, H-5A, H-5B, H-5C, H-5D, H-5E,

H-6A, H-6B, H-6C, H-6D, H-6E, H-6A%, H-

6B%, H-6C%, H-6D%, H-6E%), 3.39 (s, 3 H,

OCH₃). ¹³C NMR (D₂O): l 107.47 (C-1D),

107.20 (C-1A), 104.23 (C-1B), 103.40 (C-1C),

101.94 (C-1E), 85.51, 85.43 (C-4A, C-4D),

83.99 (2 C, C-2A, C-2D), 81.40 (C-2E), 79.61,

79.46 (C-3A, C-3D), 78.05 (2 C, C-3B, C-3C),

77.18 (C-2C), 75.96, 75.89 (C-4B, C-4C), 75.25

(C-4E), 73.37, 73.32 (C-5A, C-5D), 72.83,

68.10, 67.75 (4 C, C-3E, C-5B, C-5C, C-5E),

65.51 (2 C, C-6A, C-6D), 63.61 (3 C, C-6B,

C-6C, C-6E), 57.52 (OCH₃). Anal. Calcd for

C₃₁H₅₄O₂₆: C, 44.18; H, 6.46. Found: C, 43.81;

H, 6.22.

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Article Doi

Synthesis of galactofuranosyl-containing oligosaccharides corresponding to the glycosylinositolphospholipid of Trypanosoma cruzi.

DOI: 10.1016/S0008-6215(00)00007-0

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