The synthesis of biotinylated carbohydrates as probes for carbohydrate-recognizing proteins

Article abstract of DOI:10.1016/S0968-0896(00)00201-7

The intimate involvement of carbohydrate-protein interactions in a number of important biological processes has prompted several research efforts towards developing new methods of investigating these glycobiological interactions. Biotinylated oligosaccharides are emerging as a new and powerful tool in this area of research, primarily due to their high affinity towards streptavidin and their ease of immobilization on matrices. Here we describe a novel synthetic approach towards biotinylated saccharides which incorporate a UV absorbing group into the final compounds. The synthetic strategy described is applicable to a variety of saccharides, with examples of biotinylated mono-, di-, and trisaccharides being prepared with overall high efficiency. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00201-7

Bioorganic & Medicinal Chemistry 8 (2000) 2709±2718
The Synthesis of Biotinylated Carbohydrates as Probes for
Carbohydrate-Recognizing Proteins
Donald I. Angus, a Milton J. Kiefel a,b and Mark von Itzstein a,b,
*
aDepartment of Medicinal Chemistry, Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052, Australia
bCentre for Biomolecular Science and Drug Discovery, Grith University (Gold Coast Campus),
PMB 50 Gold Coast Mail Centre, Queensland 9726, Australia
Received 6 June 2000; accepted 18 July 2000
AbstractÐThe intimate involvement of carbohydrate±protein interactions in a number of important biological processes has
prompted several research e€orts towards developing new methods of investigating these glycobiological interactions. Biotinylated
oligosaccharides are emerging as a new and powerful tool in this area of research, primarily due to their high anity towards
streptavidin and their ease of immobilization on matrices. Here we describe a novel synthetic approach towards biotinylated sac-
charides which incorporate a UV absorbing group into the ®nal compounds. The synthetic strategy described is applicable to a
variety of saccharides, with examples of biotinylated mono-, di-, and trisaccharides being prepared with overall high eciency.
# 2000 Elsevier Science Ltd. All rights reserved.
Introduction
recognizing proteins,15,22 28 since they exploit the strong
anity of avidin and streptavidin for biotin.29 Since
each streptavidin molecule has four biotin binding sites,
the complex resulting from interaction between strepta-
vidin and biotinylated carbohydrate has the potential to
behave as a multivalent ligand.30 Thus far, applications
of biotinylated carbohydrates have included the immo-
bilization onto the surface of a sensor chip to facilitate
kinetic measurements,15,25 the production of IgG anti-
bodies directed against oligosaccharide chains,29 and
immobilization on streptavidin-coated microwells for
lectin binding studies.30,31 Generally, oligosaccharides
biotinylated at the anomeric centre of the reducing
sugar have been prepared under either reducing or non-
reducing conditions.15,22 25 The reducing methods typi-
cally involve coupling of oligosaccharides with 2-amino-
6-amidobiotinyl pyridine (BAP), whilst coupling under
non-reducing conditions generally requires biotinylated
hydrazine reagents. Unfortunately, many of these
methods result in poor yields of the oligosaccharide
being tagged.
Carbohydrate-recognizing proteins are involved in a
number of important biological processes, including
cell±cell interactions,1 3 bacterial and viral infections,4 6
in¯ammation,7 10 and metastasis.11 13 Consequently,
investigations into carbohydrate±protein interactions
are becoming increasingly important in the search for a
better understanding of these glycobiological processes.
The number of established techniques available for
investigating carbohydrate±protein interactions, includ-
ing anity chromatography, electrophoresis, and NMR
spectroscopy, is continually growing.14 17 An important
aspect of these investigations is the requirement for studies
to be carried out with structurally-de®ned oligosacchar-
ide sequences such that speci®c interactions can be ana-
lysed. Common techniques employed for carrying out
these studies include the linking of saccharides to proteins,
the linking of oligosaccharides to aminophospholipids,
the incorporation of oligosaccharides into polymers, and
the use of ¯uorescently-labelled oligosaccharides.18 21
A potential limitation of some of the reported biotiny-
lated oligosaccharides is the lack of a chromophore,
meaning, for example, that their use in the puri®cation
of oligosaccharides from natural sources is limited
because of low sensitivity of detection. Recent e€orts to
improve sensitivity have focussed on the incorporation
Recently it has been shown that biotinylated carbohy-
drates have great potential as probes for carbohydrate-
*Corresponding author. Tel.: +617-5594-8232; fax: +617-5594-8098;
e-mail: m.vonitzstein@mailbox.gu.edu.au
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00201-7

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D. I. Angus et al. / Bioorg. Med. Chem. 8 (2000) 2709±2718
of a UV tag in addition to the biotinyl group. The
resulting bifunctionally-labelled oligosaccharides, e.g.,
Here we describe a novel approach to the synthesis of
biotinylated carbohydrates that incorporate a UV active
chromophore. This method utilizes our previous exper-
tise in the preparation of thioglycosides,37 40 and is
¯exible enough to facilitate the synthesis of tagged
mono-, di- and trisaccharides in high yield.
22
1,
have both the ability to be immobilized through
the biotin portion and detected through the strong UV
absorbing group. Such compounds have the advantage in
that UV detection can be utilized during assays or pur-
i®cation procedures in the low picomolar range.15,22 24
The potential applications of tagged biotinylated oligo-
saccharides (e.g., anity chromatography, puri®cation
of complex oligosaccharides, binding assays with carbo-
hydrate-recognizing proteins) prompted us to investigate
the development of a versatile and ecient synthesis of
appropriately tagged (oligo)saccharides. Studies of car-
bohydrate interactions with glycohydrolases and other
carbohydrate-recognizing proteins, such as adhesins, are
of importance for several disease states.1,2,8,13 We
therefore felt it appropriate to prepare tagged (oligo)
saccharides of the general structure 2, in which the thio-
glycosidically linked bifunctional aglycon unit would be
stable to enzymatic hydrolysis. We have previously
demonstrated, using 1H NMR spectroscopy, that the
thioglycosidic linkage in thiosialosides such as 3 is resis-
tant to hydrolysis by sialidases.32 Others33 36 have also
shown that thioglycosides (e.g., 436) are resistant to
hydrolysis by glycohydrolases, and are therefore extre-
mely useful compounds in studies with such enzymes.
Results and Discussion
Our general strategy towards the synthesis of appro-
priately tagged (oligo)saccharides like 2 is shown in ret-
rosynthetic terms in Scheme 1. We felt the most ¯exible
and convergent approach to biotinylated (oligo)-sac-
charides like 2 would involve a ®nal coupling between 4-
aminobenzyl thioglycosides such as 5 and biotin (6),
using well established peptide coupling techniques.41
Scheme 1. Retrosynthetic approach towards biotinylated oligosaccharides.

D. I. Angus et al. / Bioorg. Med. Chem. 8 (2000) 2709±2718
2711
An ecient synthesis of appropriate 4-aminobenzyl
thioglycosides like 5 can be approached from either
`route a' or `route b' (Scheme 1). Whilst both approaches
have their merits, we felt the most ecient avenue into
4-aminobenzyl thioglycosides like 5 would be via
`route b', since we have previously described the use of
anomeric thiolacetyl compounds like 7 as precursors in
the synthesis of thioglycosides.37 40,42 In addition, we
knew that appropriately activated 4-substituted benzyl
derivatives would be commercially available, and some
of the requisite anomeric thiolacetyl derivatives 7 were
already available in our laboratories. Importantly, this
approach also has the potential to allow maximum
¯exibility in terms of incorporating di€erent carbohy-
drate units and various UV-tags whilst maintaining the
same overall strategy.
2-thiolacetyl-Neu5Ac derivative 8 with hydrazinium
acetate in the presence of benzyl bromide furnished the
benzyl thiosialoside 9 in 73% yield. Having established
the methodology for preparing benzyl thioglycosides
like 9 in high yield, we sought an appropriate glycosyl
acceptor for access towards compounds of the general
structure 5. Whilst it would have been useful to employ
4-aminobenzyl bromide as the glycosyl acceptor, pre-
liminary investigations into the preparation of such an
acceptor suggested it would be more ecient to use the
corresponding nitro derivative and subsequently reduce
the nitro group. Accordingly, hydrazinium acetate-
mediated coupling between anomeric thiolacetyl deriva-
tives 8,43 10,45 and 11, and 4-nitrobenzyl bromide pro-
ceeded smoothly to give the 4-nitrobenzyl thioglycoside
derivatives 12,44 13 and 14, respectively, in high yield.
In an initial investigation of the applicability of the
approach shown in Scheme 1 we attempted coupling
between the 2-thiolacetyl-Neu5Ac derivative 843 and
benzyl bromide. Interestingly, conditions we had pre-
viously used for the successful synthesis of thiosialo-
sides, using Et2NH promoted coupling between 8 and
primary alkyl halides,37,39 failed to furnish any of the
desired benzyl thiosialoside derivative 9. As the only
components isolated from this reaction were unreacted
benzyl bromide and the thiol corresponding to 8, we can
only assume that the thiolate derived in this way is not
reactive enough to displace a benzylic bromide.
With the 4-nitrobenzyl thioglycosides in hand, our
attention turned to reduction of the nitro group.
Although many methods for the direct reduction of
aromatic nitro groups to amines are available, the pre-
sence of the sulfur in compounds 12, 13 and 14 prevents
the use of metal-promoted catalytic hydrogenation.46 It
has been shown that the use of tin(II) chloride dihydrate
is compatible with sulfur,47 and has been successfully
employed for the reduction of a number of 4-nitro-
benzyl thioglycosides.48 Accordingly, reduction of the
nitro group in the thioglycosides 12, 13 and 14, with
.
SnCl2 2H2O in ethanol, gave the 4-aminobenzyl thio-
glycosides 15, 16 and 17, respectively, in good yield.
The coupling of the carboxylic acid group in biotin (6)
to the amine residues in the substituted benzyl thiogly-
cosides 15, 16 and 17 could be achieved using a number
of well known peptide coupling techniques.41 Whilst the
®eld of carboxylic acid activation for peptide coupling
has been traditionally dominated by the use of carbo-
diimide reagents such as DCC and EDC, these reagents
often result in the formation of side products.49 The use
of uronium salts (e.g., HBTU, TBTU) in peptide cou-
pling reactions has been shown to be fast and with lim-
ited side reactions.50 Accordingly, treatment of an N,N-
DMF solution of the 4-aminobenzyl thiosialoside deri-
vative 15 with d-biotin (6) in the presence of TBTU [O-
(1H-benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
tetra¯uoroborate]51 a€orded the desired UV-tagged
biotinylated thiosialoside 18 in 70% yield. Similarly, the
Fortunately, de-S-acetylation of 8 using hydrazinium
acetate44 proved more successful. Thus, treatment of the

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D. I. Angus et al. / Bioorg. Med. Chem. 8 (2000) 2709±2718
biotinylated glucosamine derivative 19 and the biotiny-
lated lactose derivative 20 were obtained in 92 and 86%
yields, respectively.
selective sialylation on the C-6 hydroxyl of the galactose
unit of a lactoside derivative, previous work within the
group had shown this could most eciently be achieved
if the other hydroxyl groups in the lactoside were pro-
tected.32 Towards this end, the lactoside 25 was treated
to a sequence of 4,6-O-benzylidenation, acetylation, and
subsequent de-O-benzylidenation to give the selectively
protected lactoside 26 in 68% yield from 25. Sialylation
of the lactoside 26, using a minor modi®cation (see
Experimental) of the method developed by Schmidt and
co-workers52 involving TMSOTf-mediated coupling
with the sialosyl phosphite 27, resulted in the formation
of the sialyl a(2,6)-lactoside 28 in 57% yield. Reduction
The overall yields obtained for the introduction of the
UV-tag and subsequent biotinylation of the three thiol-
acetyl glycosides 8, 10 and 11 are 26, 43, and 43%,
respectively. The protected saccharides 18, 19 and 20
were readily deprotected using standard procedures
(NaOMe or NaOH, see Experimental for details). In
this way the biotinylated saccharides 21, 22 and 23 were
obtained in good yield after chromatography.
.
of the nitro group in 28 (SnCl2 2H2O) gave the amine 29
(63%) and subsequent biotinylation in the same way as
described above gave the desired biotinylated sialyllac-
toside 30 in 85% yield. The overall yield of 30 is 31%
from the lactoside 26.
In conclusion, we have developed a new method for the
preparation of tagged biotinylated carbohydrates.
Importantly, our strategy is suciently ¯exible, utilizes
mild reagents and reaction conditions, and is ecient
enough to provide ready access to a range of suitably
functionalized biologically relevant carbohydrates. We
feel certain that compounds such as those described
herein will prove extremely useful in investigations
involving carbohydrate-recognizing proteins. We are
currently investigating the use of these compounds in an
extension of our earlier e€orts40,53 in developing highly
speci®c anity chromatography supports.
Experimental
General procedures
1H and 13C spectra were recorded using a Bruker DRX-
300 spectrometer unless indicated otherwise. Chemical
shifts are given in ppm relative to the solvent used
(CDCl3: 7.26 for 1H; 77.0 for 13C; CD3OD: 3.31 for 1H;
49.0 for 13C) or relative to external Me4Si for D2O
spectra. Two-dimensional DQF-COSY and HMQC
experiments were recorded in order to assist with spec-
tral assignment. Typically, the following parameters
were used: DQF-COSY Ð 16 scans, 512 slices, relaxa-
In an attempt to further demonstrate the general
applicability of this approach to the synthesis of biotiny-
lated (oligo)saccharides as probes for carbohydrate-
recognizing proteins we sought to prepare the tagged
sialyllactoside 24.
1
Of the many possible routes to a compound like 24, we
felt the most convergent would involve sialylation of a
4-nitrobenzyl thiolactoside, followed by reduction of the
nitro group and subsequent biotinylation. Accordingly,
the lactoside 14 was deprotected (NaOMe in MeOH) to
give the lactoside 25 in high yield (Scheme 2). For
tion delay 2.0 s; H±13C HMQC Ð 48 scans, 256 slices,
relaxation delay 2.5 s. ESI mass spectra were obtained
using a Micromass Platform II electrospray spectro-
meter. Infrared spectra were recorded as KBr discs
using a Hitachi 270-30 spectrophotometer. Reactions
were monitored by TLC (Merck silica gel plates GF254
,

D. I. Angus et al. / Bioorg. Med. Chem. 8 (2000) 2709±2718
2713
Scheme 2. Conditions: (a) NaOMe, MeOH, 0 ꢀC to rt, 2 h, 83%; (b) PhCHO, HCO2H, rt, 2 h; (c) Ac2O, pyridine,ꢀrt, 16 h, 86%; (d) 90% aq
DMF, Et3N, rt, 16 h, 85%.
ꢀ
CF3CO2H, CH2Cl2, 0 ꢀC, 2 h, 79%; (e) 27, THF, TMSOTf, 4 A sieves, 40 to 0 C, 3 h, 57%; (f) SnCl2 2H2O, EtOH, 70 C, 15 h, 63%; (g) 6, TBTU,
.
cat. #1.05554) and products were generally puri®ed by
¯ash chromatography using Merck silica gel 60 (0.040±
0.063 mm, cat. #1.09385). Microanalyses were per-
formed at the Department of Chemistry, University of
Queensland, Australia. Methyl 5-acetamido-4,7,8,9-
tetra-O-acetyl-2-S-acetyl-3,5-dideoxy-2-thio-d-glycero-a-
d-galacto-2-nonulopyranosonate (8),43 2-acetamido-3,4,
6-tri-O-acetyl-1-S-acetyl-2-deoxy-1-thio-b-d-glucopyr-
anose (10),45 and 2,3,4,6-tetra-O-benzoyl-b-d-galacto-
pyranosyl-(1,4)-O-1-S-acetyl-2,3,6-tri-O-benzoyl-1-thio-b-
d-glucopyranose (11) were all prepared by reacting the
corresponding glycosyl halides with potassium thio-
acetate using published procedures. All solvents were
distilled prior to use or were of analytical grade.
Degassed solutions were obtained using the technique
described in Vogel54 involving the bubbling of dry N2
through the solution for 20 min.
The reaction mixture was diluted with EtOAc (20 mL),
washed with HCl (0.1 M, 10 mL), H2O (10 mL), satd
NaCl (aq) (15 mL), dried (MgSO4) and concentrated.
The residue was puri®ed by column chromatography
(EtOAc:Et2O, 3:2; Rf 0.3) to give 12 (473 mg, 67%) as a
pale yellow amorphous solid: 1H NMR (CDCl3):
Neu5Ac unit: d 1.87 (3H, s, AcN), 2.05, 2.11, 2.18, 2.20
(4ÂH, 4Âs, 4ÂAcO), 2.70 (1H, dd, J3e,3a 12.7, J3e,4 4.5 Hz,
H-3e), 3.51 (3H, s, CO2Me), 3.84 (1H, d, J6,5 10.2 Hz, H-
0
0
6), 4.05±4.15 (2H, m, H-5/H-9), 4.32 (1H, d, J9 ,9 11.8, J9 ,8
2.5 Hz, H-90), 4.86 (1H, ddd, J4,3a=J4,5=10.5, J4,3e
4.5 Hz, H-4), 5.11 (1H, d, JNH,5 9.9 Hz, NH), 5.37 (1H,
d, J7,8 9.1 Hz, H-7), 5.46 (1H, m, H-8); 4-NO2-Ar unit: d
3.99 (2H, s, S-CH2), 7.54 (2H, d, ArH-2/6), 8.20 (2H, d,
ArH-3/5); 13C NMR (CDCl3): Neu5Ac unit: d 21.1, 21.5
(4ÂOC(O)Me), 23.4 (NC(O)Me), 38.1 (C-3), 47.8 (C-5),
53.1 (CO2Me), 67.1 (C-9), 67.6 (C-8), 68.5 (C-7), 69.7
(C-4), 74.4 (C-6), 83.1 (C-2), 168.5 (C-1), 170.3, 170.5,
170.8, 171.0 (4ÂOC(O)Me/NC(O)Me); 4-NO2-Ar unit:
d 32.7 (S-CH2), 124.1 (C-3/5), 130.3 (C-2/6), 145.2 (C-
4); ESIMS: 665 [M+Na] (100%), 643 [M+H] (70);
found: 643.17888; C27H35N2O14S requires: 643.18086.
Synthesis of thio-4-nitrobenzyl glycosides
Methyl (S-4-nitrobenzyl 5-acetamido-4,7,8,9-tetra-O-ace-
tyl-3,5-dideoxy-2-thio-D-glycero-ꢀ-D-galacto-2-nonulo-
pyranosid)onate (12). To a solution of 8 (600 mg,
1.09 mmol) in dry DMF (10 mL) was added hydrazi-
nium acetate (147 mg, 1.60 mmol) and the solution
degassed. After stirring for 30 min at rt, 4-nitrobenzyl
bromide (471 mg, 2.18 mmol) and Et3N (223 mL,
1.60 mmol) were added and the solution stirred for 2 h.
In a similar manner were prepared
S-(4-Nitrobenzyl) 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
1-thio-ꢁ-D-glucopyranoside (13). Prepared in 77% yield
by reaction between the thiolacetyl GlcNAc derivative 10

2714
D. I. Angus et al. / Bioorg. Med. Chem. 8 (2000) 2709±2718
and 4-nitrobenzyl bromide (chromatography: EtOAc:
1
Jgem 13.2 Hz, S-CHaHb), 4.15 (1H, d, Jgem 13.2 Hz, S-
CHaHb), 7.63 (2H, d, ArH-2/6), 8.18 (2H, d, ArH-3/5).
hexane, 3:2; Rf 0.4). H NMR (CDCl3): GlcNAc unit: d
1.96 (3H, s, AcN), 2.02, 2.11 (2Âs, 9H, 3ÂAcO), 3.59±
0
3.67 (1H, m, H-5), 4.12 (1H, dd, J6,6 12.3, J6,5 2.4 Hz,
S-(4-Nitrobenzyl) 2,3-di-O-acetyl-4,6-O-benzylidene-ꢁ-D-
galactopyranosyl-(1,4)-2,3,6-tri-O-acetyl-1-thio-ꢁ-D-gluco-
pyranoside. Benzaldehyde (5 mL) was added to a solu-
tion of the lactoside 25 (0.7 g, 1.42 mmol) in HCO2H
(5 mL) at rt under N2.55 After stirring for 2 h at rt the
mixture was concentrated and quickly chromatographed
(EtOAc:MeOH, 20:1; Rf 0.5). Without characterization,
the 4,6-O-benzylidenated product was dissolved in pyri-
dine (10 mL), Ac2O (7 mL) added, and the mixture stirred
for 16 h at rt before being concentrated. Chromato-
graphy (EtOAc; Rf 0.5) gave the title compound (0.97 g,
86%) as a pale yellow amorphous mass: 1H NMR
(CDCl3): Gal unit: d 3.57 (1H, brs, H-5), 4.02 (1H, d,
H±6), 4.20±4.26 (2H, m, H-2/H-60), 4.32 (1H, d, J1,2
10.2 Hz, H-1), 5.05±5.09 (2H, m, H-3/H-4), 5.48 (1H, d,
JNH,2 9.0 Hz, NH); 4-NO2-Ar unit: d 3.90 (1H, d, Jgem
13.0 Hz, S-CHaHb), 4.06 (1H, d, Jgem 13.0 Hz, S-CHaHb),
7.42±7.50 (2H, m, ArH-2/6), 8.15±8.20 (2H, m, ArH-3/
5), assignments con®rmed by COSY; 13C NMR
(CDCl3): GlcNAc unit: d 20.4, 20.5, 20.6 (3ÂOC(O)Me),
23.1 (NC(O)Me), 52.8 (C-2), 62.2 (C-6), 68.2 (C-4), 72.1
(C-3), 76.0 (C-5), 82.8 (C-1), 169.1, 170.0, 170.3, 171.0
(3ÂOC(O)Me/NC(O)Me); 4-NO2-Ar unit: d 32.7 (S-
CH2), 123.6 (C-3/5), 129.9 (C-2/6), 145.2 (C-1), 147.0
(C-4); ESIMS: 521 [M+Na] (28%), 516 [M+NH4]
(100), 499 [M+H] (80).
J6,6 12.0 Hz, H-6), 4.31 (1H, d, J6 ,6 12.0 Hz, H-60), 4.41
(1H, d, J1,2 9.6 Hz, H-1), 4.44 (1H, d, J4,3 3.3 Hz, H-4),
4.98 (1H, dd, J3,2 9.9, J3,4 3.3 Hz, H-3), 5.48±5.57 (1H,
m, H-2); Glc unit: d 3.65±3.77 (1H, m, H-5), 3.99 (1H,
0
0
S-(4-Nitrobenzyl) 2,3,4,6-tetra-O-benzoyl-ꢁ-D-galactopyr-
anosyl-(1,4)-2,3,6-tri-O-benzoyl-1-thio-ꢁ-D-glucopyrano-
side (14). Prepared in 74% yield by reaction between
the thiolacetyl derivative 11 and 4-nitrobenzyl bromide
(chromatography: EtOAc:hexane, 2:3; Rf 0.4). 1H NMR
(CDCl3): Gal unit: d 3.71±3.99 (3H, m, H-5/H-6/60), 4.53
(1H, d, J1,2 9.6 Hz, H-1), 5.38 (1H, dd, J3,2 10.2, J3,4
3.6 Hz, H-3), 5.68±5.75 (2H, m, H-2/H-4); Glc unit: d
0
dd, J4,3=J4,5=9.6 Hz, H-4), 4.06 (1H, dd, J6,6 12.6, J6,5
1.8 Hz, H-6), 4.08 (1H, d, J1,2 8.1 Hz, H-1), 4.30 (1H,
dd, J6 ,6 12.6, J6 ,5 1.5 Hz, H-60), 4.43 (1H, dd, J2,3 9.6,
J2,1 8.1 Hz, H-2), 5.48±5.57 (1H, m, H-3); other: d 2.05,
2.08 (2Â), 2.09 (2Â) (15H, 3Âs, 5ÂAcO), 3.96 (1H, d,
Jgem 12.9 Hz, S-CHaHb), 4.11 (1H, d, Jgem 12.9 Hz, S-
CHaHb), 5.48±5.57 (1H, m, PhCH), 7.34±7.41 (3H, m,
ArH), 7.47±7.52 (4H, m, ArH), 8.07 (2H, d, ArH-3/5).
0
0
3.71±3.99 (1H, m, H-5), 4.22 (1H, dd, J4,3=J4,5
=
0
9.6 Hz, H-4), 4.46 (1H, dd, J6,6 12.3, J6,5 4.8 Hz, H-6),
0
4.53 (1H, d, J1,2 8.1 Hz, H-1), 4.61 (1H, d, J6 ,6 12.3 Hz,
H-60), 5.51 (1H, dd, J2,3 9.6, J2,1 8.1 Hz, H-2), 5.77 (1H,
dd, J3,2=J3,4=9.6 Hz, H-3); other: d 3.71±3.99 (2H, m,
S-CH2), 7.15±8.19 (39H, m, Ar); 13C NMR (CDCl3):
Gal unit: d 61.0 (C-6), 67.4 (C-2), 69.8 (C-4), 71.7 (C-3),
77.5 (C-3), 100.8 (C-1); Glc unit: d 62.4 (C-6), 71.3 (C-5),
73.6 (C-3), 74.0 (C-2), 75.6 (C-4), 78.8 (C-1); other: d
32.7 (S-CH2), 124.2, 127.1, 127.4, 129.5, 129.8, 133.0,
133.4, (8ÂPh), 144.8 (C-4 ArNH2), 164.6, 164.8,
165.2, 165.4, 165.5, 165.9 (7ÂOC(O)Ph); ESIMS:
1245 [M+Na] (100%). Anal. calcd for C68H55NO19S:
C, 66.8; H, 4.5; N, 1.15; found: C, 66.7; H, 4.5; N,
0.95.
S-(4-Nitrobenzyl) 2,3-di-O-acetyl-ꢁ-D-galactopyranosyl-
(1,4)-2,3,6-tri-O-acetyl-1-thio-ꢁ-D-glucopyranoside (26).
To a solution of S-(4-nitrobenzyl) 2,3-di-O-acetyl-4,6-O-
benzylidene-b-d-galactopyranosyl-(1,4)-2,3,6-tri-O-acetyl-
1-thio-b-d-glucopyranoside (0.9 g, 1.14 mmol) in
CH2Cl2 (20 mL) at 0 ꢀC under N2 was added CF3CO2H
(90% aq. 2.5 mL) and the mixture stirred for 2 h at
0 ꢀC.55 The mixture was diluted with CH2Cl2 (50 mL),
washed with H2O (25 mL), satd aq NaHCO3 (25 mL),
H2O (2Â25 mL), dried (MgSO4) and concentrated.
Chromatography (EtOAc:hexanes, 5:1; Rf 0.3) gave 26
1
(0.63 g, 79%) as an o€-white solid: H NMR (CDCl3):
0
Gal unit: d 3.54±3.60 (1H, m, H-5), 3.83 (1H, dd, J6,6
12.0, J6,5 4.5 Hz, H-6), 3.85±3.97 (1H, m, H-60), 4.21
(1H, d, J4,3 2.7 Hz, H-4), 4.37 (1H, d, J1,2 9.6 Hz, H-1),
4.95 (1H, dd, J3,2 9.6, J3,4 2.7 Hz, H-3), 5.43 (1H, dd,
J2,3=J2,1=9.6 Hz, H-2); Glc unit: d 3.54±3.60 (1H, m,
H-5), 3.85±3.97 (3H, m, H-4/H-6/H-60), 4.36 (1H, d, J1,2
9.0 Hz, H-1), 4.98 (1H, dd, J2,3 9.6, J2,1 9.0 Hz, H-2),
5.44 (1H, dd, J3,4=J3,2=9.6 Hz, H-3); other: d 2.05 (6H,
s, 2ÂAcO), 2.10 (9H, s, 3ÂAcO), 3.93 (1H, d, Jgem
13.2 Hz, S-CHaHb), 4.08 (1H, d, Jgem 13.2 Hz, S-CHaHb),
7.51 (2H, d, ArH-2/6), 8.17 (2H, d, ArH-3/5).
Synthesis of sialosyllactoside 28
S-(4-Nitrobenzyl) ꢁ-D-galactopyranosyl-(1,4)-1-thio-ꢁ-D-
glucopyranoside (25). To a solution of 14 (2.0 g,
1.64 mmol) in dry MeOH (40 mL) at 0 ꢀC under N2 was
added sodium metal (ꢁ0.2 g) in portions over 10 min.
After 30 min at 0ꢀC the mixture was allowed to warm to
rt, and stirred for a further 2 h before being neutralized
(IR-120H+ resin). The resin was removed by ®ltration,
washed with MeOH (3Â40 mL) and the solvent
removed under reduced pressure. Chromatography
(EtOAc:MeOH, 4:1; Rf 0.4) gave 25 (0.67 g, 83%) as a
S-(4-Nitrobenzyl) methyl [5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-2-thio-D-glycero-ꢀ-D-galacto-2-nonu-
lopyranosid]onate-(2,6)-2,3-di-O-acetyl-ꢁ-D-galactopyra-
nosyl-(1,4)-2,3,6-tri-O-acetyl-1-thio-ꢁ-D-glucopyranoside
(28). To a solution of methyl 5-acetamido-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-d-glycero-b-d-galacto-2-nonulo-
pyranosylonate (196 mg, 0.40 mmol) in dry CH3CN
(10 mL) at rt under N2 was added EtN0Pr2 (165 mL,
0.95 mmol) and then ClP(OEt)2 (115 mL, 0.80 mmol).
After stirring for 1 h at rt the mixture was concentrated
1
pale yellow solid: H NMR (CD3OD): Gal unit: d 3.29±
3.37 (2H, m, H-2/H-5), 3.43 (1H, dd, J3,2 9.3, J3,4
3.3 Hz, H-3), 3.48 (1H, d, J6,5 6.3 Hz, H-6), 3.50 (1H, d,
J6 ,5 5.7 Hz, H-60), 3.89 (1H, d, J4,3 3.3 Hz, H-4), 4.17
0
(1H, d, J1,2 9.6 Hz, H-1); Glc unit: d 3.29±3.37 (2H, m,
H-2/H-5), 3.59 (1H, t, J3,2=J3,4=9.6 Hz, H-3), 3.69
0
(1H, dd, J6,6 11.4, J6,5 5.1 Hz, H-6), 3.70±3.76 (1H, m,
0
H-4), 3.72 (1H, d, J1,2 8.1 Hz, H-1), 3.75 (1H, dd, J6 ,6
0
11.4, J6 ,5 7.2 Hz, H-60); 4-NO2-Ar unit: d 3.97 (1H, d,

D. I. Angus et al. / Bioorg. Med. Chem. 8 (2000) 2709±2718
2715
under reduced pressure, and the residue chromato-
In a similar manner were prepared:
graphed through a short (5Â1 cm) column of silica
(toluene:acetone, 3:2; Rf 0.6) to give the unstable sialo-
syl phosphite 27 in >90% yield. To a solution of 27 in
dry THF (10 mL) at 40 ꢀC under N2 containing 4 A
sieves (ꢁ250 mg) was added the lactoside 26 (350 mg,
0.50 mmol) and then TMSOTf (10 mL). After stirring for
30 min at 40 ꢀC the mixture was allowed to warm to
0 ꢀC and stirred for a further 2 h. The mixture was neu-
tralized with Et3N, allowed to warm to rt, the sieves
removed by ®ltration, washed with THF (3Â10 mL), and
the solution concentrated. Chromatography (toluene:
acetone, 3:2, Rf 0.33; followed by CHCl3:MeOH, 25:1, Rf
0.27) gave 28 (268 mg, 57%) as an o€-white amorphous
mass: nmax 3420 (br), 1746, 1666, 1522, 1370, 1346,
S-(4-Aminobenzyl) 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
1-thio-ꢁ-D-glucopyranoside (16). Prepared in 60% yield
by reduction of 13 (chromatography: EtOAc:hexane,
1
8:1; Rf 0.2). H NMR (CDCl3): GlcNAc unit: d 1.75
(3H, s, AcN), 1.92, 2.02 (9H, 2Âs, 3ÂAcO), 3.45±3.48
(1H, m, H-5), 4.05±4.17 (4H, m, H-1/H-2/H-6/H-60),
4.87 (1H, dd, J3,2=J3,4=9.6 Hz, H-3), 4.99 (1H, dd,
J4,5=J4,3 9.6 Hz, H-4), 5.15 (1H, d, JNH,2 8.4 Hz, NH);
4-NH2-Ar unit: d 3.62 (1H, d, Jgem 13.2 Hz, S-CHaHb),
3.74 (1H, d, Jgem 13.2 Hz, S-CHaHb), 6.54 (2H, d, ArH-
3/5), 6.99 (2H, d, ArH-2/6), assignments con®rmed by
COSY; 13C NMR (CDCl3): GlcNAc unit: d 20.6, 20.7,
20.8 (3ÂOC(O)Me), 23.2 (NC(O)Me), 52.7 (C-2), 62.5
(C-6), 68.4 (C-4), 74.0 (C-3), 75.8 (C-5), 82.7 (C-1),
169.9, 171.0, 171.1 (3ÂOC(O)Me/NC(O)Me); 4-NH2-Ar
unit: d 33.4 (S-CH2), 115.3 (C-3/5), 130.1 (C-2/6), 132.5
(C-1) 145.3 (C-4); ESIMS 491 [M+Na] (11%), 469
[M+H] (7), 106.2 (100).
1
1224, 1034 cm 1; H NMR (500 MHz, CDCl3): Neu5Ac
unit: d 1.88 (3H, s, AcN), 1.97 (1H, dd, J3a,3e 13.0, J3a,4
12.0 Hz, H-3a), 2.59 (1H, dd, J3e,3a 13.0, J3e,4 4.5 Hz, H-
3e), 3.84 (3H, s, CO2Me), 4.04±4.13 (3H, m, H-5/H-6/H-
9), 4.41 (1H, dd, J9 ,9 12.5, J9 ,8 2.0 Hz, H-90), 4.88 (1H,
ddd, J4,3a 12.0, J4,5 10.0, J4,3e 4.5 Hz, H-4), 5.12 (1H, d,
JNH,5 9.5 Hz, NH), 5.32-5.41 (2H, m, H-7/H-8); Gal unit:
d 3.65±3.71 (2H, m, H-5/H-6), 3.79±3.84 (1H, m, H-60),
4.15 (1H, d, J4,3 3.0 Hz, H-4), 4.30 (1H, d, J1,2 10.0 Hz,
H-1), 4.91 (1H, dd, J3,2 9.5, J3,4 3.0 Hz, H-3), 5.32±5.41
(1H, m, H-2); Glc unit: d 3.65±3.71 (1H, m, H-4), 3.79±
3.88 (2H, m, H-5/H-6), 4.04±4.13 (2H, m, H-1/H-60),
5.32±5.41 (2H, m, H-2/H-3); other: d 2.03 (3H), 2.04
(9H), 2.10 (3H), 2.14 (6H), 2.15 (6H) (all s, 9ÂAcO),
3.79±3.88 (2H, m, SCH2), 7.52 (2H, d, ArH-2/6), 8.16
(2H, d, ArH-3/5), assignments con®rmed by COSY.
0
0
S-(4-Aminobenzyl) 2,3,4,6-tetra-O-benzoyl-ꢁ-D-galacto-
pyranosyl-(1,4)-2,3,6-tri-O-benzoyl-1-thio-ꢁ-D-glucopyr-
anoside (17). Prepared in 67% yield by reduction of 14
1
(chromatography: Et2O; Rf 0.35). H NMR (CDCl3):
0
Gal unit: d 3.75±3.83 (2H, m, H-6/60), 3.91 (1H, t, J5,6/6
6.6 Hz, H-5), 4.91 (1H, d, J1,2 8.1 Hz, H-1), 5.40 (1H,
dd, J3,2 10.5, J3,4 3.3 Hz, H-3), 5.71 (1H, d, J2,1 8.1 Hz,
H-2), 5.74 (1H, d, J4,3 3.3 Hz, H-4); Glc unit: d 3.75±3.83
(1H, m, H-5), 4.24 (1H, dd, J4,3=J4,5=9.6 Hz, H-4),
0
4.43 (1H, dd, J6,6 12.3, J6,5 5.1 Hz, H-6), 4.49 (1H, d,
0
J1,2 9.6 Hz, H-1), 4.60 (1H, d, J6,6 12.3 Hz, H-60), 5.52
(1H, dd, J2,3=J2,1=9.6 Hz, H-2), 5.77 (1H, dd, J3,2=
Reduction of nitro group
J3,4=9.6 Hz, H-3); 4-NH2-Ar unit: d 3.75±3.83 (2H, m,
S-CH2), 6.54 (2H, d, ArH-3/5), 6.97 (2H, d, ArH-2/6);
other: d 7.11±8.15 (35H, m, 7ÂPh); 13C NMR (CDCl3):
Gal unit: d 60.3 (C-6), 67.5 (C-2), 70.5 (C-4), 71.8 (C-3),
76.9 (C-5), 100.8 (C-1); Glc unit: d 62.7 (C-6), 69.9 (C-5),
71.3 (C-3), 74.0 (C-2), 76.0 (C-4), 82.2 (C-1); other: d
28.1 (S-CH2), 115.2, 126.2, 128.1, 129.1, 129.8, 129.9,
133.2, 133.4, (8ÂPh), 145.4 (C-4 ArNH2), 164.7, 165.1,
165.2, 165.3, 165.5, 165.7 (7ÂOC(O)Ph); ESIMS: 1193
[M+H] (15%), 932 (71). Anal. calcd for C68H57NO17S:
C, 68.5; H, 4.8; N, 1.2; found: C, 68.5; H, 4.8; N, 1.0.
Methyl (S-4-aminobenzyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-2-thio-D-glycero-ꢀ-D-galacto-2-nonu-
lopyranosid)onate (15). Compound 12 (266 mg, 0.41
.
mmol) was dissolved in EtOH (20 mL) and SnCl2 2H2O
(466 mg, 2.07 mmol) was added.47 The mixture was
heated at 70 ꢀC for 15 h, before being allowed to cool to
rt. The mixture was then poured onto ice/H2O (20 mL),
the pH adjusted to 8 with solid NaHCO3, extracted with
EtOAc (3Â50 mL) and dried (MgSO4). Chromato-
graphy (CH2Cl2:EtOH, 100:4; Rf 0.3) gave 15 (138 mg,
55%) as a light yellow solid: 1H NMR (CDCl3): Neu5Ac
unit: d 1.85 (3H, s, AcN), 2.00, 2.02, 2.13, 2.16 (4Â3H,
4Âs, 4 ÂAcO), 2.70 (1H, dd, J3e,3a 12.7, J3e,4 4.6 Hz, H-
3e), 3.62 (3H, s, CO2Me), 3.68 (1H, dd, J6,5 9.7, J6,7
S-(4-Aminobenzyl) methyl [5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-2-thio-D-glycero-ꢀ-D-galacto-2-nonu-
lopyranosid]onate-(2,6)-2,3-di-O-acetyl-ꢁ-D-galactopyra-
nosyl-(1,4)-2,3,6-tri-O-acetyl-1-thio-ꢁ-D-glucopyranoside
(29). Prepared in 63% yield by reduction of 28 (chro-
0
2.4 Hz, H-6), 4.07 (1H, d, J9,9 12.4, J9,8 5.5 Hz, H-9),
0
0
4.08±4.13 (1H, m, H-5), 4.34 (1H, dd, J9 ,9 12.4, J9 ,8
2.6 Hz, H-90), 4.86 (1H, ddd, J4,3a=J4,5=10.5, J4,3e
4.5 Hz, H-4), 5.11 (1H, d, JNH,5 9.2 Hz, NH), 5.34 (1H,
dd, J7,8 7.8, J7,6 2.2 Hz, H-7), 5.46 (ddd, 1H, J8,7 7.8, J8,9
matography: CHCl3:MeOH, 20:1; Rf 0.35). nmax 3400
(br), 1746, 1516, 1370, 1226, 1038 cm
1
;
1H NMR
(CDCl3): Neu5Ac unit: d 1.88 (3H, s, AcN), 2.61 (1H,
dd, J3e,3a 12.9, J3e,4 4.5 Hz, H-3e), 3.84 (3H, s, CO2Me),
0
5.5, J8,9 2.6 Hz, H-8); 4-NH2-Ar unit: d 3.88 (2H, s, S-
CH2), 6.58 (2H, d, ArH-3/5), 7.07 (2H, d, ArH-2/6); 13
C
4.03±4.15 (3H, m, H-5/H-6/H-9), 4.42 (1H, dd, J9 ,9
0
0
NMR (CDCl3): Neu5Ac unit: d 20.7, 20.8, 21.2
(4ÂOC(O)Me), 23.2 (NC(O)Me), 37.8 (C-3), 49.5 (C-5),
52.8 (CO2Me), 62.3 (C-9), 67.8 (C-8), 68.6 (C-7), 69.7
(C-4), 76.5 (C-6), 83.2 (C-2), 168.3 (C-1), 170.1, 170.9
(4ÂOC(O)Me/NC(O)Me); 4-NH2-Ar unit: d 32.7 (S-
CH2), 117.5 (C-3/5), 130.3 (C-2/6), 133.4 (C-1), 145.0
(C-4); ESIMS: 635 [M+Na] (23%), 324 (52), 301
(33).
12.4, J9 ,8 2.5 Hz, H-90), 4.89 (1H, ddd, J4,3a 11.8, J4,5
9.8, J4,3e 4.5 Hz, H-4), 5.17 (1H, d, JNH,5 9.8 Hz, NH),
5.31±5.43 (2H, m, H-7/H-8); Gal unit: d 3.62 (1H, t, J
0
6.3 Hz, H-5), 3.72 (1H, dd, J6,6 11.2, J6,5 6.3 Hz, H-6),
3.85±3.93 (1H, m, H-60), 4.03±4.15 (1H, m, H-4), 4.26
(1H, d, J1,2 10.0 Hz, H-1), 4.87 (1H, dd, J3,2 9.8, J3,4
3.0 Hz, H-3), 5.31±5.43 (1H, m, H-2); Glc unit: d 3.68±3.76
(1H, m, H-4), 3.85±3.93 (2H, m, H-5/H-6), 4.03±4.15 (2H,

2716
D. I. Angus et al. / Bioorg. Med. Chem. 8 (2000) 2709±2718
m, H-1/H-60), 4.99 (1H, dd, J2,3 9.6, J2,1 8.5 Hz, H-2),
5.31±5.43 (1H, m, H-3); other: d 2.01, 2.03, 2.04 (2Â), 2.05,
2.08, 2.14 (2Â), 2.15 (27H, 7Âs, 9ÂAcO), 3.74 (1H, d,
Jgem 12.6 Hz, SCHaHb), 3.94 (1H, d, Jgem 12.6 Hz,
SCHaHb), 6.68 (2H, d, ArH-3/5), 7.15 (2H, d, ArH-2/6).
H-2), 4.14 (1H, d, J3,4 7.6 Hz, H-3), 4.32 (1H, dd, J4,3
7.6, J4,5b 4.8 Hz, H-4), assignments con®rmed by COSY;
13C NMR (CDCl3): GlcNAc unit:
d 20.0, 20.2
(3ÂOC(O)Me), 22.0 (NC(O)Me), 52.2 (C-2), 62.3 (C-6),
68.6 (C-4), 73.8 (C-3), 75.2 (C-5), 82.1 (C-1), 170.7, 170.8,
171.2, 172.5 (3ÂOC(O)Me/NC(O)Me); Benzyl unit: d
32.9 (S-CH2), 120.1 (C-2/6), 129.2 (C-3/5), 132.6 (C-1),
137.1 (C-4); Biotin unit: d 25.1 (C-10), 27.8, 28.0 (C-20/C-
30), 36.2 (C-40), 39.9 (C-5), 55.3 (C-2), 55.9 (C-4), 61.5
(C-3), 163.8 (C-200), assignments con®rmed by HMQC;
ESIMS: 717 [M+Na] (11%), 695 [M+H] (100); found:
695.24712; C31H43N4O10S2 requires: 695.24201.
Coupling with biotin
Methyl (S-D-biotinoyl-4-aminobenzyl 5-acetamido-4,7,8, 9-
tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-ꢀ-D-galacto-2-
nonulopyranosid)onate (18). A mixture of 15 (100 mg,
0.16 mmol), TBTU (58 mg, 0.18 mmol) and d-biotin (6)
(43 mg, 0.18 mmol) was dissolved in dry DMF (5 mL)
under N2 at rt. After stirring for 20 min Et3N (34 mL,
0.25 mmol) was added and the mixture was left to stir
for 16 h. Solvents were removed in vacuo and the residue
was chromatographed (EtOAc:hexane, 10:1; Rf 0.3) to
give 18 (97 mg, 71%) as a light yellow amorphous mass:
1H NMR (500MHz, CDCl3) Neu5Ac unit: d 1.86 (3H, s,
AcN), 2.00, 2.02, 2.14, 2.16 (4Â3H, 4Âs, 4ÂAcO), 2.67
(1H, dd, J3e,3a 12.2, J3e,4 4.2 Hz, H-3e), 3.53 (3H, s,
CO2Me), 3.90 (1H, dd, J6,5 10.1, J6,7 1.5 Hz, H-6), 4.06±
S-D-Biotinoyl-4-aminobenzyl 2,3,4,6-tetra-O-benzoyl-ꢁ-
D-galactopyranosyl-(1,4)-2,3,6-tri-O-benzoyl-1-thio-ꢁ-D-
glucopyranoside (20). Prepared in 87% yield by reaction
between 17, TBTU and d-biotin (chromatography:
CH2Cl2:MeOH, 10:1; Rf 0.4). 1H NMR (600 MHz,
CDCl3): Gal unit: d 3.79±3.94 (2H, m, H-6/60), 4.02 (1H,
0
t, J5,6/6 6.6 Hz, H-5), 5.00 (1H, d, J1,2 9.0 Hz, H-1), 5.48
(1H, dd, J3,2 9.9 Hz, J3,4 3.0 Hz, H-3), 5.77 (1H, dd, J2,3
9.9, J2,1 9.0 Hz, H-2), 5.83 (1H, m, H-4); Glc unit: d 3.79±
3.94 (1H, m, H-5), 4.30 (1H, dd, J4,5=J4,3= 9.6 Hz, H-
0
4.09 (1H, m, H-5), 4.11 (1H, dd, J9,9 7.3, J9,8 3.2 Hz, H-
9), 4.33 (1H, dd, J9 ,9 7.3, J9 ,8 1.5 Hz, H-90), 4.83 (1H,
ddd, J4,3a 10.6, J4,5 9.6, J4,3e 4.2 Hz, H-4), 5.23 (1H, dd,
J7,8 8.3, J7,6 1.9 Hz, H-7), 5.42±5.47 (1H, m, H-8), 5.49
(1H, d, JNH,5 10 Hz, NH); Benzyl unit: d 3.75 (1H, d, Jgem
13.1 Hz, S-CHaHb), 3.87 (1H, d, Jgem 13.1 Hz, S-CHaHb),
7.22 (2H, d, ArH-2/6), 7.51 (2H, d, ArH-3/5); Biotin unit:
d 1.43 (2H, m, H-10), 1.63 (2H, m, H-20), 1.73 (2H, m, H-
30), 2.33 (2H, m, H-40), 2.71 (1H, d, J5a,5b 12.8 Hz, H-5a),
2.87 (1H, dd, J5b,5a 12.8, J5b,4 4.6 Hz, H-5b), 3.09±3.12
(1H, m, H-2), 4.27 (1H, dd, J3,4 7.1, J3,2 4.5 Hz, H-3),
4.47 (1H, dd, J4,3 7.1, J4,5b 5.0 Hz, H-4), 5.70 (1H, bs,
NH), assignments con®rmed by COSY; 13C NMR
(CDCl3) Neu5Ac unit: d 20.7, 20.8, 21.2, (4ÂOC(O)Me),
23.0 (NC(O)Me), 37.7 (C-3), 49.1 (C-5), 52.8 (CO2Me),
62.3 (C-9), 67.4 (C-7), 68.7 (C-8), 69.6 (C-4), 74.0 (C-6),
83.1 (C-2), 170.1, 170.2, 170.3, 170.7, 170.8 (4ÂOC(O)Me/
NC(O)Me), 171.9 (C-1); Benzyl unit: d 32.6 (S-CH2),
120.1 (C-3/5), 129.5 (C-2/6), 131.7 (C-1), 137.4 (C-4); Bio-
tin unit: d 25.5 (C-30), 27.9 (C-10), 28.1 (C-20), 36.4 (C-40),
40.4 (C-5), 55.5 (C-2), 60.2 (C-4), 62.1 (C-3), 164.1 (C-200),
168.2 (C-50), assignments con®rmed by HMQC; ESIMS:
861 [M+Na] (100%); found: 839.2847; C37H51N4O14S2
requires: 839.2842.
4), 4.58 (1H, d, J6,6 11.4, J6,5 4.8 Hz, H-6), 4.58 (1H, d,
0
0
0
0
J1,2 8.7 Hz, H-1), 4.72 (1H, d, J6 ,6 11.4 Hz, H-60), 5.57
(1H, dd, J2,3 9.6, J2,1 8.7 Hz, H-2), 5.83 (1H, m, H-3);
Biotin unit: d 1.15±1.19 (6H, m, H-10/H-20/H-30), 2.48
(2H, t, J4 ,3 7.2 Hz, H-40), 2.83 (1H, d, J5a,5b 13.2 Hz, H-
5a), 2.95 (1H, m, H-5b), 3.21 (1H, m, H-2), 4.14 (1H, d,
J3,2 6.6 Hz, H-3), 4.56 (1H, dd, J4,5a 4.8 Hz, H-4); other: d
3.79±3.94 (2H, m, S-CH2), 7.17±8.14 (39H, m, Ar),
assignments con®rmed by COSY; 13C NMR (CDCl3):
Gal unit: d 60.9 (C-6), 67.4 (C-2), 70.5 (C-4), 71.2 (C-3),
76.5 (C-5), 100.2 (C-1); Glc unit: d 60.9 (C-6), 69.8 (C-2),
71.2 (C-5), 73.8 (C-3), 76.0 (C-4), 82.4 (C-1); Biotin unit:
d 25.7 (C-30), 27.5 (C-20), 29.3 (C-10), 36.9 (C-40), 41.2 (C-
5), 54.9 (C-2), 60.3 (C-4), 62.0 (C-3), 164.2 (C-200), 168.2 (C-
50); other: d 32.6 (S-CH2), 128.1, 128.3, 128.5, 128.6, 129.4,
129.5, 129.8, 130.9, 133.2, 133.3, 133.4 (8ÂPh), 137.5
(Cipso), 164.5, 165.1, 165.2, 165.3, 165.5, 165.8 (7ÂOC(O)
Ph). ESIMS 1419 [M+H] (37%), 840 (42), 676 (100);
found: 1418.4224; C78H72N3O19S2 requires: 1418.4201.
0
0
S-D-Biotinoyl-4-aminobenzyl methyl [5-acetamido-4,7,8,9-
tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-ꢀ-D-galacto-
2-nonulopyranosid]onate-(2,6)-2,3-di-O-acetyl-ꢁ-D-galac-
topyranosyl-(1,4)-2,3,6-tri-O-acetyl-1-thio-ꢁ-D-glucopyra-
noside (30). Prepared in 85% yield by reaction between
29, TBTU and d-biotin (chromatography: CHCl3:
MeOH, 10:1; Rf 0.25). nmax 3412 (br), 1746, 1692, 1532,
In a similar manner were prepared:
S-D-Biotinoyl-4-aminobenzyl 2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-1-thio-ꢁ-D -glucopyranoside (19). Pre-
pared in 92% yield by reaction between 16, TBTU and
d-biotin (chromatography: CH2Cl2:MeOH, 10:1; Rf
1
1370, 1230, 1044 cm 1; H NMR (500 MHz, CDCl3):
Neu5Ac unit: d 1.87 (3H, s, AcN), 2.59 (1H, dd, J3e,3a
12.8, J3e,4 4.4 Hz, H-3e), 3.81 (3H, s, CO2Me), 4.01±4.13
(3H, m, H-5/H-6/H-9), 4.39±4.44 (1H, m, H-90), 4.92
(1H, ddd, J4,3a 11.2, J4,5 9.9, J4,3e 4.4 Hz, H-4), 5.27±
5.39 (2H, m, H-7/H-8), 5.75 (1H, d, JNH,5 9.5 Hz, NH);
1
0.3). H NMR (CDCl3): GlcNAc unit: d 1.71 (3H, s,
AcN), 1.81, 1.84, 1.94 (3Â3H, 3Âs, 3 ÂAcO), 3.41±3.45
(1H, m, H-5), 3.91±3.99 (2H, m, H-2/H-6), 4.05 (1H, dd,
J6 ,6 12.3, J6 ,5 5.1 Hz, H-60), 4.12 (1H, d, J1,2 10.8 Hz, H-
1), 4.84±4.87 (2H, m, H-3/H-4); Benzyl unit: d 3.58 (1H, d,
Jgem 12.9 Hz, S-CHaHb), 3.71 (1H, d, Jgem 12.9Hz, S-
CHaHb), 7.08 (2H, d, ArH-2/6), 7.35 (2H, d, ArH-3/5);
Biotin unit: d 1.31±1.61 (6H, m, H-10/H-20/H-30), 2.21 (2H,
Gal unit: d 3.62 (1H, dd, J5,6=J5,6 =5.9 Hz, H-5), 3.75
0
0
0
0
0
(1H, dd, J6,6 12.1, J6,5 5.9 Hz, H-6), 3.98 (1H, dd, J6 ,6
0
12.2, J6 ,5 5.9 Hz, H-60), 4.01±4.13 (1H, m, H-4), 4.28
(1H, d, J1,2 8.9 Hz, H-1), 4.87 (1H, dd, J3,2 9.5, J3,4
2.9 Hz, H-3), 5.27±5.39 (1H, m, H-2); Glc unit: d 3.67
(1H, dd, J4,3=J4,5=9.5 Hz, H-4), 3.86±3.92 (1H, m, H-
5), 4.04±4.13 (2H, m, H-1/H-6), 4.39±4.44 (1H, m, H-6),
t, J4 ,3 7.2 Hz, H-40), 2.54 (1H, d, J5a,5b 12.9Hz, H-5a),
2.73 (1H, dd, J5b,5a 12.9, J5b,4 4.8 Hz, H-5b), 2.98 (1H, m,
0
0

D. I. Angus et al. / Bioorg. Med. Chem. 8 (2000) 2709±2718
2717
4.84±4.98 (1H, m, H-3), 5.27±5.39 (1H, m, H-2); Biotin
unit: d 1.46±1.49 (2H, m, H-20), 1.63±1.80 (4H, m, H-10/
CHaHb), 3.86 (1H, d, J 13.2 Hz, S-CHaHb), 7.20 (2H, d,
H-2/6), 7.31 (d, 2H, H-3/5); Biotin unit: d 1.37±1.69 (6H,
H-30), 2.38 (2H, t, J4 ,3 7.1 Hz, H-40), 2.71 (1H, d, J5a,5b
12.5 Hz, H-5a), 2.89 (1H, dd, J5b,5a 12.5, J5b,4 4.2 Hz, H-
5b), 3.12±3.17 (1H, m, H-2), 4.31±4.33 (1H, m, H-3),
4.48±4.52 (1H, m, H-4); other: d 2.01, 2.02, 2.03 (3Â6H,
3Âs, 6ÂAcO), 2.07 (3H, s, AcO), 2.13 (6H, s, 2ÂAcO),
3.85±3.95 (2H, m, SCH2), 7.28 (2H, d, ArH-2/6), 7.53
(2H, d, ArH-3/5), assignments con®rmed by COSY;
ESIMS: 1103 [M 270] (100%).
m, H-10/H-20/H-30), 2.21 (2H, t, J4 ,3 7.5 Hz, H-40), 2.67
(1H, d, J5a,5b 13.2 Hz, H-5a), 2.88 (1H, dd, J5b,5a 12.9,
J5b,4 4.8 Hz, H-5b), 3.22±3.26 (1H, m, H-2), 4.31 (1H,
dd, J3,4 7.8, J3,2 4.2 Hz, H-3), 4.95 (1H, dd, J4,3 7.7, J4,5b
4.8 HZ, H-4), assignments con®rmed by COSY; 13C
NMR (D2O): GlcNAc unit: d 21.5 (NC(O)Me), 53.9 (C-
2), 60.5 (C-6), 69.3 (C-4), 74.7 (C-5), 79.5 (C-3), 82.3 (C-
1), 171.1 (NC(O)Me); Benzyl unit: d 33.0 (S-CH2), 121.8
(C-2/6), 129.3 (C-3/5), 133.7 (C-1), 137.1 (C-4); Biotin
unit: d 24.5 (C-10), 27.2, 27.4 (C-20/C-30), 35.6 (C-40),
39.1 (C-5), 54.8 (C-2), 59.8 (C-4), 61.6 (C-3), assign-
ments con®rmed by HMQC; ESIMS: 569.4 [M+H]
(100%); found: 569.21065; C25H37N4O7S2 requires:
569.21033.
0
0
0
0
Deprotection of biotinylated saccharides
S-D-Biotinoyl-4-aminobenzyl 5-acetamido-3,5-dideoxy-2-
thio-D-glycero-ꢀ-D-galacto-2-nonulopyranosidonic acid
(21). Compound 18 (67 mg, 0.08 mmol) was dissolved
in dry MeOH (2 mL) and a solution of NaOMe [1 mL,
made from Na (20 mg) in dry MeOH (3 mL)] was added
at 0 ꢀC under N2. The mixture was allowed to warm to
rt and stirred for 5 h before being concentrated under
reduced pressure. The residue was dissolved in H2O
(3 mL), the pH adjusted to 12 with NaOH (0.1 M), and
the solution was stirred for a further 16 h before being
neutralized with Dowex 50W-H+ resin and ®ltered. The
solvent was removed in vacuo and the residue chroma-
tographed (EtOAc:MeOH:H2O, 7:2:1; Rf 0.1) to give 21
S-D-Biotinoyl-4-aminobenzyl ꢁ-D-galactopyranosyl-(1,4)-
1-thio-ꢁ-D-glucopyranoside (23). To a solution of 20
(57 mg, 0.04 mmol) in MeOH (2 mL) was added NaOH
(0.1 M) to pH 13 and the mixture left to stir at rt for
16 h. Evaporation to dryness followed by column chro-
matography (EtOAc/MeOH/H2O; Rf 0.2) a€orded 23
1
(25 mg, 90%). H NMR (D2O): Gal unit: d 3.66±3.72
(1H, m, H-5), 3.80±3.83 (1H, m, H-4), 3.91±4.01 (3H, m,
H-3/H-6/H-60), 4.34 (1H, d, J1,2 9.9 Hz, H-1), 4.47 (1H,
dd, J2,1 9.9, J2,3 7.5 Hz, H-2); Glc unit: d 3.36±3.50 (3H,
1
(30 mg, 58%) as a white powder: H NMR (600 MHz,
m, H-3/H-4/H-5), 3.50 (2H, d, J6,6 12 Hz, H-6/60), 3.66±
0
CD3OD): Neu5Ac unit: d 1.75 (1H, dd, J3a,3e 12.0, J3a,4
10.8 Hz, H-3a), 1.96 (3H, s, AcN), 2.80 (1H, dd, J3e,3a
12.0, J3e,4 5.1 Hz, H-3e), 3.62±3.67 (2H, m, H-6/H-7),
3.71±3.80 (2H, m, H-5/H-9), 3.84±3.96 (3H, m, H-4/H-
8/H-90); Benzyl unit: d 4.03 (1H, d, Jgem 12.1 Hz, S-
CHaHb), 4.18 (1H, d, Jgem 12.1 Hz, S-CHaHb), 7.42 (d,
2H, H-2/6), 7.60 (d, 2H, H-3/5); Biotin unit: d 1.59±1.89
3.72 (1H, m, H-2), 3.80±3.83 (2H, m, J1,2 8.1 Hz, H-1);
Biotin unit: d 1.51±1.83 (6H, m, H-10/H-20/H-30), 2.49
(2H, t, J4 ,3 7.2 Hz, H-40), 2.80 (1H, d, J5a,5b 13.2 Hz, H-
5a), 3.03 (1H, d, J5b,5a 13.2 Hz, H-5b), 3.36±3.50 (1H,
m, H-2), 4.71 (1H, dd, J3,4 7.8, J3,2 4.4 Hz, H-3), 4.64
(1H, dd, J4,3 7.8, J4,5b 4.8 Hz, H-4); Benzyl unit: d 3.99±
4.05 (2H, m, S-CH2), 7.59±7.94 (4H, m, ArH); ESIMS:
712 [M+Na] (64%), 690.5 [M+H] (57); found:
690.23241; C29H44N3O12S2 requires: 690.23659.
0
0
(6H, m, H-10/H-20/H-30), 2.52 (2H, t, J4 ,3 7.2 Hz, H-40),
2.82 (1H, d, J5a,5b 12.6 Hz, H-5a), 3.04 (1H, dd, J5b,5a
12.6, J5b,4 4.8 Hz, H-5b), 3.33±3.40 (1H, m, H-2), 4.46
(1H, dd, J3,4 7.5, J3,2 4.5 Hz, H-3), 4.61 (1H, dd, J4,3 7.5,
0
0
J4,5b 4.8 Hz, H-4), assignments con®rmed by COSY; 13
C
NMR (500 MHz, CD3OD): Neu5Ac unit: d 22.7
(NC(O)Me), 43.1 (C-3), 54.2 (C-5), 64.7 (C-9), 69.9 (C-
4), 70.6 (C-7), 73.1 (C-8), 76.8 (C-6), 87.4 (C-2), 168.3
(C-1); Benzyl unit: d 34.7 (S-CH2), 121.3 (C-3/5), 130.9
(C-2/6), 135.1 (C-1), 138.2 (C-4); Biotin unit: d 26.9 (C-
20), 29.6 (C-10), 29.9 (C-30), 37.8 (C-40), 41.2 (C-5), 57.1
(C-2), 61.8 (C-4), 63.5 (C-3), assignments con®rmed by
HMQC; found: 657.22585; C28H41N4O10S2 requires:
657.22636.
Acknowledgements
Dr Robin Thomson is thanked for helpful discussions
and comments. We also thank Monash University for a
Monash Graduate Scholarship (to DIA), and the NIH
(Grant number PO1 A1 38084) and the ARC for ®nan-
cial support.
References
S-D-Biotinoyl-4-aminobenzyl 2-acetamido-2-deoxy-1-thio-
ꢁ-D-glucopyranoside (22). To a solution of 19 (98 mg,
0.14 mmol) in MeOH (4 mL) at 0 ꢀC was added freshly
prepared NaOMe (1 mL, prepared as above). The mix-
ture was allowed to warm to rt and then stirred for 16 h,
before being neutralized with Amberlite IR 120H+
resin. After ®ltration and concentration, the residue was
chromatographed (EtOAc:MeOH:H2O; 7:2:1; Rf 0.3) to
1. Schauer, R.; Kamerling, J. P. In Glycoproteins II; Mon-
treuil, J.; Vliengenthart, J. F. G.; Schachter, H., Eds.; Elsevier:
Amsterdam, 1997, pp. 243±402.
2. Varki, A. Glycobiology 1993, 3, 97±130.
3. Crocker, P. R.; Feizi, T. Curr. Opin. Struct. Biol. 1996, 6,
679.
4. Lis, H.; Sharon, N. Eur. J. Biochem. 1993, 218, 1.
5. Karlsson, K.-A. Curr. Opin. Struct. Biol. 1995, 5, 622.
6. Bacterial Adhesion to Cells and Tissues, Ofek, I.; Doyle, R.
J.; Eds.; Chapman Hall: New York, 1994.
7. Simanek, E. E.; McGarvey, G. J.; Jablonowski, J. A.;
Wong, C.-H. Chem. Rev. 1998, 98, 833.
1
give 22 (45 mg, 56%) as a colourless powder: H NMR
(D2O): GlcNAc unit: d 1.87 (3H, s, AcN), 3.22±3.26 (1H,
0
m, H-3), 3.32±3.35 (2H, m, H-4/H-5), 3.60 (1H, dd, J6,6
12.6, J6,5 5.7 Hz, H-6), 3.64±3.67 (1H, m, H-2), 3.76
(1H, dd, J6 ,6 12.6, J6 ,5 5.1 Hz, H60), 4.20 (1H, d, J1,2
10.5 Hz, H-1); Benzyl unit: d 3.80 (1H, d, J 13.2 Hz, S-
0
0
8. Sears, P.; Wong, C.-H. Cell. Mol. Life Sci. 1998, 54, 223.
9. Dwek, R. A. Chem. Rev. 1996, 96, 683.

2718
D. I. Angus et al. / Bioorg. Med. Chem. 8 (2000) 2709±2718
10. Rosen, S. D.; Bertozzi, C. R. Curr. Biol. 1996, 6, 261.
11. Hakomori, S. Adv. Cancer Res. 1989, 52, 257.
12. Feizi, T. Nature 1985, 314, 53.
35. Defaye, J; Gelas, J. Studies in Natural Products Chemistry,
vol 8; Atta-ur-Rahman, Ed.; Elsevier: Amsterdam, 1991, pp.
315±357
13. Sato, M.; Narita, T.; Kimura, N.; Zenita, K.; Hashimoto,
T.; Manabe, T.; Kannagi, R. Anticancer Res. 1997, 17, 3505.
14. Lee, Y. C. FASEB J. 1992, 6, 3193.
15. Shinohara, Y.; Sota, H.; Gotoh, M.; Hasebe, M.; Tosu,
M.; Nakao, J.; Hasegawa, Y. Anal. Chem. 1996, 68, 2573.
16. Honda, S.; Taga, A.; Suzuki, K.; Suzuki, S.; Kakehi, K. J.
Chromatogr. 1992, 597, 377.
17. Galanina, O. E.; Tuzikov, A. B.; Rapoport, E.; Le Pendu, J.;
Bovin, N. V. Anal. Biochem. 1998, 265, 282 and references therein.
18. Feizi, T.; Stoll, M. S.; Yuen, C.-T.; Chai, W.; Lawson, A.
M. Methods Enzymol. 1994, 230, 484.
19. Stoll, M. S.; Feizi, T.; Loveless, R. W.; Chai, W.; Lawson,
A. M.; Yuen, C.-T. Eur. J. Biochem. 2000, 267, 1795.
20. Kremsky, J. N.; Pluskal, M.; Casey, S.; Perry-O'Keefe, H.;
Kates, S. A.; Sinha, N. D. Tetrahedron Lett. 1996, 37, 4313.
21. Dohi, H.; Nishida, Y.; Mizuno, M.; Shinkai, M.;
Kobayashi, T.; Takeda, T.; Uzawa, H.; Kobayashi, K. Bioorg.
Med. Chem. Lett. 1999, 7, 2053.
36. Albrecht, B.; Putz, U.; Schwarzmann, G. Carbohydr. Res.
1995, 276, 289.
37. Bennett, S.; von Itzstein, M.; Kiefel, M. J. Carbohydr. Res.
1994, 259, 293.
38. Kiefel, M. J.; Beisner, B.; Bennett, S.; Holmes, I. D.; von
Itzstein, M. J. Med. Chem. 1996, 39, 1314.
39. Angus, D. I.; von Itzstein, M. Carbohydr. Res. 1995, 274,
279.
40. Ciccotosto, S.; Kiefel, M. J.; Abo, S.; Stewart, W.; Quelch,
K.; von Itzstein, M. Glycoconjugate J. 1998, 15, 663.
41. Bodansky, M. In Principles of Peptide Synthesis, 2nd Ed.;
Springer-Verlag: Berlin, 1993.
42. Kiefel, M. J.; Thomson, R. J.; Radovanovic, M.; von Itz-
stein, M. J. Carbohydr. Chem. 1999, 18, 937.
43. Hasegawa, A.; Nakamura, J.; Kiso, M. J. Carbohydr.
Chem. 1986, 5, 11.
44. Park, W. K. C.; Meunier, S. J.; Zanini, D.; Roy, R. Car-
bohydr. Lett. 1995, 1, 179.
22. Leteux, C.; Childs, R. A.; Chai, W.; Stoll, M. S.; Kogel-
berg, H.; Feizi, T. Glycobiology 1998, 8, 227.
45. Horton, D.; Wolfrom, M. L. J. Org. Chem. 1962, 27,
1794.
23. Rothenberg, B. E.; Hayes, B. K.; Toomre, D.; Manzi, A.
E.; Varki, A. Proc. Natl. Acad. Sci. USA 1993, 90, 11939.
24. Toomre, D. K.; Varki, A. Glycobiology 1994, 4, 653.
25. Shinohara, Y.; Sota, H.; Kim, F.; Shimizu, M.; Gotoh,
M.; Tosu, M.; Hasegawa, Y. J. Biochem. 1995, 117, 1076.
26. Sakai, T.; Naidenko, O. V.; Iijima, H.; Kronenberg, M.;
Koezuka, Y. J. Med. Chem. 1999, 42, 1836.
27. McReynolds, K. D.; Hadd, M. J.; Gervay-Hague, J. Bio-
conjugate Chem. 1999, 10, 1021.
28. Tamura, J.-i.; Miura, Y.; Freeze, H. H. J. Carbohydr.
Chem. 1999, 18, 1.
46. Hudlicky, M. Reductions in Organic Chemistry, 2nd Ed.;
ACS Publishing: Washington, 1996.
47. Cao, S. D.; Meunier, S. J.; Andersson, F. O.; Letellier, M.;
Roy, R. Tetrahedron: Asymmetry 1994, 5, 2303.
48. For a recent example see: Cao, S.; Hernandez-Mateo, F.;
Roy, R. J. Carbohydr. Chem. 1998, 17, 609.
49. Humphrey, J. M.; Chamberlin, A. R. Chem. Rev. 1997,
97, 2243.
50. See for example Bailen, M. A.; Chinchilla, R.; Dodsworth,
D. J.; Najera, C. J. Org. Chem. 1999, 64, 8936, and references
therein
29. Wilchek, M.; Bayer, E. A. Anal. Biochem. 1988, 171, 1.
30. Shao, M.-C.; Chen, L.-M.; Wold, F. Methods Enzymol.
1990, 184, 653.
31. Shao, M.-C. Anal. Biochem. 1992, 205, 77.
32. Wilson, J. C.; Kiefel, M. J.; Angus, D. I.; von Itzstein, M.
Org. Lett. 1999, 1, 443.
51. Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D.
Tetrahedron Lett. 1989, 30, 1927.
52. Martin, T. J.; Brescello, R.; Toepfer, A.; Schmidt, R. R.
Glycoconjugate J. 1993, 10, 16.
53. Abo, S.; Ciccotosto, S.; Alafaci, A.; von Itzstein, M. Car-
bohydr. Res. 1999, 322, 201.
33. Suzuki, Y.; Sato, K.; Kiso, M.; Hasegawa, A. Glyco-
conjugate J. 1990, 7, 349.
34. Sabesan, S.; Neira, S.; Davidson, F.; Duus, J. é.; Bock,
K. J. Am. Chem. Soc. 1994, 116, 1616.
54. Vogel's Textbook of Practical Organic Chemistry, 5th Ed.;
Longman Scienti®c & Technical, UK, 1989, p.122.
55. Jansson, K.; Ahlfors, S.; Frejd, T.; Kihlberg, J.; Magnus-
son, G. J. Org. Chem. 1988, 53, 5629.