The synthesis of deoxy-α-Gal epitope derivatives for the evaluation of an anti-α-Gal antibody binding

Carbohydrate Research 337 (2002) 1247–1259

www.elsevier.com/locate/carres

The synthesis of deoxy-a-Gal epitope derivatives for the evaluation

of an anti-a-Gal antibody binding

Adam J. Janczuk, Wei Zhang, Peter R. Andreana, Joshua Warrick, Peng G. Wang*

Department of Chemistry, Wayne State Uni6ersity, Detroit, MI 48202, USA

Received 6 March 2002; accepted 16 June 2002

Abstract

a-Gal epitopes (also termed as a-Gal) are carbohydrate structures bearing the a-

D

-Gal-(13)-b-D-Gal terminus 1 and are

known to be the antigen responsible for antibody-mediated hyperacute rejection in xenotransplantation. Terminal 2-, 3-, 4-, and

6-deoxy-Gal derivatives of a-Gal were synthesized. Inhibition ELISA using mouse laminin was established to determine the

binding afﬁnity of the synthesized a-Gal derivatives. 4-Deoxy-a-Gal derivative 7 showed a signiﬁcant reduction in antibody

recognition. The IC₅₀value was 15-fold poorer than the standard a-Gal epitopes a-

D

-Gal-(13)-b-

D

-Gal-(14)-b-D-Glc-NHAc

(39) and a- -Gal-(13)-b- -Gal-(14)-b- -Glc-OBn (40). A similar observation was seen with 2-deoxy-a-Gal derivative 5,

D

whose IC₅₀value was nearly tenfold higher than the standards. Interestingly, substitution at the terminal 3-position resulted in

only a fourfold decrease in antibody recognition, suggesting a possible point of future derivation. Finally, 6-deoxy-a-Gal

derivative 8 exhibited similar antibody recognition to both a-Gal epitope 39 and a-Gal epitope 40. This strongly suggests that

derivatization at the 6-position can be accomplished without loss of antibody recognition. These ﬁndings can be utilized for the

Keywords: a-Gal epitope; Deoxy sugars; Xenotransplantation; Anti-a-Gal antibody

1. Introduction

acute rejection indicated that the initial steps involve

the recognition of human natural anti-Gal antibody to

Tissue and organ transplantation has become the

preferred treatment for people suffering from organ

failure.¹Unfortunately the limited supply of suitable

organs is a serious problem associated with this proce-

dure. In 1999, roughly 72,000 Americans were on or-

gan/tissue transplant waiting lists, with only approxi-

mately 21,000 transplantations carried out.²

One novel solution to the limited availability of

organs for transplantation is the use of genetically

engineered animal organs, an idea referred to as xeno-

transplantation.³The pig is considered the primary

candidate factoring in ethical considerations, infectious

disease concerns, and size.⁴Despite this promising pro-

posal, human antibody-mediated hyperacute rejection

poses a major obstacle. Investigation into the hyper-

carbohydrate epitopes bearing an a-

Gal terminus 1 (termed as a-Gal).⁵Trisaccharides a-

Gal-(13)-b- -Gal-(14)-b- -Glc-OR (2)

a- -Gal-(13)-b- -Gal-(14)-b- -GlcNAc-OR%% (3)

and pentasaccharide a- -Gal-(13)-b- -Gal-(14)-b-

-GlcNAc-(13)-b- -Gal-(14)-b- -Glc-OR%%% (4)

have been identiﬁed as the major a-Gal epitopes re-

sponsible for the initiation of the immuonological re-

sponse. These epitopes have been found to be expressed

in most mammalian species with the exception of hu-

mans, apes and Old World primates (Fig. 1).⁶Con-

versely, anti-Gal antibodies (known as anti-a-Gal) are

among the most abundant human natural antibodies,

with concentrations of 1–2% of total serum IgG and

3–8% of total serum IgM.⁷

D

-Gal-(13)-b-

D

-

D

and

D

Several methods have been studied to prevent the

hyperacute rejection and to prolong the xenograft cell

survival through elimination or reduction of the inter-

action between a-Gal and anti-a-Gal.⁸These include

depletion through immunoadsorption of anti-a-Gal an-

* Corresponding author. Tel.: +1-313-9936759; fax: +1-

313-5775831

E-mail address: pwang@chem.wayne.edu (P.G. Wang).

PII: S0008-6215(02)00159-3

1248

A.J. Janczuk et al. / Carbohydrate Research 337 (2002) 1247–1259

Fig. 1. Major a-Gal epitopes.

tibodies using a-Gal immobilized afﬁnity columns⁹and

infusion of synthetic a-Gal epitopes to inhibit binding

of anti-a-Gal to xenograft cells.¹⁰One of the major

factors limiting the application of these methods is the

substantial amount of synthetic a-Gal oligosaccharide

needed. Studies on porcine endothelial cells bearing the

a-Gal epitope indicate that millimolar amounts of a-

Gal epitope are needed to achieve 90% inhibition of

anti-a-Gal binding.¹¹At these concentrations, applica-

tion of synthetic a-Gal epitopes cannot be considered

practical. Therefore, the design of synthetic a-Gal mim-

ics that exhibiting higher avidity becomes the next

logical area to explore.

In order to design an effective a-Gal mimic, one must

gain a better understanding of the interaction between

anti-a-Gal antibody and a-Gal. One relatively effective

way is the use of monodeoxy derivatives. A study

conducted by Lemieux et al.¹²probed the interaction of

monoclonal antibodies speciﬁc for Lewis-a (Le^a) human

blood group determinant using eight monodeoxy

derivatives. The rationale for using deoxy derivatives

was based on earlier studies that indicated that clusters

of polar groups provided key hydrophobic binding with

the antibody.¹³Evidence suggested that oligosaccha-

rides assume, if stereochemically possible, an in-

tramolecular hydrogen binding conformation exposing

key polar hydroxyl groups to the antibody binding site.

Replacing a polar hydroxyl group with nonpolar hy-

drogen essentially eliminates any potential for hydrogen

bonding which in turn affects antibody recognition.

With this approach in mind we decided to synthesize

a series of monodeoxy derivatives 5–8 (Fig. 2) of

a-Gal. Focus was placed on the crucial terminal galac-

tose residue. ELISA would then be used to evaluate

anti-a-Gal recognition of the deoxy a-Gal derivatives.

other derivatives and glycoconjugates.¹⁵A stereoselec-

tive high-yielding glycosylation¹⁶between correspond-

ing deoxy monosaccharide donors 10a–c and lactose 9

(Scheme 1) was used in the synthesis of trisaccharide 6,

7, and 8.

Synthesis of derivative 5 used a different approach

starting with peracetylated galactal 12 that was trans-

formed to 2-deoxy-2-iodo monosaccharide 13 (Scheme

2) in 62% yield.¹⁷This highly selective reaction proceeds

by the addition of an iodine atom (I) to the C-2

position of the galactal analogous to azidonitration.¹⁸

TMS–OTf was used to catalyze the glycosylation be-

tween donor 13 and lactose acceptor 9. The resulting

trisaccharide 14 was obtained in a modest 54% yield as

a 90:10 a/b mixture after approximately 1.5 h at

−30 °C. Variation in temperature and time did not

appear to signiﬁcantly improve the yield of the glycosy-

lation. Treatment of trisaccharide 14 with Bu₃SnH,

Et₃B and oxygen lead to the dehalogenated trisaccha-

ride 15. Finally, treatment with NaOMe in anhydrous

MeOH lead to the desired 2-deoxy-a-Gal derivative 5.

Originally in our synthetic approach for the synthesis

of 3-deoxy-a-Gal derivative 6, and the 4-deoxy a-Gal

derivative 7, we envisioned the use of a common inter-

Fig. 2. Terminal monodeoxy-a-Gal derivatives.

2. Results and discussion

Synthesis.—Lactose azide acceptor 9¹⁴was employed

for key glycosylations leading to the formation of the

monodeoxy derivatives 5, 6, 7, and 8. The synthetically

useful azido group at the anomeric position of lactose 9

was introduced for convenient transformation into

Scheme 1. Thioglycosylation.

A.J. Janczuk et al. / Carbohydrate Research 337 (2002) 1247–1259

1249

,

Scheme 2. (a) CAN, NaI, HOAc, MeCN, −15 °C to 25 °C; (b) TMSOTf, lactose 9, 4 A MS, CH₂Cl₂, −30 °C, 1.5 h; (c) Bu₃SnH,

Et₃B, O₂, benzene, rt, 0.5 h; (d) anhyd MeOH, NaOMe, pH 9.

mediate 21 (Scheme 3). With just a few transformations

we could synthesize both 3-deoxy-a-Gal thiophenyl

donor 22 and 4-deoxy-a-Gal thiophenyl donor 23 from

intermediate 21. Starting with commercially available

ing methods was attempted without any signiﬁcant

success. It was clear that the problem was due to the

base-sensitive acetyl group on the C-4 position. Since it

became obvious that the approach needed to be re-

designed, the decision was made to synthesize the 4-de-

oxygalactose donor using a different synthetic route.

penta-O-acetyl- -galactose (16), a glycosylation with

D

thiophenol was carried out in the presence of a Lewis

acid. Following deprotection, a selective acetonide pro-

tection of the C-3, C-4 diol gave rise to compound 19 in

50% yield.¹⁹Benzylation, followed by removal of the

3,4-O-isopropylidene moiety, gave compound 20. Next

triethyl orthoacetate was used to selectively protect the

C-4 hydroxyl group after ring opening promoted by

80% HOAc resulting in intermediate 21. Functionaliza-

tion of the C-3 hydroxyl group with phenyl chloroth-

From commercially available penta-O-acetyl-D-glu-

cose (26) (Scheme 5), glycosylation lead to the satisfac-

tory formation of thioglycoside 27. Base-catalyzed

removal of the acetyl protecting groups led to the free

thioglycoside 28, which upon acid-catalyzed treatment

with a,a-dimethoxytoluene lead to the selective 4,6-O-

benzylidene thioglucoside 29.²²The free hydroxyl

groups were then O-benzylated prior to selective reduc-

tive deprotection mediated by sodium cyanoborohy-

dride, affording the 2,3,6-tri-O-benzylated thiogluco-

side 30.²³Treatment with phenyl chlorothionocarbon-

ate, followed by free-radical deoxygenation, led to the

4-deoxythiophenyl donor 31. TfOH-catalyzed glycosy-

lation of donor 31 and lactose azide acceptor 9 gave

trisaccharide 32 in relatively modest yield. Following

the same protection and deprotection strategy resulted

in 4-deoxy-a-Gal derivative 7.

ionocarbonate

(phenoxythiocarbonyl

chloride),

employing 4-(dimethylamino)-pyridine (DMAP) as a

catalyst, afforded a O-(phenoxythiocarbonyl) ester,

which then underwent free-radical-mediated Barton–

McCombie deoxygenation with tributylstannane (trib-

utyltin hydride) with satisfactory conversion to the

thiophenyl 3-deoxy sugar donor 22 (Scheme 4).²⁰

TfOH-catalyzed N-iodosuccinamide-mediated glycosy-

lation of donor 22 and lactose azide 9 produced trisac-

charide 24 in 72% yield. Next reduction of the azide

was carried out using selective hydrogenation via

Adams’ catalyst,²¹followed by N-acylation of the free

amino group using acetic anhydride and triethylamine,

led to compound 25. Final deprotection gave rise to

3-deoxy-a-Gal derivative 6. We attempted to employ a

similar reaction strategy for 4-deoxy-a-Gal derivative 7;

however, we encountered difﬁculties in ﬁnding a suit-

able protecting group that could be used in the synthe-

sis of the thiophenyl 4-deoxy sugar donor 23. Initially

we attempted to produce the C-3 hydroxyl group using

a variety of silyl protecting groups. Under mildly basic

conditions we were able to successfully protect the

hydroxyl group. Unfortunately, removal of the acetyl-

protecting group at the C-4 position caused an exten-

sive amount of intramolecular migration of the silyl

group between the C-4 and the C-3 position, rendering

this approach unacceptable. A variety of other protect-

Scheme 3. (a) PhSH, BF₃·Et₂O, CH₂Cl₂; (b) anhyd MeOH,

NaOMe, pH 9; (c) 2,2-dimethoxypropane, TsOH, CH₂Cl₂; (d)

BnBr, NaH, DMF; (e) 80% HOAc, 90 °C; (f) MeC(OEt)₃,

TsOH, benzene; (g) 80% HOAc; (h) PTC–Cl, DMAP,

CHCl₂, 16 h; (i) Bu₃SnH, AIBN, benzene, 2 h.

1250

A.J. Janczuk et al. / Carbohydrate Research 337 (2002) 1247–1259

Scheme 4. (a) PTC–Cl, DMAP, CH₂Cl₂, 12 h; (b) AIBN, Bu₃SnH, benzene 1.25 h; (c) lactose 9, TfOH, CH₂Cl₂; NIS (d) PtO₂,

H₂, EtOH, 2 h; (e) CH₂Cl₂, Et₃N, Ac₂O, 0 °C, 2 h; (f) Pd(OH)₂–C, MeOH, H₂, 18 h; (g) MeOH, NaOMe, pH 9.

Scheme 5. (a) PhSh, BF₃·Et2O, CH₂Cl₂; (b) NaOMe, MeOH, pH 9; (c) a,a-dimethoxypropane, TsOH; MeCN, 18 h; (d) BnBr,

NaH, DMF; (e) NaCNBH₃, HCl, Et₂O, THF; (f) PTC–Cl, DMAP, CH₂Cl₂, 12 h; (g) Bu₃SnH, AIBN, benzene 1 h; (h) lactose

9, TfOH, NIS, CH₂Cl₂; (i) PtO₂, H₂, EtOH, 2 h; (j) CH₂Cl₂, Et₃N, Ac₂O, 0 °C, 2 h; (k) Pd(OH)₂–C, MeOH, H₂, 18 h; (l) NaOMe,

MeOH, pH 9.

Scheme 6. (a) Ac₂O, DMAP, pyridine; (b) PhSH, CH₂Cl₂, BF₃Et₂O; (c) NaOMe, MeOH, pH 9; (d) BnBr, DMF, NaH; (e) TfOH,

lactose 9, NIS, CH₂Cl₂; (f) Pt₂O, H₂, EtOH, 1 h; (g) Ac₂O, Et₃N; (h) Pd/C, H₂,18 h; (i) NaOMe, MeOH, pH 9.

The ﬁnal terminal 6-deoxy-a-Gal derivative was syn-

thesized starting from commercially available -fucose

34 (Scheme 6). Using our established synthetic

strategy¹⁴we were able produce thiophenyl donor 36 in

four transformations with an overall yield of 76%.

Glycosylation catalyzed by TfOH in the presence of

lactose acceptor 9 lead to trisaccharide intermediate 37.

Following established protocol lead to the formation of

6-deoxy-a-Gal derivative 8.

Binding a6idity analysis of deoxy-h-Gal deri6ati6es.—

The evaluation of the binding afﬁnities of synthesized

deoxy-a-Gal derivatives 5–8 to human anti-a-Gal anti-

D

A.J. Janczuk et al. / Carbohydrate Research 337 (2002) 1247–1259

1251

3. Conclusions

bodies was accomplished by inhibition enzyme-linked

immunosorbent assay (ELISA).²⁴Puriﬁed human

(male, blood type AB) polyclonal anti-a-Gal antibody

was used as the primary antibody, and mouse laminin

was used as the natural source of a-Gal. Studies have

suggested that anti-Gal antibodies are encoded by a

small number of conserved germline genes^7bdemon-

strating limited clonal diversity.²⁵This, in general, may

account for the speciﬁcity observed in recognizing the

In summary, four monodeoxy derivatives of the a-

Gal epitope were synthesized via facile carbohydrate

transformations. High-yielding and stereoselective gly-

cosylations were employed to incorporate the crucial

a- -Gal-(13)-Gal linkage. The derivatives were all

D

assessed for biological activity using ELISA. The re-

sults indicate that removal of the hydroxyl group at the

C-6 and the C-3 position does not appear to signiﬁ-

cantly decrease anti-a-Gal antibody recognition. This

data then suggests the potential to attach a variety of

functional groups at these positions generating a large

array of a-Gal derivatives for use in further binding

studies. Such derivatives will not only serve as tools for

structure–binding experiments but may also prove to

have practical applications in cell xenotransplantations.

a-D

-Gal-(13)-D

-Gal moiety.²⁶Therefore, it is not be-

lieved that there will be any signiﬁcant difference in

molecular recognition between the various anti-Gal an-

tibodies clones for this evaluation. The concentrations

of deoxy-a-Gal derivatives at 50% inhibition (IC₅₀) of

anti-a-Gal antibody binding to a-Gal on the mouse

laminin was measured and compared to standard com-

pounds a-

D

-Gal-(13)-b-

D

-Gal-(14)-b-

D

-GlcNHAc

(39) and a-

D

-Gal-(13)-b-

D

-Gal-(14)-b-

D-Glc-OBn

(40). Substitution of the C-6 hydroxyl group with hy-

drogen did not signiﬁcantly affect the inhibition activi-

ties of anti-a-Gal antibodies, compared to the standard

compounds 39 and 40. In contrast, removal of the polar

group at the C-4 position drastically lowered inhibition

nearly 15-fold. This result was expected since the C-4

hydroxyl group is a key polar group involved in anti-a-

Gal antibody binding to a-Gal. Hydroxyl substitution

at the C-3 position exhibited comparable inhibition

with only a relatively small decrease in the IC₅₀. As

expected substitution of the C-2 hydroxyl group also

decreased anti-a-Gal antibody recognition. The effects

were nearly tenfold as compared to the standard com-

pounds 39 and 40. These results indicate that the key

protein–carbohydrate interaction between a-Gal and

anti-a-Gal antibodies tolerates substitution at the C-6

and C-3 position of the terminal galactose residue of

a-Gal. C-4 and C-2 substitution is detrimental to the

binding between the protein and carbohydrate epitope

(Table 1).

4. Experimental

General.—¹H and ¹³C NMR spectra were recorded

on Varian 300 MHz, VXR400 and Unity 500 MHz

spectrometers. Mass spectra (ESI, FAB, EI) were run

on Kratos MS-80 and MS-50 instruments at Wayne

State University. HRMS were run at the University of

California–Riverside.

(TLC) was conducted on precoated Whatman K6F

Thin-layer

chromatography

,

Silica Gel 60 A TLC plates with a ﬂuorescent indicator.

EM Science Silica Gel 60 (230–400 Mesh) was used for

column chromatography. Optical absorptions for the

ELISA assays were determined on a BioRad microplate

reader model 550 at wavelengths of 650 and 450 nm.

Type AB human serum, peroxidase-conjugated goat

anti-human IgG antibody, and mouse laminin (a base-

ment membrane glycoprotein containing 50–70 a-Gal

epitopes per molecule) were purchased from commer-

cial sources.

Compounds 39 and 40 were synthesized using previ-

ous protocols.²⁷Lactose azide 9 was supplied from the

previous synthesis.¹⁴Size-exclusion chromatography

was performed on BioGel P2 resin and on Sephadex

G-15 resin using distilled water as the eluent. Other

reagents were obtained from commercial sources.

Table 1

ELISA assay results for deoxy-a-Gal derivatives

Compound

IC₅₀mM

1.2

a-

D

-Gal-(13)-b-

NHA (39)

-Gal-(14)-

D

-Gal-(14)-

1,3,4,6-Tetra-O-acetyl-2-deoxy-2-iodo-h-

(13).—3,4,6-Tri-O-acetyl- -galactal (12, 1.00 g, 3.67

D -talose

b-D-Glc

-

C

D

a-D

-Gal-(13)-b-

D

5.7

mmol) was dissolved in CH₃CN (20 mL) and cooled to

−15 °C. CAN (5.23 g, 9.54 mmol) and glacial HOAc

(2 mL) were added to the stirring mixture. Next NaI

(715 mg, 4.77 mmol) in CH₃CN (10 mL) was added

dropwise over approximately 30 min. The solution was

then allowed to gradually warm to rt. After 4 h the

reaction was complete, and the mixture was washed

with 10% Na₂S₂O₃, satd aq NaHCO₃, and brine. The

organic layer was then dried over anhyd Na₂SO₄, con-

centrated and puriﬁed by column chromatography (1:2

b-D-Glc

-

OBn (40)

-Gal-(13)-b-

NHAc (5)

3-Deoxy-a- -Gal-(13)-b-

b-D-Glc NHAc (6)

4-Deoxy-a- -Gal-(13)-b-

b-D-Glc NHAc (7)

6-Deoxy-a- -Gal-(13)-b-

b-D-Glc NHAc (8)

2-Deoxy-a-

D

-Gal-(14)-

50.1

21.5

92

b-D-Glc

-

D

-

D

-

D

4.0

-

1252

A.J. Janczuk et al. / Carbohydrate Research 337 (2002) 1247–1259

EtOAc–hexanes) to afford the deoxyiodotalose deriva-

tive 13 (1.18 g, 70%). H NMR (500 MHz, CDCl₃): l

61.9, 61.3, 31.4 multiple 20.9–20.7. ESIMS(+): Calcd

for C₃₆H₄₉N₃NaO₂₃, m/z 914.27; Found, 914.38.

1

6.48 (s, 1 H), 5.41 (s, 1 H), 4.87 (broad s, 1 H), 4.34 (d,

J 6.5 Hz, 1 H), 4.26 (s, 1 H), 4.17 (d, J 5 Hz, 2 H), 2.17

(s, 3 H), 2.12 (s, 3 H), 2.07 (s, 3 H), 2.02 (s, 3 H); ¹³C

NMR (125.68 MHz, CDCl₃): l 170.6, 170.1, 169.7,

168.3, 96.2 (C-1), 69.2, 65.0, 61.6, 21.1, 21.0, 20.8, 19.1.

FABMS(+): Calcd for C₁₄H₁₉IKO₉, m/z 496.97;

Found, 497.01.

2-Deoxy-h-

topyranosyl-(14)-i-

D

-lyxo-hexopyranosyl-(13)-i-

D-galac-

D

-glucopyranosyl azide (5).—

Compound 15 (160 mg, 0.18 mmol) was dissolved in

anhyd MeOH, followed by the addition of NaOMe to

pH 9. After 2 h, Dowex 50W×2-100 (H⁺) was added

to neutralize the reaction mixture. The resin was then

ﬁltered and the ﬁltrate was evaporated to afford 5 (90

mg, \95%). ¹H NMR (500 MHz, D₂O): l 5.06 (broad,

1 H), 4.32 (d, 1 H), 3.89–3.42 (m, 17 H), 3.21 (broad,

1 H), 1.78–1.72 (m, 2 H); ¹³C NMR (125.6 MHz,

D₂O): l 102.8, 93.4, 90.0, 77.9, 76.77, 75.6, 75.3, 74.5,

72.6, 71.0, 69.7, 67.6, 64.8, 64.1, 61.6, 61.1, 60.0, 31.3.

HRFABMS(+): Calcd for C₁₈H₃₁N₃NaO₁₄, m/z

536.1704; Found, 536.1714.

3,4,5-Tri-O-acetyl-2-deoxy-2-iodo-h-

yl - (13) - 2,4,6 - tri - O - acetyl - i - - galactopyranosy)-

(14)-2,3,6-tri-O-acetyl-i- -glucopyranosyl azide

(14).—A suspension of iodotaloside 13 (500 mg, 1.09

D-talopyranos-

D

,

mmol), acceptor 9 (743 mg, 1.20 mmol) and 4 A MS

(600 mg) was stirred at ambient temperature for 30 min

before being cooled to −30 °C. Next TMS-OTf (0.049

mL, 0.273 mmol) was added dropwise. After 90 min the

reaction was complete as determined by TLC, and it

was quenched with Et₃N. The mixture was then ﬁltered

through a Celite pad, diluted with CH₂Cl₂and washed.

The diluted product was then dried, ﬁltered and con-

centrated in vacuo. The crude product was then

puriﬁed by column chromatography (1:1.5 EtOAc–hex-

Phenyl 1-thio-i- -galactopyranoside (18).—Com-

D

mercially available 16 (10.0 g, 25.7 mmol) was dissolved

in anhyd CH₂Cl₂(150 mL) and cooled to 0 °C. Thio-

phenol (3.43 mL, 33.4 mmol) was added to the mixture

with stirring for 30 min. Then BF₃·Et₂O (9.77 mL, 77.0

mmol) was slowly injected into the mixture. After 4 h

the reaction was complete, and the mixture was diluted

and washed with satd aq NaHCO₃and brine. After

drying over anhyd Na₂SO₄, the solvent was removed in

vacuo, and the crude product was puriﬁed by column

chromatography (1:5 EtOAc–hexanes) to afford com-

pound 17 (9.7 g, 86%). To a mixture of compound 17

(9.7 g, 22.0 mmol) in anhyd MeOH (100 mL), NaOMe

powder was added in small portions to pH 9. After 2 h

the solution was neutralized using Dowex 50W×2-100

(H⁺). The resin was then ﬁltered off and the solvent

was removed in vacuo to afford a powdery solid 18

1

anes) to afford compound 14 (600 mg, 54%). H NMR

(300 MHz, CDCl₃): l 5.47 (s, 1 H), 5.29 (d, J 3.0 Hz, 2

H), 5.14 (t, J 9.3 Hz, 1 H), 4.98 (dd, J 10.5, 7.8 Hz, 1

H), 4.79 (t, J 9.0 Hz, 1 H), 4.60–4.57 (m, 2 H),

4.43–4.34 (m, 2 H), 4.16–3.97 (m, 7 H), 3.82–3.63 (m,

4 H), 2.11 (s, 6 H), 2.06–1.98 (m, 21 H); ¹³C NMR

(75.46 MHz, CDCl₃): l 170.7, 170.5, 170.4,170.1, 169.7,

169.5, 168.9, 101.1, 99.9, 87.8, 75.7, 74.9, 73.8, 72.6,

71.1, 70.1, 69.9, 67.5, 65.1, 65.0, 61.9, 61.7, 61.1, multi-

ple 21.1–20.6, 19.1. ESIMS(+): Calcd for C₃₆H₄₈-

IN₃NaO₂₃, m/z 1040.16; Found, 1039.95.

1

(5.99 g, \99%). H NMR (400 MHz, D₂O): l 7.58–

3,4,5-Tri-O-acetyl-2-deoxy-h-

(13)-2,4,6-tri-O-acetyl-i- -galactopyranosyl-(14)-

2,3,6-tri-O-acetyl-i- -glucopyranosyl azide (15).—To a

D

-lyxo-hexopyranosyl-

7.55 (m, 2 H), 7.42–7.35 (m, 3 H), 4.76 (d, J 9.6 Hz, 1

H, H-1), 3.97 (d, J 3.2 Hz, 1 H), 3.76–3.59 (m, 5 H);

¹³C NMR (100.61 MHz, D₂O): l 133.0, 131.4, 129.6,

88.3 (C-1), 79.3, 74.3, 69.5, 68.9, 61.2. FABMS(+):

Calcd for C₁₂H₁₆NaO₅S, m/z 295.06; Found, 295.18.

D

mixture of compound 14 (360 mg, 0.35 mmol) and

Bu₃SnH (0.24 mL, 0.885 mmol) in distilled benzene (5

mL) was added Et₃B (1 M in hexanes, 0.089 mL, 0.088

mmol). After 30 min the reaction was complete, and the

solvent was removed in vacuo. The crude mixture was

then diluted with EtOAc and worked up. The solvent

was then dried over anhyd Na₂SO₄, ﬁltered, concen-

trated and puriﬁed by column (1:1 EtOAc–hexanes) to

afford compound 15 (200 mg, 63%). ¹H NMR (500

MHz, CDCl₃): l 5.34 (d, J 3.0 Hz, 1 H), 5.29 (d, J 2.0

Hz, 1 H), 5.21–5.17 (m, 2 H), 5.04 (dd J 10.0, 8.0 Hz,

1 H), 4.98 (dt, J 11.5, 3.8 Hz, 1 H), 4.84 (t,d, J 10.0, 1.0

Hz, 1 H), 4.62 (d, J 8.5 Hz, 1 H), 4.45 (d, J 12 Hz, 1 H)

4.40 (d, J 7.0 Hz, 1 H), 4.16–3.97 (m, 7 H), 3.83–3.76

(m, 3 H), 3.71–3.68 (m, 1 H), 2.14 (s, 3 H), 2.11–2.03

(m, 25 H), 1.94 (s, 3 H) 1.70–1.65 (C-2, m, 2 H); ¹³C

NMR (125.7 MHz, CDCl₃): l 170.7, 170.6, 170.5,170.4,

170.1, 169.8, 169.7, 169.0, 101.3, 94.3, 87.8, 75.7, 75.0,

72.7, 72.4, 71.2, 70.9, 69.9, 67.5, 66.3, 65.9, 64.9, 62.2,

Phenyl

ranoside (19).—Compound 18 (5.99 g, 22.0 mmol) was

dissolved in 1:1 mixture of dry CH₂Cl₂and

3,4-O-isopropylidene-1-thio-i-D-galactopy-

a

dimethoxypropane (30 mL). TsOH·H₂O (245 mg) was

then added to the solution. After 3 h the reaction was

concentrated in vacuo and diluted with CHCl₃. The

solution was washed with satd aq NaHCO₃and brine,

then dried over Na₂SO₄. The solvent was then removed

in vacuo. Next 5% HOAc (10 mL) was added. After 30

min the solution was extracted with CHCl₃(3×20 mL)

and washed with satd aq NaHCO₃and brine. The

organic layer was then dried over Na₂SO₄and concen-

trated. Puriﬁcation by column chromatography (2:1

EtOAc–hexanes) afforded compound 19 (3.43 g,

1

50.0%). H NMR (400 MHz, CDCl₃): l 7.53–7.50 (m,

2 H), 7.33–7.26 (m, 3 H), 4.46 (d, J 10.4 Hz, 1 H, H-1),

4.17 (dd, J 5.6, 2.0 Hz, 1 H), 4.13-4.08 (m, 1 H),

A.J. Janczuk et al. / Carbohydrate Research 337 (2002) 1247–1259

1253

3.99–3.94 (dd, J 11.2, 7.2 Hz, 1 H), 3.88–3.85 (m, 1 H),

3.79 (dd, J 11.2, 3.6, 1 H), 3.56 (dd, J 10.4, 7.2 Hz, 1

H), 1.41 (s, 3 H), 1.32 (s, 3 H); ¹³C NMR (100.6 MHz,

CDCl₃): l 132.6, 132.2, 129.3, 128.3, 110.7, 87.9 (C-1),

79.5, 74.1, 71.7, 62.8, 28.6, 26.6. FABMS(+): Calcd

for C₁₅H₂₀NaO₅S m/z 335; Found 335.

for 20 min. The mixture was then concentrated in

vacuo and extracted with CH₂Cl₂. The extracts were

washed with satd aq NaHCO₃and water, and the

organic layer was dried over Na₂SO₄. The solvent was

removed in vacuo, and the crude residue was puriﬁed

by column chromatography (1:3 EtOAc–hexanes) to

1

Phenyl 2,6-di-O-benzyl-1-thio-i-

D

-galactopyranoside

afford compound 21 (3.40 g, 87.2%). H NMR (300

(20).—In a ﬂame-dried ﬂask equipped with a dropping

funnel under nitrogen compound 19 (3.35 g, 10.73

mmol) was dissolved in dry DMF (25 mL) and cooled

to 0 °C. NaH (3.43 g, in 60% oil dispersion, 8 equiv)

was added in small portions over 1 h. Afterwards

benzyl bromide (10.21 mL, 85.8 mmol) was added

dropwise to the stirring solution over a period of 1 h.

The resulting mixture was allowed to stir overnight at

ambient temperature. MeOH (10 mL) was added to

quench the excess NaH. The mixture was extracted

with CHCl₃(3×60 mL), and the extract was washed

with satd aq NaHCO₃, and brine. The organic layer

was then dried over Na₂SO₄and concentrated in vacuo.

Column chromatography (1:2 EtOAc–hexanes) of the

residue afforded the benzylated intermediate (4.55 g,

MHz, CDCl₃): l 7.60–7.57 (m, 2 H), 7.40–7.25 (m, 13

H), 5.38 (d, J 2.0 Hz, 1 H), 4.93 (d, J 10.8 Hz, 1 H),

4.68 (d, J 9.3 Hz, 1 H, H-1), 4.67 (d, J 10.5 Hz, 1 H),

4.54 (d, J 11.7 Hz, 1 H), 4.43 (d, J 11.7 Hz, 1 H),

3.86–3.76 (m, 2 H), 3.63–3.50 (m, 3 H), 2.08 (s, 3 H);

¹³C NMR (75.46 MHz, CDCl₃): l 171.4, 138.1, 137.9,

134.1, 134.1, 131.8, 129.2, 128.8, 128.6, 128.5, 128.3,

128.1, 128.0, 127.7, 87.9 (C-1), 78.2, 76.3, 75.7, 74.1,

73.8, 70.5, 68.6, 21.1; FABMS(+): Calcd for

C₂₈H₃₀NaO₆S, m/z 517; Found, 517.

Phenyl 4-O-acetyl-2,6-di-O-benzyl-3-deoxy-1-thio-i-

D

-xylo-hexopyranoside (22).—To the mixture of 21

(1.109 g, 2.24 mmol) dissolved in dry CH₂Cl₂(50 mL)

was added phenyl chlorothionocarbonate (PTC–Cl,

0.93 mL, 6.7 mmol) and cooled to 0 °C. Then DMAP

(2.46 g, 20.2 mmol) was added in small portions. The

solution was stirred overnight at ambient temperature.

The mixture was then diluted with CH₂Cl₂(50 mL) and

washed with satd aq NaHCO₃, brine and phosphate

buffer solution (pH 7.4). The organic layer was then

dried over Na₂SO₄and concentrated in vacuo. The

crude residue was then dissolved in dry benzene (80

mL) under Ar, and Bu₃SnH (6.03 mL, 22.4 mmol) was

added. The reaction mixture was then heated to 80 °C,

followed by the addition of AIBN. After nearly 2 h the

reaction was concentrated in vacuo, and the residue

was dissolved in CH₃CN (50 mL). Next the diluted

product mixture was extracted several times with hex-

anes. The CH₃CN layer was then concentrated in

vacuo, and the residue was puriﬁed by ﬂash column

chromatography (8:1 EtOAc–hexanes) to afford com-

pound 22 (0.74 g, 64.3%). ¹H NMR (400 MHz, CDCl₃):

l 7.60–7.57 (m, 2 H), 7.35–7.24 (m, 13 H), 5.14 (d, J

1.6 Hz, 1 H), 4.78 (d, J 9.2 Hz, 1 H, H-1), 4.71 (d, J

11.6 Hz, 1 H), 4.58 (d, J 11.2 Hz, 1 H), 4.55 (d, J 11.6

Hz, 1 H), 4.42 (d, J 12.0 Hz, 1 H), 3.82–3.79 (m, 1 H),

3.65–3.52 (m, 3 H), 1.98 (s, 3 H) 1.85 (C-3, m, 2H); ¹³C

NMR (100.6 MHz, CDCl₃): l 170.4, 138.1, 137.9,

134.3, 131.7, 129.0, 128.6, 128.3, 128.1, 128.0, 127.9,

127.4, 89.6 (C-1), 78.1, 73.7, 72.7, 71.8, 68.8, 68.3, 35.2,

21.2. FABMS(+): Calcd for C₂₈H₃₀KO₅S, m/z 517;

Found, 517.

1

86.0%). H NMR (400 MHz, CDCl₃): l 7.58–7.55 (m,

2 H), 7.45–7.22 (m, 13 H), 4.83 (d, J 11.6 Hz, 1 H),

4.70 (d, J 12.0 Hz, 1 H), 4.67 (d, J 10.0 Hz, 1 H, H-1),

4.61 (d, J 12.0 Hz, 1 H), 4.54 (d, J 12.0 Hz, 1 H), 4.27

(m, 1 H), 4.27 (dd, J 6.0, 1.6 Hz, 1 H), 3.96 (m, 1 H),

3.84–3.77 (m, 2 H), 3.54 (dd, J 9.6, 6 Hz, 1 H), 1.42 (s,

3 H), 1.37 (s, 3 H); ¹³C NMR (100.61 MHz, CDCl₃): l

138.5, 138.1, 134.2, 132.0, 129.0, 128.6, 128.5, 128.4,

127.9, 127.5, 110.3, 86.5 (C-1), 79.8, 78.5, 76.0, 74.1,

73.8, 73.7, 69.9, 28.0, 26.6. FABMS(+): Calcd for

C₂₉H₃₂KO₅S, m/z 531; Found, 531. The benzylated

intermediate (4.55 g, 9.24 mmol) was dissolved in 80%

HOAc (40 mL) and heated to 80 °C. After 4 h the

reaction was complete, and the solution was allowed to

cool to rt. The solution was then concentrated in vacuo,

and the residue was dissolved in CHCl₃(30 mL). After

workup, the organic layer was dried, and the solvent

was removed to afford compound 20 (3.82 g, 91.0%).

¹H NMR (300 MHz, CDCl₃): l 7.60–7.57 (m, 2 H),

7.39–7.25 (m, 13 H), 4.94 (d, J 10.8 Hz, 1 H), 4.70, (d,

J 10.2 Hz, 1 H), 4.63 (d, J 9.6 Hz, 1 H, H-1), 4.03 (d,

J 1.8 Hz, 1 H), 3.80 (s, 1 H), 3.67–3.58 (m, 3 H); ¹³C

NMR (75.46 MHz, CDCl₃): l 138.3, 137.9, 134.2,

131.9, 129.2, 128.8, 128.7, 128.6, 128.3, 128.1, 128.0,

127.7, 87.8 (C-1), 78.4, 75.6, 75.3, 74.0, 69.9, 69.8.

FABMS(+): Calcd for C₂₆H₂₈KO₅S, m/z 491; Found,

491.

Phenyl 4-O-acetyl-2,6-di-O-benzyl-1-thio-i-

D

-galac-

4-O-Acetyl-2,6-di-O-benzyl-3-deoxy-h-

pyranosyl-(13)-2,4,6-tri-O-acetyl-i- -galactopyran-

osyl-(14)-2,3,6-tri-O-acetyl-i- -glucopyranosyl azide

(24).—A suspension of acceptor 9 (0.388 g, 0.627

D-xylo-hexo-

topyranoside (21).—Compound 20 (3.82 g, 8.44 mmol)

was added to a mixture of dry benzene (50 mL), triethyl

orthoacetate (30 mL), and TsOH·H₂O (60 mg). After

approximately 1 h, Et₃N (5 mL) was added, and the

solution was concentrated in vacuo. The residue was

then dissolved in 60% HOAc (60 mL) and stirred at rt

D

,

mmol), donor 22 (0.300 g, 0.570 mmol), 4 A MS (1.2 g),

and anhyd CH₂Cl₂(8 mL) was stirred for 1 h at

ambient temperature under Ar. The mixture was then

1254

A.J. Janczuk et al. / Carbohydrate Research 337 (2002) 1247–1259

cooled to −78 °C and N-iodosuccinamide (190 mg,

0.845 mmol) was added. Next triﬂic acid (25.0 mL, 0.31

mmol) was added slowly to the mixture. After 1 h, all

of the donor was consumed, and the reaction mixture

was diluted with CH₂Cl₂(20 mL) and ﬁltered through a

Celite pad. The ﬁltrate was then washed with satd aq

NaHCO₃, 10% Na₂S₂O₃and dried over Na₂SO₄. The

solvent was then removed in vacuo, and the crude

residue was puriﬁed by ﬂash column chromatography

(1:2 EtOAc–hexanes) to afford trisaccharide 24 (407

3-Deoxy-h-

topyranosyl-(14)-N-acetyl-i-

D

-xylo-hexopyranosyl-(13)-i-

D-galac-

D

-glucopyranosylamine

(6).—Pd(OH)₂/C (10% wt, 30 mg) was added to a

mixture of 25 (238 mg, 0.237 mmol) in MeOH (30 mL).

(Caution! Extreme ﬁre hazard.) The suspension was

charged with hydrogen (50 lb/in²) and agitated

overnight. The mixture was then ﬁltered, and the

ﬁltrate was evaporated in vacuo. The residue was then

dissolved in anhyd MeOH, followed by the addition of

NaOMe powder to pH 9. After 2 h, Dowex 50W×2-

100 (H⁺) was added to neutralize the mixture. The

resin was then ﬁltered, and the ﬁltrate was evaporated

to afford 6 in quantitative yield (125 mg). Further

puriﬁcation was done by gel ﬁltration (Sephadex 25).

¹H NMR (500 MHz, D₂O): l 4.88 (d, J 3.0 Hz, 1 H),

4.79 (d, J 9.5 Hz, 1 H), 4.36 (d, J 7.5 Hz, 1 H),

4.00–3.89 (m, 4 H), 3.75 (d, J 12 Hz, 1 H), 3.69–46 (m,

11 H), 3.26 (t, J 7.0 Hz, 1 H), 1.88 (s, 3 H) 1.85 (C-3,

m, 2 H); ¹³C NMR (125.6 MHz, D₂O): l 175.6, 102.9,

94.7, 79.2, 78.1, 77.1, 76.4, 75.2, 71.5, 70.6, 69.7, 67.0,

65.0, 63.6, 63.4, 61.3, 61.1, 60.0, 32.9, 23.4. ESIMS

(+): m/z Calcd 552.19, Found 552.04. HRFABMS(+):

Calcd for C₂₀H₃₅NNaO₁₅, m/z 552.1904; Found,

522.1914.

1

mg, 72.4%). H NMR (400 MHz, CDCl₃): l 7.35–7.26

(m, 10 H), 5.47 (d, J 2.8 Hz, 1 H), 5.19 (t, J 8.4 Hz, 1

H), 5.11 (dd, J 10.0, 8.0 Hz, 1 H), 5.05 (d, J 2.8 Hz, 1

H), 4.84 (t, J 9.2 Hz, 1 H), 4.62–4.43 (m, 6 H), 4.36 (d,

J 8.0 Hz, 1 H), 4.14–4.08 (m, 2 H), 4.05 (d, J 6.4 Hz,

2 H), 3.96 (m, 1 H), 3.88–3.63 (m, 6 H), 3.46–3.39 (m,

2 H), 2.09 (s, 3 H), 2.08 (s, 3 H), 2.06 (s, 3 H), 2.04 (s,

3 H), 2.01 (s, 3 H), 1.94 (C-3, m, 2 H),1.90 (s, 3 H), 1.87

(s, 3 H); ¹³C NMR (100.61 MHz, CDCl₃): l 170.6,

170.5, 170.2, 169.8, 169.7, 128.6, 128.5, 128.0, 127.9,

127.8, 101.3 (C-1%), 93.4, (C-1%%), 87.8 (C-1), 75.7, 75.0,

73.5, 72.8, 72.6, 71.2, 71.1, 70.6, 70.4, 68.6, 68.5, 68.2,

64.6, 62.1, 61.5, 28.3, 21.1, 21.0, 20.9, 20.9, 20.8, 20.6.

ESIMS(+): Calcd for C₄₆H₅₇N₃NaO₂₁, m/z 1010.34;

Found, 1010.47.

Phenyl 2,3,4,6-tetra-O-acetyl-1-thio-i-D-glucopyran-

4-O-Acetyl-2,6-di-O-benzyl-3-deoxy-h-

pyranosyl)-(13)-2,4,6-tri-O-acetyl-i- -galactopyran-

osy)-(14)-N-acetyl-2,3,6-tri-O-acetyl-i- -glucopyra-

D-xylo-hexo-

oside (27).—Compound 26 (15.0 g, 38.43 mmol) was

dissolved in anhyd CH₂Cl₂(150 mL) and cooled to

0 °C. Then thiophenol (11.83 mL, 115.29 mmol) was

added. After 30 min BF₃·Et₂O (6.34 mL, 50.00 mmol)

was slowly injected into the mixture. After 4 h the

reaction was complete as monitored by TLC and di-

luted with CH₂Cl₂(100 mL) and washed with satd aq

NaHCO₃and brine. After drying over Na₂SO₄, the

solvent was removed in vacuo. Puriﬁcation by column

chromatography (1:5 EtOAc–hexanes) afforded com-

D

nosylamine (25).—Trisaccharide 24 (350 mg, 0.334

mmol) in EtOH was agitated with PtO₂(Adams’ cata-

lyst, 20 mg) under a hydrogen (50 lb/in²) atmosphere

for 2 h. After completion, the PtO₂was ﬁltered, and the

ﬁltrate was concentrated in vacuo. The residue was then

dissolved in dry CH₂Cl₂(10 mL) with Et₃N (1 mL) and

Ac₂O (0.3 mL) at 0 °C. The mixture was warmed to rt

and stirred for 2 h. The solution was then diluted with

CH₂Cl₂(10 mL) and washed with 0.1 M HCl, satd aq

NaHCO₃, and brine. The organic layer was then dried

over Na₂SO₄and concentrated in vacuo. The crude

reside was then puriﬁed by column chromatography

(1:1 EtOAc–hexanes) to afford compound 25 (238 mg,

71.2%).¹H NMR (500 MHz, CDCl₃): l 7.31–7.22 (m,

10 H), 5.45 (d, J 3.5 Hz, 1 H), 5.24 (t, J 9.5 Hz, 1 H),

5.18–5.09 (m, 2 H), 5.01 (broad s, 1 H), 4.79 (d, J 9 Hz,

1 H), 4.60–4.32 (m, 6 H), 4.11–3.92 (m, 4 H), 3.87 (dd,

J 10.5, 3.5 Hz, 1 H), 3.73–3.68 (m, 3 H), 3.64 (dt, J

12.0, 3.5 Hz, 1 H), 3.45–3.35 (m, 4 H), 2.06 (s, 3 H),

2.05 (s, 3 H), 2.02 (s, 3 H), 2.01 (s, 3 H), 1.99 (s, 3 H),

1.95 (s, 3 H), 1.93 (C-3, m, 2 H), 1.87 (s, 3 H), 1.84 (s,

3 H); ¹³C NMR (125.6 MHz, CDCl₃): l 171.5, 170.6,

170.57, 170.5, 170.2, 169.5, 168.9, 138.2, 138.1, 128.6,

128.5, 128.4, 128.0, 127.9, 127.8, 127.80, 127.7, 100.9

(C-1%), 93.4 (C-1%%), 78.2 (C-1), 75.7, 74.7, 73.5, 72.7,

72.6, 71.2, 71.1, 71.05, 70.5, 70.4, 68.6, 68.3, 68.2, 64.7,

62.3, 61.5, 28.6 (C-3), 21.1, 21.0, 20.9, 20.9, 20.8, 20.7,

20.6, 20.5. ESIMS(+): Calcd for C₄₈H₆₁NNaO₂₂, m/z

1026.36; Found, 1026.31.

1

pound 27 (13.33 g, 79%). H NMR (300 MHz, CDCl₃):

l 7.50–7.46 (m, 2 H), 7.32–7.25 (m, 3 H), 5.25–5.18

(m, 1 H), 5.06–4.93 (m, 2 H), C-1 4.69 (d, J 10.2 Hz, 1

H), 4.26–4.13 (m, 2 H), 3.74–3.68 (m, 1 H), 2.08 (s, 3

H), 2.07 (s, 3 H), 2.01 (s, 3 H), 1.98 (s, 3 H). ¹³C NMR

(75.46 MHz, CDCl₃): l 170.8, 170.4, 169.6, 169.5,

133.3, 131.8, 129.1, 128.6, C-1 85.9, 75.9, 74.1, 70.1,

68.4, 62.3, 20.9, 20.8. FABMS(+): Calcd for

C₂₀H₂₄NaO₉S, m/z 463; Found, 463.

Phenyl 4,6-O-benzylidene-1-thio-i-D-glucopyranoside

(28).—To a mixture of compound 27 (13.00 g, 29.5

mmol) in anhyd MeOH (150 mL), solid NaOMe was

added in small portions to pH 9. After 2 h the solution

was neutralized using Dowex 50W×2-100 (H⁺). The

resin was then ﬁltered off, and the solvent removed in

vacuo to afford a powdery solid (8.03 g). The powdery

solid was dissolved in a solution of CH₃CN (300 mL)

and a,a-dimethoxytoluene (6.6 mL 44.1 mmol). To the

suspension was added TsOH (120 mg). The reaction

mixture stirred overnight and was neutralized with

Et₃N (2 mL). The slurry was then concentrated and

A.J. Janczuk et al. / Carbohydrate Research 337 (2002) 1247–1259

1255

puriﬁed by column (20:1 CHCl₃–MeOH) to afford

compound 28 (6.85 g, 86%). ¹H NMR (400 MHz,

CDCl₃): l 7.45–7.36 (m, 4 H), 7.26–7.20 (m, 6 H) 5.42

(s, 1 H), C-1 4.55 (d, J 9.6 Hz, 1 H), 4.24 (dd, J 10.4,

4.0 Hz, 1 H), 3.77 (s, broad, 2 H), 3.69–3.60 (m, 2 H),

3.42–3.23 (m, 3 H); ¹³C NMR (100.61 MHz, CDCl₃): l

137.1, 132.7, 129.3, 129.1, 128.3, 128.2, 125.4, 101.9,

(C-1) 88.7, 80.4, 74.7, 72.9, 70.5, 68.7, 49.5, 49.3, 49.1,

48.9, 48.4; ESIMS(+) Calcd for C₁₉H₂₀NaO₅S, m/z

383.89; Found, 382.96.

Phenyl 2,3,6-tri-O-benzyl-4-deoxy-1-thio-i-

D

-xylo-

hexopyranoside (31).—To the solution of 30 (500 mg,

0.921 mmol) dissolved in dry CH₂Cl₂(50 mL) was

added DMAP (1.01 g, 8.28 mmol) in small portions.

The mixture was cooled to 0 °C, followed by the addi-

tion of phenyl chlorothionocarbonate (0.382 mL, 2.76

mmol). The solution stirred overnight at ambient tem-

perature. The mixture was then diluted with CH₂Cl₂(50

mL) and washed with satd aq NaHCO₃, brine and

phosphate buffer solution (pH 7.4). The organic layer

was then dried over Na₂SO₄and concentrated in vacuo.

The crude residue was then dissolved in dry benzene (20

mL) and charged with Ar. Bu₃SnH (1.982 mL, 7.37

mmol) was added, followed by gradual warming of the

mixture to 80 °C. A catalytic amount of AIBN (15 mg)

was added to the stirring reaction mixture. After ap-

proximately 1 h, the reaction was complete, and the

mixture was cooled and concentrated in vacuo. The

residue was then diluted with CH₃CN (50 mL) and

extracted several times with hexanes. The CH₃CN layer

was then concentrated in vacuo, and the residue was

dissolved in CH₂Cl₂. The solution was then washed

with 0.1 M HCl, NaHCO₃, and brine. Finally the

solution was dried over Na₂SO₄ﬁltered and puriﬁed by

column chromatography (1:5 EtOAc–hexanes) to af-

ford compound 31 (400 mg, 82%). ¹H NMR (400 MHz,

CDCl₃): l 7.59–7.25 (m, 20 H), 4.96–4.53 (m, 7 H),

3.79–3.38 (m, 5 H) 1.71–1.62(m, 2 H); ¹³C NMR

(100.61 MHz, CDCl₃): l 138.49, 138.28,137.9, 132.04,

129.89, 129.19, 129.08, 128.69, 128.65, 128.63, 128.54,

128.23, 128.05, 127.98, 127.76, 127.54, 87.9, 81.2, 80.63,

75.8, 75.4, 73.7, 72.6, 72.2, 33.9. ESIMS(+): Calcd for

C₃₃H₃₄NaO₄S, m/z 549.21; Found, 549.03.

Phenyl 2,3-di-O-benzyl-4,6-O-benzylidene-1-thio-i-

D

-glucopyranoside (29).—In

a

ﬂame-dried ﬂask

equipped with a dropping funnel under nitrogen com-

pound 28 (6.80 g, 18.86 mmol) was dissolved in dry

DMF (60 mL) and cooled to 0 °C. NaH (6.1 g, in 60%

oil dispersion, 8 equiv) was added in small portions

over 1 h. Benzyl bromide (18.08 mL, 152.0 mmol) was

then added dropwise to the stirring mixture over a

period of 1 h, which was allowed to stir overnight at

ambient temperature. MeOH (30 mL) was used to

quench the excess NaH. The reaction mixture was then

was poured over ice-water. The mixture was extracted

with CHCl₃(3×60 mL), and the extract was washed

with satd aq NaHCO₃, and brine. The organic layer

was then dried over Na₂SO₄and concentrated in vacuo.

Flash column chromatography (1:2 EtOAc–hexanes)

1

afforded compound 29 (8.21 g, 80.0%). H NMR (500

MHz, CDCl₃): l 7.56–7.50 (m, 4 H), 7.41–7.30 (m, 16

H), 5.61 (s, 1 H), 4.96 (d, J 11.0 Hz, 1 H), 4.89–4.77

(m, 4 H), 4.13 (dd, J 10.5, 5.0 Hz, 1 H), 3.88–3.80 (m,

2 H), 3.73 (t, J 9.0 Hz, 1 H) 3.56–3.47(m, 2 H); ¹³C

NMR (125.7 MHz, CDCl₃): l 138.5, 138.2, 137.4,

132.6, 129.2, 129.2, 128.6, 128.5, 128.4, 128.3, 128.1,

128.0, 126.2, 101.3, 88.5, 83.2, 81.7, 80.6, 76.1, 75.5,

70.4, 68.9. ESIMS(+) Calcd for C₃₃H₃₂O₅SNa⁺, m/z

563.18; Found 563.00.

2,3,6-Tri-O-benzyl-4-deoxy-h-

(13)-2,4,6-tri-O-acetyl-i- -galactopyranosyl-(14)-

2,3,6-tri-O-acetyl-i- -glucopyranosyl azide (32).—A

suspension of acceptor 8 (0.514 g, 0.835 mmol), donor

D-xylo-hexopyranosyl)-

D

Phenyl 2,3,6-tri-O-benzyl-1-thio-i-D-glucopyranoside

,

(30).—Compound 29 (1.00 g, 1.85 mmol) was dissolved

31 (0.400 g, 0.759 mmol), 4 A MS (500 mg), and anhyd

,

in a suspension of anhyd THF (30 mL) and 4 A MS

CH₂Cl₂(6 mL) was stirred for 1 h at ambient tempera-

ture under Ar. The mixture was then cooled to

−30 °C, and N-iodosuccinamide (196 mg) was added.

Next triﬂic acid (12.0 mL, 0.121 mmol) was added

slowly to the mixture. After 2 h, all of the acceptor was

consumed, and the reaction mixture was diluted with

CH₂Cl₂(20 mL) and ﬁltered through a Celite pad. The

ﬁltrate was then washed with satd aq NaHCO₃, 10%

Na₂S₂O₃and dried over Na₂SO₄. The solvent was then

removed in vacuo, and the crude residue was puriﬁed

by column chromatography (1:2 EtOAc–hexanes) to

afford trisaccharide 32 (472 mg, 60.0%). ¹H NMR

(400.1 MHz, CDCl₃): l 7.40–7.24 (m, 15 H), 5.45 (d, J

3.6 Hz, 1 H), 5.18 (t, J 8.8 Hz, 2 H), 5.11–5.06 (m, 2

H), 4.84 (t, J 9.6 Hz, 1 H), 4.70 (m, 2 H), 4.65–4.60 (m,

3 H), 4.55 (s, 2 H), 4.44 (d, J 12.4 Hz, 1 H), 4.33 (d, J

7.2 Hz, 1 H), 4.12–3.98 (m 3 H), 3.87–3.67 (m, 5 H),

3.47–3.38 (m, 3 H), 2.09 (s, 3 H), 2.07 (s, 3 H), 2.06 (s,

(1.00 g). To the stirring mixture was added HCl in Et₂O

until the evolution of gas ceased. After 20 min the

reaction was complete as monitored by TLC. The sus-

pension was diluted with CH₂Cl₂and ﬁltered through a

Celite pad. After workup the organic layer was dried

over anhyd Na₂SO₄, ﬁltered and concentrated in vacuo.

Puriﬁcation by column (1:3 EtOAc–hexanes) afforded

compound 30 (530 mg, 53%). ¹H NMR (400 MHz,

CDCl₃): l 7.59–7.25 (m, 20 H), 4.94 (d, J 2.4 Hz, 1 H),

4.92 (s, 1 H), 4.82–4.68 (m, 3 H), 4.59 (d, J 3.2 Hz, 2

H), 3.83–3.74 (m, 2 H), 3.70–3.65 (m, 1 H), 3.59–3.48

(m, 3 H), 2.61 (d, J 1.6 Hz, 1 H); ¹³C (100.61 MHz,

CDCl₃): l 138.6, 138.1, 138.16, 134.0, 132.1, 129.3,

129.2, 128.9, 128.7, 128.6, 128.5, 128.2, 128.0, 127.9,

127.7, 87.9, 86.3, 80.7, 78.3, 75.7, 75.6, 73.9, 71.9, 70.6.

ESIMS(+): Calcd for C₃₃H₃₄KO₅S, m/z 581.18;

Found, 581.29.

1256

A.J. Janczuk et al. / Carbohydrate Research 337 (2002) 1247–1259

3 H), 2.00 (s, 3 H), 1.91 (s, 3 H), 1.85 (m, 2 H), 1.80 (s,

3 H); ¹³C NMR (100 MHz, CDCl₃): l 170.6, 170.5,

170.4, 169.8, 169.7, 168.9, 138.8, 138.7, 138.2, 128.6,

128.5, 128.4, 128.0, 127.9, 127.78, 127.75, 127.70, 101.4,

94.8, 87.9, 80.3, 75.7, 75.0, 74.5, 73.5, 72.7, 72.73, 72.4,

72.3, 71.2, 71.1, 70.5, 67.5, 64.6, 62.1, 61.4, 34.1 (C-4),

21.0 (m). ESIMS(+): Calcd for C₅₁H₆₁N₃NaO₂₀, m/z

1058.38; Found, 1058.48.

(d, J 12.5 Hz, 1 H) 3.65–3.48 (m, 10 H), 3.43 (m, 1 H),

3.34 (dd, J 10.0, 3.5 Hz, 1 H), 3.29 (m, 1 H), 1.91 (s, 3

H), 1.85 (m, 2 H); ¹³C NMR (125 MHz, CDCl₃): l

171.6, 102.9, 96.1, 79.2, 78.1, 77.3, 76.4, 75.2, 73.2, 71.5,

71.1, 69.6, 68.9, 67.1, 65.0, 63.6, 61.1, 60.0, 34.1 (C-4),

22.2. FAB-HRMS(+): Calcd for C₂₀H₃₅NaO₁₅, m/z

552.1904; Found, 552.1927.

Phenyl 2,3,4-tri-O-acetyl-1-thio-i-D-fucopyranoside

2,3,6-Tri-O-benzyl-4-deoxy-h-

(13)-2,4,6-tri-O-acetyl-i- -galactopyranosyl-(14)-

N-acetyl-2,3,6-tri-O-acetyl-i- -glucopyranosylamine

D

-xylo-hexopyranosyl-

(35).—To a solution of compound 34 (1.00 g, 6.09

mmol) in pyridine (30 mL), Ac₂O(10 mL) and DMAP

(20 mg) were added. After 6 h the mixture was concen-

trated in vacuo. The crude residue was then diluted

with CHCl₃(50 mL) and washed with 0.1 M HCl,

water, satd aq NaHCO₃, and brine. After drying over

Na₂SO₄the solvent was removed in vacuo, and the

crude residue was dissolved in anhyd CH₂Cl₂and

cooled to 0 °C. Then thiolphenol (0.7 mL, 6.82 mmol)

was added. After 30 min BF₃·Et₂O (2.5 mL, 20.0 mmol)

was slowly injected into the mixture. After 4 h the

reaction mixture was diluted with CH₂Cl₂(50 mL) and

washed with satd aq NaHCO₃and brine. After drying

over Na₂SO₄the solvent was removed in vacuo. Puriﬁ-

cation by column chromatography (1:5 EtOAc–hex-

D

(33).—Trisaccharide 32 (470 mg, 0.476 mmol) in EtOH

was agitated with PtO₂(10 mg) under an atmosphere of

hydrogen (50 lb/in²) for 1 h. After the reaction was

complete, the Pt was ﬁltered and the ﬁltrate was con-

centrated in vacuo. The residue was then dissolved in

dry CH₂Cl₂(30 mL) with Et₃N (5.6 mL) and Ac₂O

(1.34 mL) at 0 °C. The mixture was then warmed to rt

and stirred for 2 h. The solution was then diluted with

CH₂Cl₂(25 mL) and washed with 0.1 M HCl, satd aq

NaHCO₃, and brine. The organic layer was then dried

over Na₂SO₄and concentrated in vacuo. The crude

reside was then puriﬁed by column chromatography

(1:1 EtOAc–hexanes) affording compound 33 (334 mg,

1

anes) afforded compound 35 (2.02 g, 87.1%). H NMR

1

70%). H NMR (400.1 MHz, CDCl₃): l 7.40–7.24 (m,

(400 MHz, CDCl₃): l 7.50 (m, 2 H), 7.30 (m, 3 H),

5.28–5.18 (m, 2 H), 5.04 (dd, J 9.6, 3.2, 1 H), 4.68 (d,

J 10.8, 1 H, H-1), 3.82 (q, J 6.3, 1 H) 2.13 (s, 3 H), 2.07

(s, 3 H), 1.95 (s, 3 H), 1.24 (d, J 6.4 Hz, 3 H, C-6); ¹³C

NMR (100 MHz, CDCl₃): l 170.8, 170.3, 169.7, 132.5,

129.0, 128.1, 86.6 (C-1), 73.3, 72.6, 70.5, 67.5, 21.0,

20.8, 20.6, 16.4 (C-6). ESIMS(+): Calcd for

C₁₈H₂₂NaO₇S, m/z 405.10; Found, 404.99.

15 H), 6.39 (broad s, 1 H), 5.45 (d, J 3.2 Hz, 1 H), 5.25

(m, 1 H), 5.18 (t, J 8.8 Hz, 1 H), 5.11–5.06 (m, 2 H),

4.80 (t, J 8.8 Hz, 1 H), 4.70–4.56 (m, 5 H), 4.53 (s, 2

H), 4.37 (d, J 11.2 Hz, 1 H), 4.30 (d, J 7.2 Hz, 1 H),

4.12–3.97 (m 3 H), 3.87–3.66 (m, 5 H), 3.43–3.33 (m,

3 H), 2.06 (s, 3 H), 2.04 (s, 3 H), 2.02 (s, 3 H), 1.99 (s,

3 H), 1.95 (s, 3 H), 1.89 (s, 3 H), 1.88 (m, 2 H), 1.79 (s,

3 H); ¹³C NMR (100 MHz, CDCl₃): l 171.5, 170.6,

170.5, 170.4, 169.8, 169.7, 168.9, 138.8, 138.7, 138.2,

128.6, 128.5, 128.4, 128.0, 127.9, 127.78, 127.75, 127.70,

101.4, 94.8, 80.3, 78.2, 75.7, 75.0, 74.5, 73.5, 72.7, 72.73,

72.4, 72.3, 71.2, 71.1, 70.5, 67.5, 64.6, 62.1, 61.4, 34.1

(C-4), 23.5, 21.0 (m). ESIMS(+): Calcd for

C₅₃H₆₅NNaO₂₁, m/z 1074.39; Found, 1074.38.

Phenyl 2,3,4-tri-O-benzyl-1-thio-i-D-fucopyranoside

(36).—To a mixture of compound 35 (1.80 g, 4.71

mmol) in anhyd MeOH (30 mL), NaOMe powder was

added in small portions to pH 9. After 2 h the solution

was neutralized using Dowex 50W×2-100 (H⁺). The

resin was then ﬁltered off, and the solvent was removed

in vacuo. The residue (1.00 g, 3.90 mmol) was dissolved

in anhyd DMF and placed into a ﬂame-dried ﬂask

equipped with a dropping funnel and maintained under

nitrogen. The solution was cooled to 0 °C, followed by

the addition of NaH (920 mg, in 60% oil dispersion, 7

equiv) over a period of 1 h. Benzyl bromide (3.0 mL,

25.2 mmol) was then slowly added dropwise to the

stirring solution. The resulting mixture was allowed to

stir overnight at ambient temperature. MeOH (3 mL)

was then added to quench the excess NaH. The mixture

was extracted with CHCl₃(3×30 mL), and the extract

was washed with satd aq NaHCO₃, and brine. The

organic layer was then dried over Na₂SO₄and concen-

trated in vacuo. Column chromatography (1:2 EtOAc–

hexanes) of the residue afforded compound 36 (1.78 g,

4-Deoxy-h-

D

-xylo-hexopyranosyl-(13)-i-D-galac-

topyranosyl-(14)-N-acetyl-i-

D

-glucopyranosylamine

(7).—Pd(OH)₂/C (10% wt, 30 mg) was added to a

mixture of 33 (330 mg, 0.328 mmol) in MeOH (30 mL).

(Caution! Extreme ﬁre hazard.) The suspension was

charged with hydrogen (50 lb/in²) and agitated

overnight. The mixture was then ﬁltered, and the

ﬁltrate was evaporated in vacuo. The residue was then

dissolved in anhyd MeOH, followed by the addition of

NaOMe powder to pH 9. After 2 h, Dowex 50W×2-

100 (H⁺) was added to neutralize the mixture. The

resin was then ﬁltered, and the ﬁltrate was evaporated

to afford 6 in quantitative yield (60 mg). Further puriﬁ-

cation was carried out by gel ﬁltration (Sephadex 25).

¹H NMR (500 Hz, CDCl₃): l 4.96 (d, J 3.5 Hz, 1 H),

4.81 (d, J 9.0 Hz, 1 H), 4.36 (d, J 8.0 Hz, 1 H) 4.05 (m,

1 H), 4.00 (d, J 3.5 Hz, 1 H), 3.89–3.83 (m, 1 H), 3.77

1

86.8%). H NMR (300 MHz, CDCl₃): l 7.67–7.24 (m,

20 H), 5.07 (d, J 11.7 Hz, 1 H), 4.83–4.75 (m, 3 H),

4.70 (d, J 9.3 Hz, 1 H, H-1), 4.63 (d, J 10.2 Hz, 1 H),

A.J. Janczuk et al. / Carbohydrate Research 337 (2002) 1247–1259

1257

3.98 (m, 1 H), 3.68–3.56 (m, 4 H), 1.32 (d, J 6.3 Hz, 3

H, H-6); ¹³C NMR (75.46 MHz, CDCl₃): l 138.8,

138.4, 134.4, 131.6, 128.8, 128.5, 128.4, 128.2, 128.0,

127.8, 127.6, 127.0, 87.6 (C-1), 84.5, 76.6, 75.6, 74.6,

72.8, 72.1, 17.4 (C-6). ESIMS(+): Calcd for

C₃₃H₃₄NaO₄S, m/z 549.21; Found, 549.05.

4.97 (m, 2 H), 4.83–4.77 (m, 2 H), 4.72–4.56 (m, 4 H),

4.34 (d, J 10.8 Hz, 1 H), 4.29 (d, J 8.0 Hz, 1 H),

4.14–3.93 (m, 6 H), 3.45 (s, 1 H), 2.07 (s, 3 H), 2.05 (s,

3 H), 2.01 (s, 3 H), 1.99 (s, 3 H), 1.94 (s, 3 H), 1.90 (s,

3 H), 1.82 (s, 3 H), 1.07 (d, J 6.4 Hz, 3 H, H-6); ¹³C

NMR (100.6 MHz, CDCl₃): l 171.4, 170.6, 170.3,

169.6, 169.4, 169.2, 168.7, 138.9, 138.8, 138.6, 128.6,

128.4, 128.4, 128.1, 128.0, 127.8, 127.7, 127.6, 101.1

(C-1%), 95.9 (C-1%%), 78.7, 78.3, 78.2, 75.9, 75.6, 75.0,

74.6, 73.8, 73.7, 73.5, 72.5, 71.3, 71.0, 70.7, 67.3, 65.4,

62.3, 61.6, 23.5, 20 (m), 16.1 (C-6). ESIMS(+): Calcd

for C₅₃H₆₅NNaO₂₁, m/z 1074.39; Found, 1074.47.

2,3,4-Tri-O-benzyl-h-

tri-O-acetyl-i- -galactopyranosyl-(14)-2,3,6-tri-O-

acetyl-i- -glucopyranosyl azide (37).—A suspension of

acceptor 9 (0.66 g, 1.06 mmol), donor 36 (0.47 g, 0.89

D

-fucopyranosyl-(13)-2,4,6-

D

,

mmol), 4 A MS (1.5 g), and anhyd CH₂Cl₂(10 mL) was

stirred for 1 h at ambient temperature under Ar. The

mixture was then cooled to −78 °C, and N-iodosucci-

namide (250 mg) was added. Next triﬂic acid (29.0 mL,

0.36 mmol) was added slowly to the mixture. After 1 h,

the donor was consumed, and the reaction mixture was

diluted with CH₂Cl₂(20 mL) and ﬁltered through a

Celite pad. The ﬁltrate was then washed with satd aq

NaHCO₃, 10% Na₂S₂O₃, brine and dried over Na₂SO₄.

The solvent was then removed in vacuo, and the crude

residue was puriﬁed by column chromatography (1:2

EtOAc–hexanes) to afford trisaccharide 37 (790 mg,

h -

D

- Fucopyranosyl - (13) - i -

D

- galactopyranosyl-

(8).—

(14)-N-acetyl-i-

D

-glucopyranosylamine

Pd(OH)₂/C (10% wt, 30 mg) was added to trisaccharide

38 (297 mg, 0.287 mmol) dissolved in MeOH (30 mL).

(Caution! Extreme ﬁre hazard.) The suspension was

charged with hydrogen (50 lb/in²) and agitated

overnight. The mixture was then ﬁltered, and the

ﬁltrate was evaporated in vacuo. The residue was then

dissolved in anhyd MeOH, followed by the addition of

NaOMe powder to pH 9. After 2 h, Dowex 50W×2-

100 (H⁺) was added to neutralize the mixture. The

resin was then ﬁltered and the ﬁltrate was evaporated to

afford 4 after puriﬁcation by gel ﬁltration (Sephadex

1

85.7%). H NMR (300 MHz, CDCl₃): l 7.36–7.24 (m,

15 H), 5.43 (d, J 2.7 Hz, 1 H), 5.20 (t, J 9.0 Hz, 1 H),

5.06 (m, 1 H), 4.97 (m, 2 H), 4.87–4.81 (m, 2 H),

4.75–4.70 (m, 2 H), 4.66–4.58 (m, 3 H), 4.46–4.42 (m,

2 H) 4.37 (d, J 7.8 Hz, 1 H), 4.13 (dd, J 12.6, 4.8 Hz,

1 H), 4.05 (d, J 6.6 Hz, 2 H), 3.89 (dd, J 10.2, 3.6 Hz,

1 H), 3.81–3.73 (m, 5 H), 3.57 (s, 1 H), 2.11 (s, 3 H),

2.07–2.06 (m, 6 H), 2.02 (s, 3 H), 1.98 (s, 3 H), 1.88 (s,

3 H), 1.09 (d, J 6.6 Hz, 3 H, H-6); ¹³C NMR (75.45

MHz, CDCl₃): l 170.1, 170.0, 169.7, 169.4, 169.2,

168.7, 138.7, 138.6, 138.4, 138.2, 128.3, 128.2, 128.0,

127.8, 127.5, 127.4, 127.3, 101.1 (C-1%), 96.0 (C-1%%), 87.7

(C-1), 78.6, 78.1, 75.8, 75.2, 74.8, 74.8, 73.5, 73.3, 72.4,

71.1, 70.9, 70.6, 70.4, 67.2, 65.3, 61.9, 61.3, 20.4, 20.2

(m), 20.1, 16.1 (C-6). ESIMS(+): Calcd for

C₅₁H₆₁N₃NaO₂₀, m/z 1058.37; Found, 1058.47.

1

25) (136 mg, 89.7%). H NMR (500 MHz, D₂O): l 4.89

(d, J 4.0 Hz, 1 H), 4.82 (d, J 10.0 Hz, 1 H), 4.36 (d, J

7.5 Hz, 1 H), 4.16 (q, J 7.5 Hz, 1 H), 3.83–3.68 (m, 2

H) 3.67–3.47 (m, 11 H), 3.30–3.26 (m, 1 H), 1.91 (s, 3

H), 1.04 (d, J 6.5 Hz, 3 H); ¹³C NMR (125.6 MHz,

D₂O): l 175.6, 103.0, 96.1, 79.2, 78.1, 77.9, 76.4, 76.3,

75.2, 75.1, 72.0, 71.5, 69.6, 68.1, 67.0, 65.3, 61.1, 59.9,

22.2, 15.3 (C-6). HRFABMS(+): Calcd for C₂₀H₃₅-

NNaO₁₅, 552.1904; Found, 552.1925.

Isolation of polyclonal anti-h-Gal antibody from hu-

man serum.—Polyclonal anti-Gal antibody was isolated

from commercially available human male type AB

serum using an a-Gal (immobilized trisaccharide [C-

Gal-(13)-b-(14)-b-GlcNAc] on Sepharose beads)

afﬁnity chromatography column.^9a,9bThe human serum

was heated at 56 °C in a water bath in order to inacti-

vate human complement. After 30 min the serum was

cooled to ambient temperature and passed through the

column to allow for binding between the anti-Gal anti-

body and the immobilized a-Gal epitope. After exten-

sive washing with phosphate-buffered saline (PBS, pH

7.4), the bound anti-Gal antibody was eluted with

glycine·HCL buffer (pH 2.8). The elute containing the

antibodies, as monitored by UV spectroscopy (280 nm),

was immediately adjusted to pH 7.2 using 0.1 M

NaOH. The resulting antibody solution was stored as

frozen aliquots (about 200 mg/mL) in PBS buffer.

ELISA inhibition assay with mouse laminin.—An

ELISA assay was conducted using mouse laminin, as

the solid-phase immobilized antigen. The puriﬁed hu-

man anti-Gal antibody was initially incubated at vary-

2,3,4-Tri-O -benzyl-h-

tri-O-acetyl-i- -galactopyranosyl-(14)-N-acetyl-

2,3,6-tri-O-acetyl-i- -glucopyranosylamine (38).—

D

-fucopyranosyl-(13)-2,4,6-

D

Trisaccharide 37 (350 mg, 0.34 mmol) in EtOH was

agitated with PtO₂(20 mg), under an atmosphere of

hydrogen (50 lb/in²) for 2 h. The Pt was then ﬁltered,

and the ﬁltrate was concentrated in vacuo. The residue

was then dissolved in dry CH₂Cl₂(10 mL) with Et₃N (1

mL) and Ac₂O (0.3 mL) at 0 °C. The mixture was then

warmed to rt and stirred for 2 h. The solution was

diluted with CH₂Cl₂(10 mL) and washed with 0.1 M

HCl, satd aq NaHCO₃, and brine. The organic layer

was then dried over Na₂SO₄and concentrated in vacuo.

The crude reside was puriﬁed by column chromatogra-

phy (1:1 EtOAc–hexanes) to afford compound 38 (260

1

mg, 72%). H NMR (400 MHz, CDCl₃): l 7.34–7.22

(m, 15 H), 6.39 (d, J 8.8 Hz, 1 H), 5.41 (d, J 3.2 Hz, 1

H), 5.24 (m, 1 H), 5.16 (t, J 8.8 Hz, 1 H), 5.06 (m, 1 H),

1258

A.J. Janczuk et al. / Carbohydrate Research 337 (2002) 1247–1259

Romano, E.; Zhudi, N. Transplant Immunol. 1993, 1,

198–205.

6. (a) Cooper, D. K. C.; Koren, E.; Oriol, R. Immunol. Re6.

1994, 141, 31–58;

ing concentrations of the terminal deoxy a-Gal deriva-

tives for 2 h at rt with gentle shaking. An aliquote (50

mL) of the mixture was then added to each microtiter

plate (Immulon 4) well which was pre-coated with

mouse laminin (50 mL/well of 10 mg/mL in 0.1 N

Na₂CO₃–NaHCO₃buffer, pH 9.5). After a 1.5-h incu-

bation period at rt, unbound antibodies were washed

out with PBS-tween (pH 7.4+0.2% Tween). Next a

secondary (1/1000 peroxidase-conjugated goat anti-hu-

man IgG; 50 mL/well) antibody was introduced and

allowed to incubate for approximately 1 h. After exten-

sive washing using PBS-tween buffer solution, the sub-

strate TMB (9:1 3,3%5,5%-tetramethylbenzidine–H₂O₂;

100 mL/well) was added. Enzymatic oxidation stained

each well in varying intensities of blue, absorption was

measured at 650 nm. The oxidation was quenched by

the addition of 1 N H₂SO₄(100 mL/well). Optical

absorption was measured at 450 nm. PBS with sec-

ondary antibody was used as background control.

Puriﬁed anti-Gal antibody with secondary antibody

was positive control as maximum staining (0% inhibi-

tion). The percent inhibition was calculated using the

following equation:

(b) Sandrin, M. S.; Vaughan, H. A.; Mckenzie, I. F. C.

Transplant Re6. 1994, 8, 134–149;

(c) Samuelsson, B. E.; Rydberg, L.; Breimer, M. E.;

Backer, A.; Gustavsson, M.; Holgersson, J.; Karlsson, E.;

Uyterwaal, A.-C.; Cairns, T.; Welsh, K. Immunol. Re6.

1994, 141, 151–168;

(d) Galili, U. Springer Semin. Immunopathol. 1993, 15,

155–171;

(e) Galili, U. Immunol. Ser. 1992, 55, 355–373;

(f) Galili, U. Blood Cells 1988, 14, 205–220;

(g) Hamadeh, R.; Galili, U.; Zhou, P.; Grifﬁss, J. Clin.

Diagn. Lab. Immunol. 1995, 2, 125–131.

7. (a) Galili, U.; Anaraki, F.; Thall, A.; Hill-Black, C.;

Radic, M. Blood 1993, 82, 2485–2493;

(b) McMorrow, I. M.; Comrack, C. A.; Sachs, D. H.;

DerSimonian, H. Transplantation 1997, 64, 501–510.

8. Galili, U. Immunol. Today 1993, 14, 480–482.

9. (a) Xu, Y.; Lorf, T.; Sablinski, T.; Gianello, P.; Bailin,

M.; Monroy, R.; Kozlowski, T.; Awwad, M.; Cooper, D.

K. C.; Sachs, D. H. Transplantation 1998, 65, 172–179;

(b) Taniguchi, S.; Neethling, F. A.; Korchagina, E. Y.;

Bovin, N.; Ye, Y.; Kobayashi, T.; Niekrasz, M.; Li, S.;

Koren, E.; Oriol, R.; Cooper, D. K. C. Transplantation

1996, 62, 1379–1384;

(c) Kroshus, T. J.; Bolman, R. M.; Dalmasso, A. P.

Transplantation 1996, 62, 5–12;

(M−S)/(M−B)=% inhibition.

(d) Leventhal, J. R.; John, R.; Fryer, J. P.; Witson, J. C.;

Derlich, J.-M.; Remiszewski, J.; Dalmasso, A. P.; Matas,

A. J.; Bolman, R. M., III Transplantation 1995, 59,

294–300.

S refers to the OD₄₅₀reading of the sample at

different concentrations of terminal deoxy a-Gal

derivatives 5, 6, 7, and 8, B is the OD₄₅₀reading of the

background, and M is the OD₄₅₀reading of the maxi-

mum staining. IC₅₀values were calculated from plot of

the % inhibition versus the concentration of the

inhibitors.

10. (a) Nagosaka, T.; Kobayashi, T.; Muramatsu, H.; Fuji-

moto, H.; Muramatsu, T.; Tokagi, H. Biochem. Biophys.

Res. Commun. 1997, 232, 731–736;

(b) Simon, P. M.; Neethling, F. A.; Taniguchi, S.; Goode,

P. L.; Zopf, D.; Hancock, W. W.; Cooper, D. K. C.

Transplantation 1998, 65, 346–353;

(c) Rieben, R.; von Allmen, E.; Korchagina, E. Y.;

Nydegger, W.; Neethling, F. A.; Kujundzic, M.; Koren,

E.; Bovin, N.; Cooper, D. K. C. Xenotransplantation

1995, 2, 98–106;

(d) Li, S.; Neethling, F.; Taniguchi, S.; Yeh, J.-C.;

Kobayashi, T.; Ye, Y.; Koren, E.; Cummings, R. D.;

Cooper, D. K. C. Transplantation 1996, 62, 1324–1331;

(e) Lambrigts, D.; Sachs, D. H.; Cooper, D. K. S.

Transplantation 1998, 66, 547–561.

Acknowledgements

This work was generously supported by NIH

(AI44040).

References

11. Galili, U.; Matta, K. L. Transplantation 1996, 62, 256–

262.

12. Lemieux, R. U.; Hindsgaul, O.; Bird, P.; Narasimhan, S.

Carbohydr. Res. 1988, 178, 293–305.

13. (a) Lemieux, R. U. Frontiers in Chemistry. In Proceed-

ings of the 28th IUPAC Congress; Laidler, K. J., Ed.;

Pergamon Press: Oxford, 1982;

1. Lafferty, K. J. Transplant Proc. 1999, 31, 11S–13S.

2. 2000 Annual Report of the U.S. Scientiﬁc Registry for

Transplant Recipients and the Organ Procurement and

Transplantation Network: Transplant Data: 1990–1999.

US Department of Health and Human Services, Health

Resources and Services Administration, Ofﬁce of Special

Programs, Division of Transplantation, Rockville, MD;

United Network for Organ Sharing, Richmond, VA.

3. Lanza, R. P.; Cooper, D. K. C.; Chick, L. C. Sci. Am.

1997, 53–59.

(b) Lemieux, R. U. In Proceedings of the VIIth Interna-

tional Symposium on Medicinal Chemistry; 1984; Vol. 1,

pp 39–351.

14. Zhang, W.; Wang, J.; Li, J.; Yu, L.; Wang, P. G. J.

Carbohydr. Chem. 1999, 18, 1009–1017.

4. Evans, R. W. In Xenotransplantation; Platt, J. L., Ed.;

ASM Press: Washington, DC, 2001; pp 29–51.

5. (a) Galili, U. Sci. Med. 1998, 5, 28–37;

15. Roy, R.; Laferriere, C. A. J. Chem. Soc., Chem. Commun.

1990, 1709–1711.

16. Norber, T. In Modern Methods in Carbohydrate Synthe-

sis; Khan, S. H.; O’Neill, R. A., Eds.; Harwood Aca-

demic: Netherlands, 1996; p 82.

(b) Cooper, D. K. C. Clin. Trans. 1992, 6, 178–183;

(c) Cooper, D. K. C.; Good, A. H.; Koren, E.; Oriol, R.;

Malcolm, A. J.; Ippolito, R. M.; Neethling, F. A.; Ye, Y.;

17. Roush, W. R.; Narayan, S. Org. Lett. 1999, 1, 899–902.

A.J. Janczuk et al. / Carbohydrate Research 337 (2002) 1247–1259

1259

18. Trahanovsky, W. S.; Robbins, M. D. J. Am. Chem. Soc.

1971, 93, 5256–5258.

19. Lemieux, R. U.; Driguez, H. J. Am. Chem. Soc. 1975, 97,

4069–4075.

23. Garegg, P. J.; Hultberg, H.; Wallin, S. A. Carbohydr. Res.

1982, 108, 97–101.

24. (a) Galili, U.; Ron, M.; Sharon, R. J. Immunol. Methods

1992, 151, 117–122;

20. (a) Barton, D. H. R.; Blundell, P.; Dorchak, J.; Jang, D.

O.; Jaszberenyi, J. C. Tetrahedron 1991, 47, 8969–8984;

(b) Barton, D. H. R.; Jang, D. O.; Jaszberenyi, J. C.

Tetrahedron 1993, 49, 2793–2804;

(b) Galili, U.; LaTemple, D. C.; Radic, M. Z. Transplan-

tation 1998, 65, 1129–1132.

25. Parker, W.; Lateef, J.; Everett, M. L.; Platt, J. L. Glycobi-

ology 1996, 6, 499–506.

26. Galili, U.; Buehler, J.; Shohet, S. B.; Macher, B. A. J. Exp.

Med. 1987, 165, 693–704.

27. (a) Wang, J.; Chen, X.; Zhang, W.; Zacharek, S.; Chen,

Y.; Wang, P. G. J. Am. Chem. Soc. 1999, 121, 8174–8181;

(b) Fang, J.; Chen, X.; Zhang, W.; Wang, J.; Andreana,

P.; Wang, P. G. J. Org. Chem. 1999, 64, 4089–4094;

(c) Andreana, P., Wang, J., Wang, P. G. unpublished

results.

(c) Robins, M. J.; Wilson, J. S. Regiospeciﬁc and Stereose-

lective 2%-Deoxygenation of a Ribonucleoside. In Nucleic

Acid Chemistry Part 2; Townsend, L. B.; Tipson, R. S.,

Eds.; Wiley: New York, 1991; pp 194–200.

21. Garg, H. G.; Jeanloz, R. W. Ad6. Carbohydr. Chem.

Biochem. 1985, 45, 135–201.

22. Jansson, K.; Ahlfors, S.; Frejd, T.; Kihlberg, J.; Magnus-

son, G. J. Org. Chem. 1988, 53, 5629–5647.

Article Doi

The synthesis of deoxy-α-Gal epitope derivatives for the evaluation of an anti-α-Gal antibody binding

DOI: 10.1016/S0008-6215(02)00159-3

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