The total synthesis of ganglioside GM3

www.elsevier.nl/locate/carres

Carbohydrate Research 328 (2000) 489–507

Richard I. Duclos Jr. *

Department of Biophysics, Boston Uni6ersity School of Medicine, 715 Albany Street, Boston, MA 02118-2526, USA

Received 1 November 1999; received in revised form 14 March 2000; accepted 20 March 2000

Abstract

Previous syntheses of ganglioside GM3 (NeuAca3Galb4Glcb1Cer) are reviewed, and both chemoenzymatic and

chemical total synthetic approaches were investigated. In a chemoenzymatic approach, (2S,3R,4E)-5§-acetyl-a-neu-

raminyl-(2§3¦)-b-galactopyranosyl-(1¦4%)-b-glucopyranosyl-(1%l1)-2-azido-4-octadecene-1,3-diol (azidoGM3)

was readily prepared utilizing recombinant b-Gal-(1¦3%/4%)-GlcNAc a-(2§3¦)-sialyltransferase enzyme, and was

evaluated as a synthetic intermediate to ganglioside GM3. The chemical total synthesis of ganglioside GM3 was

performed on one of the largest scales yet reported. The highlights of this synthesis include minimizing the steps

necessary to prepare the lactosyl acceptor as a useful anomeric mixture, which was present in excess for the highly

regioselective and fairly stereoselective sialylation with a known neuraminyl donor to give the protected GM3

trisaccharide. The synthetic methodology maximized convergence by a subsequent glycosidic coupling of the

well-characterized GM3 trisaccharide trichloroacetimidate derivative with protected ceramide. The ganglioside GM3

was nearly homogeneous as the two glycosidic couplings utilized preparative HLPC puriﬁcations, and variations in

rights reserved.

Keywords: Sialyltransferase; Ceramide; Benzyl 4-O-(2,6-di-O-benzylgalactopyranosyl)-2,3,6-tri-O-benzylglucopyranoside;

Trichloroacetimidate; Ganglioside GM3

tion (NeuAca3Galb4Glc) [23–28], protected

derivatives of this trisaccharide [29–51], and

other trisaccharide analogs [4,10,12,16,

43,45,49–57], which have utilized chemoenzy-

matic as well as chemical total synthetic

sequences.

The chemoenzymatic approaches to gan-

glioside GM3 6 have utilized one or more

enzyme-assisted conversion steps. The obvious

advantages of chemoenzymatic approaches

are the complete control of regiochemistry

and stereochemistry for the glycosidic cou-

pling reactions. The chemoenzymatic routes

have generally been demonstrated on smaller

scales, until recently [28], and have generally

required reversed-phase high-performance liq-

uid chromatography (HPLC) isolations. The

low activity of a b-Gal-(1¦3%/4%)-GlcNAc a-

1. Introduction

Ganglioside GM3 (NeuAca3Galb4Glcb1-

Cer, 6) is a ubiquitous metabolite with a vari-

ety of cellular activities that have been re-

viewed and include differentiation [1], the

modulation of several receptors and other en-

zymes [2,3], immunosupression [4], and tumor-

association [5]. The preparation of this

ganglioside is important for further biophysi-

cal and biological studies. There have already

been a number of previous syntheses of gan-

glioside GM3 6 [6–22], the trisaccharide por-

* Tel.: +1-617-6384255; fax: +1-617-6384041.

E-mail address: duclos@med-biophd.bu.edu (R.I. Duclos

Jr.).

PII: S0008-6215(00)00121-X

490

R.I. Duclos Jr. / Carbohydrate Research 328 (2000) 489–507

(2§3¦)-sialyltransferase (a-2,3-NeuAcT) for

lactosyl ceramide (Galb4Glcb1Cer, LacCer)

[58] has required additional steps to synthesize

better substrates suitable for chemoenzymatic

GM3 6 syntheses with this enzyme [13–

15,19–21,28,34,38,45,49,52,56,57].

Chemical synthetic methods offer the ﬂexi-

bility to prepare analogs as well as the

naturally occuring gangliosides. Chemical

neuraminyl glycosidic couplings to 3¦,4¦-un-

substituted lactose derivatives have already

been reported to have very high regioselectiv-

ity for the desired 3¦-position [6,8,11,22–

26,30,31,33,35,36,40–42,44,46,47,50,51].

The ganglioside GM3 (NeuAca3Galb4Glcb1-

Cer, 6) represents the next member of the

membrane lipid series under investigation in

this laboratory after ceramide (Cer) [59,60],

glucosylceramide (Glcb1Cer) and galactosyl-

ceramide (Galb1Cer) [61], and lactosylce-

ramide

(Galb4Glcb1Cer)

[62].

Similar

structural and physical studies of this homoge-

neous ganglioside GM3 6 are currently in

progress.

2. Results and discussion

Control of the a-glycosidic linkage of the neu-

raminyl residue is a continuing research inter-

est [17,39,42,44,47,50]. Recently, the coupling

of activated GM3 trisaccharide derivatives

with protected azidosphingosine derivatives

[9,10,14,16–18,22,33,41,46] rather than pro-

tected ceramide derivatives [6,8,11,13] has be-

come more commonly used when a series of

fatty acyl analogs is required.

This report details a practical chemical total

synthetic method to obtain a research quantity

of ganglioside GM3 6 with the goal of a more

concise and convergent approach. This syn-

thesis proceeds through a minimum of reac-

tion steps to yield ganglioside GM3 6 that is

optically pure and nearly homogeneous with

respect to variations in the sphingosine base

and the fatty acyl group. This synthesis uti-

lizes an easily prepared anomeric mixture of

the lactosyl acceptor [41]. Also, maximum

convergence is possible by the subsequent gly-

cosidic coupling of the GM3 trisaccharide

trichloroacetimidate with a ceramide deriva-

tive rather than an azidosphingosine analog.

Both chemoenzymatic and chemical total

synthetic approaches to ganglioside GM3 6

were investigated. A chemoenzymatic ap-

proach utilizing a sialyl transferase enzyme in

the neuraminyl glycosylation step was per-

formed. Alternatively, a chemical total synthe-

sis of ganglioside GM3 6 was developed,

which involved a minimum number of syn-

thetic steps. The purity of synthetic intermedi-

ates was assured by HPLC and all compounds

were well characterized by NMR spectroscopy

in multiple solvent systems.

Several chemoenzymatic synthetic ap-

proaches to ganglioside GM3 6 have previ-

ously

utilized

b-Gal-(1¦3%/4%)-GlcNAc

a-(2§3¦)-sialyl transferase (a-2,3-NeuAcT,

ST3N, EC 2.4.99.6) enzyme [13–

15,19,21,34,45,52,56,57]. Recombinant a-2,3-

NeuAcT enzyme was obtained from Cytel

(Noese) and was ﬁrst assayed for activity with

its natural substrate. N-Acetyllactosamine

(LacNAc, Galb4GlcNAc, 1) was readily con-

verted into the corresponding trisaccharide (a-

N-acetylneuraminyl-(2§3¦)-N-acetyllactos-

Scheme 1.

R.I. Duclos Jr. / Carbohydrate Research 328 (2000) 489–507

491

Scheme 2.

3eq§ and 4§ protons. A sample of the perme-

thylated derivative of 4 for analysis by mass

spectrometry was prepared with methyl iodide

in aqueous alkali. The methyl ester group of

the permethylated derivative was quite labile

and a sample was completely exchanged with

methyl-d₃iodide in aqueous alkali to give the

methyl-d₃ester labeled analog by mass spec-

tral analysis.

amine, a-NeuAc-(2§3¦)-LacNAc, NeuAca-

3Galb4GlcNAc, 2) (Scheme 1) in the presence

of commercially available cytidine 5%-mono-

phospho-N-acetylneuraminic acid (CMP-

NeuAc) according to the reported method

[13,63,64]. The trisaccharide product 2 was

chromatographed and demonstrated to be ho-

mogeneous in a neutral solvent system (1:3:1

ethyl

acetate–i-propanol–water),

which

Reduction of azidoGM3 4 to the known

[14,18,21,49,69–72] zwitterionic lysoGM3 5

was reported to give the amino acid 5 in yields

as high as 80% with H₂S [18]. However, the

azide reduction of azidoGM3 4 was found to

be slow and gave a mixture of products in

accordance with a related report [17]. The

subsequent acylation with hexadecanoyl chlo-

ride did not give good quality ganglioside

GM3 6.

showed that the product 2 was free of NeuAc.

Also, trisaccharide 2 was demonstrated to be

homogeneous in an acidic solvent system

(40:40:7:3 chloroform–methanol–water–ace-

tic acid), which showed that this product

trisaccharide 2 was free of starting disaccha-

1

ride 1. The H NMR and H–¹H COSY spec-

tra were consistent with a mixture of the a

and b anomers. Electrospray-ionization mass

spectral analysis of a permethylated sample

gave strong molecular ions for both the

monosodium ion at m/z 879 and the disodium

ion at m/z 451.

Ideally, the multigram preparation of gan-

glioside GM3 6 awaits the utilization of cy-

tidine

monophospho-N-acetylneuraminate:

lactosylceramide a-2,3-sialyltransferase (SAT-

1, GM3 synthase, EC 2.4.99.9) [73–76] in

combination with cytidine monophospho-N-

acetylneuraminic acid synthetase (CMP-

NeuAc synthetase, EC 2.7.7.43) [13,15,28,

34,77–82], which generates the expensive

CMP-NeuAc cofactor in situ. The availability

of SAT-1 from recombinant technology

[78,80–82] would allow conversion of the

readily synthesized substrate lactosylceramide

[62,65,83–88] directly into ganglioside GM3 6.

However, enzymatic methods are most useful

only for a few endogeneous compounds and

closely related analogs. Synthetic methods are

critical for analogs with structural variations,

which are poor enzyme substrates.

(2S,3R,4E)-b-Galactopyranosyl-(1¦4%)-b-

glucopyranosyl-(1%l1)-2-azido-4-octadecene-

1,3-diol (lactosylazidosphingosine, 3) [14,65]

was previously demonstrated to be a substrate

for the recombinant a-2,3-NeuAcT enzyme

[14]. However, this lactoside 3 was found to be

a relatively poor substrate for this enzyme.

Lactosylazidosphingosine 3 was converted

into (2S,3R,4E) - 5§ - acetyl - a - neuraminyl-

(2§3¦)-b-galactopyranosyl-(1¦4%)-b-gluco-

pyranosyl - (1%l1) - 2 - azido - 4 - octadecene -

1,3-diol (azidoGM3, 4) [10,14,18] on a 19 mg

scale. The neuraminyl glycosylation was

driven nearly to completion by the gradual

addition of CMP-NeuAc over 2 weeks. The

azidoGM3 4 was characterized by NMR spec-

The chemical total synthesis of ganglioside

GM3 6 was performed avoiding the problem-

atic azidoGM3 4 intermediate by re-investiga-

tion of a more convergent approach with the

use of a 3-O-protected ceramide in the glyco-

1

troscopy (¹H, ¹³C, H–¹H correlation, and

¹H–¹³C heterocorrelation). The a-glycosidic

linkage was demonstrated by the characteristic

[11,19,22–24,52,66–68] chemical shifts of the

492

R.I. Duclos Jr. / Carbohydrate Research 328 (2000) 489–507

sumed) and the by-product (2§b,1%a,b)-16 was

isolated in 8.7% (15% yield based on lactose

derivative a,b-14 consumed). The downﬁeld

shifts of the 3¦ protons for the coupling prod-

ucts and the correlations of the 4¦ protons to

the 4¦-OH groups in anhydrous Me₂SO-d₆

clearly showed the 2§3¦ glycosidic linkages,

and the 2§ conﬁgurations were demonstrated

by the reported [23,24,36,44] characteristic

chemical shift of 4§ and the upﬁeld shift of the

3ax§ resonance for (2§b,1%b)-16 in C₆D₆. The

synthesis of ganglioside GM3 6 via a,b-14

saves four synthetic steps otherwise necessary

to ﬁx the C-1% center, which is epimerized and

stereoselectively controlled again later in the

synthesis.

The hydrogenolysis of a,b-15 gave trisac-

charide derivative a,b-17 (Scheme 4) identical

to material prepared [36] from the longer se-

quence using the pure b-glucoside anomer.

Peracetylation [6,36] of a,b-17, selective

deacetylation [6,22] of a,b-18, and conversion

of a,b-19 into the mostly 1%a-trichloroacetimi-

date a,b-20 [6,22] was then performed.

The glycosidic coupling of the activated

GM3 trisaccharide derivative a,b-20 with a

protected ceramide 10 [90,91] was recognized

to be the most convergent synthetic approach

to GM3 6, avoiding later azide reduction and

fatty acylation reactions. The use of boron

triﬂuoride etherate catalyst for the coupling

rather than the stronger Lewis acid used for

the previously discussed neuraminyl glycosidic

coupling assured that no cis/trans isomeriza-

tion of the alkene bond would occur under the

reaction and workup conditions. The glyco-

sidic coupling step. This lipid portion was

derived (Scheme 2) from synthetic ceramide 7

[59,84,89–91] that was demonstrated to be

optically pure, 99.9% as the C18:1 sphingosine

base, and the fatty acyl group was demon-

strated to be 99.8% hexadecanoyl (C16:0).

The glycosidic coupling of the neuraminyl

donor 13 [35,36,41,46,92] with the 3¦,4¦-un-

protected lactose derivative b-14 [6,8,23–

26,35,36,41,47,93–97] was then investigated

(Scheme 3). Nearly complete regioselectivity

for sialylation at the desired 3¦-position of

b-14 has been reported [6,8,23–26,35,36,41,47]

to give the ganglioside GM3 trisaccharide

derivative (2§a,1%b)-15 [6,23,24,35,36,41,47]

and the 2§b by-product (2§b,1%b)-16 [6,23,24].

However, the easily prepared anomeric mix-

ture of lactosyl acceptor a,b-14 was recently

suggested [41] to be as useful as pure b-14 for

the neuraminyl glycosidic coupling. This has

now been demonstrated experimentally.

The a,b-hexabenzyllactose (a,b-14) synthe-

sized from b-lactose was nearly all b anomer

(3:17 a,b), in contrast to the previously re-

ported [41] 6:1 a,b mixture. A satisfactory

neuraminyl glycosidic coupling of donor 13

with the anomeric mixture of lactosyl acceptor

a,b-14 gave the ganglioside GM3 oligosaccha-

ride derivative (2§a,1%a,b)-15 as an anomeric

mixture, which was readily separated from the

anomeric mixture of by-product (2§b,1%a,b)-

16. Extra synthetic steps to improve the a,b

ratio of this glycosidic coupling were not per-

formed. The ganglioside GM3 oligosaccharide

derivative (2§a,1%a,b)-15 was isolated in 22%

(39% based on lactose derivative a,b-14 con-

Scheme 3.

R.I. Duclos Jr. / Carbohydrate Research 328 (2000) 489–507

493

Scheme 4.

total synthesis with a remarkable economy of

reaction steps proceeding through well known

ganglioside synthetic intermediates. This short

sequence utilized the lightly protected and eas-

ily prepared anomers of the lactosyl acceptor

a,b-14 to minimize synthetic steps. Also, the

highly convergent use of a 3-O-protected ce-

ramide 10 for coupling was performed to save

two synthetic steps and avoid an azide inter-

mediate. Azide reductions are extremely clean

with H₂S for neutral glycosphingolipids, but

give impure lysoganglioside, and hydrogena-

tion with the Lindlar catalyst [22] can be

substituted when necessary. Some N-acetyl

analog in the ceramide portion has been de-

tected as a by-product by mass spectrometry

in our ganglioside products when proceeding

through acetate-protected lysoganglioside in-

termediates, and is completely avoided by this

glycosidic coupling with a protected ceramide.

The use of multiple solvents for NMR char-

acterizations was also useful. Anhydrous

Me₂SO-d₆assisted with the identiﬁcation of

protons on carbons with free hydroxyl groups.

The OH resonances correlated to vicinal pro-

tons and ranged from easily assignable multi-

plets to broadened signals for certain

compounds, especially moist compounds

where some rapid exchange must occur. Sam-

ples should always be run in anhydrous

sidic coupling of trichloroacetimidate donor

a,b-20 with ceramide acceptor 10 gave a

1:1:17 mixture of a-21:a-22:b-23 in a high

coupling yield based on recovered donor and

acceptor. The 2%-hydroxyl by-product a-22 co-

eluted just ahead of the desired coupling

product b-23. A small amount of this by-

product a-22 from a putative orthoester inter-

mediate was isolated and characterized by

NMR spectroscopy in Me₂SO-d₆and in

CDCl₃. The identiﬁcation of this alcohol by-

product a-22 stimulated the ultimate puriﬁca-

tion strategy. The crude product mixture of

a-21, a-22, and b-23 was acetylated with acetic

anhydride in pyridine, and the resulting mix-

ture of a-21 and b-23 was then easily sepa-

rated by preparative HPLC. The by-product

a-21 was isolated in a coupling yield of 2.6%,

and the desired coupling product b-23 was

isolated in a glycosidic coupling yield of 41%.

The yield of the GM3 intermediate b-23 was

actually as high as 66% based on trisaccha-

ride, which was recovered by peracetylation to

give a,b-18. The ester hydrolysis of a sample

of b-23, which required the presence of water

to complete the benzoate ester hydrolysis,

gave ganglioside GM3 6 [18] in quantitative

yield.

Homogeneous ganglioside GM3 6 was read-

ily prepared on a 100 mg scale by chemical

494

R.I. Duclos Jr. / Carbohydrate Research 328 (2000) 489–507

Me₂SO-d₆before adding a few percent of

D₂O. Strikingly characteristic spectra of ben-

zylated compounds were obtained in C₆D₆

where unique conformations were observed by

solvation of the aromatic rings. The NMR

spectra of ganglioside GM3 6 were compared

with literature reports [11,19,22,66,68].

The ganglioside GM3 6 was synthesized in

only nine synthetic steps by a highly conver-

gent sequence starting from commercially

available NeuAcMe 11 in an overall yield of

2.4% (3.9% based on recovered a,b-18). The

yields of the two glycosidic coupling steps

kept the overall yield low, but there are no

scale limitations to any of these synthetic

steps. The long-term storage of ganglioside

GM3 6 in dimethyl sulfoxide below 0 °C, or as

the fully protected precursor b-23, is recom-

mended since GM3 6 is known to undergo

lactonization [22,98].

ion and selected major fragments and clusters

have been reported. More clusters with

sodium ion were seen by adding NaOH to the

sample or by using higher cone voltages where

more fragmentation was also observed. The

notations for fragment ions have been previ-

ously reported [100,101]. The primed (%) frag-

ment notation indicates either the loss of

AcOH (M_w60) or the loss of PhCO₂H (M_w

122). All solvent ratios are by volume. Hex is

hexanes, FA is fatty acyl, NeuAc is 5-acetam-

ido - 3,5 - dideoxy -

nonulopyranosylonic acid, Gal is

Glc is

or b, or a,b without a locant refers to C-1% of

the glucosyl residue.

D

- glycero - a -

D

- galacto - 2-

-galactose,

D

-Glucose, and for glycosides 14–23 a

N-Acetyl-h-neuraminyl-(2§3¦)-i-galac-

topyranosyl-(1¦4%)-N-acetylglucosamine (h-

NeuAc-(2§3¦)-LacNAc) (2).—To a 1.0 mL

solution of LacNAc 1 (2.0 mg, 5.2 mmol,

Sigma), CMP-NeuAc (4.0 mg, 5.7 mmol,

Sigma), calf intestine alkaline phosphatase

(1.6 mg, Sigma, assayed to be active with

CMP), bovine serum albumin (0.10 mg,

Sigma), containing Triton CF-54 (0.5%,

Sigma), HEPES (0.10 M), MgCl₂(5.0 mM),

MnCl₂(2.0 mM), and ZnCl₂(5.0×10⁻⁵M)

at pH 7.50 degassed with Ar was added a-2,3-

NeuT (0.10 U, as 20 mL of 5 U/mL, Cytel/

Noese) and the reaction was stirred gently at

ambient temperature for 3 days. The reaction

had become slightly heterogeneous, and the

white precipitate only partially redissolved

when the pH was adjusted from 6.60 to 7.43

with concd aq KOH. The reaction appeared

to be complete by analytical TLC (1:3:1

EtOAc–2-propanol–water, R_fof LacNAc 1

0.30, R_fof a-NeuAc-(2§3¦)-LacNAc 2 0.22,

R_fof NeuAc 0.12). After adding 4 mL of

2-propanol and 2 mL of EtOAc, the crude

reaction mixture was ﬁltered through a short

column (1:3:1 EtOAc–2-propanol–water).

The crude product was re-chromatographed

(1:3:1 EtOAc–2-propanol–water) and mi-

croﬁltered to give 2 (2.3 mg, 3.4 mmol, 65%)

[13,63,64] as an amorphous white solid that

was a 3:2 a,b mixture of anomers but was

homogeneous by TLC (40:40:7:3 CHCl₃–

MeOH–water–HOAc, R_fof LacNAc 1 0.29,

R_fof a-NeuAc-(2§3¦)-LacNAc 2 0.10, R_fof

3. Experimental

General methods.—Reactions were carried

out under argon with magnetic stirring at

ambient temperature, unless reported as bath

temperature (bt). Organic phases were dried

over Na₂SO₄, followed by paper ﬁltration and

solvent removal by rotary-evaporation under

diminished pressure. Silica gel (grade 60, 230–

400 mesh, E. Merck) was used for column

chromatography and ﬁltrations. Analytical

thin-layer chromatography (TLC) also utilized

pre-coated silica gel 60 plates, which were

visualized by charring [99]. HPLC columns

were Lichrosorb Si-60 of 5 mm particles. Re-

versed-phase HPLC columns were Delta Pak

C₁₈of 3 mm particles. Product solutions were

microﬁltered through 0.5 mm Teﬂon mem-

branes (Alltech). NMR utilized Me₄Si as an

internal reference, except for Me₂SO-d₆, where

1

the solvent peaks at l 2.50 for H and l 39.50

for ¹³C were used. Samples run in D₂O also

utilized Me₂SO-d₆as an internal reference.

Chemical shifts were assigned with the assis-

tance of COSY and HMQC spectra, and an

asterix (*) indicates a tentative assignment.

Couplings (J) for benzylic protons are gemi-

nal. Electrospray-ionization mass spectrome-

try (ESMS) was performed, and the molecular

1

NeuAc 0.07); a-2 H NMR (D₂O): l 4.98 (br

R.I. Duclos Jr. / Carbohydrate Research 328 (2000) 489–507

495

H, 2¦, 5¦, 6a¦), 3.96 (dd, 1 H, J_2¦,3¦9.7, J_3¦,4¦

2.0 Hz, 3¦), 3.65–3.70 (m, 1 H, 4¦), 3.49 (ddd,

1 H, J_5¦,6b¦6, J_6a¦,6b¦11, J_6b¦,6¦-OH5 Hz, 6b¦);

Neu, 1.35 (dd, 1 H, J_3ax§,3eq§11.5, J_3ax§,4§12

Hz, 3ax§), 2.74 (dd, 1 H, J_3eq§,4§4.3 Hz, 3eq§),

3.50–3.65 (m, 3 H, 4§, 8§, 9b§), 3.25–3.46

(m, 3 H, 5§, 6§, 9a§), 8.16 (d, 1 H, J_5§,5§-NH

12.0 Hz, 5§-NH), 1.89 (s, 3 H, 5§-NAc), 3.19

(br dd, 1 H, J_7§,7§-OH2.7, J_7§,8§8.6 Hz, 7§);

Sph, 3.50–3.65 (m, 2 H, 1a, 2), 3.65–3.72 (m,

1 H, 1b), 4.09 (ddd, 1 H, J_2,34.1, J_3,3-OH4.5,

J_3,46.7 Hz, 3), 5.43 (dd, 1 H, J_4,515.3 Hz, 4),

5.65 (dt, 1 H, J_5,66.6 Hz, 5), 1.99 (dt, 2 H, J_6,7

6.4 Hz, 6), 1.32 (m, 2 H, 7), 1.23 (m, 20 H,

8–17), 0.85 (t, 3 H, J_17,186.6 Hz, 18); ¹³C

NMR (Me₂SO-d₆): l Glc, 102.65 (1%), 73.00

(2%), 74.84* (3%), 80.48 (4%), 74.90* (5%), 60.32

(6%); Gal, 104.05 (1¦), 68.60 (2¦), 75.69 (3¦),

66.65 (4¦), 75.81 (5¦), 60.32 (6¦); Neu, 172.20

(1§-CꢀO), 99.63 (2§), 41.97 (3§), 67.37 (4§),

52.89 (5§), 72.40 (6§), 68.60 (7§), 71.07 (8§),

63.31 (9§), 170.11 (5§-NCꢀO), 22.47 (5§-

NAc); Sph, 68.13 (1), 65.70 (2), 71.25 (3),

129.24 (4), 132.68 (5), 31.61 (6), 28.60–28.96

(7–15), 31.23 (16), 22.03 (17), 13.89 (18);

ESMS (pos, 25 V) of permethylated derivative

m/z 1146 [M+Na]⁺; ESMS (pos, 25 V) after

subsequent CD₃I transesteriﬁcation m/z 1149

[M+Na]⁺.

s, 1 H, 1%), 4.33 (d, 1 H, J_1¦,2¦7.5 Hz, 1¦), 3.90

(dd, 1 H, J_2¦,3¦10, J_3¦,4¦2 Hz, 3¦), 3.40–3.80

(m, 17 H, 2%, 3%, 4%, 5%, 6a%, 6b%, 4¦, 5¦, 6a¦, 6b¦,

4§, 5§, 6§, 7§, 8§, 9a§, 9b§), 3.38 (dd, 1 H,

2¦), 2.53 (dd, 1 H, J_3ax§,3eq§12, J_3eq§,4§4 Hz,

3eq§), 1.82 (s, 3 H, 5§-NAc), 1.58 (dd, 1 H,

13

J_3ax§,4§12 Hz, 3ax§); C NMR (D₂O): l 90.39

1

(1%), 102.47 (1¦). b-2 H NMR (D₂O): l 4.50

(d, 1 H, J_1%,2%7.6 Hz, 1%), 1.81 (s, 3 H, 5§-NAc);

¹³C NMR (D₂O): l 94.78 (1%). a,b-2 ESMS

(pos, 25 V) of permethylated derivative m/z

879 [M+Na]⁺, 451 [M+2Na]⁺²

.

(2S,3R,4E)-5§-Acetyl-h-neuraminyl-(2§

3¦) - i - galactopyranosyl - (1¦4%) - i - gluco-

pyranosyl - (1%l1) - 2 - azido - 4 - octade-

cene - 1,3-diol (azidoGM3) (4).—To a 1.0 mL

solution of lactosylazidosphingosine 3 (15 mg,

23 mmol) [14,65], CMP-NeuAc (5 mg, 7.1

mmol), calf intestine alkaline phosphatase (40

U, Boehringer Mannheim), bovine serum al-

bumin (0.75 mg) containing Triton CF-54

(0.5%), HEPES (0.10 M), MgCl₂(5.0 mM),

MnCl₂(2.0 mM), and ZnCl₂(5.0×10⁻⁵M)

at pH 7.50 degassed with Ar was added a-2,3-

NeuT (0.125 U, as 25 mL of 5 U/mL) and the

mixture was stirred gently at ambient temper-

ature. The reaction was checked daily. The pH

was maintained above 7.5 with concd aq

KOH. Additional 5 mg portions of CMP-

NeuAc were added daily, and an additional

0.125 U of enzyme was added on day six. No

additional CMP-NeuAc was added after day

nine. On day 13, 2-PrOH (4 mL) was added

and the mixture ﬁltered through a short

column (4:1 2-PrOH–water). Chromatogra-

phy (3:1 CH₂Cl₂–MeOH to elute starting ma-

terial 3, followed by 4:1 2-propanol–water)

gave crude 4. The product 4 was re-chro-

matographed (5.5 g Pharmacia LH-20 Sep-

hadex 25–100 mm, MeOH), then again

re-chromatographed (4:1 2-propanol–water),

and microﬁltered (3:1 CH₂Cl₂–MeOH) to give

azidoGM3 4 (19 mg, 20 mmol, 87%) [10,14,18]

as a colorless solid, which was homogeneous

by TLC (4:1 2-propanol–water, R_fof 3 0.63,

R_fof azidoGM3 4 0.52, R_fof NeuAc 0.20); ¹H

(2S,3R,4E) - 1 - (Triphenylmethyl) - 2 - (hexa-

decanamido)-4-octadecene-1,3-diol (8).—To a

vigorously stirred solution of ceramide 7 (1.09

g, 2.03 mmol) [59,84,89–91] and ethyldiiso-

propylamine (0.530 mL, 0.393 g, 3.04 mmol)

in 20 mL of dry CH₂Cl₂, was added TrCl

(0.622 mol, 2.23 mmol) in 5 mL of CH₂Cl₂

over 20 min, while the mixture was main-

tained below 20 °C with a water bath. After

18 h, the mixture was concentrated and chro-

matographed (95:5:0.1 CH₂Cl₂–EtOAc–Et₃N,

35 °C) to give 1-tritylceramide 8 (1.34 g, 1.72

mmol, 85%) as an amorphous white solid that

was homogeneous by TLC (90:10:0.1

CH₂Cl₂–EtOAc–Et₃N, R_f0.42): mp 37–

1

38 °C; H NMR (CDCl₃): l 7.24–7.41 (m, 15

H, Ar); Sph, 3.29 (dd, 1 H, J_1a,1b9.7, J_1a,23.9

Hz, 1a), 3.39 (dd, 1 H, J_1b,23.6 Hz, 1b), 4.05

(dddd, 1 H, J_2,2-NH7.9, J_2,33.7 Hz, 2), 6.09 (d,

1 H, 2-NH), 4.18 (m, 1 H, 3), 5.25 (dd, 1 H,

J_3,46.1, J_4,515.4 Hz, 4), 5.63 (dt, 1 H, J_5,66.6

Hz, 5), 1.87–1.92 (m, 2 H, 6), 1.25–1.31 (m,

NMR (Me₂SO-d₆): l Glc, 4.19 (d, 1 H, J_1%,2%

8

Hz, 1%), 3.00 (ddd, 1 H, J_2%,2%-OH4.9, J_2%,3%8 Hz,

2%), 3.25–3.46 (m, 3 H, 3%, 4%, 5%), 3.50–3.65

(m, 1 H, 6a%), 3.70–3.75 (m, 1 H, 6b%); Gal,

4.18 (d, 1 H, J_1¦,2¦8 Hz, 1¦), 3.25–3.46 (m, 3

496

R.I. Duclos Jr. / Carbohydrate Research 328 (2000) 489–507

MeOH was added F₃B·OEt₂(81.2 mL, 93.7

mg, 0.660 mmol) dropwise over 5 min. After 4

h, 20 mL of CH₂Cl₂was added and the mix-

ture washed with 10 mL of aq NaHCO₃(0.25

g, 3.0 mmol), 10 mL of water, then dried, and

the solvent removed. Chromatography (19:1

CH₂Cl₂–MeOH) gave 3-benzoylceramide 10

(0.343 g, 0.534 mmol, 89%) [90,91] as a white

solid that was homogeneous by TLC (19:1

CH₂Cl₂–MeOH, R_f0.43): mp 88–89 °C, lit.

155–158 °C [91]; ¹H NMR (CDCl₃): l 8.03 (br

d, 2 H, J 8.4 Hz, Ar), 7.59 (br t, 1 H, J 7.4 Hz,

Ar), 7.46 (dd, 2 H, Ar); Sph, 3.69 (dd, 1 H,

J_1a,1b11.9, J_1a,23.1 Hz, 1a), 3.74 (dd, 1 H, J_1b,2

3.7 Hz, 1b), 4.28 (m, 1 H, 2), 6.06 (d, 1 H,

J_2,2-NH8.6 Hz, 2-NH), 5.54 (dd, 1 H, J_2,37.3,

J_3,47.6 Hz, 3), 5.60 (dd, 1 H, J_4,515.1 Hz, 4),

5.86 (dt, 1 H, J_5,66.7 Hz, 5), 2.04 (dt, 2 H, J_6,7

7.4 Hz, 6), 1.35 (m, 2 H, 7), 1.20–1.34 (m, 20

H, 8–17), 0.88 (t, 3 H, J_17,186.9 Hz, 18); FA,

2.17 (dt, 1 H, J_2a,2b14.5, J_2a,37.5 Hz, 2a), 2.21

(dt, 1 H, J_2b,37.5 Hz, 2b), 1.61 (quint, 2 H, J_3,4

7.5 Hz, 3), 1.20–1.34 (m, 24 H, 4–15), 0.88 (t,

3 H, J_15,166.9 Hz, 16); ¹³C NMR (CDCl₃): l

166.55 (OCꢀO); 133.45, 129.79 (2 C), 129.64,

128.51 (2 C) [Ar×6]; Sph, 61.87 (1), 53.42 (2),

74.67 (3), 124.81 (4), 137.52 (5), 32.29 (6),

28.89–29.67 (7–15), 31.91 (16), 22.67 (17),

14.09 (18); FA, 173.35 (NCꢀO), 36.89 (2),

25.72 (3), 28.89–29.67 (4–13), 31.91 (14),

22.67 (15), 14.09 (16).

22 H, 7–17), 0.88 (t, 3 H, J_17,186.9 Hz, 18);

FA, 2.21 (t, 2 H, J_2,37.6 Hz, 2), 1.61–1.65 (m,

2 H, 3), 1.25–1.31 (m, 24 H, 4–15), 0.88 (t, 3

H, J_15,166.9 Hz, 16); ¹³C NMR (CDCl₃): l

87.36 (CPh₃); 143.27 (3 C), 128.43 (6 C),

128.01 (6 C), 127.30 (3 C) [Ar×18 C]; Sph,

63.07 (1), 53.28 (2), 74.37 (3), 128.59 (4),

133.44 (5), 32.19 (6), 29.02–29.69 (7–15),

31.91 (16), 22.68 (17), 14.12 (18); FA, 173.37

(NCꢀO), 36.87 (2), 25.84 (3), 29.02–29.69 (4–

13), 31.91 (14), 22.68 (15), 14.12 (16).

(2S,3R,4E)-3-Benzoyl-1-(triphenylmethyl)-

2 - (hexadecanamido) - 4 - octadecene - 1,3 - diol

(9).—To a vigorously stirred solution of 1-

tritylceramide 8 (1.29 g, 1.65 mmol), diiso-

propylethylamine (1.46 mL, 1.08 g, 8.36

mmol) and recrystallized 4-pyrrolidinopy-

ridine (0.124 g, 0.837 mmol) in 10 mL of

anhyd PhMe was added PhCOCl (0.47 g, 3.3

mmol) over 5 min. The solvent was removed

and the residue chromatographed (97:3:0.1

CH₂Cl₂–EtOAc–Et₃N, 35 °C) to give 3-ben-

zoyl-1-tritylceramide 9 (1.40 g, 1.58 mmol,

96%) as a clear and colorless viscous liquid

that was homogeneous by TLC (97:3:0.1

CH₂Cl₂–EtOAc–Et₃N, R_f0.49); ¹H NMR

(CDCl₃): l 7.94 (br d, 2 H, J 8.3 Hz, Ar), 7.55

(br t, 1 H, J 7.4 Hz, Ar), 7.38–7.42 (m, 8 H,

Ar), 7.15–7.22 (m, 9 H, Ar); Sph, 3.20 (dd, 1

H, J_1a,1b9.5, J_1a,24.1 Hz, 1a), 3.44 (dd, 1 H,

J_1b,23.5 Hz, 1b), 4.49 (m, 1 H, 2), 5.66 (d, 1 H,

J_2,2-NH9.4 Hz, 2-NH), 5.70 (dd, 1 H, J_2,37.3,

J_3,47.5 Hz, 3), 5.45 (dd, 1 H, J_4,515.4 Hz, 4),

5.87 (dt, 1 H, J_5,66.7 Hz, 5), 1.99 (dt, 2 H, J_6,7

7.4 Hz, 6), 1.23–1.31 (m, 22 H, 7–17), 0.88 (t,

3 H, J_17,186.8 Hz, 18); FA, 2.10 (t, 2 H, J_2,3

7.5 Hz, 2), 1.56 (m, 2 H, 3), 1.23–1.31 (m, 24

H, 4–15), 0.88 (t, 3 H, J_15,166.8 Hz, 16); ¹³C

NMR (CDCl₃): l 165.32 (OCꢀO), 86.81

(CPh₃); 143.48 (3 C), 132.88, 130.27, 129.67 (2

C), 128.54 (6 C), 128.30 (2 C), 127.83 (6 C),

127.06 (3 C) [Ar×24]; Sph, 61.63 (1), 51.07

(2), 74.40 (3), 125.04 (4), 137.16 (5), 32.31 (6),

28.90–29.68 (7–15), 31.91 (16), 22.67 (17),

14.10 (18); FA, 172.42 (NCꢀO), 36.97 (2),

25.76 (3), 28.90–29.68 (4–13), 31.91 (14),

22.67 (15), 14.10 (16).

4,5,7,8,9 - Pentaacetyl - 2 - (diethylphospho)-

h,i-neuraminic acid methyl ester (a,b-13).—

Phosphite 13 [35,36,92] was readily available

in two steps from commercially available N-

acetylneuraminic acid methyl ester (Neu-

AcMe, 11) via NeuAcMe tetraacetate 12

[102–104] in at least 45% yield as a colorless

solid, which was a 1:1 a,b mixture and was

used without further puriﬁcation; TLC (3:2

PhMe–Me₂CO, R_f0.36); ¹H NMR (CDCl₃): l

4.16 (q, 4 H, J_vicinal7 Hz, CH₂), 1.37 (t, 6 H,

CH₃), 3.80 and 3.88 (s, 3 H, OMe).

Benzyl 2¦,6¦-dibenzyl-i-galactopyranosyl-

(1¦4%)-2%,3%,6%-tribenzyl-i-glucopyranoside

(b-14).—The lactoside b-14 was prepared ac-

cording to the reported [23,93,96,97] method,

and the clear and colorless viscous semi-solid

was homogeneous by TLC (22:3 CH₂Cl₂–

(2S,3R,4E)-3-Benzoyl-2-(hexadecanamido)-

4-octadecene-1,3-diol (10).—To a vigorously

stirred solution of 3-benzoyl-1-tritylceramide 9

(0.528 g, 0.597 mmol) in 10 mL of 6:4 PhMe–

1

EtOAc, R_f0.18); H NMR (Me₂SO-d₆): l

7.20–7.45 (m, 30 H, Ar); benzylic, 5.01 (d, 1

R.I. Duclos Jr. / Carbohydrate Research 328 (2000) 489–507

497

amorphous white solid that was a 1:1 a,b

H, J 10.8 Hz, a), 4.84 (d, 1 H, J 12.2 Hz, b),

4.79 (d, 1 H, J 12 Hz, c), 4.77 (ABq, 2 H, d,

d), 4.65 (d, 1 H, c), 4.63 (d, 1 H, b), 4.62 (d,

1 H, a), 4.49 (d, 1 H, J 12.2 Hz, e), 4.48 (d, 1

H, J 12.0 Hz, f), 4.40 (d, 1 H, f), 4.35 (d, 1 H,

e); Glc, 4.59 (d, 1 H, J_1%,2%7.8 Hz, 1%), 3.27 (dd,

1 H, J_2%,3%9.1 Hz, 2%), 3.59 (dd, 1 H, J_3%,4%9.1

Hz, 3%), 3.86 (dd, 1 H, J_4%,5%9.6 Hz, 4%), 3.57 (m,

1 H, 5%), 3.74 (dd, 1 H, J_5%,6a%1, J_6a%,6b%10.6 Hz,

6a%), 3.79 (dd, 1 H, J_5%,6b%4.0 Hz, 6b%); Gal,

4.45 (d, 1 H, J_1¦,2¦8 Hz, 1¦), 3.43 (dd, 1 H,

J_2¦,3¦9 Hz, 2¦), 3.43–3.52 (m, 3 H, 3¦, 5¦, 6a¦),

4.96 (d, 1 H, J_3¦,3¦-OH6.1 Hz, 3¦-OH), 3.68 (m,

1 H, 4¦), 4.74 (d, 1 H, J_4¦,4¦-OH4.5 Hz, 4¦-OH),

3.65 (dd, 1 H, J_5¦,6b¦3.7, J_6a¦,6b¦7.9 Hz, 6b¦);

¹³C NMR (Me₂SO-d₆): l 139.12, 139.07,

138.67, 138.58, 138.31, 137.69, 127.03–128.21

(30 C) [Ar×36]; 74.09, 73.98, 73.86, 72.28,

72.05, 70.09 [benzylic×6]; Glc, 101.74 (1%),

81.22 (2%), 82.07 (3%), 76.03 (4%), 74.17 (5%),

68.02 (6%); Gal, 102.23 (1¦), 79.90 (2¦), 73.02

mixture, which was homogeneous by TLC

1

(3:2:1 EtOAc–2-PrOH–water, R_f0.44); H

NMR (Me₂SO-d₆): l 1.39 (s, 3 H, Me), 1.24

(s, 3 H, Me); ¹³C NMR (Me₂SO-d₆): l 28.10

(Me), 26.30 (Me). To a stirred mixture of

NaH (9.11 g, 0.380 mol) in 60 mL of DMF

was added BnBr (59.04 g, 0.345 mol). A solu-

tion of 3¦,4¦-O-isopropylidene-a,b-lactose

(8.67 g, 22.7 mmol) in 100 mL of DMF was

added dropwise over 30 min. The exothermic

reaction was stirred for an additional 1.5 h,

then concentrated by kugelrohr distillation.

After the cautious addition of 22 mL of

MeOH, the mixture was partitioned between

250 mL of water and 200 mL of EtOAc. The

aqueous phase was extracted with additional

EtOAc (2×200 mL), and the aqueous ex-

tracts were combined, dried, and concen-

trated. The residue was chromatographed

(23:2 CH₂Cl₂–EtOAc) to give benzyl 2¦,6¦-di-

O-benzyl-3¦,4¦-O-isopropylidene-b-galacto-

pyranosyl-(1¦4%)-2%,3%,6%-tri-O-benzyl-a,b-

glucopyranoside (16.45 g, 17.8 mmol, 78%)

[41] as a faint yellow viscous semi-solid that

was a 3:17 a,b mixture identiﬁed by NMR

spectroscopy and was free of other impurities

by TLC (23:2 CH₂Cl₂–EtOAc, R_f0.45); b

1

(3¦), 68.57 (4¦), 73.53 (5¦), 68.95 (6¦); H

NMR (CDCl₃): l 7.15–7.40 (m, 30 H, Ar);

benzylic, 4.98 (d, 1 H, J 11.1 Hz, a), 4.95 (d, 1

H, J 12.1 Hz, b), 4.91 (d, 1 H, J 10.9 Hz, c),

4.79 (d, 1 H, J 11.6 Hz, d), 4.77 (d, 1 H, a),

4.72 (d, 1 H, c), 4.66 (d, 1 H, d), 4.65 (d, 1 H,

b), 4.61(d, 1 H, J 12.1 Hz, e), 4.46 (d, 1 H, e),

4.44 (d, 1 H, J 12.0 Hz, f), 4.38 (d, 1 H, f);

Glc, 4.49 (d, 1 H, J_1%,2%7.7 Hz, 1%), 3.50 (dd, 1

H, J_2%,3%9.0 Hz, 2%), 3.58 (dd, 1 H, J_3%,4%9.0 Hz,

3%), 4.02 (dd, 1 H, J_4%,5%9.7 Hz, 4%), 3.38 (ddd,

1 H, J_5%,6a%1.6, J_5%,6b%4.0 Hz, 5%), 3.76 (dd, 1 H,

J_6a%,6b%10.8 Hz, 6a%), 3.83 (dd, 1 H, 6b%); Gal,

4.44 (d, 1 H, J_1¦,2¦8 Hz, 1¦), 3.40–3.44 (m, 2

H, 2¦, 3¦), 3.92 (br d, 1 H, J_3¦,4¦1.4 Hz, 4¦),

3.35 (br dd, 1 H, J_5¦,6a¦4.9, J_5¦,6b¦6.6 Hz, 5¦),

3.49 (dd, 1 H, J_6a¦,6b¦9.9 Hz, 6a¦), 3.61 (dd, 1

1

anomer H NMR (Me₂SO-d₆): l 7.10–7.38

(m, 30 H, Ar); benzylic, 4.88 (d, 1 H, J 10.8

Hz, a), 4.84 (d, 1 H, J 12.2 Hz, b), 4.79 (d, 1

H, J 11.3 Hz, c), 4.74 (d, 1 H, J 12.1 Hz, d),

4.70 (d, 1 H, d), 4.64 (d, 1 H, c), 4.63 (d, 1 H,

b), 4.62 (d, 1 H, a), 4.53 (d, 1 H, J 12.0 Hz, e),

4.52 (d, 1 H, J 12.3 Hz, f), 4.43 (d, 1 H, e),

4.29 (d, 1 H, f); 1.31 (s, 3 H, Me), 1.28 (s, 3 H,

Me); Glc, 4.60 (d, 1 H, J_1%,2%7.8 Hz, 1%), 3.25

(dd, 1 H, J_2%,3%9.1 Hz, 2%), 3.60 (dd, 1 H, J_3%,4%

9.3 Hz, 3%), 3.86 (dd, 1 H, J_4%,5%9.7 Hz, 4%),

3.59–3.64 (m, 1 H, 5%), 3.76 (ABq, 2 H, 6a%,

6b%); Gal, 4.57 (d, 1 H, J_1¦,2¦8.1 Hz, 1¦), 3.28

(dd, 1 H, J_2¦,3¦6.3 Hz, 2¦), 4.11 (dd, 1 H, J_3¦,4¦

7.7 Hz, 3¦), 4.13 (dd, 1 H, J_4¦,5¦1.4 Hz, 4¦),

3.84 (ddd, 1 H, J_5¦,6a¦7.1, J_5¦,6b¦5 Hz, 5¦), 3.47

(dd, 1 H, J_6a¦,6b¦10.3 Hz, 6a¦), 3.62 (dd, 1 H,

6b¦); ¹³C NMR (Me₂SO-d₆): l 138.87, 138.54

(2 C), 138.31, 138.23, 137.66, 127.10–128.20

(30 C) [Ar×36]; 74.18, 73.86, 72.37, 72.26,

72.17, 70.08 [benzylic×6]; 108.91 (CMe₂),

26.28 (Me), 27.76 (Me); Glc, 101.71 (1%), 81.29

13

H, 6b¦); C NMR (CDCl₃): l 139.10, 138.51,

138.34, 138.21, 137.96, 137.47, 127.20–128.45

(30 C) [Ar×36]; 75.21, 74.93, 74.84, 73.43,

73.17, 70.91 [benzylic×6]; Glc, 102.53 (1%),

81.77 (2%), 82.80 (3%), 76.53 (4%), 75.11 (5%),

68.24 (6%); Gal, 102.45 (1¦), 80.00 (2¦), 73.47

(3¦), 68.74 (4¦), 72.85 (5¦), 68.64 (6¦).

Benzyl 2¦,6¦-dibenzyl-i-galactopyranosyl-

(1¦4%)-2%,3%,6%-tribenzyl-h,i-glucopyranoside

(a,b-14).—b-Lactose was converted into

3¦,4¦-O-isopropylidene-a,b-lactose in 31%

yield according to the reported [105] method.

3¦,4¦-O-Isopropylidene-a,b-lactose was an

498

R.I. Duclos Jr. / Carbohydrate Research 328 (2000) 489–507

(2§a,1%a,b)-15 was greater than 90% the b-gly-

coside [6,23,24,36,41] and was homogeneous

by TLC (22:3 PhMe–MeOH, R_f0.17);

(2%), 81.90 (3%), 75.58 (4%), 74.01 (5%), 67.95 (6%);

Gal, 101.01 (1¦), 80.14 (2¦), 78.47 (3¦), 73.46

1

(4¦), 71.61 (5¦), 68.73 (6¦). a Anomer H

1

NMR (Me₂SO-d₆): l Glc, 4.99 (d, 1 H, J_1%,2%

3.5 Hz, 1%), 3.39 (dd, 1 H, J_2%,3%9.7 Hz, 2%). A

solution of the isopropylidene derivative (6.45

g, 17.8 mmol) in 210 mL of water and 55 mL

of AcOH was heated at 115 °C (bt) for 1.5 h,

then cooled and concentrated at 50 °C (bt),

and given one chase with 100 mL of EtOAc.

The crude product was chromatographed (4:1

CH₂Cl₂–EtOAc) to give a,b-14 (7.58 g, 8.58

mmol, 48%) [41] as a white solid that was

homogeneous by TLC (4:1 CH₂Cl₂–EtOAc,

R_f0.40; 45:55 hex–EtOAc, R_f0.62) with the a

anomer eluting just ahead of the b anomer.

The product a,b-14 was a 3:17 a,b mixture

identiﬁed by NMR spectroscopy. Pure b-14

was already characterized, and a few a-14

(2§a,1%b)-15 H NMR (Me₂SO-d₆): l 2.03,

1.92, 1.90, 1.78, 1.68 [(s, 3 H, CH₃)×5]; 7.20–

7.45 (m, 30 H, Ar); benzylic, 4.98 (d, 1 H, J

10.7 Hz, a), 4.81 (d, 2 H, J 12 Hz, b, b%), 4.78

(d, 1 H, J 11 Hz, c), 4.68 (d, 1 H, b), 4.63 (d,

1 H, c), 4.62 (d, 1 H, a), 4.61 (d, 1 H, b%), 4.46

(d, 1 H, J 11.9 Hz, d), 4.44 (d, 1 H, J 12.1 Hz,

e), 4.41 (d, 1 H, d), 4.33 (d, 1 H, e); Glc, 4.56

(d, 1 H, J_1%,2%7.3 Hz, 1%), 3.26 (dd, 1 H, J_2%,3%9.0

Hz, 2%), 3.56 (dd, 1 H, J_3%,4%9.4 Hz, 3%), 3.82

(dd, 1 H, J_4%,5%9.3 Hz, 4%), 3.53 (m, 1 H, 5%),

3.65–3.75 (m, 2 H, 6a%, 6b%); Gal, 4.59 (d, 1 H,

J_1¦,2¦8 Hz, 1¦), 3.48 (dd, 1 H, J_2¦,3¦8 Hz, 2¦),

4.18 (dd, 1 H, J_3¦,4¦3 Hz, 3¦), 3.60 (m, 1 H,

4¦), 4.57 (d, 1 H, J_4¦,4¦-OH5 Hz, 4¦-OH), 3.51

(m, 1 H, 5¦), 3.40 (dd, 1 H, J_5¦,6a¦5.9, J_6a¦,6b¦

9.9 Hz, 6a¦), 3.61 (dd, 1 H, J_5¦,6b¦6.8 Hz, 6b¦);

Neu, 3.73 (s, 3 H, OMe§), 1.86 (dd, 1 H,

J_3ax§,3eq§12, J_3ax§,4§12.4 Hz, 3ax§), 2.56 (dd, 1

H, J_3eq§,4§4.4 Hz, 3eq§), 4.72 (ddd, 1 H, J_4§,5§

10 Hz, 4§), 3.95 (ddd, 1 H, J_5§,5-NH§9.2, J_5§,6§

10.5 Hz, 5§), 7.68 (d, 1 H, 5-NH§), 3.90 (dd,

1 H, J_6§,7§1.8 Hz, 6§), 5.18 (dd, 1 H, J_7§,8§8.1

Hz, 7§), 5.42 (ddd, 1 H, J_8§,9a§5.5, J_8§,9b§3.0

Hz, 8§), 3.93 (dd, 1 H, J_9a§,9b§12.0 Hz, 9a§),

1

peaks were assigned; a-14 H NMR (Me₂SO-

d₆): l Glc, 5.00 (d, 1 H, J_1%,2%3.7 Hz, 1%), 3.43

(dd, 1 H, J_2%,3%9.5 Hz, 2%), 3.76 (dd, 1 H, J_3%,4%

9.2 Hz, 3%).

Benzyl [methyl (4§,5§,7§,8§,9§-penta-O-

acetyl-h-neuramin)ate]yl-(2§3¦)-2¦,6¦-di-

O-benzyl-i-galactopyranosyl-(1¦4%)-2%,3%,6%-

tri-O-benzyl-h,i-glucopyranoside (a,b-15).—

A stirred solution of phosphite donor 13 (1.21

g, 1.98 mmol) [35,36,41,46,92] and hexaben-

zyllactose acceptor a,b-14 (2.82 g, 3.19 mmol)

[41] in 6 mL of dry CH₃CN was cooled to

−41 °C (bt, CH₃CN/N₂) for 15 min, then a

solution of Me₃SiOTf (75 mg, 0.34 mmol) in

0.5 mL of CH₃CN was added dropwise over 2

min. The reaction was maintained at −41 °C

(bt) for 1 h, then quenched with Et₃N (0.64

mL, 0.46 g, 4.5 mmol). The solvent was re-

moved and the crude product chro-

matographed (3:2 PhMe–Me₂CO) to recover

starting hexabenzyllactose (a,b-14, 1.82 g, 2.06

mmol), followed by the crude mixture of

products. Due to the presence of impurities

just above and below the two coupling prod-

ucts, the products were best isolated by injec-

tion of the crude mixture in seven portions

onto a preparative HPLC (97:3 PhMe–

MeOH). The homogeneous by-product

(2§b,1%a,b)-16 (0.235 g, 1.73 mmol, 8.7%)

eluted before the homogeneous desired cou-

pling product (2§a,1%a,b)-15 (0.602 g, 0.444

mmol, 22%). The desired trisaccharide analog

13

4.19 (dd, 1 H, 9b§); C NMR (Me₂SO-d₆): l

169.96, 169.55, 169.36, 169.18, 169.08, 168.31

[CꢀO×6]; 22.56, 20.88, 20.55, 20.44, 20.30

[CH₃×5]; 139.23, 139.07, 138.56 (2 C),

138.40, 137.68, 126.90–128.30 (30 C) [Ar×

36]; 74.13, 74.02, 73.85, 72.21, 72.00, 70.10

[benzylic×6]; Glc, 101.68 (1%), 81.28 (2%),

82.02 (3%), 76.05 (4%), 74.10 (5%), 68.15 (6%); Gal,

101.80 (1¦), 78.06 (2¦), 75.51 (3¦), 66.66 (4¦),

72.76 (5¦), 68.42 (6¦); Neu, 52.79 (OMe§),

97.64 (2§), 36.88 (3§), 69.35 (4§), 47.77 (5§),

71.62 (6§), 66.95 (7§), 68.15 (8§), 61.66 (9§);

¹H NMR (CDCl₃): l 2.09, 2.01, 1.98, 1.90,

1.87 [(s, 3 H, CH₃)×5]; 7.10–7.45 (m, 30 H,

Ar); benzylic, 4.98 (d, 1 H, J 10.8 Hz, a), 4.93

(d, 1 H, J 12.0 Hz, b), 4.90 (d, 1 H, J 10.8 Hz,

c), 4.78 (d, 1 H, J 11.7 Hz, d), 4.74 (d, 1 H, a),

4.72 (d, 1 H, c), 4.68 (d, 1 H, d), 4.64 (d, 1 H,

b), 4.54 (d, 1 H, J 12.3 Hz, e), 4.50 (d, 1 H, e),

4.44 (d, 1 H, J 12.0 Hz, f), 4.34 (d, 1 H, f);

Glc, 4.46 (d, 1 H, J_1%,2%7.7 Hz, 1%), 3.47 (dd, 1

H, J_2%,3%9 Hz, 2%), 3.55 (dd, 1 H, J_3%,4%9 Hz, 3%),

R.I. Duclos Jr. / Carbohydrate Research 328 (2000) 489–507

499

13

1 H, 9b§); C NMR (C₆D₆): l 170.26, 170.12,

170.09, 169.98, 169.47, 169.21 [CꢀO×6];

22.93, 21.08, 20.62, 20.34 (2 C) [CH₃×5];

140.15, 139.74, 139.65, 139.31, 139.23, 138.38,

127.10–128.80 (30 C) [Ar×36]; 75.58, 75.34,

75.01, 73.44, 73.34, 70.78 [benzylic×6]; Glc,

102.89 (1%), 82.41 (2%), 83.24 (3%), 76.74 (4%),

75.73 (5%), 68.84 (6%); Gal, 102.89 (1¦), 79.24

(2¦), 76.93 (3¦), 68.08 (4¦), 73.12 (5¦), 68.84

(6¦); Neu, 52.55 (OMe§), 98.81 (2§), 37.20

(3§), 69.36 (4§), 49.00 (5§), 73.34 (6§), 67.52

(7§), 69.91 (8§), 62.87 (9§). (2§a,1%a,b)-15

ESMS (pos, 20 V) m/z 1357 [M+H]⁺, 816

[B₂]⁺, 710 [M+2Na+water]⁺²; (2§a,1%a,b)-

15 ESMS (pos, 60 V) m/z 1379 [M+Na]⁺,

816 [B₂]⁺, 474 [B₁]⁺, 414 [B₁%]⁺.

3.99 (dd, 1 H, J_4%,5%9.8 Hz, 4%), 3.35 (ddd, 1 H,

J_5%,6a%2, J_5%,6b%4 Hz, 5%), 3.72–3.78 (m, 2 H, 6a%,

6b%); Gal, 4.58 (d, 1 H, J_1¦,2¦7.7 Hz, 1¦), 3.51

(dd, 1 H, J_2¦,3¦9.5 Hz, 2¦), 4.06 (dd, 1 H, J_3¦,4¦

3.2 Hz, 3¦), 3.83 (br d, 1 H, 4¦), 3.48–3.51 (m,

2 H, 5¦, 6a¦), 3.68 (dd, 1 H, J_5¦,6b¦9.2, J_6a¦,6b¦

11.6 Hz, 6b¦); Neu, 3.75 (s, 3 H, OMe§), 2.06

(dd, 1 H, J_3ax§,3eq§13, J_3ax§,4§10 Hz, 3ax§),

2.50 (dd, 1 H, J_3eq§,4§4.6 Hz, 3eq§), 4.86 (ddd,

1 H, J_4§,5§10 Hz, 4§), 4.09 (ddd, 1 H, J_5§,5§-NH

10.0, J_5§,6§10 Hz, 5§), 5.20 (d, 1 H, 5§-NH),

4.00 (dd, 1 H, J_6§,7§2.1 Hz, 6§), 5.31 (dd, 1 H,

J_7§,8§7.9 Hz, 7§), 5.40 (ddd, 1 H, J_8§,9a§5.9,

J_8§,9b§2.5 Hz, 8§), 3.96 (dd, 1 H, J_9a§,9b§12.4

Hz, 9a§), 4.31 (dd, 1 H, 9b§); ¹³C NMR

(CDCl₃): l 170.76, 170.53, 170.26, 169.97,

169.88, 168.34 [CꢀO×6]; 23.09, 21.06, 20.76,

20.66, 20.49 [CH₃×5]; 139.08, 138.91, 138.59,

138.48, 138.36, 137.53, 127.10–128.30 (30 C)

[Ar×36]; 75.34, 74.91 (2 C), 73.27, 73.01,

70.87 [benzylic×6]; Glc, 102.39 (1%), 81.82

(2%), 82.93 (3%), 76.42 (4%), 75.10 (5%), 68.41 (6%);

Gal, 102.28 (1¦), 78.40 (2¦), 76.31 (3¦), 67.87

(4¦), 72.44 (5¦), 68.41 (6¦); Neu, 52.97

(OMe§), 98.39 (2§), 36.42 (3§), 69.07 (4§),

49.21 (5§), 72.71 (6§), 67.17 (7§), 68.84 (8§),

Benzyl [methyl (4§,5§,7§,8§,9§-penta-O-

acetyl - i - neuramin)ate]yl - (2§3¦) - 2¦,6¦-

dibenzyl-i-galactopyranosyl-(1¦4%)-2%,3%,6%-

tri-O-benzyl-h,i-glucopyranoside (a,b-16).—

The trisaccharide by-product (2§b,1%a,b)-16

was also greater than 90% the b-benzyl glu-

coside [23,24] by NMR spectroscopy and was

homogeneous by TLC (22:3 PhMe–MeOH,

1

R_f0.24); (2§b,1%b)-16 H NMR (Me₂SO-d₆): l

2.03, 1.95, 1.93, 1.79, 1.73 [(s, 3 H, CH₃)×5];

7.20–7.50 (m, 30 H, Ar); benzylic, 5.00 (d, 1

H, J 11.5 Hz, a), 4.82 (d, 1 H, J 12.2 Hz, b),

4.78 (d, 1 H, J 11.3 Hz, c), 4.72 (d, 1 H, J 11.7

Hz, d), 4.64 (d, 1 H, c), 4.63 (d, 1 H, d), 4.62

(d, 1 H, b), 4.61 (d, 1 H, a), 4.48 (d, 1 H, J 11

Hz, e), 4.45 (d, 1 H, J 12 Hz, f), 4.43 (d, 1 H,

e), 4.32 (d, 1 H, f); Glc, 4.56 (d, 1 H, J_1%,2%7.6

Hz, 1%), 3.24 (dd, 1 H, J_2%,3%9.2 Hz, 2%), 3.56

(dd, 1 H, J_3%,4%8 Hz, 3%), 3.88 (m, 1 H, 4%), 3.49

(m, 1 H, 5%), 3.72 (dd, 1 H, J_5%,6a%1, J_6a%,6b%11

Hz, 6a%), 3.76 (dd, 1 H, J_5%,6b%3 Hz, 6b_%); Gal,

4.47 (d, 1 H, J_1¦,2¦8 Hz, 1¦), 3.61 (dd, 1 H,

J_2¦,3¦9 Hz, 2¦), 3.92 (m, 1 H, 3¦), 3.84 (br dd,

1 H, J_3¦,4¦3, J_4¦,4¦-OH6.1 Hz, 4¦), 4.74 (d, 1 H,

4¦-OH), 3.57 (m, 1 H, 5¦), 3.52 (dd, 1 H, J_5¦,6a¦

6.6, J_6a¦,6b¦10 Hz, 6a¦), 3.64 (dd, 1 H, J_5¦,6b¦

4.3 Hz, 6b¦); Neu, 3.47 (s, 3 H, OMe§), 1.99

(dd, 1 H, J_3ax§,3eq§13, J_3ax§,4§13 Hz, 3ax§),

2.61 (dd, 1 H, J_3eq§,4§4.5 Hz, 3eq§), 5.21 (ddd,

1 H, J_4§,5§8.8 Hz, 4§), 3.91 (ddd, 1 H, J_5§,5§-NH

9.5, J_5§,6§10.4 Hz, 5§), 7.43 (d, 1 H, 5§-NH),

4.37 (br d, 1 H, 6§), 5.33 (br s, 1 H, 7§), 5.16

(br d, 1 H, J_8§,9a§9.1 Hz, 8§), 4.01 (dd, 1 H,

J_9a§,9b§12.1 Hz, 9a§), 5.00 (br d, 1 H, 9b§);

¹³C NMR (Me₂SO-d₆): l 169.95, 169.88,

1

62.23 (9§); H NMR (C₆D₆): l 2.03, 1.90,

1.71, 1.64, 1.63 [(s, 3 H, CH₃)×5]; 7.00–7.70

(m, 30 H, Ar); benzylic, 5.28 (d, 1 H, J 10.9

Hz, a), 5.01 (d, 1 H, J 11.4 Hz, b), 4.94 (d, 1

H, a), 4.91 (d, 1 H, J 12 Hz, c), 4.87 (d, 1 H,

J 13 Hz, d), 4.84 (d, 1 H, d), 4.81 (d, 1 H, b),

4.59 (d, 1 H, J 12.1 Hz, e), 4.58 (d, 1 H, c),

4.54 (d, 1 H, e), 4.43 (d, 1 H, J 12 Hz, f), 4.30

(d, 1 H, f); Glc, 4.48 (d, 1 H, J_1%,2%7.2 Hz, 1%),

3.64 (dd, 1 H, J_2%,3%8.5 Hz, 2%), 3.67 (dd, 1 H,

J_3%,4%9.4 Hz, 3%), 4.36 (dd, 1 H, J_4%,5%9.4 Hz, 4%),

3.37 (ddd, 1 H, J_5%,6a%1.4, J_5%,6b%4.3 Hz, 5%), 3.83

(dd, 1 H, J_6a%,6b%10.8 Hz, 6a%), 3.98 (dd, 1 H,

6b%); Gal, 4.90 (d, 1 H, J_1¦,2¦8 Hz, 1¦), 3.84

(dd, 1 H, J_2¦,3¦9 Hz, 2¦), 4.40 (m, 1 H, 3¦),

4.12 (br s, 1 H, 4¦), 3.79 (br dd, 1 H, J_5¦,6a¦5.3,

J_5¦,6b¦7.6 Hz, 5¦), 3.62 (dd, 1 H, J_6a¦,6b¦9.4 Hz,

6a¦), 3.96 (dd, 1 H, 6b¦); Neu, 3.40 (s, 3 H,

OMe§), 2.14 (dd, 1 H, J_3ax§,3eq§12.8, J_3ax§,4§

12.1 Hz, 3ax§), 2.70 (dd, 1 H, J_3eq§,4§4.5 Hz,

3eq§), 4.86 (m, 1 H, 4§), 4.38–4.47 (m, 2 H,

5§, 5§-NH), 4.02 (dd, 1 H, J_5§,6§10.4, J_6§,7§

2.3 Hz, 6§), 5.47 (dd, 1 H, J_7§,8§7.5 Hz, 7§),

5.79 (ddd, 1 H, J_8§,9a§6.7, J_8§,9b§2.4 Hz, 8§),

4.21 (dd, 1 H, J_9a§,9b§12.3 Hz, 9a§), 4.70 (dd,

500

R.I. Duclos Jr. / Carbohydrate Research 328 (2000) 489–507

H, J 12.1 Hz, d), 4.85 (d, 1 H, c), 4.79 (d, 1 H,

b), 4.59 (d, 1 H, d), 4.59 (s, 2 H, e, e), 4.43 (d,

1 H, J 12.4 Hz, f), 4.38 (d, 1 H, f); Glc, 4.48

169.82, 169.38, 169.17, 166.25 [CꢀO×6];

22.66, 20.69, 20.64, 20.56, 20.26 [CH₃×5];

139.13, 138.59 (2 C), 138.25 (2 C), 137.69,

126.90–128.70 (30 C) [Ar×36]; 74.39, 74.07,

73.90, 72.32, 72.12, 70.13 [benzylic×6]; Glc,

101.75 (1%), 81.16 (2%), 81.88 (3%), 75.68 (4%),

73.96 (5%), 67.96 (6%); Gal, 101.83 (1¦), 77.05

(2¦), 76.80 (3¦), 68.22 (4¦), 73.35 (5¦), 69.08

(6¦); Neu, 52.17 (OMe§), 99.34 (2§), 37.64

(3§), 68.96 (4§), 47.96 (5§), 71.76 (6§), 68.87

(7§), 72.36 (8§), 62.07 (9§); ¹H NMR

(CDCl₃): l 2.11, 2.09, 1.99, 1.98, 1.74 [(s, 3 H,

CH₃)×5]; 7.15–7.50 (m, 30 H, Ar); benzylic,

4.99 (d, 1 H, J 10.9 Hz, a), 4.94 (d, 1 H, J 12.1

Hz, b), 4.90 (d, 1 H, J 10.9 Hz, c), 4.89 (d, 1

H, J 11.1 Hz, d), 4.77 (d, 1 H, a), 4.72 (d, 1 H,

c), 4.67 (d, 1 H, d), 4.64 (d, 1 H, b), 4.61 (d,

1 H, J 12 Hz, e), 4.57 (d, 1 H, e), 4.46 (d, 1 H,

J 12 Hz, f), 4.40 (d, 1 H, f); Glc, 4.47 (d, 1 H,

J_1%,2%7.5 Hz, 1%), 3.49 (dd, 1 H, J_2%,3%9 Hz, 2%),

3.58 (dd, 1 H, J_3%,4%9 Hz, 3%), 4.06 (dd, 1 H,

J_4%,5%9 Hz, 4%), 3.39 (dt, 1 H, J_5%,6%2 Hz, 5%), 3.84

(d, 2 H, 6a%, 6b%); Gal, 4.58 (d, 1 H, J_1¦,2¦8 Hz,

1¦), 3.64–3.69 (m, 2 H, 2¦, 6b¦), 3.81 (dd, 1 H,

J_2¦,3¦9.4, J_3¦,4¦2.9 Hz, 3¦), 3.89 (dd, 1 H, J_4¦,5¦

(d, 1 H, J_1%,2%7.3 Hz, 1%), 3.64 (dd, 1 H, J_2%,3%

9

Hz, 2%), 3.65 (dd, 1 H, J_3%,4%8 Hz, 3%), 4.37 (dd,

1 H, J_4%,5%9 Hz, 4%), 3.32 (ddd, 1 H, J_5%,6a%1,

J_5%,6b%3.9 Hz, 5%), 3.87 (dd, 1 H, J_6a%,6b%9 Hz,

6a%), 4.01 (dd, 1 H, 6b%); Gal, 4.86 (d, 1 H,

J_1¦,2¦7.8 Hz, 1¦), 3.98 (dd, 1 H, J_2¦,3¦9.1 Hz,

2¦), 4.09 (dd, 1 H, J_3¦,4¦3 Hz, 3¦), 4.24 (br d,

1 H, 4¦), 3.59 (br dd, 1 H, J_5¦,6a¦5.1, J_5¦,6b¦5.6

Hz, 5¦), 3.69 (dd, 1 H, J_6a¦,6b¦10.0 Hz, 6a¦),

3.81 (dd, 1 H, 6b¦); Neu, 3.29 (s, 3 H, OMe§),

1.94 (dd, 1 H, J_3ax§,3eq§13.5, J_3ax§,4§12 Hz,

3ax§), 2.61 (dd, 1 H, J_3eq§,4§4.2 Hz, 3eq§),

5.46 (ddd, 1 H, J_4§,5§10 Hz, 4§), 4.53 (ddd, 1

H, J_5§,5§-NH10, J_5§,6§10.6 Hz, 5§), 4.37 (d, 1

H, 5§-NH), 4.72 (dd, 1 H, J_6§,7§2.3 Hz, 6§),

5.67 (m, 1 H, 7§), 5.69 (m, 1 H, 8§), 4.35 (dd,

1 H, J_8§,9a§8.0, J_9a§,9b§12.2 Hz, 9a§), 5.36 (dd,

13

1 H, J_8§,9b§1.8 Hz, 9b§); C NMR (C₆D₆): l

170.48, 170.22 (3 C), 169.60, 167.82 [CꢀO×6];

23.06, 20.84, 20.69, 20.43 (2 C) [CH₃×5];

140.09, 139.54, 139.29, 139.19, 138.83, 138.32,

127.30–128.70 (30 C) [Ar×36)]; 75.71, 75.41,

75.03, 73.76, 73.25, 70.83 [benzylic×6]; Glc,

103.00 (1%), 82.39 (2%), 83.01 (3%), 76.59 (4%),

75.56 (5%), 68.90 (6%); Gal, 102.94 (1¦), 78.96

(2¦), 78.75 (3¦), 68.94 (4¦), 73.34 (5¦), 69.74

(6¦); Neu, 52.44 (OMe§), 100.08 (2§), 36.72

(3§), 69.06 (4§), 49.05 (5§), 72.94 (2 C, 6§,

8§), 69.12 (7§), 63.33 (9§). (2§b,1%a,b)-16

ESMS (pos, 20 V) m/z 1357 [M+H]⁺, 816

[B₂]⁺, 710 [M+2Na+water]⁺²; (2§b,1%a,b)-

16 ESMS (pos, 60 V) m/z 1379 [M+Na]⁺,

816 [B₂]⁺, 474 [B₁]⁺, 414 [B₁%]⁺.

1 Hz, 4¦), 3.42 (ddd, 1 H, J_5¦,6a¦5.0, J_5¦,6b¦

5

Hz, 5¦), 3.54 (dd, 1 H, J_6a¦,6b¦9.8 Hz, 6a¦);

Neu, 3.65 (s, 3 H, OMe§), 2.01 (dd, 1 H,

J_3ax§,3eq§13.5, J_3ax§,4§11 Hz, 3ax§), 2.55 (dd, 1

H, J_3eq§,4§4.2 Hz, 3eq§), 5.18 (ddd, 1 H, J_4§,5§

10 Hz, 4§), 4.09 (ddd, 1 H, J_5§,5§-NH9.1, J_5§,6§

10.6 Hz, 5§), 4.69 (d, 1 H, 5§-NH), 4.32 (dd,

1 H, J_6§,7§2.2 Hz, 6§), 5.26 (dd, 1 H, J_7§,8§

Hz, 7§), 5.20 (ddd, 1 H, J_8§,9a§7.8, J_8§,9b§

4

1

Hz, 8§), 4.02 (dd, 1 H, J_9a§,9b§12.3 Hz, 9a§),

4.77 (dd, 1 H, 9b§); ¹³C NMR (CDCl₃): l

170.56, 170.47, 170.17, 170.06 (2 C), 167.70

[CꢀO×6]; 23.06, 21.03, 20.79, 20.69 (2 C)

[CH₃×5]; 139.02, 138.54, 138.49, 138.43,

138.08, 137.45, 127.10–128.50 (30 C) [Ar×

36]; 75.22, 75.18, 74.89, 73.39, 72.99, 70.90

[benzylic×6]; Glc, 102.41 (1%), 81.78 (2%),

82.59 (3%), 76.19 (4%), 75.06 (5%), 68.34 (6%); Gal,

102.33 (1¦), 78.38 (2¦), 77.84 (3¦), 67.81 (4¦),

72.57 (5¦), 68.87 (6¦); Neu, 52.90 (OMe§),

99.28 (2§), 35.42 (3§), 68.65 (4§), 48.56 (5§),

71.84 (6§), 68.38 (7§), 71.49 (8§), 62.61 (9§);

¹H NMR (C₆D₆): l 1.96, 1.89, 1.67, 1.65, 1.59

[(s, 3 H, CH₃)×5]; 7.00–7.65 (m, 30 H, Ar);

benzylic, 5.24 (d, 1 H, J 11.2 Hz, a), 5.00 (d, 2

H, J 11 Hz, b, c), 4.95 (d, 1 H, a), 4.90 (d, 1

[Methyl (4§,5§,7§,8§,9§-penta-O-acetyl-h-

neuramin)ate]yl - (2§3¦) - i - galactopyranos-

yl-(1¦4%)-h,i-glucopyranoside (a,b-17).—

Hydrogenation of a,b-15 (0.602 g, 0.444

mmol) in 18 mL of 9:1 MeOH–AcOH with 95

mg of 10% Pd–C under H₂(40 PSI) was

performed with mechanical shaking on a Parr

apparatus for 19 h. The catalyst was removed

by micropore ﬁltration. The solvent was re-

moved to give a quantitative yield of a,b-17

[36] as an amorphous white solid that was a

1:1 a,b mixture of anomers identiﬁed by

NMR spectroscopy and was used without fur-

ther puriﬁcation; b-17 ¹H NMR (Me₂SO-d₆): l

7.68 (d, 1 H, J_5§,5§-NH8.6 Hz, 5§-NH), 5.25

R.I. Duclos Jr. / Carbohydrate Research 328 (2000) 489–507

501

(ddd, 1 H, J_7§,8§7.5, J_8§,9a§6.2, J_8§,9b§2.5 Hz,

8§), 5.14 (br d, 1 H, 7§), 4.76 (ddd, 1 H,

J_3ax§,4§12, J_3eq§,4§4.6, J_4§,5§9.1 Hz, 4§), 4.25–

4.35 (m, 3 H, 1%, 1¦, 9b§), 4.03 (dd, 1 H,

J_9a§,9b§12.3 Hz, 9a§), 3.91 (br d, 1 H, J_2¦,3¦9.8

Hz, 3¦), 3.80–3.90* (m, 2 H, 5§, 6§), 3.74 (s,

3 H, OMe§), 3.10–3.80 (m, 10 H, 3%, 4%, 5%, 6a%,

6b%, 2¦, 4¦, 5¦, 6a¦, 6b¦), 2.95 (br dd, 1 H, J_1%,2%

8, J_2%,3%9 Hz, 2%), 2.48 (dd, 1 H, J_3ax§,3eq§12 Hz,

3eq§), 2.04 (dd, 1 H, 3ax§), 2.05, 2.01, 1.97,

[CH₃×12]; Glc, 91.65 (1%), 70.71 (2%), 73.20

(3%), 75.76 (4%), 73.64 (5%), 61.97 (6%); Gal,

100.99 (1¦), 69.87 (2¦), 71.43 (3¦), 67.32 (4¦),

70.56 (5¦), 61.60 (6¦); Neu, 53.16 (OMe§),

96.82 (2§), 37.44 (3§), 69.34 (4§), 49.24 (5§),

72.06 (6§), 66.88 (7§), 67.80 (8§), 62.17 (9§);

b-18 ESMS (pos, 20 V) m/z 1110 [M+H]⁺,

1050 [M+H%]⁺, 586.5 [M+2Na+water]⁺²

;

b-18 ESMS (pos, 60 V) m/z 1132 [M+Na]⁺,

1050 [M+H%]⁺, 762 [B₂]⁺, 474 [B₁]⁺, 414

1

[B₁%]⁺. a-18 H NMR (CDCl₃): l 2.25, 2.18,

1.93, 1.68 [(s, 3 H, CH₃)×5]. a-17 H NMR

(Me₂SO-d₆): l 4.99 (dd, 1 H, J_1%,2%4, J_1%,1%-OH

Hz, 1%).

5

2.16, 2.10 (2 Me), 2.08, 2.07 (2 Me), 2.05, 2.01

(2 Me), 1.86 [(s, 3 H, CH₃)×12]; Glc, 6.25 (d,

1 H, J_1%,2%3.7 Hz, 1%), 5.02 (dd, 1 H, J_2%,3%10.3

Hz, 2%), 5.47 (dd, 1 H, J_3%,4%9.2 Hz, 3%), 3.91

(dd, 1 H, J_4%,5%10.0 Hz, 4%), 3.95–4.05 (m, 1 H,

5%), 4.22 (dd, 1 H, J_5%,6a%4.4, J_6a%,6b%12.1 Hz,

6a%), 4.39 (dd, 1 H, J_5%,6b%1.7 Hz, 6b%); Gal,

4.66 (d, 1 H, J_1¦,2¦8.0 Hz, 1¦), 4.95 (dd, 1 H,

J_2¦,3¦10.1 Hz, 2¦), 4.52 (dd, 1 H, J_3¦,4¦3.3 Hz,

3¦), 4.89 (br d, 1 H, 4¦), 3.87 (m, 1 H, 5¦),

3.95–4.05 (m, 2 H, 6a¦, 6b¦); Neu, 3.85 (s, 3

H, OMe§), 1.68 (dd, 1 H, J_3ax§,3eq§12.6, J_3ax§,4§

12.2 Hz, 3ax§), 2.58 (dd, 1 H, J_3eq§,4§4.6 Hz,

3eq§), 4.90 (ddd, 1 H, J_4§,5§10.2 Hz, 4§), 4.02

(ddd, 1 H, J_5§,5§-NH10.2, J_5§,6§10.7 Hz, 5§),

5.08 (d, 1 H, 5§-NH), 3.64 (dd, 1 H, J_6§,7§2.7

Hz, 6§), 5.41 (dd, 1 H, J_7§,8§9.3 Hz, 7§), 5.51

(ddd, 1 H, J_8§,9a§4.4, J_8§,9b§2.8 Hz, 8§), 3.99

(dd, 1 H, J_9a§,9b§12.7 Hz, 9a§), 4.43 (dd, 1 H,

9b§); ¹³C NMR (CDCl₃): l 170.80, 170.58 (2

C), 170.34, 170.31, 170.24, 170.18, 169.94,

169.56, 169.51 (2 C), 168.97, 167.91 [CꢀO×

13]; 23.15, 21.50, 20.93, 20.92, 20.80, 20.73 (5

C), 20.59, 20.49 [CH₃×12]; Glc, 89.04 (1%),

69.50 (2%), 69.95 (3%), 75.77 (4%), 70.68 (5%),

61.72 (6%); Gal, 101.07 (1¦), 69.90 (2¦), 71.37

(3¦), 67.22 (4¦), 70.47 (5¦), 61.52 (6¦); Neu,

53.12 (OMe§), 96.73 (2§), 37.37 (3§), 69.27

(4§), 49.13 (5§), 71.97 (6§), 66.73 (7§), 67.71

(8§), 62.00 (9§); a-18 ESMS (pos, 20 V) m/z

1110 [M+H]⁺, 1050 [M+H%]⁺, 586.5 [M+

2Na+water]⁺²; a-18 ESMS (pos, 60 V) m/z

1132 [M+Na]⁺, 1050 [M+H%]⁺, 762 [B₂]⁺,

474 [B₁]⁺, 414 [B%₁]⁺.

[Methyl (4§,5§,7§,8§,9§-penta-O-acetyl-h-

neuramin)ate]yl - (2§3¦) - 2¦,4¦,6¦ - tri - O-

acetyl-i-galactopyranosyl-(1¦4%)-1%,2%,3%,6%-

tetraacetyl-h,i-glucopyranose (a,b-18).—To a

stirred solution of a,b-17 (0.362 g, 0.444

mmol) in 6 mL of pyridine was added 4 mL of

Ac₂O dropwise over 5 min. After 21 h, the

solvent was removed and the residue chro-

matographed (92.5:7.5 EtOAc–MeOH). The b

anomer eluted just ahead of the a anomer. All

fractions were combined to give a,b-18 (0.461

g, 0.415 mmol, 93%) [6,36] as an amorphous

white solid that was a 1:1 a,b mixture, which

was homogeneous by TLC (92.5:7.5 EtOAc–

MeOH, R_f0.40; 1:1 PhMe–Me₂CO, R_f0.29):

1

b-18 mp 120–122 °C; H NMR (CDCl₃): l

2.24, 2.16, 2.10, 2.09 (2 Me), 2.08 (2 Me), 2.07,

2.04, 2.03, 2.01, 1.86 [(s, 3 H, CH₃)×12]; Glc,

5.68 (d, 1 H, J_1%,2%8.5 Hz, 1%), 5.05 (dd, 1 H,

J_2%,3%9.3 Hz, 2%), 5.23 (dd, 1 H, J_3%,4%9.3 Hz, 3%),

3.93 (dd, 1 H, J_4%,5%9.6 Hz, 4%), 3.76 (ddd, 1 H,

J_5%,6a%5.0, J_5%,6b%2 Hz, 5%), 4.20 (dd, 1 H, J_6a%,6b%

12 Hz, 6a%), 4.42 (dd, 1 H, 6b%); Gal, 4.65 (d,

1 H, J_1¦,2¦8.0 Hz, 1¦), 4.93 (dd, 1 H, J_2¦,3¦10.2

Hz, 2¦), 4.51 (dd, 1 H, J_3¦,4¦3.3 Hz, 3¦), 4.88

(br d, 1 H, 4¦), 3.85 (m, 1 H, 5¦), 4.01 (m, 2 H,

6a¦, 6b¦); Neu, 3.84 (s, 3 H, OMe§), 1.68 (dd,

1 H, J_3ax§,3eq§12.6, J_3ax§,4§12.2 Hz, 3ax§), 2.58

(dd, 1 H, J_3eq§,4§4.6 Hz, 3eq§), 4.89 (ddd, 1 H,

J_4§,5§10 Hz, 4§), 4.03 (m, 1 H, 5§), 5.09 (d, 1

H, J_5§,5§-NH10.2 Hz, 5§-NH), 3.63 (dd, 1 H,

J_5§,6§10.8, J_6§,7§2.7 Hz, 6§), 5.41 (dd, 1 H,

J_7§,8§9.4 Hz, 7§), 5.52 (ddd, 1 H, J_8§,9a§4.6,

J_8§,9b§2.9 Hz, 8§), 3.98 (dd, 1 H, J_9a§,9b§12

Hz, 9a§), 4.42 (dd, 1 H, 9b§); ¹³C NMR

(CDCl₃): l 170.89, 170.66, 170.64, 170.42,

170.39, 170.31, 170.26, 169.65 (3 C), 169.48,

168.87, 168.01 [CꢀO×13]; 23.20, 21.55, 20.96,

20.82 (3 C), 20.80 (3 C), 20.71, 20.63 (2 C)

[Methyl (4§,5§,7§,8§,9§-penta-O-acetyl-h-

neuramin)ate]yl - (2§3¦) - 2¦,4¦,6¦ - tri - O-

acetyl - i - galactopyranosyl - (1¦4%) - 2%,3%,6%-

tri-O-acetyl-h,i-glucopyranose (a,b-19).—To

a stirred solution of a,b-18 (0.461 g, 0.415

mmol) in 5 mL of anhyd DMF, was added

502

R.I. Duclos Jr. / Carbohydrate Research 328 (2000) 489–507

hydrazine acetate (0.050 g, 0.54 mmol) and

the mixture was heated for 1 h at 50 °C (bt).

After adding 10 mL of EtOAc, the organic

phase was washed with satd aq NaCl (3×10

mL), dried, and the solvent removed to give

the crude product, which was chro-

matographed (23:2 EtOAc–MeOH). After the

elution of 7 mg of starting material a,b-18, the

product a,b-19 (0.335 g, 0.314 mmol, 76%)

[6,22] eluted, followed by 52 mg of over-hy-

drolysis by-product. The deacetylation

product a,b-19 was an amorphous white solid

that was a 13:7 a,b mixture of anomers, which

was homogeneous by TLC (23:2 EtOAc–

a,b-20 mp 114 °C (soften) 132–133 °C (melt).

1

a-20 H NMR (CDCl₃): l 2.24, 2.16, 2.09,

2.08, 2.07, 2.06 (2 Me), 2.05, 2.00 (2 Me), 1.85

[(s, 3 H, CH₃)×11]; Glc, 6.49 (d, 1 H, J_1%,2%3.8

Hz, 1%), 5.08 (dd, 1 H, J_2%,3%10.2 Hz, 2%), 5.55

(dd, 1 H, J_3%,4%9.3 Hz, 3%), 3.95 (dd, 1 H, J_4%,5%

10.3 Hz, 4%), 4.12 (ddd, 1 H, J_5%,6a%4.6, J_5%,6b%1.8

Hz, 5%), 4.24 (dd, 1 H, J_6a%,6b%12.1 Hz, 6a%), 4.43

(dd, 1 H, 6b%); Gal, 4.67 (d, 1 H, J_1¦,2¦8.0 Hz,

1¦), 4.96 (dd, 1 H, J_2¦,3¦10.1 Hz, 2¦), 4.52 (dd,

1 H, J_3¦,4¦3.3 Hz, 3¦), 4.90 (br d, 1 H, 4¦), 3.86

(m, 1 H, 5¦), 4.03 (m, 2 H, 6a¦, 6b¦); Neu, 3.85

(s, 3 H, OMe§), 1.68 (dd, 1 H, J_3ax§,3eq§12.7,

J_3ax§,4§12.1 Hz, 3ax§), 2.58 (dd, 1 H, J_3eq§,4§

4.6 Hz, 3eq§), 4.91 (ddd, 1 H, J_4§,5§10 Hz,

4§), 4.02 (ddd, 1 H, J_5§,5§-NH10.2, J_5§,6§10.8

Hz, 5§), 5.10 (d, 1 H, 5§-NH), 3.64 (dd, 1 H,

J_6§,7§2.7 Hz, 6§), 5.41 (dd, 1 H, J_7§,8§9.3 Hz,

7§), 5.50 (ddd, 1 H, J_8§,9a§4.4, J_8§,9b§2.8 Hz,

8§), 4.00 (dd, 1 H, J_9a§,9b§12.5 Hz, 9a§), 4.42

(dd, 1 H, 9b§), 8.65 (s, 1 H, Cl₃CꢁCꢀNꢁH);

¹³C NMR (CDCl₃): l 170.80, 170.58 (2 C),

170.29 (2 C), 170.23, 170.19, 170.08, 169.50,

169.45, 169.31, 167.92 [CꢀO×12]; 23.14,

21.49, 21.00, 20.78, 20.72 (3 C), 20.65 (2 C),

20.58, 20.45 [CH₃×11]; 161.10 (CꢀN), 90.71

(CCl₃); Glc, 93.00 (1%), 70.04 (2%), 69.90 (3%),

75.78 (4%), 70.97 (5%), 61.67 (6%); Gal, 101.12

(1¦), 69.90 (2¦), 71.51 (3¦), 67.25 (4¦), 70.50

(5¦), 61.54 (6¦); Neu, 53.11 (OMe§), 96.74

(2§), 37.37 (3§), 69.27 (4§), 49.15 (5§), 71.99

(6§), 66.74 (7§), 67.73 (8§), 61.99 (9§). b-20

¹H NMR (CDCl₃): l 5.85 (d, 1 H, J_1%,2%7.7 Hz,

1%), 5.20 (dd, 1 H, J_2%,3%10.0 Hz, 2%), 5.27 (dd,

1 H, J_3%,4%7.6 Hz, 3%).

1

MeOH, R_f0.39); a-19 H NMR (CDCl₃): l

2.24, 2.16, 2.11, 2.10, 2.09, 2.08, 2.07, 2.06,

2.04, 2.01, 1.86 [(s, 3 H, CH₃)×11]; Glc, 5.38

(d, 1 H, J_1%,2%3.6 Hz, 1%), 4.86 (dd, 1 H, J_2%,3%

10.3 Hz, 2%), 5.51 (dd, 1 H, J_3%,4%9 Hz, 3%), 3.87

(dd, 1 H, J_4%,5%10 Hz, 4%), 3.64 (ddd, 1 H, J_5%,6a%

5.2, J_5%,6b%2 Hz, 5%), 4.19 (dd, 1 H, J_6a%,6b%12 Hz,

6a%), 4.43 (dd, 1 H, 6b%); Gal, 4.66 (d, 1 H,

J_1¦,2¦8.0 Hz, 1¦), 4.95 (dd, 1 H, J_2¦,3¦10.2 Hz,

2¦), 4.51 (dd, 1 H, J_3¦,4¦3.2 Hz, 3¦), 4.88 (m, 1

H, 4¦), 3.86 (m, 1 H, 5¦), 3.98–4.06 (m, 2 H,

6a¦, 6b¦); Neu, 3.85 (s, 3 H, OMe§), 1.68 (dd,

1 H, J_3ax§,3eq§12.6, J_3ax§,4§12.2 Hz, 3ax§), 2.58

(dd, 1 H, J_3eq§,4§4.6 Hz, 3eq§), 4.89 (m, 1 H,

4§), 3.98–4.06 (m, 2 H, 5§, 9a§), 5.06 (d, 1 H,

J_5§,5§-NH10.2 Hz, 5§-NH), 3.63 (dd, 1 H, J_5§,6§

10.7, J_6§,7§2.6 Hz, 6§), 5.41 (dd, 1 H, J

9.4

7§,8§

Hz, 7§), 5.52 (m, 1 H, 8§), 4.42 (dd, 1 H,

1

J_8§,9b§3, J_9a§,9b§12 Hz, 9b§). b-19 H NMR

(CDCl₃): l Glc, 4.72 (d, 1 H, J_1%,2%8 Hz, 1%),

4.80 (dd, 1 H, J_2%,3%10 Hz, 2%), 5.24 (dd, 1 H,

J_3%,4%9 Hz, 3%).

[Methyl (4§,5§,7§,8§,9§-penta-O-acetyl-h-

neuramin)ate]yl - (2§3¦) - 2¦,4¦,6¦ - tri - O-

acetyl - i - galactopyranosyl - (1¦4%) - 2%,3%,6%-

tri-O-acetyl-h,i-glucopyranosyl trichloroace-

timidate (a,b-20).—To a stirred solution of

a,b-19 (0.335 g, 0.314 mmol) and Cl₃CCN

(0.944 mL, 1.36 g, 9.42 mmol) in 4 mL of dry

CH₂Cl₂, was added DBU (47 mL, 47.8 mg,

0.314 mmol) dropwise over 1 min. After 1.5 h,

the solvent was removed and the residue chro-

matographed (9:1 EtOAc–Me₂CO) to give

trichloroacetimidate a,b-20 (321 mg, 0.265

mg, 84%) [6,22] as an amorphous white solid

that was a 19:1 a,b mixture of anomers iden-

tiﬁed by NMR spectroscopy but was homoge-

neous by TLC (9:1 EtOAc–Me₂CO, R_f0.29):

(2S,3R,4E)-[Methyl (4§,5§,7§,8§,9§-pen-

ta - O - acetyl - h - neuramin)ate]yl - (2§3¦)

-2¦,4¦,6¦ - tri - O - acetyl - i - galactopyranosyl-

(1¦4%)-2%,3%,6%-tri-O-acetyl-h-glucopyran-

osyl-(1%l1) - 3 - benzoyl - 2 - (hexadecanamido) -

4-octadecene-1,3-diol (a-21).—A solution of

trichloroacetimidate a,b-20 (0.321 g, 0.265

mmol), alcohol 10 (0.326 g, 0.508 mmol), and

,

dried 4 A molecular sieves (1 g) in 5 mL of

dry CH₂Cl₂was stirred for 2 h, then F₃B·OEt₂

(65.2 mL, 75.2 mg, 0.530 mmol) was added

over 2 min. After 3 h, the reaction was

quenched with 5 mL of 0.5 M aq NaHCO₃.

The organic phase was dried, concentrated,

and chromatographed (24:1 EtOAc–MeOH)

R.I. Duclos Jr. / Carbohydrate Research 328 (2000) 489–507

503

7–17), 0.88 (t, 3 H, J_17,187.0 Hz, 18); FA,

2.15–2.32 (m, 2 H, 2), 1.60–1.71 (m, 2 H, 3),

1.19–1.35 (m, 24 H, 4–15), 0.88 (t, 3 H, J_15,16

7.0 Hz, 16); ¹³C NMR (CDCl₃): l 172.92,

170.83, 170.61, 170.56, 170.45, 170.34, 170.29,

170.27, 170.18, 169.66, 169.58 (2 C), 167.94,

165.05 [CꢀO×14]; 23.17, 21.50, 20.96, 20.75

(4 C), 20.68 (4 C) [CH₃×11]; 133.14, 129.93,

129.59 (2 C), 128.48 (2 C) [Ar×6]; Glc, 96.91

(1%), 70.49 (2 C, 2%, 3%), 77.20 (4%), 68.47 (5%),

62.06 (6%); Gal, 101.26 (1¦), 69.89 (2¦), 71.42

(3¦), 67.30 (4¦), 70.49 (5¦), 61.54 (6¦); Neu,

53.13 (OMe§), 96.76 (2§), 37.40 (3§), 69.27

(4§), 49.18 (5§), 72.01 (6§), 66.83 (7§), 67.75

(8§), 62.12 (9§); Sph, 67.45 (1), 50.63 (2),

73.37 (3), 124.98 (4), 138.39 (5), 32.31 (6),

28.90–29.70 (7–15), 31.91 (16), 22.67 (17),

14.09 (18); FA, 36.90 (2), 25.78 (3), 28.90–

29.70 (4–13), 31.91 (14), 22.67 (15), 14.09

(16); ESMS (pos, 20 V) m/z 1692 [M+H]⁺,

eluting 232 mg of ceramide derivatives, which

were recovered, then 273 mg of glycosidic

coupling products, followed by 40 mg of un-

coupled trisaccharide, which was combined

with recovered trisaccharide from the synthe-

sis of a,b-19, then re-acetylated, and puriﬁed

to recover a,b-18 (0.112 g, 0.101 mmol). The

crude glycosidic coupling mixture was injected

in four portions onto a preparative HPLC

column (4:1 PhMe–Me₂CO) to elute some

nearly pure a-21, followed by 200 mg of an

intractable mixture of 3:47 a-22, b-23. The

mixture was peracetylated with Ac₂O–pyri-

dine and chromatographed (2:1 PhMe–

Me₂CO). The resulting mixture of a-21 and

b-23 was then easily separated by HPLC

(80.5:19.5 PhMe–Me₂CO) eluting a-21, fol-

lowed by b-23 (184 mg, 0.109 mmol, 41%).

The HPLC fractions, conﬁrmed to be pure by

NMR spectroscopy, were combined to give

by-product a-21 (11.5 mg, 6.80 mmol, 2.6%) as

an amorphous white solid that was homoge-

neous by TLC (2:1 PhMe–Me₂CO, R_f0.23):

mp 40–55 °C (soften) 78–80 °C (melt); [h]_D²²

878 [M+2Na+water]⁺², 846.5 [M+2 H]⁺²

;

ESMS (pos, 60 V) m/z 1692 [M+H]⁺, 1570

[M+H%]⁺, 1097 [Y₂+2 H%]⁺, 878 [M+

2Na+water]⁺², 414 [B%₁]⁺.

1

+39° (c 0.699, CH₂Cl₂); H NMR (CDCl₃): l

(2S,3R,4E)-[Methyl (4§,5§,7§,8§,9§-pen-

ta-O-acetyl-h-neuramin)ate]yl-(2§3¦)-2¦,4¦,

6¦-tri-O-acetyl-i-galactopyranosyl-(1¦4%)-

3%,6%-di-O-acetyl-h-glucopyranosyl-(1%l1)-

3-benzoyl-2-(hexadecanamido)-4-octadecene-

1,3-diol (a-22).—A small amount of a-22 was

separated and was an amorphous white solid

that was homogeneous by TLC (2:1 PhMe–

2.24, 2.15, 2.11, 2.09, 2.08, 2.06 (2 Me), 2.05 (2

Me), 2.01, 1.85 [(s, 3 H, CH₃)×11]; 7.98 (d, 2

H, J 8.2 Hz, Ar), 7.56 (t, 1 H, J 7.4 Hz, Ar),

7.43 (dd, 2 H, Ar); Glc, 4.79 (d, 1 H, J_1%,2%3.8

Hz, 1%), 4.84 (dd, 1 H, J_2%,3%10.2 Hz, 2%), 5.47

(dd, 1 H, J_3%,4%9.4 Hz, 3%), 3.78 (dd, 1 H, J_4%,5%

9.4 Hz, 4%), 3.85 (ddd, 1 H, J_5%,6a%5.4, J_5%,6b%1.7

Hz, 5%), 4.17 (dd, 1 H, J_6a%,6b%11.9 Hz, 6a%), 4.38

(dd, 1 H, 6b%); Gal, 4.63 (d, 1 H, J_1¦,2¦8.0 Hz,

1¦), 4.94 (dd, 1 H, J_2¦,3¦10.1 Hz, 2¦), 4.51 (dd,

1 H, J_3¦,4¦3.3 Hz, 3¦), 4.88 (m, 1 H, 4¦), 3.83

(m, 1 H, 5¦), 4.02 (m, 2 H, 6a¦, 6b¦); Neu, 3.84

(s, 3 H, OMe§), 1.68 (dd, 1 H, J_3ax§,3eq§12.7,

J_3ax§,4§12.3 Hz, 3ax§), 2.58 (dd, 1 H, J_3eq§,4§

4.6 Hz, 3eq§), 4.89 (m, 1 H, 4§), 4.02 (ddd, 1

H, J_4§,5§10, J_5§,5§-NH10.2, J_5§,6§10.7 Hz, 5§),

5.01 (d, 1 H, 5§-NH), 3.63 (dd, 1 H, J_6§,7§2.7

Hz, 6§), 5.40 (dd, 1 H, J_7§,8§9.4 Hz, 7§), 5.52

(m, 1 H, 8§), 3.98 (dd, 1 H, J_8§,9a§4.9, J_9a§,9b§

12.7 Hz, 9a§), 4.42 (dd, 1 H, J_8§,9b§2.6 Hz,

9b§); Sph, 3.61 (dd, 1 H, J_1a,1b10.6, J_1a,22.8

Hz, 1a), 3.77 (dd, 1 H, J_1b,22.8 Hz, 1b), 4.52

(m, 1 H, 2), 5.81 (d, 1 H, J_2,2-NH9.6 Hz,

2-NH), 5.54 (dd, 1 H, J_2,38, J_3,48 Hz, 3), 5.49

(dd, 1 H, J_4,514.9 Hz, 4), 5.88 (dt, 1 H, J_5,66.7

Hz, 5), 2.05 (m, 2 H, 6), 1.19–1.35 (m, 22 H,

1

Me₂CO, R_f0.19); H NMR (DMSO-d₆): l

2.14, 2.10, 2.04, 2.01, 1.99 (2 Me), 1.98, 1.95,

1.90, 1.65 [(s, 3 H, CH₃)×10]; 7.90–8.00 (m,

2 H, Ar), 7.63–7.66 (m, 1 H, Ar), 7.50 (dd, 2

H, J 8.4, 7.2 Hz, Ar); Glc, 4.75 (d, 1 H, J_1%,2%

3.6 Hz, 1%), 3.44 (ddd, 1 H, J_2%,2%-OH8.1, J_2%,3%9.4

Hz, 2%), 4.88 (d, 1 H, 2%-OH), 5.02 (dd, 1 H,

J_3%,4%9.5 Hz, 3%), 3.62 (m, 1 H, 4%), 3.75 (m, 1

H, 5%), 4.03 (dd, 1 H, J_5%,6a%4.2, J_6a%,6b%11.8 Hz,

6a%), 4.29 (dd, 1 H, J_5%,6b%1.0 Hz, 6b%); Gal,

4.52 (d, 1 H, J_1¦,2¦8.0 Hz, 1¦), 4.66 (dd, 1 H,

J_2¦,3¦10 Hz, 2¦), 4.45 (dd, 1 H, J_3¦,4¦3.5 Hz,

3¦), 4.87 (br d, 1 H, 4¦), 3.79 (m, 1 H, 5¦), 3.96

(m, 2 H, 6a¦, 6b¦); Neu, 3.77 (s, 3 H, OMe§),

1.34 (dd, 1 H, J_3ax§,3eq§12.2, J_3ax§,4§12.2 Hz,

3ax§), 2.45 (dd, 1 H, J_3eq§,4§4.4 Hz, 3eq§),

4.73 (ddd, 1 H, J_4§,5§10 Hz, 4§), 3.83 (ddd, 1

H, J_5§,5§-NH10, J_5§,6§10 Hz, 5§), 7.62 (d, 1 H,

5§-NH), 3.64 (m, 1 H, 6§), 5.24 (dd, 1 H,

504

R.I. Duclos Jr. / Carbohydrate Research 328 (2000) 489–507

J_6§,7§2.5, J_7§,8§9.1 Hz, 7§), 5.36 (ddd, 1 H,

J_8§,9a§4.3, J_8§,9b§2.6 Hz, 8§), 3.94 (dd, 1 H,

product b-23 (184 mg, 0.109 mmol, 41%) was

an amorphous white solid that was homoge-

neous by TLC (2:1 PhMe–Me₂CO, R_f0.18):

mp 84–86 °C; [h]²_D²0.00° (c 0.701, CH₂Cl₂),

[h]²_D²0.00° (c 2.41, CH₂Cl₂); ¹H NMR

(CDCl₃): l 2.22, 2.16, 2.08 (2 Me), 2.07 (2

Me), 2.04, 2.02, 2.00, 1.94, 1.85 [(s, 3 H,

CH₃)×11]; 8.00 (d, 2 H, J 8.4 Hz, Ar), 7.56

(t, 1 H, J 7.4 Hz, Ar), 7.43 (dd, 2 H, Ar); Glc,

4.44 (d, 1 H, J_1%,2%7.9 Hz, 1%), 4.85–4.90 (m, 1

H, 2%), 5.17 (dd, 1 H, J_2%,3%9.3, J_3%,4%9.3 Hz, 3%),

3.85 (dd, 1 H, J_4%,5%9.8 Hz, 4%), 3.56 (ddd, 1 H,

J_5%,6a%5.5, J_5%,6b%1.6 Hz, 5%), 4.05 (dd, 1 H, J_6a%,6b%

11.6 Hz, 6a%), 4.35 (dd, 1 H, 6b%); Gal, 4.64 (d,

1 H, J_1¦,2¦8.0 Hz, 1¦), 4.91 (dd, 1 H, J_2¦,3¦10.1

Hz, 2¦), 4.52 (dd, 1 H, J_3¦,4¦3.3 Hz, 3¦),

4.85–4.90 (m, 1 H, 4¦), 3.84 (m, 1 H, 5¦),

3.98–4.05 (m, 2 H, 6a¦, 6b¦); Neu, 3.84 (s, 3

H, OMe§), 1.67 (dd, 1 H, J_3ax§,3eq§12.5, J_3ax§,4§

12.4 Hz, 3ax§), 2.57 (dd, 1 H, J_3eq§,4§4.5 Hz,

3eq§), 4.85–4.90 (m, 1 H, 4§), 3.98–4.05 (m, 2

H, 5§, 9a§), 5.15 (d, 1 H, J_5§,5§-NH10.2 Hz,

5§-NH), 3.64 (dd, 1 H, J_5§,6§10.7, J_6§,7§2.7

Hz, 6§), 5.40 (dd, 1 H, J_7§,8§9.3 Hz, 7§), 5.52

(m, 1 H, 8§), 4.41 (dd, 1 H, J_8§,9b§2.6, J_9a§,9b§

12.6 Hz, 9b§); Sph, 3.63 (dd, 1 H, J_1a,1b11,

J_1a,23 Hz, 1a), 3.98–4.05 (m, 1 H, 1b), 4.45

(m, 1 H, 2), 5.77 (d, 1 H, J_2,2-NH9.2 Hz,

2-NH), 5.54 (dd, 1 H, J_2,36.7, J_3,47.5 Hz, 3),

5.46 (dd, 1 H, J_4,515.2 Hz, 4), 5.86 (dt, 1 H,

J_5,66.7 Hz, 5), 2.05 (m, 2 H, 6), 1.19–1.35 (m,

22 H, 7–17), 0.88 (t, 3 H, J_17,186.9 Hz, 18);

FA, 2.14 (m, 2 H, 2), 1.59 (m, 2 H, 3),

1.19–1.35 (m, 24 H, 4–15), 0.88 (t, 3 H, J_15,16

6.9 Hz, 16); ¹³C NMR (CDCl₃): l 175.19,

172.67, 170.81, 170.56, 170.32, 170.27, 170.23,

170.16, 169.70, 169.67, 169.56, 169.50, 167.90,

165.17 [CꢀO×14]; 23.12, 21.47, 20.87, 20.79,

20.71 (3 C), 20.62 (2 C), 20.58, 20.52 [CH₃×

11]; 132.98, 130.20, 129.56 (2 C), 128.35 (2 C)

[Ar×6]; Glc, 100.35 (1%), 71.81 (2%), 72.99 (3%),

76.04 (4%), 72.79 (5%), 62.06 (6%); Gal, 100.92

(1¦), 69.81 (2¦), 71.31 (3¦), 67.26 (4¦), 70.44

(5¦), 61.52 (6¦); Neu, 53.09 (OMe§), 96.72

(2§), 37.35 (3§), 69.27 (4§), 49.08 (5§), 71.99

(6§), 66.84 (7§), 67.77 (8§), 62.15 (9§); Sph,

67.38 (1), 50.69 (2), 74.11 (3), 124.62 (4),

137.40 (5), 32.29 (6), 28.90–29.63 (7–15),

31.87 (16), 22.63 (17), 14.06 (18); FA, 36.80

(2), 25.68 (3), 28.90–29.63 (4–13), 31.87 (14),

22.63 (15), 14.06 (16); ESMS (pos, 20 V) m/z

J_9a§,9b§12.6 Hz, 9a§), 4.25 (dd, 1 H, 9b§); Sph,

3.60 (m, 1 H, 1a), 3.68 (m, 1 H, 1b), 4.36 (m,

1 H, 2), 7.95 (d, 1 H, J_2,2-NH9.0 Hz, 2-NH),

5.46 (dd, 1 H, J_2,36.3, J_3,47.2 Hz, 3), 5.53 (dd,

1 H, J_4,515.3 Hz, 4), 5.75 (dt, 1 H, J_5,66.5 Hz,

5), 2.05 (m, 2 H, 6), 1.15–1.35 (m, 22 H,

7–17), 0.85 (t, 3 H, J_17,186.8 Hz, 18); FA, 2.15

(m, 2 H, 2), 1.50 (m, 2 H, 3), 1.15–1.35 (m, 24

1

H, 4–15), 0.85 (t, 3 H, J_15,166.8 Hz, 16); H

NMR (CDCl₃): l 2.25, 2.16, 2.14, 2.10, 2.08,

2.07, 2.05 (2 Me), 2.01, 1.86 [(s, 3 H, CH₃)×

10]; 8.02 (d, 2 H, J 8.4 Hz, Ar), 7.57 (t, 1 H,

J 7.4 Hz, Ar), 7.44 (dd, 2 H, Ar); Glc, 4.71 (d,

1 H, J_1%,2%3.7 Hz, 1%), 3.55 (dd, 1 H, J_2%,3%9.4

Hz, 2%), 5.29 (dd, 1 H, J_3%,4%9.4 Hz, 3%), 3.72

(dd, 1 H, J_4%,5%9.7 Hz, 4%), 3.80–3.90 (m, 1 H,

5%), 4.17 (dd, 1 H, J_5%,6a%5.7, J_6a%,6b%11.0 Hz,

6a%), 4.36 (br d, 1 H, 6b%); Gal, 4.67 (d, 1 H,

J_1¦,2¦8.0 Hz, 1¦), 4.96 (dd, 1 H, J_2¦,3¦10.2 Hz,

2¦), 4.52 (dd, 1 H, J_3¦,4¦3.0 Hz, 3¦), 4.89 (br d,

1 H, 4¦), 3.80–3.90 (m, 1 H, 5¦), 4.01 (dd, 1

H, J_5¦,6a¦6.4, J_6a¦,6b¦11.4 Hz, 6a¦), 4.07 (dd, 1

H, J_5¦,6b¦6.8 Hz, 6b¦); Neu, 3.85 (s, 3 H,

OMe§), 1.69 (dd, 1 H, J_3ax§,3eq§12.6, J_3ax§,4§

12.4 Hz, 3ax§), 2.59 (dd, 1 H, J_3eq§,4§4.2 Hz,

3eq§), 4.90 (ddd, 1 H, J_4§,5§10 Hz, 4§), 4.04

(ddd, 1 H, J_5§,5§-NH10.2, J_5§,6§10.7 Hz, 5§),

5.01 (d, 1 H, 5§-NH), 3.63 (dd, 1 H, J_6§,7§2.1

Hz, 6§), 5.39 (dd, 1 H, J_7§,8§9.3 Hz, 7§), 5.54

(m, 1 H, 8§), 3.96 (dd, 1 H, J_8§,9a§5.2, J_9a§,9b§

12.7 Hz, 9a§), 4.42 (dd, 1 H, J_8§,9b§1.5 Hz,

9b§); Sph, 3.57 (dd, 1 H, J_1a,1b11, J_1a,23 Hz,

1a), 3.80–3.90 (m, 1 H, 1b), 4.51 (m, 1 H, 2),

5.83 (d, 1 H, J_2,2-NH9.1 Hz, 2-NH), 5.71 (dd,

1 H, J_2,38.1, J_3,48.1 Hz, 3), 5.53 (dd, 1 H, J_4,5

15.3 Hz, 4), 5.92 (dt, 1 H, J_5,66.7 Hz, 5), 2.05

(m, 2 H, 6), 1.20–1.40 (m, 22 H, 7–17), 0.88

(t, 3 H, J_17,186.8 Hz, 18); FA, 2.21 (m, 2 H, 2),

1.62 (m, 2 H, 3), 1.20–1.40 (m, 24 H, 4–15),

0.88 (t, 3 H, J_15,166.8 Hz, 16); ESMS (pos, 20

V) m/z 1650 [M+H]⁺, 825.5 [M+2 H]⁺²

;

ESMS (pos, 60 V) m/z 1650 [M+H]⁺, 1528

[M+H%]⁺, 1054 [Y₂+2 H%]⁺, 856 [M+

2Na+water]⁺², 762 [B₂]⁺, 414 [B₁%]⁺.

(2S,3R,4E)-[Methyl (4§,5§,7§,8§,9§-pen-

ta-O-acetyl-h-neuramin)ate]yl-(2§3¦)-2¦,4¦,

6¦-tri-O-acetyl-i-galactopyranosyl-(1¦4%)-

2%,3%,6%-tri-O-acetyl-i-glucopyranosyl-(1%l1)-

3 - benzoyl - 2 - (hexadecanamido) - 4 - octa-

decene-1,3-diol (b-23).—The desired coupling

R.I. Duclos Jr. / Carbohydrate Research 328 (2000) 489–507

505

1692 [M+H]⁺, 846.5 [M+2 H]⁺²; ESMS

(pos, 60 V) m/z 1692 [M+H]⁺, 1570 [M+

H%]⁺, 1097 [Y₂+2 H%]⁺, 414 [B₁%]⁺.

103.52 (1%), 73.23 (2%), 74.47 (3%), 80.47 (4%),

74.89 (5%), 60.32 (6%); Gal, 104.07 (1¦), 68.55

(2¦), 75.87 (2 C, 3¦, 5¦), 66.67 (4¦), 60.36 (6¦);

Neu, 172.36 (1§-CꢀO), 99.65 (2§), 41.84 (3§),

67.06 (4§), 53.00 (5§), 72.23 (6§), 68.55 (7§),

71.12 (8§), 63.25 (9§), 170.68 (5§-NCꢀO),

22.43 (5§-NAc); Sph, 69.16 (1), 53.00 (2),

70.67 (3), 131.45 (4), 131.29 (5), 31.75 (6),

28.71–29.08 (7–15), 31.29 (16), 22.07 (17),

13.88 (18); FA, 171.80 (NCꢀO), 35.60 (2),

25.38 (3), 28.71–29.08 (4–13), 31.29 (14),

22.07 (15), 13.88 (16); ESMS (pos, 20 V) m/z

1154 [M+H]⁺; ESMS (neg, 60 V) m/z 1152

[M−H]⁻.

(2S,3R,4E)-5§-Acetyl-h-neuraminyl-(2§

3¦)-i-galactopyranosyl-(1¦4%)-i-glucopyra-

nosyl - (1%l1) - 2 - (hexadecanamido) - 4-octade-

cene-1,3-diol (ganglioside GM3) (6).—To a

stirred solution of b-23 (72.2 mg, 42.7 mmol)

in 3 mL of MeOH was added 0.20 mL of 1.3

M NaOMe in MeOH. After 3 h, 1 mL of

water was added and the reaction mixture

stirred for an additional 19 h. The reaction

mixture was cooled to 0 °C (bt), and 0.11 g of

Amberlite IRC-50 resin was added slowly over

1 min. The resin was removed by ﬁltration

through glass wool, washed with MeOH, and

the ﬁltrate concentrated. The residue was

chromatographed (60:35:8 CHCl₃–MeOH–

water) to give ganglioside GM3 6 (49.0 mg,

42.5 mmol, 100 mol%) [18] as an amorphous

white solid that was homogeneous by TLC

(60:35:8 CHCl₃–MeOH–water, R_f0.24) and

by reversed-phase HPLC (17:3 MeOH–wa-

ter): mp 180–190 °C (soften), \220 °C (de-

comp.); [h]²_D²+0.890.8° (c 1.2, 1:1

CHCl₃–MeOH), lit. [h]²_D²+1.5° (c 1.2, 1:1

Acknowledgements

This research was supported by Research

Grant HL-57405 (to G.G.S.) from the Na-

tional Institutes of Health, and the author is

grateful to G.G. Shipley for mentorship. The

Shipley Group and the Biophysics Depart-

ment are gratefully acknowledged, especially

C.J. McKnight and J.M. Vural for assistance

with NMR, and M.S. Gigliotti for GC and

other assistance. ESMS data were obtained by

H. Perreault at the University of Manitoba,

Winnipeg, Canada, and by C.E. Costello, S.

Ye, and S.Y. Chen at the Boston University

School of Medicine Mass Spectrometry Re-

source, which is supported by NIH Grant RR

10888 (to C.E.C.). J.C. Paulson and R.J.

Bayer of Cytel (Noese) provided a generous

amount of recombinant sialyltransferase en-

zyme (a-2,3-NeuAcT, ST3N, EC 2.4.99.6).

Special thanks to S.-L. Wu, S. Lin, D.M.

Small, H. Kadowaki, and P.H. Seeberger.

1

CHCl₃–MeOH) [18]; H NMR (Me₂SO-d₆): l

Glc, 4.17 (d, 1 H, J_1%,2%7.7 Hz, 1%), 3.04 (dd, 1

H, J_2%,3%8.0, 2%), 3.30–3.40 (m, 2 H, 3%, 4%), 3.28

(m, 1 H, 5%), 3.62 (m, 1 H, 6a%), 3.74 (m, 1 H,

6b%); Gal, 4.19 (d, 1 H, J_1¦,2¦7.6 Hz, 1¦),

3.30–3.40 (m, 2 H, 2¦, 5¦), 3.94 (dd, 1 H, J_2¦,3¦

9.8, J_3¦,4¦2.1 Hz, 3¦), 3.72 (m, 1 H, 4¦), 3.44

(dd, 1 H, J_5¦,6a¦6.6, J_6a¦,6b¦10.7 Hz, 6a¦), 3.50

(dd, 1 H, J_5¦,6b¦4.9 Hz, 6b¦); Neu, 1.38 (dd, 1

H, J_3ax§,3eq§11.3, J_3ax§,4§11.6 Hz, 3ax§), 2.73

(dd, 1 H, J_3eq§,4§3.8 Hz, 3eq§), 3.55–3.65 (m,

1 H, 4§), 3.30–3.40 (m, 3 H, 5§, 6§, 9a§), 8.53

(br s, 1 H, 5§-NH), 1.89 (s, 3 H, 5§-NAc),

3.21 (br d, 1 H, J_7§,8§8.9 Hz, 7§), 3.55–3.65

(m, 1 H, 8§), 3.55–3.65 (m, 1 H, 9b§); Sph,

3.41 (dd, 1 H, J_1a,1b9.7, J_1a,24 Hz, 1a), 4.00

(dd, 1 H, J_1b,24.1 Hz, 1b), 3.78 (m, 1 H, 2),

7.52 (d, 1 H, J_2,2-NH9.0 Hz, 2-NH), 3.88 (dd,

1 H, J_2,38.0, J_3,47.1 Hz, 3), 5.34 (dd, 1 H, J_4,5

15.2 Hz, 4), 5.52 (dt, 1 H, J_5,66.7 Hz, 5), 1.92

(m, 2 H, 6), 1.32 (m, 2 H, 7), 1.15–1.35 (m, 20

H, 8–17), 0.85 (t, 3 H, J_17,186.9 Hz, 18); FA,

2.02 (t, 2 H, J_2,37.1 Hz, 2), 1.43 (m, 2 H, 3),

1.15–1.35 (m, 24 H, 4–15), 0.85 (t, 3 H, J_15,16

6.9 Hz, 16); ¹³C NMR (Me₂SO-d₆): l Glc,

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The total synthesis of ganglioside GM3

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