Synthesis of lacto-N-neotetraose and lacto-N-tetraose using the dimethylmaleoyl group as amino protective group

Carbohydrate Research 316 (1999) 121–132

Synthesis of lacto-N-neotetraose and lacto-N-tetraose

using the dimethylmaleoyl group as amino protective

group

Mohamed R.E. Aly, El-Sayed I. Ibrahim ¹, El-Sayed H. El Ashry ²,

Richard R. Schmidt *

Fakulta¨t Chemie, Uni6ersita¨t Konstanz, M 725, D-78457 Konstanz, Germany

Received 9 November 1998; accepted 16 February 1999

Abstract

The disaccharide donor O-[2,3,4,6-tetra-O-acetyl-b-

dimethylmaleimido-a,b- -glucopyranosyl] trichloroacetimidate (7) was prepared by reacting O-(2,3,4,6-tetra-O-

acetyl-a- -galactopyranosyl) trichloroacetimidate with tert-butyldimethylsilyl 3,6-di-O-benzyl-2-deoxy-2-dimethyl-

D

-galactopyranosyl)-(14)-3,6-di-O-benzyl-2-deoxy-2-

D

maleoylamido-glucopyranoside to give the corresponding disaccharide 5. Deprotection of the anomeric center and

then reaction with trichloroacetonitrile afforded 7. Reaction of 7 with 3%-O-unprotected benzyl (2,4,6-tri-O-benzyl-b-

D

-galactopyranosyl)-(14)-2,3,6-tri-O-benzyl-b-

benzyl (2,3,4,6-tetra-O-acetyl-b- -galactopyranosyl)-(14)-(3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-b-

copyranosyl)-(13)-(2,4,6-tri-O-benzyl-b- -galactopyranosyl)-(14)-2,3,6-tri-O-benzyl-b- -glucopyranoside. Re-

placement of the N-dimethylmaleoyl group by the acetyl group, O-debenzylation and ﬁnally O-deacetylation gave

lacto-N-neotetraose. Similarly, reaction of O-[(2,3,4,6-tetra-O-acetyl-b- -galactopyranosyl)-(13)-4,6-O-benzyli-

dene-2-deoxy-2-dimethylmaleimido-a,b- -glycopyranosyl] trichloroacetimidate as donor with 8 as acceptor afforded

the desired tetrasaccharide benzyl (2,3,4,6-tetra-O-acetyl-b- -galactopyranosyl)-(13)-(4,6-benzylidene-2-deoxy-2-

dimethylmaleimido-b- -glucopyranosyl)-(13)-(2,4,6-tri-O-benzyl-b- -galactopyranosyl)-(14)-2,3,6-tri-O-benzyl-

b- -glucopyranoside. Removal of the benzylidene group, replacement of the N-dimethylmaleoyl group by the acetyl

D

-glucopyranoside (8) as acceptor afforded the desired tetrasaccharide

D

-glu-

D

group and then O-acetylation afforded tetrasaccharide intermediate 15, which carries only O-benzyl and O-acetyl

protective groups. O-Debenzylation and O-deacetylation gave lacto-N-tetraose (1). Additionally, known tert-

butyldimethylsilyl (2,3,4,6-tetra-O-acetyl-b-

mido-b- -glucopyranoside was transformed into O-[2,3,4,6-tetra-O-acetyl-b-

di-O-acetyl-2-deoxy-2-dimethylmaleimido-a,b- -glucopyranosyl] trichloroacetimidate as glycosyl donor, to afford

D

-galactopyranosyl)-(13)-4,6-O-benzylidene-2-deoxy-2-dimethylmalei-

D

-galactopyranosyl)-(13)-4,6-

D

with 8 as acceptor the corresponding tetrasaccharide 22, which is transformed into 15, thus giving an alternative

Keywords: Lacto-N-tetraose; Lacto-N-neotetraose; Protecting groups, dimethylmaleoyl; Trichloroacetimidate; Glucosamine

1. Introduction

* Corresponding author. Tel.: +49-7531-882538; fax: +

49-7531-883135.

The tetrasaccharides lacto-N-tetraose (1)

¹Present address: Chemistry Department, Faculty of Sci-

and lacto-N-neotetraose (2) are amongst the

ence, Suez Canal University, Ismailia, Egypt.

²Present address: Chemistry Department, Faculty of Sci-

ence, Alexandria University, Alexandria, Egypt.

oligosaccharides isolated from human milk

[1,2]; the former was the ﬁrst aminodeoxy

PII: S0008-6215(99)00051-8

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M. R.E. Aly et al. / Carbohydrate Research 316 (1999) 121–132

was prepared by the phthalimide method

[20,21]. The benzyl glycoside of 2 [22] as well

as the ceramide of 1 [23] were prepared by

using 2-azido-2-deoxy glucose as a precursor

for the N-acetylglucosamine moiety. The

methyl lacto-N-neotetraoside and derivatives

thereof were prepared by the phthalimide

method [24–26] and the oxazoline method

[27,28]. The latter method was also used for

the synthesis of lacto-N-tetraose by a route

oligosaccharide that was found to occur free

in nature [3–5]. Oligosaccharides 1 and 2 rep-

resent core structural elements of more com-

plex oligosaccharides in human milk [1,2], in

glycolipids, such as paragloboside and sialo-

sylparagloboside, and in glycoproteins. These

oligosaccharides are useful in the recognition

of the acceptor speciﬁcity of glycosyltrans-

ferases, the substrate speciﬁcity of glycosi-

dases, and the structure of antigenic

determinants [1]. Thus, the sequence con-

tained in lacto-N-neotetraose had been recog-

nized by an antibody bound to granulocytes

that was obtained by immunization with nor-

mal human granulocytes [6]. Paragloboside, a

lactoneotetraosyl ceramide, was ﬁrst isolated

from human erythrocytes [7]; it was also iden-

tiﬁed as a speciﬁc surface component of virus

polyoma-transformed hamster embryo ﬁbro-

blast cells [8] and thus it may act as surface

antigen associated with this tumor [9]. The

Neu5Ac a-(26)-lacto-N-neotetraose was

found to cause inhibition of the bacterial in-

fection of a human patient suffering from

pneumonia [10]. Both 1 and 2 act as bacterial

receptors for pneumococci [11,12]. The pen-

tasaccharide HSO₃-3Gal-b-(13)-GlcNAc-b-

(13)-Gal-b-(14)Glc is one of the most

potent oligosaccharide ligands for human E-

selectin [13]. It is a strong supporter of the

adhesion of E-selectin-expressing cells and an

inhibitor of E-selectin adhesion. Such in-

hibitors may be used for the treatment of

disorders of inﬂammation and for minimizing

risk of metastasis after surgery reactions of

tumors [13–15]. Partially protected lacto-N-

neotetraoses were prepared as intermediates

for the synthesis of blood group I-active bi-

antennary neolacto-type decaosyl ceramide

[16,17].

utilizing the oxazoline derivative of b-

D-galac-

topyranosyl-(13)- -glucosamine (lacto-N-

D

biose I) as a donor for a suitably protected

lactose acceptor [29–31]. Also the 2-O-methyl

derivative of the galactose moiety at the non-

reducing end has been prepared via the oxazo-

line method [32]. Recently, we introduced the

dimethylmaleoyl (DMM) group as a new

amino protecting group for glucosamine [33].

Its ease of attachment and subsequent cleav-

age as well as its electron-withdrawing prop-

erty make the

D-glucosamine derivative a

good glycosyl donor with an enforced capabil-

ity of forming a b-linkage. Moreover, versatile

glycosyl acceptors containing the dimethylma-

leoylamido group could also be generated and

successfully employed in glycosylation reac-

tions [33]. Owing to these perspectives of the

DMM group, we report herein synthetic

routes, using this group, to the biologically

important lacto-N-neotetraose (2) and lacto-

N-tetraose (1). The generated building blocks

also allow their further use as donors or ac-

ceptors for the preparation of various

oligosaccharides.

2. Results and discussion

For the synthesis of the target tetrasacchar-

ides, ﬁnal linkage of two disaccharides has

been designed. Thus, the disaccharide donor

will have the glucosamine residue containing

the DMM group at the reducing end. The

required disaccharide donor 7 was synthesized

in three steps from known acceptor 4 [33] by

its galactosidation with known trichloroacet-

One of the crucial points in the syntheses of

oligosaccharides incorporating 2-amino-2-de-

oxy- -glucose necessitates its availability as a

D

donor that requires a suitable protection of

the amino group. Various protecting groups

for such purposes are now available. Thus, the

2-deoxy-2-phthalimido sugars were used as

glycosyl donors for the synthesis of lacto-N-

neotetraose derivatives [16,17], of p-nitro-

phenyl lacto-N-tetraoside [18], and of benzyl

lacto-N-neotetraoside [19]. Also lacto-N-biose

imidate

3

[34] under the catalysis of

trimethylsilyl triﬂuoromethanesulfonate (Me₃-

SiOTf) to give the b-linked disaccharide 5 in

84% yield. Its H NMR spectrum supported

1

M. R.E. Aly et al. / Carbohydrate Research 316 (1999) 121–132

123

the assignment; two doublets for H-1 and H-1%

at l 5.10 and 4.61 with a J value of 8.0 Hz for

each indicated the axial nature of H-1 and

H-2 in both rings. The tert-butyldimethylsilyl

(TBDMS) group was cleaved from 5 with

tetrabutylammonium ﬂuoride (TBAF) to give

6, which can be readily transformed into the

trichloroacetimidate donor 7. Its reaction with

the known disaccharide acceptor benzyl

presence of four signals at

l

100.20,

100.70, 102.70, and 102.80 corresponding to

the four b-linked anomeric carbons. Deblock-

ing of the DMM group from 9 with NaOH to

open the DMM ring followed by treatment

with HCl to cleave the presumably formed

butenolide and then intermediate acetylation

gave 10 in 71% yield. On the other hand,

using hydrazine hydrate as a deblocking agent

followed by acetylation led to the formation

of 10 in 54% yield. The success of this trans-

(2,4,6 - tri - O - benzyl - b -

D

- galactopyranosyl)-

-glucopyran-

(14)-2,3,6-tri-O-benzyl-b-

D

1

oside (8) [35] gave the desired tetrasaccharide

formation was readily conﬁrmed by H NMR

spectroscopy, which exhibited the disappear-

9; the ¹³C NMR spectrum showed the

Scheme 1.

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M. R.E. Aly et al. / Carbohydrate Research 316 (1999) 121–132

hydrogenation led to deblocking of the benzyl

groups which, upon acetylation, gave 11 in

95% yield as the peracetyl derivative of 2. The

¹H NMR spectrum of 11 indicated its presence

as a mixture of a (l 6.23) and b (l 5.64)

anomers in a ratio of 3:2. Almost the same

ratio was also reported based on the calcula-

1

tion of speciﬁc rotations [28] and H NMR

spectroscopy [21]. Deacylation of 11 gave 2 in

91% yield. The ¹³C NMR data of 2 were in

accordance with published values [35]; reacety-

lation of 2 led again to compound 11, thus

conﬁrming the structure of 2 (Scheme 1).

For the synthesis of the lacto-N-tetraose 1,

two approaches were designed. Both methods

were based on the coupling of a disaccharide

donor with the disaccharide acceptor 8 [36].

The designation of these two routes has been

developed in order to allow for the presence of

temporary blocking groups on the resulting

tetrasaccharide thus making it suitable as ac-

ceptor for other mono- and oligosaccharide

donors. In the ﬁrst approach known donor 12

[33] was coupled with 8 to give 13 in 76% yield.

1

Although its H NMR spectrum showed a

doublet at l 5.27 for the newly formed glyco-

sidic bond with a J value of 8.4 Hz conﬁrming

its b-conﬁguration, the signals for the

anomeric protons are hidden under other sig-

nals. However, their coupling with H-2 can be

depicted from the signals for H-2. On the other

hand, the ¹³C NMR spectrum showed signals

at l_c100.30, 100.50, and 102.80 corresponding

to the four b-linked anomeric carbons. Deben-

zylidenation of 13 with p-TsOH gave 14 whose

NMR spectra showed the absence of the ben-

zylidene CH. The transformation of the DMM

group to an acetyl group was achieved by the

action of alkali and then acidiﬁcation followed

by acetylation to give 15 in 76% yield. Cata-

lytic hydrogenation of 15, followed by acetyla-

tion, gave the peracetyl derivative 16 as a

mixture of a and b anomers in a ratio of 1:2,

respectively (Scheme 2).

Scheme 2.

ance of the methyl signals of the dimethylmale-

oyl group and the appearance of an NH signal

at l 5.8 and of a new methyl signal. Catalytic

Alternatively, the required disaccharide

donor 21 was prepared from known 17 [33] by

debenzylidenation with p-TsOH to give 18

whose O-acetylation gave 19. Subsequent re-

moval of the TBDMS group with TBAF gave

1-O-unprotected 20, thus being suitable for the

activation with trichloroacetonitrile in the

presence of DBU to give trichloroacetimidate

Scheme 3.

M. R.E. Aly et al. / Carbohydrate Research 316 (1999) 121–132

125

out on silica gel (Baker, 30–60 (mm) and

Lichroprep NH₂, particle size 40–63 mm (E.

Merck). Medium-pressure liquid chromatog-

raphy (MPLC): LiChroprep Si 60 (E. Merck;

size 15–25 mm), detection by differential re-

fractometer. Optical rotations were deter-

mined at 25 °C with a Perkin–Elmer 241/MC

polarimeter (1 dm cell). NMR spectra were

recorded with Bruker AC 250 and 600 DRX

instruments, using tetramethylsilane as inter-

21. Coupling of 21 with 8 under standard

conditions gave tetrasaccharide 22 in 75%

1

yield. Its H NMR spectrum showed for the

H-1c a doublet at l 5.16 with a coupling

constant of 8.4 Hz, thus conﬁrming the b-

conﬁguration. Moreover, its ¹³C NMR spec-

trum showed signals at l_c99.90, 100.50, 102.8

(2C), conﬁrming the presence of the four

anomeric centers. Removal of the DMM

group from 22 and subsequent acetylation

afforded 15, which was identical to the mate-

rial obtained via the ﬁrst approach (Scheme

3).

In conclusion, lacto-N-tetraose (1) and

lacto-N-neotetraose (2) were successfully syn-

thesized by the elaboration of the newly devel-

oped protection of amino sugars with

dimethylmaleic anhydride providing the

dimethylmaleimido group. Cleavage by addi-

tion of alkali followed by acid can be success-

fully performed as exhibited in our ﬁrst report

[33], using this group for amino sugars, and in

other reports, using it for amino acids and

proteins [37–41]. Its combination with the

trichloroacetimidate leaving group [42] at C-1

of a glucosamine residue either in monomeric

form or as part of an oligosaccharide provides

good glycosyl donors in order to form b-link-

ages. Moreover, the DMM group tolerates

manipulations of the common temporary pro-

tective groups required during oligosaccharide

synthesis and, in addition, it can be selectively

removed under quite mild conditions. The

partially protected target tetrasaccharides pre-

pared in this paper, as well as their precursors,

can be used for the elaboration of various

higher oligosaccharides.

1

nal standard. The assignments of H NMR

spectra were based on chemical shift correla-

tion (DQFCOSY) and rotating frame nuclear

Overhauser effect spectroscopy (ROESY). The

assignments of ¹³C NMR spectra were based

on carbon–proton shift-correlation heteronu-

clear multiple quantum coherence (HMQC).

MS spectra were recorded with MALDI-

Kompakt (Kratos), EI and FAB with Finnin-

gen MAT 312/AMD. Microanalyses were

performed in the Microanalysis Unit at the

Fakulta¨t fu¨r Chemie, Universita¨t Konstanz.

tert-Butyldimethylsilyl

acetyl-i- -galactopyranosyl)-(14)-3,6-di-O-

benzyl-2-deoxy-2-dimethylmaleimido-i- -glu-

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

D

copyranoside (5).—A solution of 3 (0.25 g,

0.52 mmol) and 4 (0.24 g, 0.4 mmol) in dry

CH₂Cl₂(2 mL) was stirred under nitrogen at

rt for 10 min while Me₅SiTf (0.01 M in

CH₂Cl₂, 0.46 mL) was added dropwise. After

45 min, the reaction mixture was neutralized

with Et₃N and evaporated under reduced pres-

sure. The residue was puriﬁed by ﬂash chro-

matography (2:1 petroleum EtOAc) to yield 5

(0.32 g, 84%) as an oil. TLC (2:1 petroleum

EtOAc): R_f0.34, [h]_D+19.8° (c 0.6, CHCl₃);

¹H NMR (600 MHz, CDCl₃): l 7.36–7.10 (m,

10 H, 2 Ph), 5.26 (d, 1 H, J_3,42.9 Hz, H-4b),

5.13 (dd, 1 H, J_1,28.0, J_2,310.4 Hz, H-2b),

5.10 (d, 1 H, J_1,28.0 Hz, H-1a), 4.86 (dd, 1 H,

H-3b), 4.61 (d, 1 H, H-1b), 4.73, 4.50 (2 d, 2

H, J_gem12.2 Hz, CH₂Ph), 4.85, 4.41 (2 d, 2 H,

J_gem12.4 Hz, CH₂Ph), 4.12 (dd, 1 H, J_2,310.8,

J_3,48.8 Hz, H-3a), 3.98–3.93 (m, 3 H, H-4a,

H-6b, H-6%b), 3.84 (dd, 1 H, H-2a), 3.73 (dd, 1

H, J_gem11.0, J_5,6%3.4 Hz, H-6%a), 3.68–3.65

(m, 2 H, H-5b, H-6a), 3.44 (m, 1 H, H-5a),

2.05, 1.99, 1.98, 1.95 (4 s, 12 H, 4 CH₃CO),

1.80 (br.s, 6 H, 2 CH₃), 0.72 [s, 9 H,

SiC(CH₃)₃], 0.02 (s, 3 H, SiCH₃), −0.09 (s, 3

3. Experimental

General methods.—Solvents were puriﬁed in

the usual way. TLC was performed on plastic

plates Silica Gel 60 F₂₅₄and on HPTLC plates

NH₂F₂₅₄S (E. Merck, layer thickness 0.2

mm). The detection was achieved by treatment

with a solution of 20 g ammonium molybdate

and 0.4 g cerium(IV) sulfate in 400 mL 10%

H₂SO₄or with 15% H₂SO₄, and heating at

150 °C. Flash chromatography was carried

126

M. R.E. Aly et al. / Carbohydrate Research 316 (1999) 121–132

[MNaI]Na⁺. Anal. Calcd for C₄₂H₄₇N₂O₁₆Cl₃

(942.18): C, 53.53; H, 5.02; N, 2.97. Found: C,

53.18; H, 5.02; N, 3.09.

13

H, SiCH₃). C NMR (150.9 MHz, CDCl₃): l

170.31, 170.20, 170.03, 169.17 (4 CH₃CO, 2

CO), 139.10–136.69, 128.51–127.03 (2 Ph),

100.38 (C-1b), 93.43 (C-1a), 78.03 (C-4a),

74.76 (C-3a), 74.08 (C-5a), 73.58 (CH₂Ph),

71.03 (CH₂Ph), 70.48 (C-3b), 69.53 (C-5b),

67.72 (C-2b), 67.06 (C-6a), 66.95 (C-4b), 60.85

(C-6b), 57.54 (C-2a), 25.32 [SiC(CH₃)₃], 20.75,

20.64, 20.59, 20.54 (4 CH₃CO), 17.57

[SiC(CH₃)], 8.00 (2 CH₃), −4.24 (SiCH₃),

−5.62 (SiCH₃). FABMS (positive mode,

NBOH/NaI-matrix): m/z 934 [MNa⁺]. Anal.

Calcd for C₄₆H₆₁NO₁₆Si (912.04): C, 60.57; H,

6.74; N, 1.53. Found: C, 60.17; H, 6.94; N,

1.50.

Benzyl (2,3,4,6-tetra-O-acetyl-i-

pyranosyl)-(14)-(3,6-di-O-benzyl-2-deoxy-

2 - dimethylmaleimido - i - - glucopyranosyl)-

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

osyl)-(14)-2,3,6-tri-O-benzyl-i- -glucopyr-

D-galacto-

D

anoside (9).—A mixture of 7 (0.414 g, 0.439

mmol) and 8 (0.333 g, 0.342 mmol) in dry

MeCN (2 mL) was stirred under nitrogen at rt

for 10 min while Me₃SiOTf (0.01 M in MeCN,

0.4 mL) was added dropwise. After 2 h, the

solution was neutralized with triethylamine

and evaporated under reduced pressure. The

residue was puriﬁed by ﬂash chromatography

(2:1 petroleum ether–EtOAc) to yield 9 (0.501

g, 83%) as a white foam. TLC (2:1 petroleum

ether–EtOAc): R_f0.14; [h]_D+11.2° (c 0.4,

CHCl₃); ¹H NMR (600 MHz, CDCl₃): l

7.34–7.07 (m, 45 H, 9 Ph), 5.26 (d, 1 H, J_3,4

3.0 Hz, H-4d), 5.21 (d, 1 H, J_1,28.4 Hz, H-1c),

5.14 (dd, 1 H, J_1,28.4, J_2,310.2 Hz, H-2d),

5.04 (d, 1 H, J_gem11.4 Hz, CHHPh), 4.92 (d,

1 H, J_gem10.5 Hz, CHHPh), 4.88–4.82 (m, 4

H, H-3d, 1.5 CH₂Ph), 4.69 (d, 1 H, J_gem10.8

Hz, CHHPh), 4.66 (d, 1 H, J_gem11.8 Hz,

CHHPh), 4.60–4.56 (m, 3 H, H-1d, CH₂Ph),

4.48 (d, 1 H, J_gem11.4 Hz, CHHPh), 4.47 (d,

1 H, J_gem12.1 Hz, CHHPh), 4.44 (d, 1 H, J_gem

11.8 Hz, CHHPh), 4.39–4.26 (m, 7 H, H-1b,

H-1a, 2.5 CH₂Ph), 4.21–4.17 (m, 2 H, H-3c,

CHHPh), 4.03–4.01 (m, 3 H, H-2c, H-4b,

H-4c), 3.93 (m, 2 H, H-6d, H-6%d), 3.88 (m, 1

H, H-4a), 3.77–3.71 (m, 2 H, H-6c, H-6%c),

3.63 (m, 1 H, H-5d), 3.55–3.51 (m, 5 H, H-5c,

H-6%b, H-2b, H-3b, H-6%a), 3.40–3.35 (m, 5 H,

H-6b, H-6a, H-5b, H-2a, H-3a), 3.01 (m, 1 H,

H-5a), 2.07, 1.99, 1.98, 1.97 (4 s, 12 H, 4

CH₃CO), 1.56 (br.s, 6 H, 2 CH₃). ¹³C NMR

(150.9 MHz, CDCl₃): l 102.80 (C-1a), 102.70

(C-1b), 100.70 (C-1d), 100.20 (C-1c), 83.40

(C-3a), 82.50 (C-3b), 82.00 (C-2a), 79.30 (C-

2b), 78.20 (C-4c), 77.30 (C-3c), 76.90 (C-4b),

76.40 (C-4a), 75.80 (CH₂Ph), 75.30 (2

CH₂Ph), 75.20 (C-5a), 74.70 (C-5, CH₂Ph),

74.60 (CH₂Ph), 74.20 (CH₂Ph), 73.80

(CH₂Ph), 73.50 (CH₂Ph), 73.30 (C-5b), 71.30

(CH₂Ph, C-3d), 70.80 (C-5d), 69.90 (C-2d),

68.60 (C-6b), 68.30 (C-6c), 68.20 (C-6a), 67.20

(C-4d), 60.90 (C-6d), 56.40 (C-2c). MALDI-

O-[(2,3,4,6-Tetra-O-acetyl-i-

anosyl)-(14)-3,6-di-O-benzyl-2-deoxy-2-di-

methylmaleimido - h,i - - glucopyranosyl tri -

D

-galactopyr-

D

chloroacetimidate (7).—A solution of 5 (3.0 g,

3.28 mmol) in dry THF (8 mL) in an ice-salt

bath was treated with glacial AcOH (0.21 mL,

3.54 mmol) and TBAF (0.1 M, 3.6 mL, 3.6

mmol) with stirring. After 1 h, the ice bath

was removed and the solution was stirred

overnight then diluted with a saturated NaCl

solution (20 mL) and extracted with CH₂Cl₂

(3×20 mL). The organic layer was separated,

dried over anhydrous magnesium sulfate and

evaporated under reduced pressure. The

residue was puriﬁed by ﬂash chromatography

(1.5:1 EtOAc–petroleum ether) to yield 6 (2.2

g, 83%) as a white foam. A mixture of 6 (2.2

g, 2.75 mmol), trichloroacetonitrile (0.6 mL,

5.8 mmol) and 1,8-diazabicyclo(5,4,0)undec-7-

ene (0.02 mL, 0.12 mmol) in dry CH₂Cl₂(6

mL) was stirred at rt for 8 h and then concen-

trated under reduced pressure. The residue

was puriﬁed by ﬂash chromatography (1:1

petroleum ether–EtOAc+1% triethylamine)

to yield 7 (2.23 g, 85%) as a pale yellow foam

in the a:b ratio 1:3. TLC (1:1 petroleum

ether–EtOAc+1% triethylamine): R_f0.5 (b-

1

form) and R_f0.6 (a-form); H NMR (250

MHz, d₆-Me₂SO): l 9.92 (br.s, 0.75 H, NH_b),

8.92 (s, 0.25 H, NH_a), 7.40–7.06 (m, 10 H, 2

Ph), 6.11 (d, 0.75 H, J_1,28.5 Hz, H-1a_b), 5.84

(d, 0.25 H, J_1,24.5 Hz, H-1a_a), 5.25 (d, 1 H,

J_3,42.7 Hz, H-4b), 5.10–3.41 (m, 16 H), 2.07,

2.00, 1.96, 1.92, 1.75 (5 s, 18 H, 2 CH₃, 4

COCH₃). FABMS (positive mode, NBOH/

NaI-matrix): m/z 965 [MNa⁺], 1115

M. R.E. Aly et al. / Carbohydrate Research 316 (1999) 121–132

127

(CH₂Ph), 73.60 (CH₂Ph), 73.30 (CH₂Ph),

73.20 (CH₂Ph), 73.10 (CH₂Ph), 73.00 (C-5b),

70.80 (CH₂Ph), 70.60 (C-3d), 70.40 (C-5d),

69.30 (C-2d), 68.30 (C-6c), 68.10 (C-6b), 68.00

(C-6a), 66.70 (C-4d), 60.50 (C-6d), 55.10 (C-

2c). MALDIMS (positive mode, DHB/THT-

matrix): m/z 1711 [MNa⁺]. Anal. Calcd for

C₉₇H₁₀₇N O₂₅(1686.83): C, 69.06; H, 6.39; N,

0.83. Found: C, 68.64; H, 6.55; N, 0.71.

MS (positive mode, DHB/THF-matrix): m/z

1776 [MNa⁺]. Anal. Calcd for C₁₀₁H₁₀₉NO₂₆

(1752.89): C, 69.20; H, 6.26; N, 0.79. Found:

C, 69.26; H, 6.68; N, 0.78.

Benzyl (2,3,4,6-tetra-O-acetyl-i-

pyranosyl)-(14)-(2-acetamido- 2-deoxy-3,6-

di-O-benzyl-i- -glucopyranosyl)-(13)-(2,4,-

6-tri-O-benzyl-i- -galactopyranosyl)-(14)-

2,3,6-tri-O-benzyl-i- -glucopyranoside (10)

D

-galacto-

D

Method A. A mixture of 9 (0.542 g, 0.309

mmol) and NaOH (1.5 g, 0.037 mol) in

MeOH–dioxane–water (5:2:1, 16 mL) was

stirred overnight. Then, the solution was neu-

tralized with 1:1 concd HCl–H₂O and the pH

was adjusted to 5 by N HCl. After 24 h, the

solution was neutralized with ethanolamine

and dried well under reduced pressure. The

residue was treated with pyridine–AcOH (2:1,

24 mL). After 20 h, the reaction mixture was

co-evaporated with toluene under reduced

pressure. The residue was diluted with water

(30 mL) and extracted with CH₂Cl₂(3×30

mL). The organic layers were combined, dried

over MgSO₄and evaporated under reduced

pressure. The residue was puriﬁed by ﬂash

chromatography (1.5:1 petroleum ether–

EtOAc) and the unreacted material was

treated similarly to yield 10 (0.370 g, 71%) as

a colorless oil. TLC (1.5:1 petroleum ether–

EtOAc): R_f0.09; [h]_D−6.2° (c 0.93, chloro-

form); ¹H NMR (600 MHz, CDCl₃): l

7.34–7.17 (m, 45 H, 9 Ph), 5.80 (br.s, 1 H,

NH), 5.28 (d, 1 H, J_3,43.0, H-4d), 5.12 (dd, 1

H, J_1,2=J_2,39.0 Hz, H-2d), 5.01 (d, 1 H, J_gem

11.6 Hz, CHHPh), 4.98 (d, 1 H, J_gem10.6 Hz,

CHHPh), 4.92–4.31 (m, 20 H, H-1b, H-1a,

H-1d, H-3d, H-1c, 7.5 CH₂Ph), 4.18 (d, 1 H,

J_gem11.6 Hz, CHHPh), 4.01–3.90 (m, 5 H,

H-6d, H-4a, H-4b, H-6%d, H-4c), 3.78–3.72

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

3.67–3.58 (m, 4 H, H-3b, H-5d, H-6a, H-2b),

3.52–3.49 (m, 3 H, H-3a, H-6%b, H-5c), 3.45–

3.41 (m, 2 H, H-5b, H-2a), 3.35 (m, 1 H,

H-6b), 3.24 (2 m, 1 H, H-5a), 2.01, 1.99, 1.98,

1.53 (4 s, 15 H, 5 CH₃CO). ¹³C NMR (150.9

MHz, CDCl₃): l 102.40 (C-1b), 102.30 (C-1a),

101.70 (C-1c), 99.80 (C-1d), 82.70 (C-3a),

81.70 (C-3b), 81.60 (C-2a), 79.70 (C-2b), 78.40

(C-3c), 76.30 (C-4c), 76.10 (C-4b), 75.80 (C-

4a), 75.30 (CH₂Ph), 74.90 (C-5a), 74.80

(CH₂Ph), 74.70 (CH₂Ph), 74.60 (C-5c), 74.50

Method B. A mixture of 9 (0.153 g, 0.087

mmol), hydrazine hydrate (3.096 g, 0.06 mol,

3.0 mL) in dry MeOH (6 mL) was heated

under reﬂux. After 9 h, the solution was evap-

orated under reduced pressure and treated

with Ac₂O and pyridine as described in

Method A to yield 10 (0.08 g, 54%).

(2,3,4,6-Tetra-O-acetyl-i-

syl)-(14)-(2-acetamido-2-deoxy-3,6-di-O-

acetyl-i- -glucopyranosyl)-(13)-(2,4,6-tri-

O-acetyl-i- -galactopyranosyl)-(14)-1,2,3,-

6-tetra-O-acetyl-h,i- -glucopyranose (11).—

D

-galactopyrano-

D

A solution of 10 (0.1 g, 0.059 mmol) in 1:1:1

AcOH–MeOH–dioxane (6 mL) was stirred

under hydrogen in the presence of palladium

on carbon (10% Pd, 0.1 g) at rt. After 48 h,

the reaction mixture was ﬁltered through

Celite and washed with MeOH. The combined

ﬁltrates were concentrated under reduced

pressure. The residue was treated with

pyridine (10 mL), Ac₂O (5 mL) and stirred at

rt. After 20 h, the reaction was worked up as

described for 10. The residue was puriﬁed by

ﬂash chromatography (2:1.5 toluene–acetone)

to yield 11 (0.071 g, 95%) as an amorphous

mass in the ratio of a/b 3:2. TLC (2:1.5

toluene–acetone): R_f0.19; ¹H NMR (600

MHz, CDCl₃): l 6.23 (d, 0.6 H, J_1,2=J_1,2=

3.5 Hz, H-1a_a), 5.63 (d, 0.4 H, J_1,28.3 Hz,

H-1a_b), 5.4 (t, 0.6 Hz, J_2,3=J_3,49.7 Hz, H-

3a_a), 5.32 (m, 2 H, H-4d, NH), 5.28 (m, 1 H,

J_3,43.3 Hz, H-4b), 5.18–5.15 (m, 1.4 H, H-3c,

H-3a_b), 5.8 (dd, 1 H, J_1,2=J_2,38.2 Hz, H-2d),

5.02 (dd, 0.4 H, J_1,28.3, J_2,39.2 Hz, H-2a_b),

4.99–4.95 (m, 2.63 H, H-3d, H-2b, H-2a_a),

4.76 (m, 1 H, H-6%c), 4.65 (m, 1 H, H-1c), 4.52

(d, 1 H, J_1,27.9 Hz, H-1d), 4.39–4.37 (m, 1 H,

H-6%a_b, H-6%a_a), 4.31 (d, 1 H, J_1,27.9 Hz,

H-1b), 4.13–4.02 (m, 5 H, H-6b, H-6%b, H-6d,

H-6%d, H-6a_b, H-6a_a), 3.96–3.94 (m, 1.6 H,

H-6c, H-5a_a), 3.85 (br.t, 1 H, H-5d), 3.78–

3.71 (m, 4.4 H, H-3b, H-5a_b, H-4a_a, H-5b,

128

M. R.E. Aly et al. / Carbohydrate Research 316 (1999) 121–132

1

0.12; [h]_D−8.6° (c 0.5, CHCl₃): H NMR

(600 MHz, CDCl₃): l 7.40–7.10 (m, 40 H, 8

Ph), 5.57 (s, 1 H, CHPh), 5.27 (d, 1 H, J_1,28.4

Hz, H-1c), 5.22 (dd, 1 H, J_3,42.6 Hz, H-4d),

5.00 (dd, 1 H, J_1,28.0, J_2,310.3 Hz, H-2d),

4.94 (d, 1 H, J_gem11.3 Hz, CHHPh), 4.92 (d,

1 H, J_gem10.5 Hz, CHHPh), 4.86 (d, 1 H, J_gem

12.1 Hz, CHHPh), 4.85 (d, 1 H, J_gem10.8 Hz,

CHHPh), 4.81 (dd, 1 H, J_3,43.4 Hz, H-3d),

4.70 (d, 1 H, J_gem10.8 Hz, CHHPh), 4.65 (dd,

1 H, J_2,3=J_3,49.5 Hz, H-3c), 4.61 (d, 1 H, J_gem

10.5 Hz, CHHPh), 4.57 (d, 1 H, J_gem10.5 Hz,

H-4a_b, H-4c), 3.49–3.47 (m, 2 H, H-5c, H-2c),

2.16–1.88 (several s, 42 H, 14 CH₃CO). ¹³C

NMR (150.9 MHz, CDCl₃): l 101.40 (C-1d),

101.10, 100.70 (C-1b), 100.50 (C-1c), 91.80

(C-1a_b), 89.20 (C-1a_a), 76.00 (C-3b), 75.90 (C-

4c), 75.40 (C-4a_a), 75.30 (C-4a_b), 73.90 (C-

5a_b), 73.00 (C-5c), 72.60 (C-3a_b), 72.10 (C-3c),

71.40 (C-5b), 71.30 (C-2a_a), 71.10 (C-3d),

71.00 (C-5d), 70.70 (C-2a_b), 69.60 (C-3a_a),

69.50 (C-2b), 69.40 (C-2d), 69.10 (C-4b), 66.90

(C-4d), 62.10 (C-6a_a), 61.80 (C-6a_b, C-6b),

61.00 (C-6d), 60.50 (C-6c), 55.20 (C-2c).

MALDIMS (positive mode, DHB/THT-ma-

trix): m/z 1277 [MNa⁺]. Anal. Calcd for

C₅₂H₇₁NO₃₄(1254.09): C, 49.79; H, 5.70; N,

1.11. Found: C, 49.81; H, 6.22; N, 1.15.

CHHPH), 4.54–4.51 (m,

2

H, H-1d,

CHHPh), 4.48 (m, 2 H, CH₂Ph), 4.39 (dd, 1

H, J_gem10.5, J_5,6%4.8 Hz, H-6%c), 4.36–4.28 (m,

4 H, H-1a, H-1b, CH₂Ph), 4.27 (d, 1 H, J_gem

12.1 Hz, CHHPh), 4.22 (d, 1 H, J_gem11.8 Hz,

CHHPh), 4.14 (dd, 1 H, J_2,39.5 Hz, H-2c),

4.05 (dd, 1 H, J_gem11.0, J_5,6%8.1 Hz, H-6%d),

3.91–3.83 (m, 4 H, H-6c, H-6d, H-4b, H-4a),

3.77 (dd, 1 H, J_3,4=J_4,59.5 Hz, H-4c), 3.60–

3.48 (m, 6 H, H-5d, H-6%b, H-6%a, H-2b, H-3b,

H-5c), 3.41–3.35 (m, 5 H, H-6a, H-6b, H-5b,

H-3a, H-2a), 2.98 (m, 1 H, H-5a), 2.09, 1.93,

1.90, 1.81 (4 s, 12 H, 4 CH₃CO), 1.55 (br.s, 6

(i -

mido - 2 - deoxy - i -

(i- -galactopyranosyl)-(14)-

D

- Galactopysranosyl)(14) - (2 - aceta -

- glucopyranosyl)(13) -

-glucopyran-

D

ose (2).—A mixture of 11 (0.041 g, 0.0326

mmol), MeOH (6.85 mL) and MeONa soln

(0.195 M, 0.45 mL, 0.087 mmol) was stirred at

rt. After 3 days, the mixture was neutralized

with Amberlite IR 120 resin (H⁺form),

ﬁltered, evaporated under reduced pressure,

and then lyophilized from water to yield 2

(0.021 g, 91%). TLC (1:1:0.5 CH₂Cl₂–

MeOH–AcOH), R_f0.08; HPTLC (4:1 EtOH–

water), R_f0.31. MALDIMS (positive mode,

DHB/THF-matrix): m/z 732 [MNa⁺]. Anal.

Calcd for C₂₆H₄₅NO₂₁·2H₂O (743.64): C,

41.99; H, 6.64; N, 1.88. Found: C, 41.94; H,

6.64; N, 1.68. The ¹H NMR spectroscopy data

(600 MHz, D₂O) are in accordance with litera-

ture data [35].

13

H, 2 CH₃). C NMR (150.9 MHz, CDCl₃): l

102.80 (C-1a, C-1b), 101.90 (C-CHPh), 100.50

(C-1d), 100.30 (C-1c), 83.40 (C-3a), 82.10 (C-

2a), 81.80 (C-3b), 81.20 (C-4c), 79.40 (C-2b),

76.80 (C-4b), 76.30 (C-4a), 75.70 (CH₂Ph),

75.40 (C-3c, CH₂Ph), 75.30 (CH₂Ph), 75.20

(C-5a), 74.50 (CH₂Ph), 73.80 (CH₂Ph), 73.50

(CH₂Ph), 73.40 (C-5b), 71.40 (C-3d), 71.20

(CH₂Ph), 70.70 (C-5d), 69.50 (C-2d), 69.10

(C-6c), 68.50 (C-6b), 68.10 (C-6a), 67.00 (C-

4d), 66.30 (C-5c), 61.20 (C-6d), 56.00 (C-2c).

MALDIMS (positive mode, DHB/THT-ma-

trix): m/z 1684 [MNa⁺]. Anal. Calcd for

C₉₄H₁₀₁NO₂₆(1660.75): C, 67.97; H, 6.13; N,

0.84. Found: C, 68.06; H, 6.41; N, 0.64.

Benzyl (2,3,4,6-tetra-O-acetyl-i-

pyranosyl)-(13)-(4,6-benzylidene-2-deoxy-2-

dimethylmaleimido - i - - glucopyranosyl) -

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

osyl)-(14)-2,3,6-tri-O-benzyl-i- -glucopyr-

D

-galacto-

D

anoside (13).—A mixture of 12 (0.13 g, 0.15

mmol) and 8 (0.123 g, 0.126 mmol) in dry

CH₂Cl₂(1 mL) was stirred under nitrogen

at−30 °C for 10 min while Me₃SiOTf (0.01 M

in CH₂Cl₂, 0.17 mL) was added dropwise.

After 40 min, the solution was neutralized

with triethylamine and evaporated under re-

duced pressure. The residue was puriﬁed by

ﬂash chromatography (2:1 petroleum ether–

EtOAc) to yield 13 (0.16 g, 76%) as a colorless

oil. TLC (2:1 petroleum ether–EtOAc): R_f

Benzyl

topyranosyl) - (13) - (2 - acetamido - 4,6 - di-O-

acetyl-2-deoxy-i- -glucopyranosyl)-(13)-

(2,4,6 - tri - O - benzyl - i - - galactopyranosyl) -

(14)-2,3,6-tri-O-benzyl-i- -glucopyranoside

(2,3,4,6-tetra-O-acetyl-i-

D-galac-

D

(15).—(a) From 13. A mixture of 13 (0.165 g,

0.099 mmol), p-TsOH (0.005 g, 0.026 mmol)

and ethanethiol (0.1 g, 1.62 mmol, 0.12 mL) in

dry CH₂Cl₂(1 mL) was stirred at rt. After 24

h, the solution was neutralized with Et₃N and

evaporated under reduced pressure. The

M. R.E. Aly et al. / Carbohydrate Research 316 (1999) 121–132

129

(b) From 22. Compound 22 (0.169 g, 0.102

mmol) was treated as described for 14 to yield

15 (0.116 g, 71%).

residue was puriﬁed by ﬂash chromatography

(1:1 petroleum ether–EtOAc) to yield 14

(0.118 g, 75%) as an oil; TLC (1:1 petroleum

ether–EtOAc): R_f0.11. A mixture of the

aforementioned oil (0.118 g, 0.075 mmol) and

NaOH (0.17 g, 4.25 mmol) in a dioxane–wa-

ter mixture (5:0.5, 5.5 mL) was stirred at rt.

After 24 h, the pH of the solution was ad-

justed and kept at 5 by N HCl. After 24 h, the

solution was neutralized with K₂CO₃and

dried under reduced pressure in the presence

of ethanolamine (0.2 mL). The residue was

treated with Ac₂O (4 mL) and pyridine (8 mL)

and stirred at rt. After 15 h, the solution was

worked up as described for 10. The residue

was puriﬁed by ﬂash chromatography (1:1

petroleum ether–EtOAc) and the un-reacted

material was separated and deprotected again

to yield 15 (0.091 g, 76%) as a colorless oil.

TLC (1:1 petroleum ether–EtOAc): R_f0.09;

(2,3,4,6-Tetra-O-acetyl-i-

osyl)-(13)-(2-acetamido-2-deoxy-4,6-di-O-

acetyl-i- -glucopyranosyl)-(13)-(2,4,6-tri-

O-acetyl-i- -galactopyranosyl)-(14)-1,2,3,-

6-tetra-O-acetyl-h/i- -glucopyranose (16).—

D

-galactopyran-

D

According to the procedure described for the

preparation of 11, compound 15 (0.111 g, 0.07

mmol) was dissolved in 1:1:1 AcOH–MeOH–

dioxane (4.5 mL) and hydrogenolized in the

presence of Pd/C (10% Pd, 0.1 g) at rt for 48

h, then worked up and the residue was acetyl-

ated. The residue was puriﬁed by MPLC

(1.5:1 toluene–acetone) to yield 16 (0.075 g,

86%) as an amorphous mass in the ratio of

a:b 1:2. TLC (1.5:1 toluene–acetone): R_f0.18;

¹H NMR (600 MHz, CDCl₃): l 6.23 (d, 0.33

H, J_1,23.5 Hz, H-1a_a), 5.65–5.63 (m, 1.7 H,

NH, H-1a_b), 5.41 (t, 0.3 H, J_2,3=J_3,49.8 Hz,

H-3a_a), 5.32–5.31 (m, 2 H, H-4b, H-4d), 5.20

(t, 0.7 H, H-3a_b), 5.11 (t, 1 H, H-1c), 5.02–

4.91 (m, 5 H, H-4c, H-3d, H-2a_a, H-2b, H-2d,

H-2a_b), 4.54 (t, 1 H, J_2,3=J_3,49.8 Hz, H-3c),

4.44–4.38 (m, 3 H, H-6%a_a, H-6%a_b, H-6%c, H-

1d), 4.33, 4.32 (2 d, 1 H, J_1,27.8 Hz, H-1b),

4.14 (dd, 1 H, J_gem12.1, J_5,62.9 Hz, H-6a_a,

H-6a_b), 4.07–4.01 (m, 4 H, H-6b, H-6%b, H-

6d, H-6%d), 3.98–3.96 (m, 1.3 H, H-5a_a, H-6c),

3.82 (t, 1 H, H-5d), 3.77–3.73 (m, 3.7 H,

H-3b, H-5a_b, H-5b, H-4a_a, H-4a_b), 3.59 (br.d,

1 H, H-5c), 2.80 (br.t, 1 H, H-2c), 2.16–1.94

1

[h]_D−12.0° (c 0.15, CHCl₃); H NMR (600

MHz, CDCl₃): l 7.40–7.10 (m, 35 H, 7 Ph),

5.33 (br.s, 1 H, H-4d), 4.99–4.84 (m, 9 H,

NH, H-4c, H-1c, H-3d, H-2d, 2.5 CH₂Ph),

4.73 (d, 1 H, J_gem10.9 Hz, CHHPh), 4.69 (d,

1 H, J_gem10.5 Hz, CHHPh), 4.63–4.57 (m, 3

H, 1.5 CH₂Ph), 4.50 (d, 1 H, J_gem11.7 Hz,

CHHPh), 4.44–4.40 (m, 3 H, H-1b, H-1a,

CHHPh), 4.34–4.33 (m, 2 H, H-1d, CHHPh),

4.21–4.15 (m, 5 H, H-3c, H-6%d, H-6c, H-6%c,

CHHPh), 4.07 (m, 1 H, H-6d), 3.98 (m, 1 H,

H-4a), 3.92 (m, 1 H, H-4b), 3.83 (m, 1 H,

H-5d), 3.76–3.74 (m, 2 H, H-5c, H-6%a), 3.67–

3.66 (m, 2 H, H-6a, H-2b), 3.54–3.45 (m, 5 H,

H-2c, H-2a, H-6%b, H-3a, H-3b), 3.40 (m, 1 H,

H-5b), 3.36 (m, 1 H, H-6b), 3.28 (m, 1 H,

H-5a), 2.13, 2.10, 2.05, 1.99, 1.95, 1.52 (6 s, 21

H, 7 CH₃CO). ¹³C NMR (150.9 MHz,

CDCl₃): l 102.80 (C-1a, C-1b), 101.20 (C-1c),

101.10 (C-1d), 83.10 (C-3a), 82.10 (C-2a, C-

3b), 80.70 (C-2b), 77.10 (C-3c), 76.70 (C-4a),

76.40 (C-4b), 75.50 (C-5a, CH₂Ph), 75.40

(CH₂Ph), 75.20 (CH₂Ph), 75.10 (CH₂Ph),

73.70 (2 CH₂Ph), 73.60 (C-5b), 72.10 (C-5c),

71.30 (C-3d, CH₂Ph), 70.80 (C-5d), 69.80 (C-

4c), 69.40 (C-2d), 68.60 (C-6a), 68.40 (C-6b),

67.20 (C-4d), 63.00 (C-6c), 61.40 (C-6d), 57.40

(C-2c). FABMS (positive mode, NBOH/NaI-

matrix): m/z 1613 [MNa⁺]. Anal. Calcd for

C₈₇H₉₉NO₂₇(1590.66): C, 65.68; H, 6.27; N,

0.88. Found: C, 65.77; H, 6.58; N, 0.71.

13

(several s, 42 H, 14 CH₃CO). C NMR (150.9

MHz, CDCl₃): l 101.30, 100.90 (C-1b), 101.00

(C-1d), 99.00 (C-1c), 91.90 (C-1ab), 89.30 (C-

1aa), 76.50 (C-3b), 76.30 (C-3c), 75.50 (C-

4aa), 75.30 (C-4ab), 74.00 (C-5ab), 72.70

(C-3ab), 72.20, 71.60 (C-5c), 71.40 (C-3d),

71.20 (C-2aa) 71.10 (C-5aa), 70.90 (C-5d),

70.70 (C-2ab), 69.90 (C-2d), 69.70 (C-3aa, C-

2b), 69.50 (C-4d), 69.20 (C-4c), 67.20 (C-4b),

62.00 (C-6a), 61.80 (C-6b), 61.70 (C-6c), 61.20

(C-6d), 59.30 (C-2c). MALDIMS (positive

mode, DHB/THT-matrix): m/z 1278 [MNa⁺].

Anal. Calcd for C₅₂H₇₁NO₃₄(1254.09): C,

49.79; H, 5.70; N, 1.11. Found: C, 49.85; H,

6.23; N, 1.08.

i-

2 - deoxy - i -

galactopyranosyl-(14)-

D

-Galactopyranosyl-(13)-2-acetamido-

- glucopyranosyl - (13) -i -

-glucopyranose (1).

D

-

D

130

M. R.E. Aly et al. / Carbohydrate Research 316 (1999) 121–132

(positive mode, NBOH/NaI-matrix): m/z 754

[MNa⁺]. Anal. Calcd for C₃₂H₄₉NO₁₆Si

(731.80): C, 52.51; H, 6.74; N, 1.91. Found: C,

52.63; H, 6.91; N, 1.77.

—A mixture of 16 (0.053 g, 0.033 mmol),

MeOH (3.4 mL) and NaOMe (0.195 M in

MeOH, 0.45 mL) was stirred at rt. After 3 h,

water (3.4 mL) and NaOMe (0.195 M, 0.90

mL) were added. After 3 days, the reaction

mixture was worked up as described for 2.

The residue was puriﬁed by ﬂash chromatog-

raphy on amino-phase Lichroprep (3:1

EtOH–water) to yield 1 (0.018 g, 78%) as a

foam. HPTLC (4:1 EtOH–water): R_f0.19;

[h]_D+23.9° (c 0.5, H₂O, 3 h); lit. [h]²_D⁴+25.2°

(ﬁnal value) (c 1.5, H₂O) [5]. MALDIMS (pos-

itive mode, DHB/NaI-matrix): m/z 731.6

tert-Butyldimethylsilyl(2,3,4,6-tetra-O-acet-

yl - i -

D

- galactopyranosyl) - (13) - 4,6 - di - O-

acetyl - 2 - deoxy - 2 - dimethylmaleimido - i -

D

-

glucopyranoside (19).—A mixture of 18 (1.51

g, 1.43 mmol), pyridine (15 mL) and acetic

anhydride (7.5 mL) was stirred at rt. After 18

h, the solution was worked up as described for

10. The residue was puriﬁed by ﬂash chro-

matography (1.5:1 petroleum ether–EtOAc)

to yield 19 (1.141 g, 97%) as a white foam.

TLC (1.5:1 petroleum ether–EtOAc: R_f0.11;

1

[MNa⁺]. The H NMR spectroscopy data

(600 MHz, D₂O) were in accordance with

literature data [35].

1

[h]_D−6.6° (c 0.5, CHCl₃), H NMR (600

tert-Butyldimethylsilyl(2,3,4,6-tetra-O-acet-

MHz, CDCl₃): l 5.26 (d, 1 H, J_3,4Hz, H-4b),

5.06 (d, 1 H, J_1,28.1 Hz, H-1a), 4.93 (m, 1 H,

H-2b), 4.92 (m, 1 H, H-4a), 4.84 (dd, 1 H, J_2,3

10.4, J_3,43.4 Hz, H-3b), 4.54 (dd, 1 H, J_2,3

10.8, J_3,49.3 Hz, H-3a), 4.20 (d, 1 H, J_1,27.8

Hz, H-1b), 4.13 (m, 2 H, H-6a, H-6%a), 4.07

(m, 2 H, H-6b, H-6%b), 3.95 (dd, 1 H, J_2,310.8

Hz, H-2a), 3.76 (m, 1 H, H-5b), 3.70 (m, 1 H,

H-5a), 2.10, 2.05, 2.04, 2.03, 2.02, 1.97, 1.91,

1.89 (8 s, 24 H, 2 CH₃, 6 CH₃CO), 0.73 [s, 9

H, SiC(CH₃)₃], 0.02 (s, 3 H, SiCH₃), −0.06 (s,

yl-i-

D

-galactopyranosyl)-(13)-2-deoxy-2-

-glucopyranoside (18).

dimethylmaleimido-i-

D

—A mixture of 17 (1.51 g, 1.84 mmol), p-

TsOH (0.008 g, 0.42 mmol) and ethanethiol

(0.84 g, 13.46 mmol, 1.0 mL) in dry CH₂Cl₂

(15 mL) was stirred at rt. After 20 h, the

solution was neutralized with triethylamine

and evaporated under reduced pressure. The

residue was puriﬁed by ﬂash chromatography

(1:1 petroleum ether–EtOAc) to yield 18

(1.096 g, 81%) as a white foam. TLC (1:1

petroleum ether–EtOAc): R_f0.14; [h]_D−6.4°

13

3 H, SiCH₃). C NMR (150.9 MHz, CDCl₃):

l 170.32, 170.19, 169.29, 169.10 (6 CH₃CO, 2

CO), 100.37 (C-1b), 93.26 (C-1a), 75.08 (C-

3a), 71.86 (C-5a), 70.84 (C-3b), 70.47 (C-5b),

69.70 (C-4a), 68.97 (C-2b), 66.76 (C-4b), 62.70

(C-6a), 60.76 (C-6b), 57.69 (C-2a), 25.27

[SiC(CH₃)₃], 20.78, 20.65, 20.53 (6 CH₃CO),

8.83 (2 CH₃), −4.24 (SiCH₃), −5.60 (SiCH₃).

FABMS (positive mode, NBOH/NaI-matrix):

m/z 838 [MNa⁺], 988 [MNaI]Na⁺. Anal.

Calcd for C₃₆H₅₃NO₁₈Si (815.87): C, 52.99; H,

6.54; N, 1.71. Found: C, 52.87; H, 6.73; N,

1.32.

1

(c 0.7, chloroform); H NMR (600 MHz,

CDCl₃): l 5.31 (d, 1 H, J_3,42.6 Hz, H-4b),

5.13 (dd, 1 H, J_1,28.0, J_2,310.5 Hz, H-2b),

5.07 (d, 1 H, J_1,28.1 Hz, H-1a), 4.91 (dd, 1 H,

J_3,42.6 Hz, H-3b), 4.35 (d, 1 H, H-1b), 4.32

(dd, 1 H, J_2,310.8, J_3,48.4 Hz, H-3a), 4.09 (m,

2 H, H-6b, H-6%b), 3.95 (m, 1 H, H-5b),

3.91–3.88 (m, 2 H, H-6%a, H-2a), 3.75 (dd, 1

H, J_gem11.5, J_5,65.6 Hz, H-6a), 3.54 (dd, 1 H,

J_3,48.4, J_4,59.2 Hz, H-4a), 3.46 (m, 1 H,

H-5a), 2.12, 2.05, 1.95, 1.92, 1.81 (5 s, 18 H, 2

CH₃, 4 CH₃CO), 0.73 [s, 9 H, SiC(CH₃)₃], 0.02

(s, 3 H SiCH₃), −0.07 (s, 3 H, SiCH₃). ¹³C

NMR (150.9 MHz, CDCl₃): l 170.38, 170.10,

170.05, 168.68 (4 CH₃CO, 2 CO), 137.41 (2

C-DMM), 101.03 (C-1b), 93.41 (C-1a), 81.78

(C-3a), 75.36 (C-5a), 71.20 (C-5b), 70.76 (C-

4a), 70.64 (C-3b), 68.60 (C-2b), 66.87 (C-4b),

63.17 (C-6a), 61.48 (C-6b), 56.87 (C-2a), 25.24

[SiC(CH₃)₃], 20.58, 20.53, 20.47, 20.27 (4

CH₃CO), 17.48 [SiC[CH₃)], 8.73 (2 C–CH₃),

−4.10 (SiCH₃), −5.61 (SiCH₃). FABMS

O-[(2,3,4,6-Tetra-O-acetyl-i-

anosyl)-(13)-4,6-di-O-acetyl-2-deoxy-2-di-

methylmaleimido - h/i - - glucopyranosyl]tri -

D

-galactopyr-

D

chloroacetimidate (21).—A mixture of 19

(1.047 g, 1.28 mmol), glacial AcOH (0.084 g,

1.408 mmol, 85.0 mL) and TBAF (0.368 g,

1.41 mmol) in dry THF (10 mL) was reacted

and worked up as described for 6. The residue

was puriﬁed by ﬂash chromatography (2:1

EtOAc–petroleum ether) to yield 20 (0.9 g,

quantitative) as a white foam. TLC (2:1

M. R.E. Aly et al. / Carbohydrate Research 316 (1999) 121–132

131

2.6 Hz, H-4b), 3.91 (m, 1 H, H-4a), 3.75 (m, 1

H, H-5c), 3.71 (m, 1 H, H-5d), 3.60–3.50 (m,

4 H, H-6%b, H-3b, H-2b, H-6%a), 3.41–3.36 (m,

5 H, H-6b, H-5b, H-6a, H-2a, H-3a), 2.99 (m,

1 H, H-5a), 2.10–1.90 (6 s, 18 H, 6 CH₃CO),

1.70 (br.s, 3 H, CH₃), 1.50 (br.s, 3 H, CH₃).

¹³C NMR (150.9 MHz, CDCl₃): l 102.80 (C-

1a, C-1b), 100.50 (C-1d), 99.90 (C-1c), 83.30

(C-3a), 82.60 (C-3b), 82.00 (C-2a), 79.00 (C-

2b), 76.70 (C-4b), 76.30 (C-4a), 75.70

(CH₂Ph), 74.50 (CH₂Ph), 75.20 (C-5a,

CH₂Ph), 74.60 (C-3c), 74.30 (CH₂Ph), 73.70

(CH₂Ph), 73.60 (CH₂Ph), 73.40 (C-5b), 71.90

(C-5c), 71.10 (C-3d, CH₂Ph), 70.80 (C-5d),

69.70 (C-4c), 69.30 (C-2d), 68.50 (C-6b), 68.10

(C-6a), 67.00 (C-4d), 62.80 (C-6c), 60.90 (C-

6d), 56.20 (C-2c). MALDIMS (positive mode,

DHB/THT-matrix): m/z 1679 [MNa⁺]. Anal.

Calcd for C₉₁H₁₀₁NO₂₈(1656.72): C, 65.96; H,

6.14; N, 0.84. Found: C, 65.55; H, 5.96; N,

0.78.

EtOAc–petroleum ether): R_f0.24. A solution

of the aforementioned foam (0.9 g, 1.28

mmol) in dry CH₂Cl₂(5 mL) was treated by

trichloroacetonitrile (0.83 mL, 8.1 mmol) and

DBU (0.03 mL, 1.8 mmol) with stirring. After

9 h, the solution was evaporated under re-

duced pressure. The residue was puriﬁed by

ﬂash

chromatography

(1.5:1

EtOAc–

petroleum ether+1% Et₃N) to yield 21 (0.932

g, 85%) as a ﬂuorescent foam in the ratio of

a:b 1:5. TLC (1.5:1 EtOAc–petroleum

1

ether+1% Et₃N): R_f0.28; H NMR (250

MHz, CDCl₃): l 8.66 (s, 0.15 H, NH_a), 8.62

(s, 0.85 H, NH_b), 6.19 (d, 0.15 H, J_1,23.2 Hz,

H-1a_a), 6.15 (d, 0.85 H, J_1,28.9 Hz, H-1a_b),

5.31–3.76 (m, 13 H), 2.13, 2.11, 2.08, 2.07,

1.98, 1.94, 1.81 (7 s, 24 H, 2 CH₃, 6 CH₃CO).

FABMS (positive mode, NBOH/NaI-matrix):

m/z 869 [MNa⁺], 1019 [MNaI]Na⁺. Anal.

Calcd for C₃₂H₃₉N₂Cl₃O₁₈(846.02): C, 45.42;

H, 4.64; N, 3.31. Found: C, 44.86; H, 4.49; N,

3.43.

Benzyl (2,3,4,6-tetra-O-acetyl-i-

pyranosyl)-(13)-(4,6-di-O-acetyl-2-deoxy-

2 - dimethylmaleimido - i - - glucopyranosyl) -

(13)-(2,4,6-tri-O-benzyl-i- -galactopyrano-

syl)-(14)-2,3,6-tri-O-benzyl-i- -glucopyran-

D

-galacto-

Acknowledgements

D

This work was supported by the European

Community (grant No. ERB 4061 PL 95-

0372), the Deutsche Forschungsgemeinschaft

and the Fonds der Chemischen Industrie.

M.R.E.A. is grateful for a stipend within the

channel program of the Egyptian Govern-

ment. The help of Dr Armin Geyer in struc-

tural assignments is gratefully acknowledged.

D

oside (22).—A mixture of 21 (0.16 g, 0.177

mmol) and 8 (0.15 g, 0.154 mmol) in dry

MeCN (1 mL) was stirred under nitrogen at rt

for 10 min while Me₃SiOTf (0.01 M in MeCN,

0.2 mL) was added dropwise and the mixture

was stirred overnight, then neutralized with

Et₃N and evaporated under reduced pressure.

The residue was puriﬁed by ﬂash chromatog-

raphy (1.5:1 petroleum ether–EtOAc) to yield

22 (0.192 g, 75%) as a white foam. TLC (1.5:1

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Article Doi

Synthesis of lacto-N-neotetraose and lacto-N-tetraose using the dimethylmaleoyl group as amino protective group

DOI: 10.1016/S0008-6215(99)00051-8

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