Design and synthesis of a multivalent homing device for targeting to murine CD22

Bioorganic & Medicinal Chemistry 9 (2001) 85±97

Design and Synthesis of a Multivalent Homing Device for

Targeting to Murine CD22

Leo A. J. M. Sliedregt,^a,ySabine M. W. van Rossenberg,^aReshma Autar,^a

A. Rob P. M. Valentijn,^bGijs A. van der Marel,^bJacques H. van Boom,^b

Christina Piperi,^cP. Anton van der Merwe,^cJohan Kuiper,^a

Theo J.C. van Berkel^aand Erik A. L. Biessen^a,*

^aLeiden/Amsterdam Center for Drug Research, Division of Biopharmaceutics, Sylvius Laboratories, Leiden University,

PO Box 9503, 2300 RA Leiden, The Netherlands

^bLeiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands

^cSir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK

Received 9 February 2000; accepted 9 August 2000

AbstractÐCD22 is a cell-surface glycoprotein uniquely located on mature B-cells and B-cell derived tumour cells. Current evidence

suggests that binding of endogenous ligands to CD22 leads to modulation of B-cell activation by antigen. Incidentally, however, B-

cell activation may derail, and lead to an undesired immune response, for example in cases of allergy, rheumatoid arthritis and

Crohn's disease. In this situation, synthetic high-anity ligands for CD22 may be of therapeutic value as inhibitors of B-cell acti-

vation. Recent studies have revealed that natural ligands for CD22 contain the trisaccharide NeuAca-2,6-Lac as the basic binding

motif. In addition, it has been demonstrated that binding to CD22 is strongly enhanced by multivalent presentation of the basic

binding motif (cluster eect). In this paper, the stepwise development of a novel multivalent high-anity ligand for CD22 is

described. In the ®rst stage, a series of monovalent NeuAca-2,6-Glc(Y)X type binding motifs was prepared, and their anity for

murine CD22 was monitored, to obtain more insight into the eect of separate structure elements on ligand recognition. In the

second stage, we prepared a trivalent cluster, based on the monovalent motif that displayed the highest anity for CD22, NeuAca-

2,6-GlcNBzNO₂OMe (7). This cluster, TRIS(NeuAca-2,6-GlcNBzNO₂)₃(52), displayed a more than 58-fold higher anity for

CD22 than the reference structure NeuAca-2,6-LacOMe (10). To our knowledge, the cluster 52 is one of the most potent antago-

Introduction

therapeutic value as inhibitors of B-cell activation. The

structural features that determine the anity of (syn-

thetic) ligands for human and/or murine CD22 have

been the subject of a number of studies.^{7 13}From these

studies, it appeared that CD22 binds a-2,6-sialylated

glycoproteins, whereas it does not recognise a-2,3-sialy-

lated glycoproteins.^7,8The basic monovalent binding

motif in natural ligands for CD22 was identi®ed as a-2,6-

sialyl-N-acetyllactosamine (Siaa-2,6-LacNAc).⁹Unlike

in the case of the selectins,¹⁴the presence of the Sia

moiety, and in particular the C₇±C₈±C₉glycerol side

arm and the axial anomeric carboxyl function, is

imperative for binding to CD22. The presence of a so-

called cluster eect for CD22 was also suggested, as

multiple sialylated structures were bound more avidly

than monovalent sialosides.^9,10While sharing the above

mentioned binding characteristics, human and murine

CD22 dier in their preference for the substituent at the

C-5 position of Sia. Human CD22 binds equally well to

Sialoadhesins, or siglecs,¹are a group of cell adhesion

molecules within the immunoglobulin superfamily

(IgSF) that bind to glycans with terminal sialic acid (Sia)

residues. The siglec CD22 is a cell-surface glycoprotein

uniquely located on mature B-cells and B-cell derived

tumour cells.^2,3Binding of endogenous ligands to CD22

triggers a cascade of intracellular events, leading to B-cell

activation and proliferation.^{4 6}Incidentally, however, B-

cell activation may derail, and lead to an undesired

immune response, for example in cases of allergy, rheu-

matoid arthritis and Crohn's disease. In this situation,

synthetic high-anity ligands for CD22 may be of

*Corresponding author. Tel.: +31-71-5276040; fax: +31-71-5276032;

e-mail: biessen@lacdr.leidenuniv.nl

^yCurrent address: Solvay Pharmaceuticals, PO Box 900, 1380 DA

Weesp, The Netherlands.

PII: S0968-0896(00)00224-8

86

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97

N-acetyl (NeuAc) and N-glycoloyl (NeuGc) sialic acid-

containing glycans, whereas murine CD22 prefers

NeuGc to NeuAc conjugates.¹²In a recent report, Kelm

et al.¹³investigated the binding of human and murine

CD22 to erythrocytes which presented dierent N-

modi®ed sialyl glycans. Remarkably, replacement of the

respective native N-acyl group on the erythrocyte Sia

moiety by a halogenated acetyl, a formyl, or an ami-

noacetyl group did not aect binding by either human

or murine CD22. In contrast, in the same study,

binding to the siglec myelin associated glycoprotein

(MAG) was shown to improve by a factor 17 by sub-

stitution of the N-acetyl by an N-¯uoroacetyl group.

The most extensive QSAR study for CD22 was

reported by Powell et al.,¹⁰revealing that the minimal

motif required for recognition by human CD22 is a

Figure 1. Possible variations in NeuAca-2,6-Glc(Y)X type ligands for

CD22.

Figure 3. Structures of sialoside donor 14 and acceptors 15±26.

Figure 2. Structures of monovalent ligands 1±13.

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97

87

Table 1. Structures, yields and a/b ratios of protected di- and trisaccharides prepared from donor 14 and acceptors 15±26

Acceptor

Product

X

Y

Yield^a(a/b)^b

15

16

17

18

27

28

29

30

OMe

N₃

O(4-NO₂)Ph

OMe

NHAc

N₃

37% (5/1)

41% (4/1)

28% (6/1)

50% (5/1)

19

20

21

22

31

32

33

34

Ð

NHBz

NHOct

NH(2-Napht)

NH(4-NO₂)Bz

42% (3/1)

30% (3/1)

25% (5/1)

44% (4/1)

23

24

35

36

OMe

N₃

Ð

26% (2/1)

33% (3/1)

25

26

37

38

OMe

O(4-NO₂)Ph

Ð

47% (6/1)

52% (4/1)

^aa/b Mixture.

^bAs gauged by ¹³C NMR analysis.

disaccharide NeuAca-2,6-Hex(NAc), Hex(NAc) being

Gal, GalNAc or GlcNAc. The most potent synthetic

binding motifs in this study displayed a threefold higher

anity for CD22 than the reference structure NeuAca-

2,6-Lac.

mido, benzoylamido, azido) of the glucosamine. In the

second stage, a trivalent cluster, based on the mono-

valent motif that displayed the highest anity for

murine CD22, was prepared and tested, to establish

whether the anity of a synthetic oligosaccharide could

be further enhanced by multivalent presentation.

In summary, from the literature it appears that (a) the

presence of a complete Sia ring structure is essential for

binding to CD22, (b) the group at the C-5 position of

the Sia moiety does not have a dramatic eect on bind-

ing, and (c) the anity for CD22 can be enhanced by

further optimisation of the (carbohydrate) structure

attached to the reducing end of the Sia moiety.

We were interested in developing a novel synthetic high-

anity ligand for CD22. The molecular structure of this

ligand should warrant maximal (selective) binding to

CD22, but also be easily accessible from a synthetic

point of view. These two requirements can be met by

preparing a trivalent glycoconjugate, based on a TRIS

dendritic core used by our group in earlier studies,¹⁵and

a NeuAca-2,6-Hex(NAc) derived minimal motif as pro-

posed by Powell et al., in which (a) the terminal NeuAc

moieties are intact and unmodi®ed, and (b) glucosamine

serves as the basic skeleton for the underlying carbohy-

drate. In search of this high-anity ligand, a two-stage

approach was pursued. In the ®rst stage, a series of

monovalent NeuAca-2,6-Glc(Y)X type binding motifs

(Fig. 1) was prepared, and their anity for murine CD22

was monitored, to obtain insight into the eect of mod-

i®cations at both the C-1 position (X=methoxy, azido,

nitrophenoxy) and at the C-2 position (e.g., Y=aceta-

Scheme 1. Protecting group exchange of NeuAca-2,6-GalX type

disaccharides. Reagents: (i) NaOMe; (ii) Ac₂O.

88

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97

Results and Discussion

with the sialyl xanthate 14¹⁶(Fig. 3) in combination

with N-iodosuccinimide/tri¯ic acid (NIS/TfOH)^17,18as

promoter system. The results of the glycosylation reac-

tions are depicted in Table 1.

For the ®rst phase of our study, nine NeuAca-2,6-

Glc(Y)X type ligands were prepared (1±9, see Fig. 2),

together with, as a frame of refererence, two NeuAca-2,6-

LacX (10 and 11) and two NeuAca-2,6-GalX (12 and

13) type ligands.

The a-isomers of the di- and trisaccharides 27±36 were

identi®ed by the characteristic ¹H NMR chemical

shifts of the H-3eq [d(a-isomer)>d(b-isomer)] and H-4

(dÆ4.8 ppm) of the NeuAc moieties, and were separated

from the b-isomers by silica gel chromatography. For the

galactose derived disaccharides 37 and 38, separation of

The synthesis of the di- and trisaccharides 1±13 involved

sialylation of the partially protected acceptors 15±26

Table 2. Anity data of oligosaccharides 1±13^a

Compound

Relative IC₅₀

1

2

3

4

5

6

7

8

NeuAca-2,6-GlcNAcOMe

NeuAca-2,6-GlcN₃OMe

NeuAca-2,6-GlcNH₂OMe

NeuAca-2,6-GlcNHBzOMe

NeuAca-2,6-GlcNHOctOMe

NeuAca-2,6-GlcNHNaphtOMe

NeuAca-2,6-GlcNHBzNO₂OMe

NeuAca-2,6-GlcNAcN₃

NeuAca-2,6-GlcNAcOPhNO₂

NeuAca-2,6-LacOMe

NeuAca-2,6-LacN₃

NeuAca-2,6-GalOMe

NeuAca-2,6-GalOPhNO₂

6.9

1.2

2.0

2.2

1.6

1.8

9.2

2.1

7.9

1.0

2.2

2.4

3.6

9

10

11

12

13

^aThe relative IC₅₀was calculated by dividing the average IC₅₀found

for compound 10 by the average IC₅₀of the respective compound. A

relative IC₅₀>1 therefore represents a better binding to CD22 than the

reference compound 10.

Scheme 2. Synthesis of 2-amino disaccharide 3. Reagents: (i) NaOMe;

(ii) NaOH; (iii) 1,3-propanedithiol.

Scheme 3. Synthesis of disaccharide ®t for coupling to TRIS dendritic core. Reagents: (i) HOCH₂CH₂OCH₂CH₂N₃, TfOH; (ii) 1 M NaOH, re¯ux;

(iii) excess 4-NO₂BzCl; (iv) NaOMe; (v) tBDMS-Cl; (vi) Ac₂O; (vii) p-TsOH; (viii) NIS, TfOH; (ix) P(Ph)₃, H₂O.

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97

89

the respective a- and b-isomers could only be accom-

plished after replacing the benzoyl groups on the galac-

tose ring by acetyl groups (see Scheme 1). Having the

fully protected a-isomers of 27±36, 39 and 40 in hand,

two-step basic hydrolysis of the protecting groups then

gave the respective target ligands 1±2 and 4±13.

is slightly lower for murine CD22 than for human

CD22.¹²The anity of 10 was de®ned as 1 and the IC₅₀

values of all other ligands were expressed relative to that

of 10 (see Table 2).

From Table 2 it can be seen that, in line with the results

of Powell et al.,¹⁰the glucosamine derived dimers 1±9 all

have a comparable or higher anity for CD22 than the

trisaccharide 10. Remarkably, substitution of an anome-

ric methoxy by an azido group appears to have a negative

eect (1 versus 8, respectively) at the disaccharide level,

but a bene®cial eect at the trisaccharide level (10 versus

11, respectively). An anomeric nitrophenoxy instead of

a methoxy group has a positive eect on binding, both

in the case of glucosamine- (9 versus 1, respectively) and

galactose-derived (13 versus 12, respectively) disac-

charides. Focusing on the C-2 position of the glucoside

in the NeuAca-2,6-Glc(Y)X template, substitution of

the acetamide in 1 by an azide or an amine impairs

binding to CD22 (2 and 3 versus 1, respectively). With

Finally, the 2-amino ligand 3 was prepared from the

deprotected 2-azido ligand 2 by mild reduction with 1,3-

propanedithiol¹⁹(see Scheme 2).

The monovalent ligands 1±13 were tested on their a-

nity for CD22 in an in vitro competition assay based on

the binding of porcine erythrocytes to immobilised

murine CD22D1-3-Ig.^20,21The NeuAca-2,6-LacOMe

trisaccharide 10, which was selected as the reference

compound, displayed an average IC₅₀of 244 mM. This

compares well with the value reported by Powell et al.¹⁰

for binding of NeuAca-2,6-Lac to human CD22, taking

into account that the anity of NeuAc glycoconjugates

Scheme 4. Synthesis of the cluster based on 7. Reagents: (i) HBTU, HOBT; (ii) NaOMe; (iii) NaOH.

90

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97

respect to 3, it should be noted that Kelm et al.¹³also

reported that the presence of a free amine reduced the

anity for CD22, as well as for the other siglecs sia-

loadhesin (Sn) and MAG. By contrast, azido and amino

groups at the C-2 position of the glucose ring of sialyl-

LewisX were found²²to enforce the binding to E-selec-

tin by a factor of 4±6. Introduction of a more lipophilic

moiety at the C-2 position of a disaccharide by repla-

cing the N-acetyl in 1 by either an aromatic (benzoyl,

naphthoyl) or aliphatic (octanoyl) group negatively

aects binding to CD22 (4, 5 and 6 versus 1, respec-

tively). The latter result may look rather unexpected in

view of recent reports in which lipophilic groups confer

stronger binding in many dierent ligand±receptor sys-

tems.^{23 29}On the other hand, addition of a single nitro

group to the aromatic benzoyl ring leads to a dramatic

increase in anity for CD22 (7 versus 1, 7 versus 4). The

nitrobenzoyl disaccharide 7 displayed an almost 10-fold

higher anity than the reference 10, establishing 7 as

the most potent ligand of all the monovalent carbohy-

drates tested in this study. The disaccharide 7 was

therefore selected as the basic motif for the multivalent

ligand to be prepared in the second phase of our study.

structure 10, a 58.1-fold improvement in binding was

observed. It cannot be excluded that the presence of the

lipophilic benzyloxycarbonyl group in 52 contributes to

the binding of the TRIS cluster. However, the results

found for the lipophilic monovalent ligands 19 and

21 suggest that the eect of a single phenyl ring on bind-

ing will not be very signi®cant. Therefore, we assume

that the strong gain in anity found for the multivalent

sialoside 52 is mainly due to the cluster eect.

Conclusion

We have prepared 13 sialoside oligosaccharides that

carry dierent structural features on the hexose moieties

at the reducing terminus, and tested these oligosacchar-

ides on their ability to bind to CD22. The main result of

these tests is that the presence of 4-nitroaryl groups at

either the C-1 or C-2 position of the reducing hexose

enhances binding, while the presence of either lipophilic

or positively charged groups at C-2 does not. The most

potent disaccharide, the nitrobenzoyl derivative 7,

displays an almost 10-fold higher anity for CD22 than

the reference structure, NeuAca-2,6-LacOMe. Based on

disaccharide 7, a trivalent cluster (52) was prepared. This

trivalent sialoside 52 displayed a 6.3-fold higher anity

for CD22 than disaccharide 7, thereby providing strong

evidence that ligand valency is indeed an important

determinant for high-anity binding to CD22.^9,10To

our knowledge, the cluster 52 is one of the most potent

ligands for CD22 yet synthesised, and may ®nd appli-

cation as (ant)agonist or homing device for CD22-

expressing B-cells.

The synthesis of the multivalent ligand based on 7 started

with the introduction of a masked diethylene glycol

amino spacer moiety at the anomeric position of the

glucosamine ring. This spacer should allow smooth

attachment of the disaccharide to the TRIS dendritic

core, and warrant an optimal resemblance of the result-

ing cluster to the native multivalent ligand structure, in

terms of spacing of the terminal sialic acid residues.

To this end, the azide 42 was prepared by the addition

of 2-(2-azidoethoxy)ethanol to the known³⁰oxazoline

41 of glucosamine (see Scheme 3). Conversion into the

nitrobenzoyl derivative 45 proceeded via a sequence

of straightforward protection and deprotection steps.

Sialylation of 45 with the methyl thiosialoside 46³¹and

subsequent Staudinger³²reduction of the azido group of

the dimer 47 aorded the amine 48, ®t for coupling to

the TRIS dendritic core.

Experimental

General

All solvents were of analytical grade. Dry solvents were

stored over 4 A molecular sieves. Merck Kieselgel 60

F₂₅₄DC Alufolien was used for TLC analysis. Carbo-

hydrate compounds were visualised by charring with

sulfuric acid:ethanol (1:4, v/v). Compounds containing

NH functions were visualised by charring with a solution

of 4,4⁰-tetramethyldiamino-diphenylmethane (TDM)³³

after treatment of the TLC plates with chlorine, or

visualised by charring with a 0.3% solution of ninhy-

drine in acetic acid:n-butanol (3:100, v/v). Column

chromatography was performed with Kieselgel 60, 230±

400 mesh (Merck). Gel ®ltration was performed with

Sephadex LH-20 (Pharmacia). ¹H NMR spectra

(200 MHz) and ¹³C{¹H} NMR spectra (50.1 MHz) were

recorded with a Jeol JNM-FX200 spectrometer. ¹H

NMR spectra (300 MHz) were recorded with a Bruker

WM-300 spectrometer. Matrix Assisted Laser Desorp-

tion Ionisation (MALDI) Time-of-Flight (TOF) mass

spectrometry³⁴was performed with a Perkin Elmer/

PerSeptive Biosystems Voyager-DE-RP MALDI-TOF

mass spectrometer equipped with delayed extraction.³⁵

Exact masses were obtained by nano±ESI±TOF±MS on

a Micromass LCT with a resolution of 5000 FWHM

using pentaphenylalanine as reference mass. Murine

Condensation of TRIS-derived tricarboxylate 49¹⁵with

48 (Scheme 4), under standard peptide coupling condi-

tions, furnished the protected cluster 50 in an excellent

yield. Finally, mild two-step removal of the protective

ester groups aorded the desired sialoside 52.

It was found that the multivalent sialoside 52 displayed

a 6.2-fold higher anity for CD22 than 7 (see Table 3).

Compared to our initial NeuAca-2,6-LacOMe reference

Table 3. Anity data of oligosaccharides 10, 7 and the cluster 52^a

Compound

Relative IC₅₀

10

7

52

NeuAca-2,6-LacOMe

NeuAca-2,6-GlcNHBzNO₂OMe

TRIS(NeuAca-2,6-GlcNHBzNO₂)₃

1.0

9.2

58.1

^aThe relative IC₅₀was calculated by dividing the average IC₅₀found

for compound 10 by the average IC₅₀of the respective compound. A

relative IC₅₀>1 therefore represents a better binding to CD22 than the

reference compound 10.

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97

91

CD22/IgG was puri®ed from a stable transfected CHO

cell line (kindly provided by Dr. P. Crocker, Dundee,

Scotland). Cells were grown in GMEM (First link UK)

containing 5% IgG-poor FCS (Gibco), 20 U pen/strep

and 400 mM MSX (Sigma). Production of CD22 was

induced by addition of 2 mM sodium butyrate (Sigma),

and medium was collected for 3 weeks. Murine CD22

was routineously puri®ed from the pooled media by

protein-A Sepharose chromatography (yieldÆ200 mg/

mL). Purity was routinely checked by PAGE analysis.

(CH_arombenzoyl), 129.3±128.7 (C_qbenzoyl), 99.5 (C-2⁰),

88.9 (C-1), 73.6, 73.1, 72.5, 69.9. 68.6, 67.6, 67.3 (C-

3,4,5, C-4⁰,6⁰,7⁰,8⁰), 65.0, 63.2 (C-6, C-9⁰), 53.7 (C-2),

52.7 (OCH₃ester), 48.7 (C-5⁰), 37.2 (C-3⁰), 22.9 (N-ace-

tyl), 20.9, 20.8, 20.6 (CH₃O-acetyl). b-Isomer d 98.0 (C-

2 ). H NMR (CDCl₃): d 8.05±7.33 (m, 10H, CH_arom),

6.52 (d, 1H, NH acetyl, J_NH-2=9.8 Hz), 5.65 (dd, 1H,

H-3, J_{3 4}=9.6 Hz), 5.57 (dd, 1H, H-4, J_{4 5}=9.5 Hz),

0

1

5.37 (ddd, 1H, H-8⁰, J_{8 9 a}=2.7 Hz, J₈

7.1 Hz), 5.23

0

9 b

(dd, 1H, H-7⁰, J_{6 7}=1.2 Hz, J_{7 8}=9.5 Hz), 4.90 (d,

0

1H, H-1, J_{1 2}=9.3 Hz), 4.72 (ddd, 1H, H-4⁰, J₄

0

=

5

10.0 Hz), 4.35 (dd, 1H, H-9⁰a, J_{9 a 9 b}=12.2 Hz), 4.30

(ddd, 1=H, H-2, J_{2 3}=9.9 Hz), 4.19 (m, 3H, H-

0

Methyl 2-acetamido-3,4-di-O-benzoyl-2-deoxy-6-O-(methyl

4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-^D-neuraminate-2-yl)-

ꢁ-^D-glucopyranoside (27). A mixture of acceptor 15

(145 mg; 327 mmol), donor 14 (130 mg; 218 mmol)¹⁶and

powdered molecular sieves (3 A) in acetonitrile (3 mL)

was stirred for 1 h under argon atmosphere. The tem-

perature of the mixture was lowered to 50 ^ꢀC, and a

solution of NIS (74 mg; 0.33 mmol) and TfOH (3 mL;

0.03 mmol) in acetonitrile (1 mL) was added. The mix-

ture was gradually warmed to room temperature over a

period of 3 h. The reaction was quenched by the addi-

tion of triethylamine (0.1 mL). The mixture was ®ltered

over Hy¯o Super Cel (Fluka), diluted with ethyl acetate

(50 mL), washed with aq Na₂S₂O₃(1 M; 50 mL), aq

NaHCO₃(1 M; 50 mL) and water (50 mL), dried (MgSO₄),

concentrated, and applied to a silica gel column. Elution

was performed with DCM:methanol (1:0!96:4, v/v).

The fractions containing the product were pooled, con-

centrated, and applied to a Sephadex LH-20 column.

The disaccharide 27 was obtained as a/b mixture. Yield:

73 mg (80 mmol, 37%, a/b 5:1). The a-isomer was iso-

lated from the latter mixture by silica gel chromato-

graphy, with toluene:ethanol (1:0!8:2, v/v) as eluent, in

a yield of 45 mg (49 mmol). ¹³C{¹H} NMR (CDCl₃): d

171.1±128.3 (CO acetyl, benzoyl, C-1⁰), 133.2±128.3

(CH_arombenzoyl), 128.9 (C_qbenzoyl), 101.9 (C-1), 99.2

(C-2⁰), 73.4, 72.3, 71.8, 70.0, 68.8, 67.8, 67.1 (C-3,4,5, C-

4⁰,6⁰,7⁰,8⁰), 64.6, 62.8 (C-6, C-9⁰), 56.5 (OCH₃), 52.6

(OCH₃ester), 52.2 (C-2), 48.9 (C-5⁰), 37.3 (C-3⁰), 23.1

(N-acetyl), 20.9, 20.6 (CH₃O-acetyl). b-Isomer d 98.0 (C-

5⁰,6⁰,9⁰b), 3.82 (dd, 1H, H-6a, J_{5 6a}=7.4 Hz, J_{6a 6b}

12.2 Hz), 3.78±3.70 (m, 2H, H-5,6b), 3.75 (s, 3H, OCH₃

=

ester), 2.33 (dd, 1H, H-3⁰eq, J_{3 eq 4}=4.7 Hz, J_{3 eq 3 ax}

=

0

12.8 Hz), 2.16, 2.14, 2.04, 1.95, 1.88, 1.86 (6Âs, 18H,

CH₃acetyl), 1.42 (dd, 1H, H-3⁰ax, J_{3 ax 4}=12.3 Hz).

0

4-Nitrophenyl 2-acetamido-3,4-di-O-benzoyl-2-deoxy-6-

O-(methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-^D-neurami-

nate-2-yl)-ꢁ-^D-glucopyranoside (29). Compound 29 was

prepared from acceptor 17 (180 mg; 327 mmol) and

donor 14 (130 mg; 218 mmol) as described above for the

synthesis of 27. Yield: 61 mg (60 mmol, 28%, a/b 6:1).

Isolated yield of a-isomer: 23 mg (22 mmol). ¹³C{¹H}

NMR (CDCl₃): d 171.9±164.9 (CO acetyl, benzoyl, C-

1⁰), 161.6, 142.6 (C_qphenyl), 133.6±128.0 (CH_aromben-

zoyl), 125.6, 116.7 (CH_aromphenyl), 99.9 (C-2⁰), 98.5 (C-

1), 72.5, 71.7, 70.3, 68.5, 67.2 (C-3,4,5, C-4⁰,6⁰,7⁰,8⁰),

65.5, 63.7 (C-6, C-9⁰), 54.2 (C-2), 52.8 (OCH₃ester),

48.7 (C-5⁰), 37.0 (C-3⁰), 23.2 (N-acetyl), 20.6 (CH₃O-

0

1

acetyl). b-Isomer d 98.1 (C-2 ). H NMR (CDCl₃): d

8.21±7.29 (m, 14H, CH_arom), 6.56 (d, 1H, NH acetyl,

J_NH-2=9.4 Hz), 5.81 (dd, 1H, H-3, J_{3 4}=9.7 Hz), 5.61

(dd, 1H, H-4, J_{4 5}=9.6 Hz), 5.56 (ddd, 1H, H-8⁰,

0

J_{8 9 a}=2.8 Hz, J_{8 9 b}=8.0 Hz), 5.50 (d, 1H, H-1,

0

J_{1 2}=8.2 Hz), 5.23 (dd, 1H, H-7⁰, J_{7 8}=9.8 Hz), 5.1₀6

0

(d, 1H, NH acetyl, J_NH-5=9.6 Hz), 4.68 (ddd, 1H, H-4 ,

0

J_{4 5}=9.3 Hz), 4.66 (ddd, 1H, H-2, J_{2 3}=10.3 Hz), 4.46

0

(dd, 1H, H-9⁰a, J_{9 a 9 b}=12.1 Hz), 4.33 (ddd, 1H, H-5,

0

2 ). H NMR (CDCl₃): d 8.01±7.33 (m, 10H, CH_arom),

6.19 (d, 1H, NH acetyl, J_NH-2=9.3 Hz), 5.62 (dd, 1H, H-

3, J_{3 4}=9.5 Hz), 5.55 (dd, 1H, H-4, J_{4 5}=9.6 Hz), 5.33

J_{5 6a}=6.7 Hz, J_{5 6b}=5.0 Hz), 4.10 (ddd, 1H, H-5⁰,

1

0

J_{5 6}=10.6 Hz), 4.03 (dd, 1H, H-6 , J_{6 7}=1.7 Hz),

3.90±3.82 (m, 2H, H-6a, H-9⁰b), 3.71 (s, 3H, OCH₃

ester), 3.68 (dd, 1H, H-6b, J_{6a 6b}=13.0 Hz), 2.26 (dd,

(ddd, 1H, H-8⁰, J_{8 9 a}=2.6 Hz, J_{8 9 b}=6.6 Hz), 5.24 (dd,

0

1H, H-7⁰, J_{6 7}=1.2 Hz, J_{7 8}=10.0 Hz), 4.73 (ddd, 1H,

1H, H-3⁰eq, J_{3 eq 4}=4.7 Hz, J_{3 eq 3 ax}=12.9 Hz), 2.20,

2.19, 1.94, 1.88, 1.86 (5Âs, 18H, CH₃acetyl), 1.29 (dd,

0

H-4⁰, J_{4 5}=9.9 Hz), 4.65 (d, 1H, H-1, J_{1 2}=8.4 Hz),

0

4.29 (dd, 1H, H9⁰a, J_{9 a 9 b}=12.3 Hz), 4.23 (ddd, 1H,

H-2, J_{2 3}=10.1 Hz), 4.05±3.95 (m, 3H, H-5⁰,6⁰,9⁰b),

3.86±3.71 (m, 3H, H-5,6a,6b), 3.74 (s, 3H, OCH₃ester),

1H, H-3⁰ax, J_{3 ax 4}=12.7 Hz).

0

Methyl 2-azido-3,4-di-O-benzoyl-2-deoxy-6-O-(methyl 4,

7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-^D-neuraminate-2-yl)-ꢁ-D-

glucopyranoside (30). Compound 30 was prepared from

acceptor 18 (128mg; 300 mmol) and donor 14 (119mg;

200 mmol) as described above for the synthesis of 27.

Yield: 90mg (100mmol, 50%, a/b 5:1). Isolated yield of a-

isomer: 51mg (56 mmol). ¹³C{¹H} NMR (CDCl₃): d

170.7±164.8 (CO acetyl, benzoyl, C-1⁰), 133.1±128.2

(CH_arombenzoyl), 129.2, 129.0 (C_qbenzoyl), 102.9 (C-

1), 98.6 (C-2⁰), 73.0, 72.5, 72.4, 69.2, 69.0, 68.4, 67.1,

64.1 (C-2,3,4,5, C-4⁰,6⁰,7⁰,8⁰), 63.4, 62.1 (C-6, C-9⁰), 57.1

(OCH₃), 52.4 (OCH₃ester), 49.1 (C-5⁰), 37.4 (C-3⁰), 23.0

(N-acetyl), 20.6 (CH₃O-acetyl). b-Isomer d 98.1 (C-2⁰).

¹H NMR (CDCl₃): d 8.04±7.29 (m, 10H, CH_arom), 5.51

3.54 (s, 3H, OCH₃), 2.39 (dd, 1H, H-3⁰eq, J_{3 eq 4}

0

=

4.6 Hz, J_{3 eq 3 ax}=12.7 Hz), 2.11, 2.03, 1.96, 1.86, 1.85

0

(5Âs, 18H, CH₃acetyl), 1.55 (dd, 1H, H-3⁰ax, J_{3 ax 4}

0

=

12.3 Hz).

2-Acetamido-3,4-di-O-benzoyl-2-deoxy-6-O-(methyl

4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-^D-neuraminate-2-yl)-

ꢁ-^D-glucopyranosyl azide (28). Compound 28 was pre-

pared from acceptor 16 (149 mg; 327 mmol) and donor

14 (130mg; 218 mmol) as described above for the synthesis

of 27. Yield: 83mg (89 mmol, 41%, a/b 4:1). Isolated yield

of a-isomer: 54mg (58 mmol). ¹³C{¹H} NMR (CDCl₃): d

171.5±164.6 (CO acetyl, benzoyl, C-1⁰), 133.3±128.3

92

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97

(dd, 1H, H-4, J_{4 5}=9.6 Hz), 5.40 (dd, 1H, H-3, J_{3 4}=

9.6 Hz), 5.24 (dd, 1H, H-7⁰, J_{7 8}=8.6 Hz), 5.19 (ddd,

4.48 (d, 1H, H-1, J_{1 2}=8.3 Hz), 4.31 (dd, 1H, H-9⁰a,

0

0 0

J_{9a 9 b}=12.4 Hz), 4.04 (ddd, 1H, H-2, J_{2 3}=10.4 Hz),

1H, H-8⁰, J_{8 9 a}=2.5 Hz, J_{8 9 b}=5.0 Hz), 4.79 (ddd,

4.03 (dd, 1H, H-9⁰b), 3.97 (dd, 1H, H-6⁰, J_{5 6}=10.8 Hz,

0

1H, H-4⁰, J_{4 5}=9.8 Hz), 4.45 (d, 1H, H-1, J_{1 2}

=

0

(s, 3H, OCH₃ester), 3.46 (s, 3H, OCH₃), 2.61 (dd, 1H,

0

J_{6 7}=2.0 Hz), 3.94±3.56 (m, 4H, H-5 , H-5,6a,6b), 3.79

8.0 Hz), 4.10 (dd, 1H, H-9⁰a, J_{9 a 9 b}=12.5 Hz), 3.99 (dd,

0

1H, H-6⁰, J_{5 6}=6.0 Hz, J_{6 7}=1.4 Hz), 3.97±3.94 (m,

2H, H-5⁰, H-6a), 3.86±3.82 (m, 1H, H-5), 3.80 (dd, 1H,

H-9⁰b), 3.65 (dd, 1H, H-2, J_{2 3}=10.1 Hz), 3.61 (dd, 1H,

H-6b, J_{5 6b}=3.0 Hz, J_{6a 6b}=12.1 Hz), 3.74, 3.63 (2Âs,

H-3⁰eq, J_{3 eq 4}=4.7 Hz, J_{3 eq 3 ax}=12.7 Hz), 2.14, 2.13,

2.05, 2.03, 2.02, 2.00, 1.86 (7Âs, 21H, CH₃acetyl), 1.94

0

(dd, 1H, H-3⁰ax, J_{3 ax 4}=12.4 Hz), 1.56 (t, 2H, CH₂

octanoyl), 1.25 (broad, 10H, CH₂octanoyl), 0.86 (t, 3H,

CH₃octanoyl).

0

6H, OCH₃), 2.49 (dd, 1H, H-3⁰eq, J_{3 eq 4}=4.7 Hz,

0

J_{3 eq 3 ax}=12.6 Hz), 2.08, 2.02, 2.00, 1.99, 1.83 (5Âs,

15H, CH₃acetyl), 1.78 (dd, 1H, H-3⁰ax, J_{3 ax 4}

12.5 Hz).

Methyl 2-naphthoylamido-3,4-di-O-acetyl-2-deoxy-6-O-

(methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-^D-neurami-

nate-2-yl)-ꢁ-^D-glucopyranoside (33). Compound 33 was

prepared from acceptor 21 (177 mg; 410 mmol) and

donor 14 (160 mg; 268 mmol) as described above for the

synthesis of 27. Yield: 63 mg (69 mmol, 25%, a/b 5:1).

Isolated yield of a-isomer: 39 mg (43 mmol). ¹³C{¹H}

NMR (CDCl₃): d 171.3±167.3 (CO acetyl, naphtoyl,

C-1⁰), 134.5±131.0 (C_qnaphtoyl), 128.6±123.3 (CH_arom

naphtoyl), 101.6 (C-1), 98.4 (C-2⁰), 73.0, 72.0, 71.8, 69.2,

68.9, 68.2, 67.2 (C-3,4,5, C-4⁰,6⁰,7⁰,8⁰), 63.0, 62.3 (C-6,

C-9⁰), 56.3 (OCH₃), 54.1 (C-2), 52.2 (OCH₃ester), 48.5

(C-5⁰), 37.3 (C-3⁰), 22.0 (N-acetyl), 20.1 (CH₃O-acetyl).

0

=

Methyl 2-benzoylamido-3,4-di-O-acetyl-2-deoxy-6-O-

(methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-^D-neurami-

nate-2-yl)-ꢁ-^D-glucopyranoside (31). Compound 31 was

prepared from acceptor 19 (164 mg; 430 mmol) and

donor 14 (170 mg; 285 mmol) as described above for the

synthesis of 27. Yield: 102 mg (119 mmol, 42%, a/b 3:1).

Isolated yield of a-isomer: 70 mg (81 mmol). ¹³C{¹H}

NMR (CDCl₃): d 171.0±166.9 (CO acetyl, benzoyl, C-

1⁰), 134.1 (C_qbenzoyl), 131.5±126.9 (CH_arombenzoyl),

102.0 (C-1), 98.7 (C-2⁰), 73.0, 72.4, 72.3, 69.0, 68.3, 67.4

(C-3,4,5, C-4⁰,6⁰,7⁰,8⁰), 63.6, 62.6 (C-6, C-9⁰), 56.5

(OCH₃), 54.5 (C-2), 52.6 (OCH₃ester), 49.3 (C-5⁰), 37.6

(C-3⁰), 23.0 (N-acetyl), 20.7 (CH₃O-acetyl). b-Isomer d

98.3 (C-2⁰). ¹H NMR (CDCl₃): d 7.75±7.38 (m, 5H,

CH_arom), 6.59 (d, 1H, NH benzoyl, J_NH-2=9.1 Hz), 5.40

b-Isomer d 98.1 (C-2⁰). H NMR (CDCl₃): d 8.27±7.48

1

(m, 7H, CH_arom), 6.73 (d, 1H, NH naftanoyl, J_NH-2=

=

9.0 Hz), 5.41 (ddd, 1H, H-8⁰, J_{8 9 a}=2.5 Hz, J₈

6.3 Hz), 5.40 (dd, 1H, H-3, J_{3 4}=9.6 Hz), 5.34 (d, 1H,

0

9 b

NH acetyl, J_NH-5=9.5 Hz), 5.30 (dd, 1H, H-7⁰,

0

(ddd, 1H, H-8⁰, J_{8 9 a}=2.6 Hz, J_{8 9 b}=6.1 Hz), 5.35

0

J_{7 8}=8.7 Hz), 5.23 (dd, 1H, H-4, J_{4 5}=9.5 Hz), 4.88

0

(dd, 1H, H-3, J_{3 4}=9.6 Hz), 5.28 (dd, 1H, H-7⁰,

(ddd, 1H, H-4⁰, J_{4 5}=9.8 Hz), 4.60 (d, 1H, H-1, J_{1 2}

=

0

8.3 Hz), 4.37 (dd, 1H, H-9⁰a, J_{9 a 9 b}=12.3 Hz), 4.28

0

J_{7 8}=8.7 Hz), 5.20 (dd, 1H, H-4, J_{4 5}=9.5 Hz), 4.88

0

(ddd, 1H, H-4⁰, J_{4 5}=9.7 Hz), 4.57 (d, 1H, H-1,

(ddd, 1H, H-2, J_{2 3}=10.2 Hz), 4.08 (ddd, 1H, H-5⁰₀,

0

J_{1 2}=8.3 Hz), 4.36 (dd, 1H, H-9⁰a, J_{9 a 9 b}=12.4 Hz),

0

J_{5 6}=10.7 Hz), 4.04 (dd, 1H, H-9 b), 4.02 (dd, 1H, H-6 ,

0

4.22 (ddd, 1H, H-2, J_{2 3}=10.2 Hz), 4₀.07 (ddd, 1H, H-5⁰,

J_{6 7}=1.9 Hz), 3.90 (dd, 1H, H-6a, J_{6a 6b}=11.5 Hz,

J_{5 6a}=4.1 Hz), 3.81 (s, 3H, OCH₃ester), 3.76±3.70 (m,

1H, H-5), 3.65 (dd, 1H, H-6b, J_{5 6b}=3.0 Hz), 3.49 (s,

0

J_{5 6}=10.7 Hz), 4.02 (dd, 1H, H-9 b), 4.00 (dd, 1H,

H-6⁰, J_{6 7}=1.9 Hz), 3.88 (dd, 1H, H-6a, J_{5 6a}=4.1 Hz,

J_{6a 6b}=11.4 Hz), 3.81 (s, 3H, OCH₃ester), 3.74±3.68

(m, 1H, H-5), 3.65 (dd, 1H, H-6b, J_{5 6b}=3.0 Hz), 3.47

0

3H, OCH₃), 2.64 (dd, 1H, H-3⁰eq, J_{3 eq 4}=4.6 Hz,

0

J_{3 eq 3 ax}=12.7 Hz), 2.16, 2.15, 2.09, 2.04, 2.02, 1.97,

(s, 3H, OCH₃), 2.63 (dd, 1H, H-3⁰eq, J_{3 eq 4}=4.6 Hz,

1.87 (7Âs, 21H, CH₃acetyl), 1.95 (dd, 1H, H-3⁰ax,

0

J_{3 ax 4}=12.3 Hz).

0

J_{3 eq 3 ax}=12.7 Hz), 2.15, 2.14, 2.08, 2.03, 2.02, 1.97 1.86

0

(7Âs, 21H, CH₃acetyl), 1.94 (dd, 1H, H-3⁰ax, J_{3 ax 4}

0

=

12.3 Hz).

Methyl 2-(4-nitrobenzoyl)amido-3,4-di-O-acetyl-2-deoxy-

6-O-(methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-^D-neura-

minate-2-yl)-ꢁ-^D-glucopyranoside (34). Compound 34

was prepared from acceptor 22 (180 mg; 422 mmol) and

donor 14 (160 mg; 268 mmol) as described above for the

synthesis of 27. Yield: 109 mg (121 mmol, 44%, a/b 4:1).

Isolated yield of a-isomer: 67 mg (74 mmol). ¹³C{¹H}

NMR (CDCl₃): d 171.1±165.3 (CO acetyl, benzoyl, C-

1⁰), 149.6, 139.6 (C_qbenzoyl), 128.3, 123.6 (CH_arom

benzoyl), 101.7 (C-1), 98.8 (C-2⁰), 73.0, 72.5, 72.2, 69.0,

68.9, 68.3, 67.4 (C-3,4,5, C-4⁰,6⁰,7⁰,8⁰), 63.7, 62.7 (C-6,

C-9⁰), 56.5 (OCH₃), 54.8 (C-2), 52.6 (OCH₃ester), 49.3

(C-5⁰), 37.6 (C-3⁰), 22.9 (N-acetyl), 20.6 (CH₃O-acetyl).

Methyl 2-octanoylamido-3,4-di-O-acetyl-2-deoxy-6-O-

(methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-^D-neurami-

nate-2-yl)-ꢁ-^D-glucopyranoside (32). Compound 32 was

prepared from acceptor 20 (122 mg; 302 mmol) and

donor 14 (119 mg; 200 mmol) as described above for the

synthesis of 27. Yield: 53 mg (60 mmol, 30%, a/b 3:1).

Isolated yield of a-isomer: 34 mg (38 mmol). ¹³C{¹H}

NMR (CDCl₃): d 173.3±167.7 (CO acetyl, octanoyl,

C-1⁰), 101.8 (C-1), 98.7 (C-2⁰), 72.9, 72.5, 72.3, 69.0,

68.4, 67.4 (C-3,4,5, C-4⁰,6⁰,7⁰,8⁰), 63.4, 62.5 (C-6, C-9⁰),

56.5 (OCH₃), 54.2 (C-2), 52.6 (OCH₃ester), 49.4 (C-5⁰),

37.6, 36.7 (C-3⁰, CH₂octanoyl), 31.6±22.5 (CH₂octa-

noyl), 23.0 (N-acetyl), 20.6 (CH₃O-acetyl), 13.9 (CH₃

b-Isomer d 98.4 (C-2⁰). H NMR (CDCl₃): d 8.29 (m,

1

4H, CH_arom), 7.17 (d, 1H, NH nitrobenzoyl, J_NH-2=

9.2 Hz), 5.48 (d, 1H, NH acetyl, J_NH-5=9.5 Hz), 5.40

octanoyl). b-Isomer d 98.3 (C-2⁰). H NMR (CDCl₃): d

1

0

(ddd, 1H, H-8⁰, J_{8 9 a}=2.4 Hz, J_{8 9 b}=6.4 Hz), 5.34

0

5.64 (d, 1 H, NH octanoyl, J_NH-2=9.0 Hz), 5.36 (ddd,

1H, H-8⁰, J_{8 9 a}=2.7 Hz, J_{8 9 b}=5.9 Hz), 5.31 (d, 1H,

(dd, 1H, H-3, J_{3 4}=9.6 Hz), 5.30 (dd, 1H, H-7⁰, J₇

0

=

8

0

8.6 Hz), 5.21 (dd, 1H, H-3, J_{3 4}=9.4 Hz), 5.12 (dd, 1H,

0

NH acetyl, J_NH-5=8.4 Hz), 5.29 (dd, 1H, H-7 , J₇

0

0 0

8.6 Hz, J_{6 7}=1.4 Hz), 5.18 (dd, 1H, H-4, J_{4 5}=9.5 Hz),

=

8

4.88 (ddd, 1H, H-4⁰, J_{4 5}=9.8 Hz), 4.62 (d, 1H, H-1,

0 0

H-4, J_{4 5}=9.4 Hz), 4.85 (ddd, 1H, H-4⁰, J_{4 5}=9.7 Hz),

J_{1 2}=8.3 Hz), 4.40 (dd, 1H, H-9⁰a, J_{9 a 9 b}=12.3 Hz),

0 0

0

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97

93

4.25 (ddd, 1H, H-2, J_{2 3}=10.1 Hz), 4.07 (ddd, 1H, H-5⁰,

1H, H-3⁰, J_{3 4}=3.4 Hz), 5.37 (dd, 1H, H-2, J_{2 3}

=

0

9.5 Hz), 5.33 (dd, 1H, H-7⁰⁰, J₆=1.7 Hz, J₇

00 00 00

7

0

J_{5 6}=10.6 Hz), 4.06±4.03 (m, 1H, H-6 ), 4.02 (dd, 1H,

0

00

8

H-9⁰b), 3.86 (dd, 1H, H-6a, J_{5 6a}=4.5 Hz, J_{6a 6b}

11.5 Hz), 3.81 (s, 3H, OCH₃ester), 3.78±3.74 (m, 1H,

=

8.3 Hz), 5.04 (d, 1H, H-1⁰, J_{1 2}=7.8 Hz), 4.85 (d, 1H,

0

H-1, J_{1 2}=8.9 Hz), 4.79 (ddd, 1H, H-4⁰⁰, J₄

9.6 Hz), 4.68 (dd, 1H, H-6a, J_{5 6a}=1.9 Hz, J_{6a 6b}

=

00

5

H-5), 3.64 (dd, 1H, H-6b, J_{5 6b}=3.0 Hz), 3.50 (s, 3H,

OCH₃), 2.63 (dd, 1H, H-3⁰eq, J_{3 eq 4}=4.6 Hz, J_{3 eq 3 ax}

0

=

12.8 Hz), 2.15, 2.12, 2.08, 2.05, 2.01, 1.97, 1.88 (7Âs,

12.3 Hz), 4.59 (dd, 1H, H-6b, J_{5 6b}5.5 Hz), 4.44 (dd,

H-9⁰⁰a, J_{9 a 9 b}=12.1 Hz), 4.39 (dd, H-4, J_{4 5}=9.6 Hz),

00

21H, CH₃acetyl), 1.94 (dd, 1H, H-3⁰ax, J_{3 ax 4}

12.3 Hz).

4.12 (dd, 1H, H-9⁰⁰b), 4.16 (m, 4H, H-5⁰⁰5,6⁰⁰, H-5⁰, H-5),

0

=

3.53 (dd, 1H, H-6⁰a, J₅

0

0 0

6⁰a

5.6 Hz, J_{6 a 6 b}=10.0 Hz),

3.43 (s, 3H, OCH₃ester), 2.90 (dd, 1H, H-6⁰b,

J_{5 6 b}=9.6 Hz), 2.42 (dd, 1H, H-3⁰⁰eq, J_{3 eq 4}=4.5 Hz,

0

Methyl 2,3,6-tri-O-benzoyl-4-O-(2,3,4-tri-O-benzoyl-6-O-

(methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-^D-neuraminate

-2-yl)-ꢁ-^D-galactopyranosyl)-ꢁ-D-glucopyranoside (35).

Compound 35 was prepared from acceptor 23 (131 mg;

129 mmol) and donor 14 (149 mg; 250 mmol) as described

above for the synthesis of 27. Yield: 49 mg (34 mmol,

26%, a/b 2:1). Isolated yield of a-isomer: 23 mg

(15 mmol). ¹³C{¹H} NMR (CDCl₃): d 170.8±164.9 (CO

acetyl, benzoyl, C-1⁰⁰), 133.6±128.2 (CH_arombenzoyl),

129.0 (C_qbenzoyl), 101.7, 100.6 (C-1, C-1⁰), 98.9 (C-2⁰),

75.8, 73.4, 72.9, 72.1, 71.9, 71.8, 70.2, 68.6, 67.6, 67.3

(C-2,3,4,5, C-2⁰,3⁰,4⁰,5⁰, C-4⁰,6⁰,7⁰,8⁰), 62.9, 62.7, 61.6 (C-

6, C-6⁰, C-9⁰), 56.7 (OCH₃), 52.5 (OCH₃ester), 49.5 (C-

5⁰), 37.9 (C-3⁰), 23.1 (N-acetyl), 20.7 (CH₃O-acetyl).

00

J_{3 eq 3 ax}=13.0 Hz), 2.20, 2.17, 2.09, 2.00, 1.88 (5Âs,

15H, CH₃acetyl), 1.83 (dd, 1H, H-3⁰⁰ax, J_{3 ax 4}

12.5 Hz).

00

=

Methyl 2,3,4-tri-O-benzoyl-6-O-(methyl 4,7,8,9-tetra-O-

acetyl-N-acetyl-ꢀ-^D-neuraminate-2-yl)-ꢁ-D-galactopyra-

noside (37). Compound 37 was prepared from acceptor

25 (152 mg; 300 mmol) and donor 14 (119 mg; 200 mmol)

as described above for the synthesis of 27. Yield: 92 mg

(94 mmol, 47%, a/b 6:1). ¹³C{¹H} NMR (CDCl₃): d

170.8±165.1 (CO acetyl, benzoyl, C-1⁰), 133.8±128.1

(CH_arombenzoyl), 129.0 (C_qbenzoyl), 102.3 (C-1), 99.0

(C-2⁰a-isomer), 72.4, 71.9, 71.7, 69.9, 68.7, 68.4, 67.7,

67.4 (C-2,3,4,5, C-4⁰,6⁰,7⁰,8⁰), 62.7, 62.3 (C-6, C-9⁰), 57.1

(OCH₃), 52.5 (OCH₃ester), 49.4 (C-5⁰), 37.9 (C-3⁰), 23.0

(N-acetyl), 20.6 (CH₃O-acetyl). b-Isomer d 98.8 (C-2⁰).

b-Isomer d 99.1 (C-2⁰). H NMR (CDCl₃): d 8.10±7.16

1

(m, 30H, CH_arom), 5.78 (dd, 1H, H-3, J_{3 4}=9.5 Hz),

5.75 (d, 1H, H-4⁰), 5.61 (dd, 1H, H-2⁰, J_{2 3}=10.2 Hz),

0

5.49 (ddd, 1H, H-8⁰⁰, J_{8 9 a}=2.9 Hz, J_{8 9 b}=6.7 Hz),

00

5.44 (dd, 1H, H-3⁰, J_{3 4}=3.1 Hz), 5.38 (dd, 1H, H-2,

Methyl 2,3,4-tri-O-acetyl-6-O-(methyl 4,7,8,9-tetra-O-

acetyl-N-acetyl-ꢀ-^D-neuraminate-2-yl)-ꢁ-D-galactopyra-

noside (39). Compound 37 (78 mg; 79 mmol) was dis-

solved in a methanolic solution of sodium methanolate

(0.05 M; 2 mL), and stirred overnight. The solution was

neutralised by the addition of Dowex/H⁺and ®ltered.

The ®ltrate was evaporated to dryness, and dissolved in

a mixture of pyridine and acetic anhydride (1:1, v/v;

10 mL). This mixture was stirred overnight and then

concentrated, evaporated with toluene (2Â10 mL) and

ethanol (10 mL), and applied to a silica gel column.

Elution with ethanol:toluene (0:1!1:9, v/v) aorded the

pure a-isomer of 39. Isolated yield of a-isomer: 37 mg

(47 mmol). ¹H NMR (CDCl₃): d 5.44 (dd, 1H, H-4,

J_{4 5}=0.8 Hz, J_{3 4}=3.3 Hz), 5.36 (ddd, 1H, H-8⁰,

0

J_{2 3}=9.6 Hz), 5.32 (dd, 1H, H-7⁰⁰, J₇=8.4 Hz), 5.21

00

8

0

J_{1 2}=7.8 Hz), 4.78 (ddd, 1H, H-4 , J₄=10.1 Hz),

00

(d, 1H, NH acetyl, J_NH-5= 9.7 Hz), 5.00 (d, 1H, H-1 ,

00

4.66 (dd, 1H, H-6a, J_{5 6a}= 1.7 Hz, J_{6a 6b}=12.0 Hz),

0

00

5

4.60 (d, 1H, H-1, J_{1 2}= 7.9 Hz), 4.55 (dd, 1H, H-6b,

J_{5 6b}=5.7 Hz), 4.43 (dd, 1H, H-9⁰⁰a, J_{9 a 9 b}=12.3 Hz),

00

4.35 (dd, 1H, H-4, J_{4 5}=9.6 Hz), 4.11 (dd, 1H, H-9⁰⁰b),

00

4.10 (dd, 1H, H-6⁰⁰, J₆=1.8 Hz), 4.03 (dd, 1H, H-5 ,

00

7

0

00

J₅=10.6 Hz), 3.91±3.86 (m, 2H, H-5, H-5 ), 3.51 (dd,

6

1H, H-6⁰a, J_{5 6 a}=5.5 Hz, J_{6 a 6 b}=9.9 Hz), 3.42, 3.41

0

(2Âs, 6H, OCH₃), 2.87 (dd, 1H, H-6⁰b, J_{5 6 b}=9.5 Hz),

0

H-3⁰⁰eq,

00 00

J_{3 eq 4}=4.6 Hz,

2.40

00

(dd,

1H,

00

J_{3 eq 3 ax}=12.8 Hz), 2.18, 2.16, 2.09, 2.00, 1.89 (5Âs,

15H, CH₃acetyl), 1.81 (dd, 1H, H-3⁰⁰ax, J_{3 ax 4}

12.5 Hz).

00

J_{8 9 a}=2.7 Hz, J_{8 9 b}=5.7 Hz), 5.28 (dd, 1H, H-7⁰,

0

J_{7 8}=8.8 Hz), 5.23 (d, 1H, NH acetyl, J_NH-5=9.6 Hz),

5.18 (dd, 1H, H-2, J_{2 3}=10.5 Hz), 5.05 (dd, 1H, H-3,

2,3,6-Tri-O-benzoyl-4-O-(2,3,4-tri-O-benzoyl-6-O-

(methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-ꢀ-^D-neuraminate-

2-yl)-ꢁ-^D-galactopyranosyl)-ꢁ-D-glucopyranosyl azide

(36). Compound 36 was prepared from acceptor 24

(131 mg; 129 mmol) and donor 14 (149 mg; 250 mmol) as

described above for the synthesis of 27. Yield: 98 mg

(67 mmol, 33%, a/b 3:1). Isolated yield of a-isomer:

59 mg (40 mmol). ¹³C{¹H} NMR (CDCl₃): d 170.8±164.9

(CO acetyl, benzoyl, C-1⁰⁰), 133.3±128.3 (CH_aromben-

zoyl), 128.9 (C_qbenzoyl), 100.3 (C-1⁰), 98.9 (C-2⁰), 87.9

(C-1), 75.6, 75.3, 72.8, 72.4, 71.9, 71.5, 70.1, 68.6, 68.5,

67.6, 67.2 (C-2,3,4,5, C-2⁰,3⁰,4⁰,5⁰, C-4⁰,6⁰,7⁰,8⁰), 62.8,

61.6 (C-6, C-6⁰, C-9⁰), 52.5 (OCH₃ester), 49.4 (C-5⁰⁰),

37.8 (C-3⁰⁰), 23.0 (N-acetyl), 20.7 (CH₃O-acetyl). b-Iso-

J_{3 4}=3.3 Hz), 4.85 (ddd, 1H, H-4⁰, J_{4 5}=9.7 Hz), 4.46

0

(d, 1H, J_{1 2}=7.9 Hz), 4.33 (dd, 1H, H-9⁰a, J_{9 a 9 b}

0

=

12.4 Hz), 4.09 (dd, 1H, H-6⁰, J_{6 7}=1.9 Hz), 4.06 (dd,

0

1H, H-9⁰b), 4.03 (ddd, 1H, H-5⁰, J_{5 6}=10.8 Hz), 3.95±

3.91 (bt, 1H, H-5, J_{5 6a}=6.0 Hz, J_{5 6b}=7.6 Hz), 3.83

(dd, 1H, H-6a, J_{6a 6b}=10.1 Hz), 3.79 (s, 3H, OCH₃

ester), 3.53 (s, 3H, OCH₃), 3.40 (dd, 1H, H-6b), 2.53

0

(dd, 1H, H-3⁰eq, J_{3 eq 4}=4.6 Hz, J_{3 eq 3 ax}=12.9 Hz),

2.18, 2.14, 2.13, 2.06, 2.03, 2.02, 1.97, 1.88 (8Âs, 24H,

0

CH₃acetyl), 1.91 (dd, 1H, H-3⁰ax, J_{3 ax 4}=12.3 Hz).

0

4-Nitrophenyl 2,3,4-tri-O-benzoyl-6-O-(methyl 4,7,8,9-

tetra-O-acetyl-N-acetyl-ꢀ-^D-neuraminate-2-yl)-ꢁ-D-

galactopyranoside (38). Compound 38 was prepared

from acceptor 26 (184 mg; 300 mmol) and donor 14

(119 mg; 200 mmol) as described above for the synthesis

of 27. Yield: 114 mg (105 mmol, 52%, a/b 4:1). ¹³C{¹H}

mer d 99.1 (C-2⁰). H NMR (CDCl₃): d 8.11±7.16 (m,

1

30H, CH_arom), 5.84 (dd, 1H, H-3, J_{3 4}=9.6 Hz), 5.77 (d,

1H, H-4⁰), 5.62 (dd, 1H, H-2⁰, J_{2 3}=10.3 Hz), 5.50

0

(ddd, 1H, H-8⁰⁰, J_{8 9 a}=3.1 Hz, J_{8 9 b}6.8 Hz), 5.46 (dd,

00

94

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97

1

NMR (CDCl₃): d 170.9±165.0 (CO acetyl, benzoyl, C-

1⁰), 161.6, 142.8 (CH_aromphenyl), 133.8±128.2 (CH_arom

benzoyl), 129.2 (C_qbenzoyl), 125.8, 117.0 (C_qphenyl),

99.8 (C-2⁰a-isomer), 98.4 (C-1), 72.8, 72.4, 71.5, 69.5,

68.5, 67.7, 67.4 (C-2,3,4,5, C-4⁰,6⁰,7⁰,8⁰), 63.7, 62.9 (C-6,

C-9⁰), 52.7 (OCH₃ester), 49.2 (C-5⁰), 37.7 (C-3⁰), 23.0

(N-acetyl), 20.7 CH₃O-acetyl). b-Isomer d 98.8 (C-2⁰).

(41 mmol, 51%). H NMR (D₂O): d 7.78±7.49 (m, 5H,

CH_arom), 4.57 (d, 1H, J_{1 2}=8.5 Hz), 3.50 (s, 3H,

OCH₃), 2.76 (dd, 1H, H-3⁰eq, J_{3 eq 4}=4.4 Hz,

0

J_{3 eq 3 ax}=12.3 Hz), 2.03 (s, 3H, CH₃acetyl), 1.73 (dd,

0

1H, H-3⁰ax, J_{3 ax 4}=12.0 Hz). HRMS: calculated for

0

[C₂₅H₃₆N₂O₁₄+Na⁺] 611.2064, found 611.2079.

Methyl 2-octanoylamido-2-deoxy-6-O-(N-acetyl-ꢀ-^D-

neuraminate-2-yl)-ꢁ-^D-glucopyranoside (5). Yield: 17 mg

4-Nitrophenyl 2,3,4-tri-O-acetyl-6-O-(methyl 4,7,8,9-

tetra-O-acetyl-N-acetyl-ꢀ-^D-neuraminate-2-yl)-ꢁ-D-

galactopyranoside (40). Compound 38 (93 mg, 85 mmol)

was treated with sodium methanolate and then acetyl-

ated as described above for the synthesis of 39. Silica gel

chromatography, with ethanol:toluene (1:0!1:9, v/v) as

eluent, aorded the pure a-isomer of 40. Isolated yield

of a-isomer: 38 mg (42 mmol). ¹H NMR (CDCl₃): d 8.21±

7.21 (m, 4H, CH_arom), 5.59 (d, 1H, H-4), 5.52 (dd, 1H,

1

(28 mmol, 74%). H NMR (D₂O): d 4.39 (d, 1H, H-1,

J_{1 2}=8.4 Hz), 3.48 (s, 3H, OCH₃), 2.72 (dd, 1H, H-3⁰eq,

0

J_{3 eq 4}=4.6 Hz, J_{3 eq 3 ax}=12.4 Hz), 2.25 (t, 2H,

CH₂CO octanoyl, J=7.2 Hz), 2.01 (s, 3H, CH₃ace-

tyl), 1.70 (dd, 1H, H-3⁰ax, J_{3 ax 4}=12.0 Hz), 1.58 (bt,

2H, CH₂octanoyl), 1.27 (bs, 8H, CH₂octanoyl), 0.84 (t,

3H, CH₃octanoyl, J=6.8 Hz). HRMS: calculated for

[C₂₆H₄₆N₂O₁₄+Na⁺] 633.2847, found 633.2863.

0

H-2, J_{2 3}=10.0 Hz), 5.50 (ddd, 1H, H-8⁰, J_{8 9 a}=3.0Hz,

0

J_{8 9 b}=7.1 Hz), 5.44 (d, 1H, H-1, J_{1 2}=7.9Hz), 5.29

Methyl 2-naphthoylamido-2-deoxy-6-O-(N-acetyl-ꢀ-^D-

neuraminate-2-yl)-ꢁ-^D-glucopyranoside (6). Yield: 19 mg

(dd, 1H, H-3, J_{3 4}=3.5 Hz), 5.24 (dd, 1H, H-7⁰,

0

1

0

J_{7 8}=9.9Hz), 4.85 (ddd, 1H, H-4 , J_{4 5}=10.0 Hz), 4.37

0

(30 mmol, 70%). H NMR (D₂O): d 8.32±7.61 (m, 7H,

CH_arom), 4.65 (d, 1H, H-1, J_{1 2}=8.5 Hz), 3.56 (s,

(dd, 1H, H-9⁰a, J_{9 a 9 b}=12.2 Hz), 4.34 (dd, 1H, H-5,

0

J_{5 6a}=6.5 Hz, J_{5 6b}=8.5 Hz), 4.16 (dd, 1H, H-6⁰,

3H, OCH₃), 2.80 (dd, 1H, H-3⁰eq, J_{3 eq 4}=4.5 Hz,

0

0 0

J_{3 eq 3 ax}=12.3 Hz), 2.07 (s, 3H, CH₃acetyl), 1.78 (dd,

J_{5 6}=10.7 Hz, J_{6 7}=1.8 Hz), 4.14±4.05 (m, 1H, H-5 ),

3.98 (dd, 1H, H-9⁰b), 3.81 (s, 3H, OCH₃ester), 3.67 (dd,

1H, H-6a, J_{6a 6b}=11.5 Hz), 3.55 (dd, 1H, H-6b), 2.53

1H, H-3⁰ax, J_{3 ax 4}=12.0 Hz). HRMS: calculated for

0

[C₂₉H₃₈N₂O₁₄+Na⁺] 661.2221, found 661.2201.

(dd, 1H, H-3⁰eq, J_{3 eq 4}=4.6 Hz, J_{3 eq 3 ax}=12.9 Hz),

2.24, 2.22, 2.17, 2.08, 2.03, 2.01, 1.91, 1.90 (8Âs, 24H,

0

Methyl 2-(4-nitrobenzoylamido)-2-deoxy-6-O-(N-acetyl-

ꢀ-^D-neuraminate-2-yl)-ꢁ-D-glucopyranoside (7). Yield:

CH₃acetyl), 1.93 (dd, 1H, H-3⁰ax, J_{3 ax 4}=12.3 Hz).

0

1

35 mg (55 mmol, 74%). H NMR (D₂O): d 8.33 (d, 2H,

CH_arom, J=9.0 Hz), 7.95 (d, 2H, CH_arom), 4.58 (d, 1H,

Methyl 2-acetamido-2-deoxy-6-O-(N-acetyl-ꢀ-^D-neura-

minate-2-yl)-ꢁ-^D-glucopyranoside (1). Compound 27

(45 mg; 49mmol) was dissolved in a methanolic solution of

sodium methanolate (0.05 M; 2 mL), and stirred over-

night. TLC analysis (2-propanol:water 4:1, v/v) revealed

the formation of a single product. The solution was neu-

tralised by the addition of Dowex/H⁺and ®ltered. The

®ltrate was evaporated to dryness and dissolved in a

solution of NaOH (0.1 M) in 1,4-dioxane:water (3:1, v/v;

2 mL). The solution was carefully neutralised by the

addition of Dowex/H⁺and ®ltered. The ®ltrate was

evaporated to dryness and applied to an HW40 S gel

®ltration column, with water as eluent. The product

fractions were pooled, concentrated and lyophilised.

Yield of 1: 23 mg (44 mmol, 90%). Characteristic ¹H

NMR (D₂O) signals: d 4.40 (d, 1H, H-1, J_{1 2}=8.4 Hz),

H-1, J_{1 2}=8.5 Hz), 3.51 (s, 3H, OCH₃), 2.75 (dd, 1H,

0

H-3⁰eq, J_{3 eq 4}=4.6 Hz, J_{3 eq 3 ax}=12.3 Hz), 2.02 (s, 3H,

0

CH₃acetyl), 1.73 (dd, 1H, H-3⁰ax, J_{3 ax 4}=12.0 Hz).

Mass: calculated for C25H35N3O16 m/e 633.2, found

656.6 [M+Na⁺].

0

2-Acetamido-2-deoxy-6-O-(N-acetyl-ꢀ-^D-neuraminate-2-

yl)-ꢁ-^D-glucopyranosyl azide (8). Yield: 27 mg (50 mmol,

=

90%). ¹H NMR (D₂O): d 4.73 (d, 1H, H-1, J_{1 2}

9.2 Hz), 2.73 (dd, 1H, H-3⁰eq, J_{3 eq 4}=4.4 Hz,

0

J_{3 eq 3 ax}=12.3 Hz), 2.04, 2.03 (2Âs, 6H, CH₃acetyl),

1.69 (dd, 1H, H-3⁰ax, J_{3 ax 4}=11.9 Hz). HRMS: calcd

0

for [C₁₉H₃₁N₅O₁₃+Na⁺] 560.1816, found 560.1823.

4-Nitrophenyl 2-acetamido-2-deoxy-6-O-(N-acetyl-ꢀ-^D-

neuraminate-2-yl)-ꢁ-^D-glucopyranoside (9). Yield: 11 mg

(17 mmol, 79%). ¹H NMR (D₂O): d 8.24, 7.18 (2Âd, 4H,

CH_arom, J=9.3 Hz), 5.28 (d, 1H, H-1, J_{1 2}=8.4 Hz),

3.47 (s, 3H, OCH₃), 2.72 (dd, 1H, H-3⁰eq, J_{3 eq 4}

0

=

0

4.7 Hz, J_{3 eq 3 ax}=12.4 Hz), 2.01, 2.00 (2Âs, 6H, CH₃

acetyl), 1.69 (dd, 1H, H-3⁰ax, J_{3 ax 4}=12.0 Hz). HRMS:

calculated for [C₂₀H₃₄N₂O₁₄+Na⁺] 549.1908, found

549.1918.

0

2.73 (dd, 1H, H-3⁰eq, J_{3 eq 4}=4.4 Hz, J_{3 eq 3 ax}

=

0

12.3 Hz), 2.00 (s, 6H, CH₃acetyl), 1.65 (dd, 1H, H-3⁰ax,

0

J_{3 ax 4}=12.0 Hz). HRMS: calcd for [C₂₅H₃₅N₃O₁₆+

Methyl 2-azido-2-deoxy-6-O-(N-acetyl-ꢀ-^D-neuraminate-

2-yl)-ꢁ-^D-glucopyranoside (2). Yield: 13 mg (25 mmol,

=

Na⁺] 656.1915, found 656.1931.

89%). ¹H NMR (D₂O): d 4.41 (d, 1H, H-1, J_{1 2}

Methyl 4-O-(6-O-(N-acetyl-ꢀ-^D-neuraminate-2-yl)-ꢁ-D-

galactopyranosyl)-ꢁ-^D-glucopyranoside (10). Yield: 10 mg

8.2 Hz), 3.57 (s, 3H, OCH₃), 3.28 (dd, 1H, H-2,

J_{2 3}=9.4 Hz), 2.72 (dd, 1H, H-3⁰eq, J_{3 eq 4}=4.5 Hz,

(15 mmol, 99%). H NMR (D₂O): d 4.40 (d, 1H, H-1⁰,

1

0

0 0

J_{1 2}=7.8 Hz), 4.39 (d, 1H, H-1, J_{1 2}=8.0 Hz), 3.55 (s,

J_{3 eq 3 ax}=12.3 Hz), 2.01 (s, 3H, CH₃acetyl), 1.69 (dd,

0

1H, H-3⁰ax, J_{3 ax 4}=12.1 Hz). HRMS: calculated for

3H, OCH₃), 3.51 (dd, 1H, H-2⁰, J_{2 3}=10.0 Hz), 3.31 (dd,

0

[C₁₈H₃₀N₄O₁₃+Na⁺] 533.1707, found 533.1679.

1H, H-2, J_{2 3}=8.6 Hz), 2.69 (dd, 1H, H-3⁰⁰eq, J_{3 eq 4}

00

=

4.7 Hz, J_{3 eq 3 ax}=12.3 Hz), 2.00 (s, 3H, CH₃acetyl),

00

1.72 (dd, 1H, H-3⁰⁰ax, J_{3 ax 4}=12.1 Hz). HRMS: calcd

00

Methyl 2-benzoylamido-2-deoxy-6-O-(N-acetyl-ꢀ-^D-neur-

aminate-2-yl)-ꢁ-^D-glucopyranoside (4). Yield: 24 mg

for [C₂₄H₄₁NO₁₉+Na⁺] 670.2170, found 670.2165.

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97

95

4-O-(6-O-(N-Acetyl-ꢀ-D-neuraminate-2-yl)-ꢁ-D-galacto-

pyranosyl)-ꢁ-^D-glucopyranosyl azide (11). Yield: 26 mg

(10.9 mmol, 72%). ¹³C{¹H} NMR (CDCl₃): d 170.4±168.8

(CO acetyl), 99.9 (C-1), 71.8, 70.6, 68.1 (C-3,4,5), 69.1,

69.0, 68.0 (CH₂O glycol, C-6), 53.3 (C-2), 49.7 (CH₂N₃),

21.7 (CH₃N-acetyl), 19.4 (CH₃O-acetyl).

1

(39 mmol, 99%). H NMR (D₂O): d 4.75 (d, 1H, H-1,

J_{1 2}=8.8 Hz), 4.40 (d, 1H, H-1⁰, J_{1 2}=7.8 Hz), 3.51

0

(dd, 1H, H-2⁰, J_{2 3}=9.9 Hz), 3.32 (dd, 1H, H-2, J_{2 3}

=

0

8.9 Hz), 2.68 (dd, 1H, H-3⁰eq, J_{3 eq 4}=4.7 Hz, J_{3 eq 3 ax}

12.3 Hz), 2.01 (s, 3H, CH₃acetyl), 1.71 (dd, 1H, H-3⁰ax,

1-Azido-3-oxopentyl 2-amino-2-deoxy-ꢁ-D-glucopyrano-

side (43). A solution of the azido derivative 42 (1.38 g;

3.0 mmol) in aq NaOH (1 M; 150 mL) was re¯uxed over-

night. The mixture was allowed to cool to room tempera-

ture, and was neutralised by the addition of aq HCl (1 M).

The mixture was concentrated, evaporated with methanol

(3Â50mL) and ®ltered. The ®ltrate, containing the crude

amine 43, was concentrated and led over a silica gel col-

umn, with ethyl acetate:methanol:water (7:2:1, v/v/v) as

eluent. The product, still contaminated with salts, was

used immediately for the synthesis of 45.

0

J_{3 ax 4}=12.1 Hz). HRMS: calcd for [C₂₂H₃₈N₄O₁₈+

Na⁺] 681.2079, found 681.2118.

Methyl 6-O-(N-acetyl-ꢀ-^D-neuraminate-2-yl)-ꢁ-D-galac-

topyranoside (12). Yield: 13 mg (26 mmol, 55%). ¹H

NMR (D₂O): d 4.27 (d, 1H, H-1, J_{1 2}=7.9 Hz), 3.53 (s,

3H, OCH₃), 3.45 (dd, 1H, H-2, J_{2 3}=10.0 Hz), 2.69 (dd,

1H, H-3⁰eq, J_{3 eq 4}=4.6 Hz, J_{3 eq 3 ax}=12.3 Hz), 1.99 (s,

0

3H, CH₃acetyl), 1.66 (dd, 1H, H-3⁰ax, J_{3 ax 4}

12.0 Hz). HRMS: calcd for [C₁₈H₃₁NO₁₄+Na⁺]

508.1642, found 508.1639.

0

=

1-Azido-3-oxopentyl 2-(4-nitrobenzoyl)amido-3,4-di-O-

acetyl-2-deoxy-ꢁ-^D-glucopyranoside (45). The crude

amine 43 (ꢁ3 mmol) was evaporated with dry toluene

(2Â20 mL), and dissolved in pyridine (30 mL). The

solution was placed in an ice bath. Triethylamine

(0.42 mL; 3.0 mmol) and 4-nitrobenzoyl chloride (2.22 g;

12 mmol) were added, and the mixture was stirred

overnight. TLC analysis (ethanol:toluene 1:9, v/v)

revealed the absence of 43. The reaction was quenched

by the addition of water (1 mL), the mixture was con-

centrated, taken up in DCM (50 mL), washed with aq

NaHCO₃(50 mL) and water (50 mL), dried (MgSO₄)

and evaporated with toluene (2Â20 mL) and ethanol

(20 mL). The product mixture, obtained as a viscous oil,

was dissolved in a methanolic solution of sodium

methanolate (0.1 M; 50 mL), and stirred overnight.

According to TLC analysis (toluene:methanol 1:1, v/v)

one product was formed. The solution was neutralised

by the addition of Dowex/H⁺and ®ltered. The ®ltrate,

containing the crude 4-nitrobenzoylamido glucopyr-

anoside 44, was concentrated, evaporated with dry

toluene (2Â20 mL), and dissolved in pyridine (30 mL).

tBDMS-Cl (0.46 g; 3.0 mmol) was added and the mix-

ture was stirred for 3 h. TLC analysis (toluene:methanol

1:1, v/v) indicated complete conversion of the starting

material. Acetic anhydride (2 mL) was added and the

mixture was stirred overnight. TLC analysis (hexanes/

ethyl acetate) showed the acetylation to be complete.

The mixture was concentrated, taken up in DCM

(100 mL), washed with aq NaHCO₃(1 M, 100 mL) and

water (100 mL), dried over MgSO₄and evaporated with

toluene (2Â10 mL) and ethanol (10 mL). The fully

protected product thus obtained was dissolved in

acetonitrile:water (7:1, v/v; 16 mL) and the pH of the

mixture was adjusted to 3 by the addition of para-toluene-

sulfonic acid monohydrate. After 2 h, TLC analysis

(DCM:methanol 95:5, v/v) showed the removal of the

tBDMS group to be complete. The reaction mixture was

diluted with ethyl acetate (50 mL), washed with aq

NaHCO₃(1 M; 50 mL) and water (50 mL), dried

(MgSO₄) and concentrated. The crude product was

applied to a silica gel column, and eluted with DCM:

MeOH (1:0!9:1, v/v). Yield: 0.51 g (0.97 mmol, 32%

based on 42). ¹³C{¹H} NMR (CDCl₃): d 170.6±166.1

(CO acetyl, benzoyl), 149.1, 139.5 (C_qbenzoyl), 127.9,

123.1 (CH_arombenzoyl), 100.2 (C-1), 73.9, 72.5, 68.7

4-Nitrophenyl 6-O-(N-acetyl-ꢀ-^D-neuraminate-2-yl)-ꢁ-D-

galactopyranoside (13). Yield: 12 mg (20 mmol, 91%).

¹H NMR (D₂O): d 8.25 (d, 2H, CH_arom, J=9.3 Hz),

7.23 (d, 2H, CH_arom), 5.16 (d, 1H, H-1, J_{1 2}=7.3 Hz),

2.73 (dd, 1H, H-3⁰eq, J_{3 eq 4}=4.2 Hz, J_{3 eq 3 ax}

=

0

12.3 Hz), 1.97 (s, 3H, CH₃acetyl), 1.61 (dd, 1H, H-3⁰ax,

0

J_{3 ax 4}=11.8 Hz). HRMS: calcd for [C₂₃H₃₂N₂O₁₆+

Na⁺] 615.1650, found 615.1627.

Methyl 2-amino-2-deoxy-6-O-(N-acetyl-ꢀ-^D-neuraminate-

2-yl)-ꢁ-^D-glucopyranoside (3). Compound 2 (14 mg;

28 mmol) was dissolved in dry methanol (140 mL). To

this solution were added triethylamine (20 mL; 140 mmol)

and 1,3-propanedithiol (14 mL; 140 mmol). The mixture

was left at room temperature, until TLC analysis (2-

propanol:water 4:1, v/v) indicated complete conversion of

the starting material into a ninhydrine-positive product.

The mixture was ®ltered, the ®ltrate was concentrated and

then evaporated with 1,4-dioxane (1Â10 mL). The crude

product thus obtained was lyophilised from 1,4-dioxane,

and then applied to an HW40 S gel ®ltration column,

with water as eluent. The product fractions were pooled,

concentrated and lyophilised. Yield: 2.4 mg (5.0 mmol,

18%). ¹H NMR (D₂O): d 4.61 (d, 1H, H-1, J_{1 2}=8.1 Hz),

3.57 (s, 3H, OCH₃), 2.73 (dd, 1H, H-3⁰eq,

0

J_{3 eq 4}=4.5 Hz, J_{3 eq 3 ax}=12.3 Hz), 2.02 (s, 3H, CH₃

0

acetyl), 1.70 (dd, 1H, H-3⁰ax, J_{3 ax 4}=12.1 Hz). HRMS:

calcd for [C₁₈H₃₂N₂O₁₃+H⁺] 485.1983, found 485.1984.

0

1-Azido-3-oxopentyl 2-acetamido-3,4,6-tri-O-acetyl-2-

deoxy-ꢁ-^D-glucopyranoside (42). To a solution of the

known³⁰oxazoline of glucosamine (41) (4.94 g;

15 mmol) and 2-(2-azidoethoxy)ethanol (2.95 g; 22.5

mmol) in 1,2-dichloroethane (DCE, 50 mL) was added,

under an argon atmosphere, a catalytic amount of

TfOH (130 mL, 1.5 mmol). The reaction was stirred

overnight at 40 ^ꢀC. According to TLC analysis (DCM:

methanol 9:1, v/v), the starting material was completely

converted into a product with lower mobility. The mix-

ture was taken up in DCM (50 mL), washed with aq

NaHCO₃(1 M, 50 mL) and water (50 mL), dried over

MgSO₄and concentrated. The crude oil thus obtained

was puri®ed by silica gel column chromatography, with

DCM:methanol (100:0!96:4, v/v) as eluent. Yield: 5.02g

96

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97

(C-3,4,5), 69.7, 69.3, 68.4 (OCH₂glycol), 60.6 (C-6),

54.4 (C-2), 50.1 (CH₂N₃), 19.8 (CH₃O-acetyl). ¹H

NMR (CDCl₃): d 8.00±7.67 (m, 4H, CH_arombenzoyl),

5.13 (dd, 1H, H-3, J_{3 4}=9.6 Hz), 4.81 (dd, 1H, H-4,

J_{4 5}=8.9 Hz), 4.59 (d, 1H, H-1, J_{1 2}=8.6 Hz), 3.93 (dd,

1H, H-2, J_{2 3}=10.5 Hz), 3.78 (m, 1H, H-5), 3.55±2.95

(m, 10H, H-6a,6b, CH₂glycol), 1.79, 1.71 (2Âs, 6H,

CH₃acetyl).

20 mL; 250 mmol) was added, followed by 1,4-dioxane

(10 mL). The mixture was concentrated, and evaporated

with 1,4-dioxane (2Â10 mL). The crude amine 48 thus

obtained was used immediately for the synthesis of the

cluster 50.

[Ac₄NeuAcMeAc₂GlcN(BzNO₂)O(CH₂)₂O(CH₂)₂NHC

(O)(CH₂)₂OCH₂]₃CNHC(O)CH₂NHZ (50). The amine

48 (90 mmol) and the tris-carboxylic acid 49 (11 mg;

20 mmol) were dissolved in DMF (5 mL), and DIPEA

(41 mL; 0.24 mmol) and HOBT (10 mg; 75 mmol) were

added. To this mixture was added HBTU (28 mg;

75 mmol). The reaction was stirred for 3 h, when TLC

analysis (DCM:methanol 9:1, v/v) revealed the reaction to

be complete. The mixture was taken up in DCM (40 mL),

washed with dil. H₃PO₄(1 M; 50 mL), aq NaHCO₃(1 M;

50 mL), and water (50 mL), dried (Mg SO₄), concentrated

and evaporated with toluene (2Â20mL) and ethanol

(20 mL). The crude product was puri®ed by silica gel

column chromatography, with DCM:methanol (1:0!

92:8, v/v) as eluent, followed by Sephadex LH-20 gel

®ltration, with DCM:methanol (2:1, v/v) as eluent.

Yield: 44 mg (13.0 mmol, 65%). ¹³C{¹H} NMR (CDCl₃):

d 171.7±165.8 (CO acetyl, benzoyl, C-1⁰), 156.7 (CO

Z), 149.6, 140.0 (C_qbenzoyl), 136.3 (C_qZ), 128.5±123.7

(CH_arombenzoyl, Z), 101.0 (C-1), 98.8 (C-2⁰), 72.9, 72.5,

69.2, 69.0, 68.3, 67.5 (C-3,4,5, C-4⁰,6⁰,7⁰,8⁰), 70.1 69.7,

69.4, 68.6 66.9 (OCH₂TRIS, propanoyl, glycol, CH₂Z),

63.6, 62.6 (C-6, C-9⁰), 60.0 (C_qTRIS), 55.1 (C-2), 52.7

(OCH₃ester), 49.4 (C-5⁰), 44.7 (CH₂Gly), 39.3, 37.6,

36.5 (C-3, CH₂CO propanoyl, CH₂NH), 23.0 (CH₃N-

1-Azido-3-oxopentyl 2-(4-nitrobenzoyl)amido-3,4-di-O-

acetyl-2-deoxy-6-O-(methyl 4,7,8,9-tetra-O-acetyl-N-ace-

tyl-ꢀ-^D-neuraminate-2-yl)-ꢁ-D-glucopyranoside (47). A

mixture of acceptor 45 (262 mg; 0.500 mmol) and donor

46 (521 mg; 1.00 mmol)³¹and powdered molecular

sieves (3 A) in acetonitrile (10 mL) was stirred for 1 h

under argon atmosphere. The temperature of the mix-

ture was lowered to 50 ^ꢀC, and a solution of NIS

(225 mg; 1.00 mmol) and TfOH (10 mL; 0.11 mmol) in

acetonitrile (1 mL) was added. The mixture was gradu-

ally warmed to room temperature over a period of 3 h.

The reaction was quenched by the addition of triethyl-

amine (0.1 mL). The mixture was ®ltered over Hy¯o,

diluted with ethyl acetate (50 mL), washed with aq

Na₂S₂O₃(1 M; 50 mL), aq NaHCO₃(1 M; 50 mL) and

water (50 mL), dried (MgSO₄), concentrated, and

applied to a silica gel column. Elution was performed

with DCM:methanol (1:0!96:4, v/v). The fractions

containing the product were pooled, concentrated, and

applied to a Sephadex LH-20 column. The disaccharide

47 was obtained as a/b mixture. Yield: 214 mg

(214 mmol, 45%, a/b 3:1). The a-isomer was isolated

from the latter mixture by silica gel chromatography,

with toluene:ethanol (1:0!9:1, v/v) as eluent. Yield:

143 mg (143 mmol). ¹³C{¹H} NMR (CDCl₃): d 171.0±

165.4 (CO acetyl, benzoyl, C-1⁰), 149.5, 139.5 (C_q

benzoyl), 128.2, 123.6 (CH_arombenzoyl), 101.2 (C-1),

98.7 (C-2⁰), 72.9, 72.3, 68.9, 68.3, 67.4 (C-3,4,5, C-

4⁰,6⁰,7⁰,8⁰), 70.6, 69.6, 68.5 (OCH₂glycol), 63.6, 62.5 (C-

6, C-9⁰), 54.8 (C-2), 52.6 (OCH₃ester), 50.7 (CH₂N₃),

49.2 (C-5⁰), 37.5 (C-3⁰), 22.9 (CH₃N-acetyl), 20.6 (CH₃

1

acetyl), 21.0, 20.6 (CH₃O-acetyl). H NMR (CDCl₃):

d 8.30±7.19 (m, 12H, CH_arombenzoyl), 7.31 (bs, 5H,

CH_aromZ), 6.87 (d, 3H, NH benzoyl, J=9.3 Hz), 5.39

(ddd, 3H, H-8⁰, J_{8 9 a}=2.7 Hz, J_{8 9 b}=6.2 Hz), 5.30±

0

5.17 (m, 11H, CH₂Z, H-3,4, H-7⁰), 4.87 (ddd, 3H, H-4⁰,

0

J_{4 5}=9.7 Hz, J_{3 ax 4}=12.3 Hz), 4.76 (d, 3H, H-1,

J_{1 2}=8.4 Hz), 4.35 (dd, 3H, H-9⁰a, J_{9 a 9 b}=12.3 Hz),

4.27 (ddd, 3H, H-2, J_{2 3}=9.3 Hz, J_{3 4}=9.4 Hz), 4.10±

3.18 (m, 62H, H-5,6a,6b, H-5⁰,6⁰,9⁰b, CH₂Gly, OCH₂

propanoyl, CH₂glycol, OCH₂TRIS, CH₂CO propa-

noyl), 3.81 (s, 9H, OCH₃ester), 2.63 (dd, 3H, H-3⁰eq,

0

O-acetyl). b-Isomer: d 98.3 (C-2⁰). H NMR (CDCl₃): d

1

8.25±7.96 (m, 4H, CH_arombenzoyl), 5.61 (dd, 1H, H-3,

J_{3 4}=9.6 Hz), 5.38 (ddd, 1H, H-8⁰, J_{8 9 a}=2.5 Hz,

0

J_{3 eq 4}=4.6 Hz, J_{3 eq 3 ax}=12.8 Hz), 2.15, 2.09, 2.04,

2.02, 1.98, 1.88 (6Âs, 63H, CH₃acetyl), 1.94 (dd, 3H,

H-3⁰ax).

J_{8 9 b}=6.0 Hz), 5.30 (dd, 1H, H-7⁰, J_{7 8}=8.7 Hz,

0

J_{6 7}=11.5 Hz), 5.17 (dd, 1H, H-4, J_{4 5}=9.6 Hz), 4.88

(m, 1H, H-4⁰), 4.84 (d, 1H, H-1, J_{1 2}=8.5 Hz), 4.34 (dd,

1H, H-9⁰a, J_{9 a 9 b}=9.9 Hz), 4.15 (ddd, 1H, H-2,

J_{2 3}=9.0 Hz), 4.07±3.16 (m, 14H, H-5,6a,6b, H-5⁰,6⁰,9⁰b,

CH₂glycol), 3.80 (s, 3H, OCH₃ester), 2.62 (dd, 1H, H-

[NeuAcGlcN(BzNO₂)O(CH₂)₂O(CH₂)₂NHC(O)(CH₂)₂

OCH₂]₃CNHC(O)CH₂NHZ (52). Compound 50

(44 mg; 13 mmol) was dissolved in a methanolic solution

of sodium methanolate (0.05 M; 2 mL), and stirred

overnight. According to TLC analysis (2-propanol:

water 4:1, v/v) one product was formed. The solution

was neutralised by the addition of Dowex/H⁺and ®l-

tered. The ®ltrate was evaporated to dryness and dis-

solved in a solution of NaOH (0.1 M) in 1,4-dioxane:

water (3:1, v/v; 2 mL). The solution was carefully neutral-

ised by the addition of Dowex/H⁺and ®ltered. The ®l-

trate was evaporated to dryness and applied to an

HW40 S gel ®ltration column, with water as eluent. The

product fractions were pooled, concentrated and

lyophilised. Yield: 16 mg (6 mmol, 47%). ¹H NMR

(D₂O): d 8.26±7.91 (m, 12 H, CH_arombenzoyl), 7.30 (bs,

5H, CH_aromZ), 5.02 (s, 2H, CH₂Z), 4.68 (d, 3H, H-1,

0

3⁰eq, J_{3 eq 4}=4.5 Hz, J_{3 eq 3 ax}=12.7 Hz), 2.14, 2.13,

2.07, 2.03, 2.01, 1.96, 1.88 (7Âs, 21H, CH₃acetyl), 1.93

(dd, 1H, H-3⁰ax).

0

1-Amino-3-oxopentyl 2-(4-nitrobenzoylamido)-2-deoxy-6-

O-(N-acetyl-ꢀ-^D-neuraminate-2-yl)-ꢁ-D-glucopyranoside

(48). The azido disaccharide 47 (122 mg; 122 mmol) was

dissolved in dry THF (2 mL). To the solution was added

triphenylphosphine (40 mg; 0.15 mmol), and the mixture

was stirred for 48 h. Water (6 mL; 350 mmol) was added,

and the mixture was stirred for another 24 h. According

to TLC analysis (toluene:methanol 1:1, v/v), the starting

material was completely converted into a lower-running

ninhydrine-positive product. Tri¯uoroacetic acid (TFA,

L. A. J. M. Sliedregt et al. / Bioorg. Med. Chem. 9 (2001) 85±97

97

J_{1 2}=8.4 Hz), 2.73 (dd, 3H, H-3⁰eq, J_{3 eq 4}=4.5Hz,

6. Doody, G. M.; Dempsey, P. W.; Fearon, D. T. Curr. Opin.

Immun. 1996, 8, 378.

7. Sgroi, D.; Varki, A.; Braesch Andersen, S.; Stamenkovic, I.

J. Biol. Chem. 1993, 268, 7011.

8. Powell, L. D.; Sgroi, D.; Sjoberg, E. R.; Stamenkovic, I.;

Varki, A. J. Biol. Chem. 1993, 268, 7019.

0

J_{3 eq 3 ax}= 12.3 Hz), 2.01 (s, 9H, CH₃acetyl), 1.71 (dd,

0

3H, H-3⁰ax, J_{3 ax 4}=12.0 Hz). Mass: calcd for

C₁₀₇H₁₅₂N₁₄O₆₀m/e 2592.9, found 2592.7.

0

9. Powell, L. D.; Varki, A. J. Biol. Chem. 1994, 269, 10628.

10. Powell, L. D.; Jain, R. K.; Matta, K. L.; Sabesan, S.;

Varki, A. J. Biol. Chem. 1995, 270, 7523.

11. Sjoberg, E. R.; Powell, L. D.; Klein, A.; Varki, A. J. Cell

Biol. 1994, 126, 549.

Erythrocyte solid-phase binding assay

The anities of the glycosides for CD22 were deter-

mined in a solid phase binding assay, based on the

binding of porcine erythrocytes to immobilised murine

CD22D1-3-IgG (Costar, high binding). In short, multi-

well plates were coated with Goat anti Human Fc

(0.75 mg/well) by overnight incubation in 0.1 M sodium

bicarbonate buer (pH=9.6) at 4 ^ꢀC. Subsequently, the

wells were blocked with 5% skimmed milk (w/w) for 1 h

at 37 ^ꢀC. After thorough washing, the wells were incu-

bated with mCD22 for 3 h at 4 ^ꢀC at a concentration of

0.01 mg/well in PBS+0.25% BSA. The wells were again

thoroughly washed and incubated for 1 h at 20 ^ꢀC with

porcine erythrocytes (1% dilution in PBS+0.25% BSA)

in the absence or presence of 1.0±1500 mM of the inhi-

bitor. The peroxidase activity of haemoglobulin in the

bound erythrocytes was determined using o-phenylene-

diamine hydrochloride as substrate in 50 mM phosphate

citrate buer (pH=5)+0.014% H₂O₂after thorough

washing with PBS+0.25% BSA. CD22-speci®c binding

was de®ned as the dierential binding in the absence

and the presence of an excess of Orosomucoid (Sigma)

(3mM). The CD22-speci®c erythrocyte binding amounted

to a reproducible 40±60% of the total binding. From

the competition curves, the IC₅₀(de®ned as the con-

centration of inhibitor giving 50% reduction of speci®c

erythrocyte binding) and the Hill coecient (which is

indicative of the cooperativity of ligand binding) could

be calculated by non-linear regression analysis (Prism,

GraphPad Software Inc.). Data are means of a triplicate

experiment.

12. Kelm, S.; Schauer, R.; Manuguerra, J.-C.; Gross, H.-J.;

Crocker, P. R. Glycoconjugate J. 1994, 11, 576.

13. Kelm, S.; Brossmer, R.; Isecke, R.; Gross, H. J.; Strenge,

K.; Schauer, R. Eur. J. Biochem. 1998, 255, 663.

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1993, 3, 633.

15. Sliedregt, L. A. J. M.; Rensen, P. C. N.; Rump, E. T.; van

Santbrink, P. J.; Bijsterbosch, M. K.; Valentijn, A. R. P. M.;

Van der Marel, G. A.; Van Boom, J. H.; van Berkel, T. J. C.;

Biessen, E. A. L. J. Med. Chem. 1999, 42, 609.

16. Marra, A.; Sinay, P. Carbohydr. Res. 1989, 187, 35.

17. Veeneman, G. H.; Van Leeuwen, S. H.; Van Boom, J. H.

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18. Nifant'ev, N. E.; Tsvetkov, Y. E.; Shashkov, A. S.;

Kononov, L. O.; Menshov, V. M.; Tuzikov, A. B.; Bovin, N.

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20. Nath, D.; Van der Merwe, P. A.; Kelm, S.; Brad®eld, P.;

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21. Van der Merwe, P. A.; Crocker, P. R.; Vincent, M.;

Barclay, A. N.; Kelm, S. J. Biol. Chem. 1996, 271, 9273.

22. Nelson, R. M.; Dolich, S.; Aruo, A.; Cecconi, O.;

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1520.

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Acknowledgement

The ®nancial support from Snow Brand Milk Products

(Japan) is gratefully acknowledged

27. Hayashi, M.; Tanaka, M.; Itoh, M.; Miyauchi, H. J. Org.

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Article Doi

Design and synthesis of a multivalent homing device for targeting to murine CD22

DOI: 10.1016/S0968-0896(00)00224-8

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Full text of DOI:10.1016/S0968-0896(00)00224-8

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