The synthesis of 16-mercaptohexadecanyl glycosides for biosensor applications

Article abstract of DOI:10.1016/S0008-6215(98)00053-6

The p(k) trisaccharide and the central disaccharide element of asialo GM₁ activated as their trichloroacetimidates were each used to glycosylate 16-(p-toluensulfonyloxy)hexadecanol 1. Displacement of the tosyl group by thiocyanate followed by s

Full text of DOI:10.1016/S0008-6215(98)00053-6

Carbohydrate Research 307 (1998) 361±369
Note
The synthesis of 16-mercaptohexadecanyl
glycosides for biosensor applications
Pavel I. Kitov, Craig Railton, David R. Bundle *
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada
Received 1 October 1997; accepted 12 January 1998
Abstract
The Pk trisaccharide and the central disaccharide element of asialo GM1 activated as their
trichloroacetimidates were each used to glycosylate 16-(p-toluensulfonyloxy)hexadecanol 1.
Displacement of the tosyl group by thiocyanate followed by sodium borohydride reduction and
saponi®cation a€orded oligosaccharide 16-mercaptohexadecanyl glycosides that were isolated
as the corresponding disul®des 6 and 17 unless oxygen was rigorously excluded from the solvents
used for work-up. Dithiothreitol reduction of disul®des and subsequent isolation under an inert
atmosphere with degassed solvents gave the thiols 7 and 18. Chemisorption of !-glycosyl alka-
nethiols and alkanethiols onto gold electrodes produces self-assembled monolayers that can act as
amperometric biosensors for the detection of proteins that bind to the immobilized oligosacchar-
ide epitope. # 1998 Elsevier Science Ltd. All rights reserved
Keywords: 16-mercaptohexadecanyl glycosides; Pk trisaccharide; Asialo GM1; Asialo GM2; Amperometric
biosensors
Carbohydrates serve as attachment sites for tox-
ins, bacteria, and viruses, and can, therefore, be
employed for the detection of those agents in phy-
siological samples. However, the e€ective design of
a rapid and sensitive assay requires the coupling of
the protein±sugar association process to a change
in a physical quantity such as an electrical current.
Amperometric biosensors [1] utilize the electron
transfer properties of self-assembled monolayers
(SAMs) created by chemisorption of alkanethiols
and !-functionalized alkanethiols mixtures from
ethanol solution onto a gold surface [2±4]. If the
chain length of the alkanethiol and !-functiona-
lized alkanethiols are matched in the biosensor, the
ligand does not perturb the dielectric monolayer
until a speci®c binding event occurs between it and
a protein receptor. Two recognition systems that
involve the interaction of bacteria or their toxins
with glycolipids were chosen for development of
amperometric biosensors. The disaccharide ꢀ-d-
GalpNAc-(1!4)-ꢀ-d-Galp, a fragment of some
human glycosphingolipids (GM1, GM2, for
instance), represents the minimal structural ele-
ment recognized by adhesins, proteins with sugar
binding sites, located on ®lamentous structures of
Pseudomonas aeruginosa and Candida albicans, a
dimorphic yeast [5±7]. The Pk trisaccharide, the
carbohydrate portion of the glycosphingolipid
Gb3, is a receptor for toxins [8±12] secreted by
pathogenic bacteria such as Shigella dysenteriae [8]
* Corresponding author. Fax: (403) 492-7705.
0008-6215/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00053-6

362
P. I. Kitov et al./Carbohydrate Research 307 (1998) 361±369
and certain strains of uropathogenic Eshericia coli.
Covalent attachment of a neoglycoprotein to gold
surfaces via thiols has been reported for biosensor
detection of P-®mbriated E. coli bacteria [13].
The syntheses of 16-mercaptohexadecanyl gly-
cosides of the disaccharide ꢀ-d-GalpNAc-(1!4)-ꢀ-
d-Galp and Pk trisaccharide ꢁ-d-Galp-(1!4)-ꢀ-d-
Galp-ꢀ-d-(1!4)-Glcp are reported by a method
that is general and represents one possibility for the
fabrication of a carbohydrate-based biosensor by
immobilizing carbohydrate-ligands on the surface
of gold electrodes [3]. The scheme uses an aglycone
prepared from commercially available 1,16-dihy-
droxyhexadecane. Controlled tosylation gave the
monotosylated diol 1 in 42% yield, and following
O-glycosylation of this tether, displacement of the
tosyl group by thiocyanate introduced a latent
thiol function. Reduction of the thiocyanate with
sodium borohydride gave a thiol and simulta-
neously hydrolyzed, partially or completely, the
ester-protecting groups of the carbohydrate.
Manipulation in solvents without exclusion of dis-
solved oxygen resulted in almost quantitative oxi-
dation of thiol to disul®de. Thiols were prepared
immediately prior to use by reduction under con-
trolled conditions. In fact, either thiol or disul®de
are known to undergo chemisorption to gold sur-
faces [2±4,14].
Trisaccharide 2, prepared according to published
procedures [15], was converted into an anomeric
mixture of trichloroacetimidates 3 and reacted with
alcohol 1 in CH2Cl2 in the presence of a catalytic
amount of trimethylsilyl tri¯uoromethanesulf-
onate. The tosyloxy group of the trisaccharide
glycoside 4 was displaced by thiocyanate to pro-
vide the protected trisaccharide glycoside 5.
Reduction of thiocyanate by the action of sodium
borohydride [16] followed by saponi®cation of acyl
groups gave the trisaccharide ligand 6. The dis-
ul®de 6 was converted to the thiol 7 by reduction
with dithiothreitol (DTT) under argon followed by
reverse-phase high-performance liquid chromato-
graphy (HPLC).
by heterogeneous activators leads mainly to a ꢀ-
(1!4) glycosidic linkage. This method has some
advantages, since it avoids using a comparatively
expensive galactosamine starting material, and the
absence of a protecting group at O-3 of the accep-
tor avoids steric and electronic in¯uences on the
reactivity of the 4-hydroxyl group.
Acetonation of 2-(trimethylsilyl)ethyl galacto-
pyranoside followed by benzylation of 8 [15] gave 9
and then the 3,4-diol acceptor 10 [20,21] after
acetal hydrolysis. Glycosylation of 10 by glycosyl
bromide 11 [22,23] in dichloromethane in the pre-
sence of silver silicate on alumina [24] as the pro-
moter provided disaccharide 12 in 74% yield. The
site of galactosylation was con®rmed after reduc-
tion of the azido function and removal of benzyl
groups followed by per-acetylation to give the
1
acetamido derivative 13. Comparison of the H
NMR spectra of disaccharides 12 and 13 shows
that, after acetylation, the signal of H-3 had shifted
down®eld by more than 1 ppm from the 3.68±
3.57 ppm area to 4.91 ppm, whereas the chemical
shift of H-4 did not change signi®cantly.
The 2-(trimethylsilyl)ethyl glycoside 13 was con-
verted into the trichloroacetimidate 14. Glycosyla-
tion of 1 by 14 gave the disaccharide glycoside 15.
Nucleophilic displacement of the tosyloxy group
by thiocyanate was carried out as described for the
synthesis of 5. Reduction of thiocyanate 16
accompanied by deacetylation a€orded the dis-
accharide 17. This disul®de was reduced to the
thiol 18 with DTT and isolated by HPLC.
The synthetic scheme compares favourably with
other procedures used to generate substituted
alkanethiols [2,3,25]. However, !-glycosyl alka-
nethiols exhibit a strong propensity for oxidation
to the corresponding disul®des and any isolation
protocol that employed solvents that had not been
degassed resulted in virtually quantitative yields of
the disul®des 6 and 17. In order to monitor the
oxidation state of the sulfur, 1H NMR of
the -CH2S- group was diagonstic. Methanol-d4 was
the preferred solvent since D2O solution gave only
broad signals, presumably owing to the formation
of micelles. The chemical shift of the -CH2SH
methylene protons were observed at ꢂ 2.48 ppm,
while the those of the corresponding protons in
disul®de derivatives -CH2S-SCH2- had ꢂ 2.68 ppm.
This was particularly useful since mass spectral
measurements in the commonly employed Cle-
land's matrix (dithiothreitol:dithioerythitol, 5:1)
reduces any disul®de to thiol. Electrospray mass
There are two synthetic strategies to build the ꢀ-
GalpNAc(1!4)Galp disaccharide linkage. One
involves glycosylation of 4-O-unprotected galac-
tose derivatives with various N-acetyl and N-
phthaloyl galactosamine donors [17]. Another
route is selective glycosylation of a galactose 3,4-
diol derivative 10 by a 2-azido-2-deoxy-galacto-
pyranosyl bromide derivative 11. According to
Paulsen [18] and Sinay [19], the reaction promoted

P. I. Kitov et al./Carbohydrate Research 307 (1998) 361±369
363
ꢀ
spectra were recorded to observe the disul®des. The
deacylated !-glycosyl alkanethiols 7 and 18 or
their disul®de derivatives exhibited rather poor
solubility characteristics and were poorly soluble in
water and ethanol. Warm methanol was the most
e€ective protic solvent.
cell at ambient temperature (22‹2 C). Analytical
thin-layer chromatography (TLC) was performed
on Silica Gel 60-F254 (Merck) with detection by
quenching of ¯uorescence and/or by charring with
10% H2SO4 in ethanol solution followed by heat-
ꢀ
ing at 180 C. Millex-HV (0.45 ꢃM) ®lter units
Amperometric biosensors, prepared by incor-
poration of these glycoconjugates into SAMs, are
able to detect verotoxin binding to the Pk tri-
saccharide 6, and pilin protein or pilin peptide
binding to the asialo GM1 disaccharide 17 [1b].
were from Millipore (Missisuaga, ON). Column
chromatography was performed on Silica Gel 60
(Merck, 40±60 ꢃm), and solvents were distilled
prior to use. Sep-Pak C18 reverse-phase cartridges
(Waters, Missisuaga, ON) were conditioned prior
to use by washing with MeOH (10 mL) and water
(20 mL). 1H NMR spectra were recorded at
360 MHz (Bruker WM-360) or at 500 MHz (Varian
Unity 500) in CDCl3 (referenced to residual CHCl3
at 7.24 ppm), CD3OD (referenced to residual
CD2HOD at 3.3 ppm), or D2O (referenced to
1. Experimental
General methods.ÐOptical rotations were mea-
sured on a Perkin-Elmer 241 polarimeter in a 1 dm

364
P. I. Kitov et al./Carbohydrate Research 307 (1998) 361±369
internal or external acetone at 2.225 ppm). Mass
spectrometric analysis was performed by positive
mode electrospray ionization on a Micromass
ZabSpec Hybrid Sector-TOF. The liquid carrier
was infused into the electrospray source by means
of a Harvard syringe pump at a ¯ow rate of 10 ꢃL/
min. The sample solution was introduced via a
1 ꢃL loop injector. Prepuri®ed nitrogen was used
as a spray pneumatic aid andꢀ®ltered nitrogen as
the bath gas, heated at ca. 80 C. For low resolu-
tion, the mass spectra were acquired by magnet
scan at a rate of 10 s/decade. For exact mass mea-
surements, the spectra were obtained by voltage
scan at a rate of 10 s/decade. Data acquisition and
processing was achieved by using the OPUS soft-
ware package on a Digital Alpha station with VMS
operating system. All commercial reagents were
used as supplied and solvents were distilled from
appropriate desiccants prior to use [26].
mmol) in dry pyridine (10 mL). After 2 h the mix-
ture was concentrated, diluted with acetone
(20 mL), silica gel (5 g) was added and acetone was
removed in vacuum. The solid was slurred onto a
silica gel column and eluted with pentane±etꢀhyl
acetate (2:1) to yield 1 (748 mg, 42%), m.p. 58 C.
1H NMR: ꢂH 7.78 and 7.32 (2d, 4 H, J=8.1 Hz,
3
arom.), 3.99 (t, 2H, J=6.6 Hz, CH2OTs), 3.62 (t,
3
2 H, J=6.5 Hz, CH2OH), 2.43 (s, 3 H, CH3),
1.63±1.50 (m, 2 H, CH2CH2OTs), 1.32±1.20 (m, 26
H, 13 CH2). Anal calcd for C23H40SO4 (412.63): C,
66.95; H, 9.77; S, 7.77. Found: C, 66.96; H, 9.90; S,
7.73.
16-(p-Toluenesulfonyloxy)hexadecanyl 4-O-[6-O-
acetyl-2,3-di-O-benzoyl-4-O-(2,3,4,6-tetra-O-acetyl-
a-d-galactopyranosyl)-b-d-galactopyranosyl]-2,3,6-
tri-O-benzoyl-b-d-glucopyranoside (4).ÐTri¯uoro-
acetic acid (2 mL) was added to a solution of
trisaccharide 2 [15] (445 mg, 0.33 mmol) in CH2Cl2
(4 mL). After 45 min, EtOAc (2 mL) and toluene
(2 mL) were added and the mixture was con-
centrated, co-evaporated with toluene three times,
16-(p-Toluenesulfonyloxy)hexadecanol
(1).Ð
Tosyl chloride (0.8 g, 4.21 mmol) was added to a
solution of 1,16-dihydroxyhexadecane (1.1 g, 4.25

P. I. Kitov et al./Carbohydrate Research 307 (1998) 361±369
365
0
00
0
(dd, 1 H, J1 ,2 =7.8 Hz, J2 ,3 =10.8 Hz, H-2 ), 5.45
0
0
0
and dried in vacuum. A mixture of the residue with
Cl3CCN (1 mL) in CH2Cl2 (5 mL) was cooled at
0 ꢀC and DBU (40 ꢃL) was added. After 30 min the
mixture was concentrated and chromatographed
on silica gel with toluene±acetone (3:1) to give an
ꢁ=ꢀ mixture of imidates 3 (396 mg, 86%). 1H
NMR: ꢂH 8.59 (ꢀ-NH), 8.53 (ꢁ-NH).
The imidate mixture 3 (100 mg, 72 ꢃmol),
monotosylated diol 1 (36 mg, 87 ꢃmol), and 4A
molecular sieves (200 mg) in dry CH2Cl2 (4 mL)
were stirred for 1 h. Then TMSOTf (8 ꢃL,
40 ꢃmol) was added. After 2 h, triethylamine
(0.1 mL) was added and the solids were removed
by ®ltration. The ®ltrate was concentrated and
dried in vacuum. Chromatography of the residue
on silica gel with pentane±ethyl acetate (3:2) gave 4
(84 mg, 71%), [ꢁ]d+75.2ꢀ (c. 0.9; CHCl3). 1H
NMR: ꢂH 8.02±7.16 (m, 29 H, arom.), 5.73 (t, 1 H,
00 00
(dd, 1 H, J3 ,4 =3.2 Hz, J4 ,5 =1.1 Hz, H-4 ), 5.33
00 00
00 00
(dd, 1 H, J1,2=7.8 Hz, H-2), 5.28 (dd, 1 H, J2 ,3
00 00
0
=11.1 Hz, H-3), 5.08 (dd, 1 H, J 1 ,2 = 3.6 Hz, H-
200), 5.02 (dd, 1 H, J3 ,4 =2.8 Hz, H-3 ), 3.92 (d, 1
H, H-100), 4.80 (d, 1 H, H-10), 4.64 (d, 1 H, H-1),
4.59 (dd, 1 H, J6a,6b=12.0 Hz, J5,6a=2.0 Hz, H-
6a), 4.44 (dd, 1 H, J5,6b=4.7 Hz, H-6b), 4.41
0
0
0
(broad t, 1 H, H-500), 4.35 (t, 1 H, J3 ,4 'J4 ,5
0
0
0
0
9.4 Hz, H-40), 4.12 (d, 1 H, H-40), 3.83 (ddd, 1 H,
H-5), 3.80±3.70 (m, 3H, H-600a, CH2O, H-60a), 3.66
(dd, 1 H, J6 a,6 b=11.1 Hz, J5 ,6 b= 7.4 Hz, H-60b),
0
0
0
0
00 00
00
00
3.54 (dd, 1 H, J5 ,6 b= 5.7 Hz, J6 a,6 b=10.8 Hz,
H-600b), 3.49 (broad t, 1 H, H-50), 3.38 (dt, 1 H,
2J=9.7 Hz, 3J=6.8 Hz, CH2O), 2.91 (t, 2 H,
3J=7.3 Hz, CH2SCN), 2.02, 1.97, 1.95, 1.92, 1.91
(5s, 15 H, 5 Ac), 1.79 (p, 2 H, 3J=7.3 Hz,
CH2CH2SCN), 1.40±0.86 (m, 26 H, 13 CH2). Anal.
calcd for C80H93 SNO26 (1516.65): C, 63.35; H,
6.18; N, 0.92; S, 2.11. Found: C, 63.41; H, 5.82; N,
0.91; S, 2.70.
0
0
=
7.8 Hz, J2 ,3 =10.8 Hz, H-2 ), 5.45 (dd, 1 H,
J2,3'J3,4=9.2 Hz, H-3), 5.61 (dd, 1 H, J1 ,2
0
0
0
00
00 00
00 00
J3 ,4 =3.2 Hz, J4 ,5 =1.1 Hz, H-4 ), 5.33 (dd, 1 H,
00 00
J1,2=7.8 Hz, H-2), 5.28 (dd, 1 H, J2 ,300 =11.1 Hz,
Bis{16-[4-O-[4-O-(a-d-galactopyranosyl)-b-d-
galactopyranosyl]-b-d-glucopyranosyloxy]hexadec-
anyl} disul®de (6).ÐTo a solution of 5 (60 mg,
39 ꢃmol) in dry MeOH (4 mL) solid sodium boro-
hydride (ꢁ40 mg) was added under argon. After
00 00
H-3), 5.08 (dd, 1 H, J1 ,2 =3.6 Hz, H-2 ), 5.02 (dd,
0
00
0
0
1 H, J3 ,4 =2.8 Hz, H-3 ), 3.92 (d, 1 H, H-1 ), 4.80
(d, 1 H, H-10), 4.64 (d, 1 H, H-1), 4.59 (dd, 1 H,
J6a,6b=12.0 Hz, J5,6a=2.0 Hz, H-6a), 4.44 (dd, 1
H, J5,6b=4.7 Hz, H-6b), 4.41 (broad t, 1 H, H-500),
stirring for 2 h at 45 C, the mixture was con-
ꢀ
0
0
0
0
0
4.35 (t, 1 H, J3 ,4 'J4 ,5 =9.4 Hz, H-4 ), 4.12 (d, 1
centrated and dissolved under gentle re¯ux in a
solution of NaOH (50 mg) in water (10 mL). After
0
3
H, H-4 ), 4.00 (t, 2 H, J=6.5 Hz, CH2OTs), 3.83
(ddd, 1 H, H-5), 3.80±3.70 (m, 3 H, H-600a, CH2O,
stirring overnight at 45 C, the mixture was neu-
ꢀ
H-60a), 3.66 (dd, 1 H, J6 a,6 b=11.1 Hz, J5 ,6 b
tralized with Dowex resin 50Â8-400 (H+ form)
and then applied to a Sep-Pak (C-18) cartridge
(Waters). The cartridge was washed with 20, 40,
60, and 80% solution of MeOH in water, then with
pure MeOH. The methanol fractions containing
sugar were concentrated to give 6 (24.4 mg, 81%),
0
0
0
0
=
7.4 Hz, H-60b), 3.54 (dd, 1 H, J5 ,6 b=5.7 Hz,
00 00
J6 a,6 b=10.8 Hz, H-600b), 3.49 (broad t, 1 H, H-50),
00
00
2
3
3.38 (dt, 1 H, J=9.7 Hz, J=6.8 Hz, CH2O), 2.42
(s, 3 H, Me), 2.02, 1.97, 1.95, 1.92, 1.91 (5s, 15 H, 5
Ac), 1.60 (p, 2 H, J=7.3 Hz, CH2CH2OTs), 1.40±
0.86 (m, 26 H, 13 CH2). Anal. calcd for C86H100SO29
(1629.79) C, 63.38; H, 6.18; S, 1.97. Found: C,
63.42; H, 6.12; S, 2.00.
3
[ꢁ]d +43.5ꢀ (c. 0.8; H2O). H NMR (CD3OD,
1
ꢀ
00
00 00
45 C): ꢂH 4.95 (d, 1 H, J1 ,2 =3.8 Hz, H-1 ), 4.42
(m, X of ABX system, 1 H, H-10), 4.27 (d, 1 H,
00 00
J1,2=7.8 Hz, H-1), 4.24 (ddd, 1 H, J4 ,5 =1.2 Hz,
16-(Thiocyano)hexadecanyl
4-O-[6-O-acetyl-
2,3-di-O-benzoyl-4-O-(2,3,4,6-tetra-O-acetyl-a-d-
galactopyranosyl)-b-d-galactopyranosyl]-2,3,6-tri-
O-benzoyl-b-d-glucopyranoside (5).ÐA solution of
tosylate 4 (84 mg, 51 ꢃmol) and KSCN (50 mg,
J5 ,6 a=5.0 Hz, J5 ,6 b 6.6 Hz, H-500), 3.98 (broad
00 00
00 00
0
0
d, 1 H, J4 ,5 =1.2 Hz, H-4 ), 3.91 (dd, 1 H,
00 00
0
00
J3 ,4 =3.2 Hz, H-4 ), 3.90±3.66 (m 10 H, H-6a,
H-6b, H-50, H-60a, H-60b, H-200, H-300, H-600a, H-
600b, CH2O), 3.58±3.49 (m, 5 H, H-3, H-4, H-20, H-
30, CH2O), 3.38 (dt, 1 H, J5,6a=J5,6b=9.5 Hz,
J5,4= 3.8 Hz, H-5), 3.23 (dd, 1 H, J2,3=9.0 Hz, H-
2), 2.68 (t, 2 H, 3J=7.3 Hz, CH2S-SCH2), 1.67 (p, 2
H, 3J=7.0 Hz, CH2CH2S), 1.61 (p, 3J=6.9 Hz,
CH2CH2O), 1.42±1.26 (m, 24 H, 12 CH2). Electro-
spray MS: 1541.8 (calcd for C68H126S2O32Na
1541.8).
ꢀ
0.5 mmol) in DMF (2 mL) was stirred at 80 C for
2 h. The mixture was concentrated, dissolved in
CH2Cl2 (30 mL), washed with water, and con-
centrated again. Chromatography of the residue on
silica gel with pentane±ethyl acetate (3:2) gave 5
(67.4 mg, 86%), [ꢁ]d+81.2ꢀ (c. 0.6; CHCl3). IR
1
2153.6 (ꢄc-scn). H NMR: ꢂH 8.02±7.16 (m, 25 H,
arom.), 5.73 (t, 1 H, J2,3'J3,4=9.2 Hz, H-3), 5.61

366
P. I. Kitov et al./Carbohydrate Research 307 (1998) 361±369
16-(Mercapto)hexadecanyl 4-O-[4-O-(a-d-gal-
CH2Si), 0.0035 (s, 9 H, SiMe3). Anal. calcd. for
C14H28SiO6 (320.45): C, 52.47; H, 8.81. Found: C,
52.30; H, 9.08.
actopyranosyl)-b-d-galactopyranosyl]-b-d-glucopyr-
anoside (7).ÐDithiothreitol (10 mg, 70 ꢃmol) was
added to a solution of 6 (10.8 mg, 14 ꢃmol) in
degassed water (5 mL) and the pH was adjusted to
8 with a sat. solution of NH4HCO3. After stirring
for 24 h under argon, the mixture was heated to
dissolve the precipitated residue, ®ltered through a
Millex-HV 0.45 ꢃm membrane ®lter (Millipore),
and chromatographed on a C-18 reverse phase
HPLC column with a step gradient of 20, 40, 60,
80, and 100% aqueous MeOH. The methanol
fractions containing sugar were concentrated to
give faster moving 7 (2 mg, 18.5%), [ꢁ]d +40.1ꢀ
2-(Trimethylsilyl)ethyl 2,6-di-O-benzyl-3,4-O-iso-
propylidene-b-d-galactopyranoside (9).ÐA mixture
of 8 (550 mg, 1.71 mmol) and sodium hydride
(80%, 130 mg, 4.3 mmol) in DMF (4 mL) was stir-
red for 0.5 h, then benzyl bromide (0.61 mL,
5.1 mmol) was added dropwise. After 16 h MeOH
(1 mL) was added, the mixture was diluted with
CH2Cl2 (100 mL), and the organic solution was
washed with water and concentrated. Chromato-
graphy of the residue on silica gel in pentane-etꢀhyl
acetate (6:1) gave 9 (700 mg, 82%), m.p. 93±94 C,
[ꢁ]d +19.2ꢀ (c. 1.6; CHCl3) (ref. 21 [31ꢁ]d
+25.4ꢀ). 1H NMR: ꢂH 7.39±7.23 (m, 10 H, arom.),
1
ꢀ
(c. 0.5; MeOH). H NMR (CD3OD, 45 C): ꢂH
00
00 00
4.95 (d, 1 H, J1 ,2 =3.8 Hz, H-1 ), 4.42 (m, X of
ABX system, 1 H, H-10), 4.27 (d, 1 H, J1,2=7.8 Hz,
2
4.83 and 4.78 (2d, 2 H, J=11.7 Hz, CH2Ph), 4.62
2
00 00
00 00
H-1), 4.24 (ddd, 1 H, J4 ,5 =1.2 Hz, J5 ,6 a
00 00
and 4.54 (2d, 2 H, J=11.8 Hz, CH2Ph), 4.29 (d, 1
=5.0 Hz, J5 ,6 b=6.6 Hz, H-500), 3.98 (broad d, 1
H, J1,2=8.1 Hz, H-1), 4.12±4.11 (m, 2 H, H-3, H-
4), 4.05±3.97 (m, 1 H, CH2O), 3.89 (ddd, 1 H,
J4,5=1.3 Hz, J5,6a=5.2 Hz, J5,6b=6.9 Hz, H-5),
3.79 (dd, 1 H, J6a,6b=10.1 Hz, H-6a), 3.76 (dd, 1
H, H-6b), 3.6±3.52 (m, 1 H, CH2O), 3.35 (m, 1 H,
H-2), 1.33 and 1.3 (2s, 6 H, isopropylidene), 1.03
(m, 2 H, CH2Si), 0.002 (s, 9 H, SiMe3). Anal. calcd
for C28H40SiO6 (500.70): C, 67.17; H, 8.05. Found:
C, 67.07; H, 8.22.
H, J4 ,5 =1.2 Hz,00 H-40), 3.91 (dd,
1
H,
0
0
00 00
J3 ,4 =3.2 Hz, H-4 ), 3.90±3.66 (m 10 H, H-6a, H-
6b, H-50, H-60a, H-60b, H-200, H-300, H-600a, H-600b,
CH2O), 3.58±3.49 (m, 5H, H-3, H-4, H-20, H-30,
CH2O), 3.38 (dt,
J5,4=3.8 Hz, H-5), 3.23 (dd, 1 H, J2,3=9.0 Hz, H-
1
H, J5,6a=J5,6b=9.5 Hz,
3
2), 2.48 (t, 2 H, J=7.2 Hz, CH2S), 2.02 (s, 3 H,
Ac), 1.65±1.53 (m, 4 H, CH2CH2S, CH2CH2O),
1.42±1.28 (m, 24 H, 12 CH2). Electrospray
MS:783.3823 (calcd. for C34H64SO16Na 783.3812).
A slower moving fraction of disul®de 6 (2.1 mg,
19.4%) was also recovered. Due to the poor solu-
bility of 6 and 7 in water, most of the material was
retained on the Millex 0.45 ꢃm ®lter and recovered
by washing with MeOH to provide a mixture of 6
and 7 (1:3 by NMR, 6.7 mg, 62%).
2-(Trimethylsilyl)ethyl 2,6-di-O-benzyl-b-d-gal-
actopyranoside (10).ÐA solution of 9 (630 mg,
1.26 mmol) in aqueous 80% acetic acid (5 mL) was
ꢀ
stirred at 60 C for 6 h. The mixture was con-
centrated, co-evaporated with toluene, and chro-
matographed on silica gel in pentane±ethyl acetate
(2:1) to yield 10 (550 mg, 93%), m.p. 61±62 ꢀC, [ꢁ]d
+2.8ꢀ (c. 3.1; CHCl3) (ref. 21. [ꢁ]d +7.5ꢀ). H
1
2-(Trimethylsilyl)ethyl 3,4-O-isopropylidene-b-d-
galactopyranoside (8).ÐA solution of trimethylsi-
NMR: ꢂH 7.26±7.35 (m, 10 H, arom.), 4.96 (d, 1 H,
2
2J=11.5 Hz, CH2Ph), 4.67 (d, 1 H, J=11.5 Hz,
lylethyl ꢀ-d-galactopyranoside [15]
(4.86 g,
CH2Ph), 4.57 (s, 2 H, CH2Ph), 4.36 (d, 1 H,
J1,2=7.6 Hz, H-1), 4.02 (m, 1 H, CH2O), 4.96 (dd,
1 H, J3,4=3.3 Hz, J4,5=0.9 Hz, H-4), 3.78 (dd, 1 H,
J5,6a=5.7 Hz, J6a,6b=10.0 Hz, H-6a), 3.74 (dd, 1
H, J5,6b=5.7 Hz, H-6b), 3.61±3.55 (m, 3 H, H-3,
H-5, CH2O), 3.46 (dd, 1 H, J2,3 9.4 Hz, H-2), 1.02
(m, 2 H, CH2Si), 0.014 (s, 9 H, SiMe3). Anal. calcd
for C25H36SiO6 (470.63): C, 65.19; H, 7.88. Found:
C, 65.14; H, 7.88.
2-(Trimethylsilyl)ethyl 4-O-(3,4,6-tri-O-acetyl-
2-azido-2-deoxy-b-d-galactopyranosyl)-2,6-di-O-
benzyl-b-d-galactopyranoside (12).ÐA mixture of
10 (4 g, 8.5 mmol), Ag-silicate on alumina (8 g),
17.3 mmol) and 2,2-dimethoxypropane (2 mL) in
acetone (30 mL) in the presence of p-toluene-
sulfonic acid (50 mg) was stirred for 4 h. Then trie-
thylamine (1 mL) was added, the mixture was
concentrated and co-evaporated with toluene.
Chromatography of the residue on silica gel with
pentane±ethꢀyl acetate (7:3) gave 8 (3.63 g, 63%),
m.p. 93±94 C, [ꢁ]d +6.3ꢀ (c. 1.9; CHCl3) (ref. 20
m.p. 88±89.5 ꢀC, [ꢁ]d +9.6ꢀ) 1H NMR: ꢂH 4.18 (d,
1 H, J1,2=8.3 Hz, H-1), 4.14 (dd, 1 H, J3,4=5.5 Hz,
J4,5=2.1 Hz, H-4), 4.08 (dd, 1 H, J2,3=7.4 Hz, H-
3), 4.01±3.95 (m, 2 H, H-6a, CH2O), 3.86±3.83 (m,
2 H, H-5, H-6b), 3.54±3.49 (m, 2 H, H-2, CH2O),
1.50, 1.05 (2s, 6 H, isopropylidene), 0.99 (m, 2 H,
4A molecular sieves (10 g) in dry CH2Cl2
(50 mL) was stirred for 1 h, then cooled at

P. I. Kitov et al./Carbohydrate Research 307 (1998) 361±369
367
15 ꢀC, and a solution of 3,4,6-tri-O-acetyl-2-
azido-2-deoxy-ꢁ-d-galactopyranosyl bromide 11
[22,23] (3.5 g, 8.88 mmol) in CH2Cl2 (20 mL) was
added dropwiseꢀover 2 h. The mixture was stirꢀred
for 5 h at 15 C and for 16 h at 15 to 0 C.
After ®ltration through Celite followed by con-
centration, the residue was chromatographed
twice on silica gel with toluene±ethyl acetate (4:1)
as the solvent to yield 12 (4.91 g, 74%) as a
6.5 Hz, H-50), 3.70 (broad t, 1 H, J5 ,6 ab=6.1 Hz,
H-5), 3.53 (ddd, 1 H, 3J=7.0 Hz, 3J=9.8 Hz,
CH2O), 3.32 (ddd, 1 H, H-20), 2.10, 2.08, 2.016,
2.012, 2.008, 1.96Â2 (6s, 21 H, 7 Ac), 1.01±0.83 (m,
2 H, SiCH2), 0.02 (s, 9 H, SiMe3). Anal. calcd for
C31H49NSiO17 (735.80): C, 50.60; H, 6.71; N, 1.90.
Found: C, 50.88; H, 6.79; N, 1.96.
0
0
4-O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-
d-galactopyranosyl)-2,3,6-tri-O-acetyl-a-d-galacto-
pyranose trichloroacetimidate (14).ÐA solution of
13 (1.16 g, 1.57 mmol) in CH2Cl2 (10 mL) and
CF3COOH (5 mL) was stirred for 15 min. Then
EtOAc (5 mL) was added, the mixture was con-
centrated, co-evaporated with toluene (2Â5 mL),
and dried for 2 h under vacuum. To a solution of
the residue in CH2Cl2 (6 mL) trichloroacetonitrile
(1.15 mL) was added, the mixture was cooled to
syrup, [ꢁ]d 18.0ꢀ (c. 1.46; CHCl3). H NMR: ꢂH
1
7.37±7.26 (m, 10 H, arom.), 5.27 (dd, 1 H,
0
0
0
0
0
J3 ,4 =3.3 Hz, J4 ,5 =0.3 Hz, H-4 ), 4.97 (d, 1 H,
2J=11.5 Hz,
CH2Ph),
4.79
(dd,
1
H,
J2 ,3 =10.7 Hz, H-30), 4.68 (d, 2J=11.5 Hz,
0
0
0
0
0
CH2Ph), 4.39 (d, 1 H, J1 ,2 =8.1 Hz, H-1 ), 4.55
(s, 2 H, CH2Ph), 4.38 (d, 1 H, J1,2=7.3 Hz, H-1),
4.08±14.02 (m, 3 H, H-4, H-60a, CH2O), 3.94 (dd,
1
H, J5 ,6 b=6.2 Hz, J6 a,6 b=11.1 Hz, H-60b),
0 C, and DBU (0.3 mL) was added. After 1 h of
ꢀ
0
0
0
0
3.68±3.57 (m, 8 H, H-2, H-20, H-3, H-5, H-50, H-
6a, H-6b, CH2O), 2.1, 2.03, 1.96 (3s, 9 H, 3 Ac),
1.043 (m, 2 H, CH2Si), 0.007 (s, 9 H, SiMe3).
Anal. calcd for C37H51N3SiO13 (773.90): C, 57.42;
H, 6.64; N, 5.43. Found: C, 57.39; H, 6.68; N,
5.35.
stirring at room temperature, MeOH (0.5 mL) was
added, and the mixture was concentrated. Chro-
matography of the residue on silica gel with pen-
tane±acetone (1:1) yielded 14 (1.095 g, 89%) as a
foam, [ꢁ]d +51.1 (c. 0.9; CHCl3). H NMR: ꢂH
8.62 (s, 1 H, C=NH), 6.48 (d, 1 H, J1,2=3.7 Hz,
ꢀ
1
0
0
0
0
2-(Trimethylsilyl)ethyl 4-O-(2-acetamido-3,4,6-
tri-O-acetyl-2-deoxy-b-d-galactopyranosyl)-2,3,6-
tri-O-acetyl-b-d-galactopyranoside (13).ÐA mix-
ture of 12 (1.43 g, 1.85 mmol) and Pd(OH)2/C
(250 mg) in EtOH (10 mL) containing water
(0.5 mL) was stirred for 3 h under hydrogen. After
®ltration followed by concentration, the residue
was dissolved in MeOH (10 mL). Acetic anhydride
(2 mL) was added and after 16 h the mixture was
concentrated. A solution of the residue in acetic
acid (10 mL) in the presence of 10% Pd/C (1 g)
H-1), 5.82 (dd, 1 H, J2 ,3 =11.3 Hz, J3 ,4 =3.5 Hz,
0
H-30), 5.63 (d, 1 H, JNH,2 =7.2 Hz, NH), 5.53 (dd,
1 H, J2,3=10.8 Hz, H-2), 5.36 (d, 1 H, H-40), 5.33
(dd, 1 H, J3,4 2.6 Hz, H-3), 5.07 (d, 1 H, J1 ,2
8.2 Hz, H-10), 4.32±4.26 (m, 3 H, H-4, H-5, H-6a),
0
0
=
4.16 (dd, 1 H, J5,6b=8.3 Hz, J6a,6b 12.8 Hz, H-6b),
4.04 (d, 2 H, J5 ,6 a'J5 ,6 b=6.8 Hz, H-60a, H-60b),
3.90 (broad t, H-50), 3.40 (ddd, 1 H, H-20), 2.14,
2.12, 2.02, 2.00, 1.99, 1.98, 1.94 (7s, 21 H, 7 Ac).
Anal. calcd. for C28H37Cl3N2O17 (779.95): C, 43.12;
H, 4.78; N, 3.59. Found: C, 43.40; H, 4.78; N, 3.53.
16-(Thiocyano)hexadecanyl 4-O-(2-acetamido-
3,4,6-tri-O-acetyl-2-deoxy-b-d-galactopyranosyl)-
0
0
0
0
ꢀ
under 17.2 MPa of hydrogen was shaken at 40 C
for 24 h, then ®ltered, concentrated, and acetylaꢀted
with Ac2O (4 mL) in pyridine (10 mL) at 45 C.
After 4 h MeOH (1 mL) was added, and the mix-
ture was concentrated. Chromatography of the
residue on silica gel with pentane±acetone (2:1)
gave 13 (1.18 g, 87%) as a foam, [ꢁ]d 20.2ꢀ (c.
2,3,6-O-acetyl-b-d-galactopyranoside
(16).ÐA
mixture of the imidate 15 (100 mg, 0.128 mmol),
monotosylated diol 1 (68 mg, 0.165 mmol), and 4A
molecular sieves (100 mg) in CH2Cl2 (5 mL) was
stirred for 1 h. Then TMSOTf (5 ꢃL, 64 ꢃmol) was
added. After 2 h triethylamine (1 mL) was added,
and the solid was removed by ®ltration. The ®ltrate
was concentrated and dried under vacuum. A
solution of the residue and KSCN (70 mg,
0.72 mmol) in DMF (3 mL) was stirred at 80 ꢀC for
2 h. The mixture was concentrated, dissolved in
CH2Cl2 (30 mL), and the solution was washed with
water and concentrated. Chromatography of the
residue on silica gel with pentane±acetone (2:1)
gave 16 (82 mg, 70%), [ꢁ]d 15.3ꢀ (c. 1.8; CHCl3).
1.1; CHCl3). 1H NMR: ꢂH 5.92 (dd, 1 H,
0
0
0
0
0
J2 ,3 =11.4 Hz, J3 ,4 =3.4 Hz, H-3 ), 5.69 (d, 1 H,
0
0
JNH,2 =6.9 Hz, NH), 5.36 (d, 1 H, H-4 ), 5.22 (dd,
1 H, J1,2=7.9 Hz, 0J2,3=10.4 Hz, H-2), 5.12 (d, 1 H,
0
J1 ,2 =8.2 Hz, H-1 ), 4.91 (dd, 1 H, J3,4=2.7 Hz, H-
0
3), 4.43 (d, 1 H, H-1), 4.27 (d, 2 H, J5,6a'J5,6b=
6.1 Hz, H-6a, H-6b), 4.10 (d, 1 H, J4,5=2.2 Hz, H-
4), 4.02 (d, 2H, J5 ,6 a'J5 ,6 b=6.5 Hz, H-60a, H-
0
0
0
0
60b), 3.98 (ddd, 1 H, 2J=14.8 Hz, 3J=9.8 Hz,
3J=5.1 Hz, CH2O), 3.88 (broad t, 1 H, J5 ,6 ab
0
0
=

368
P. I. Kitov et al./Carbohydrate Research 307 (1998) 361±369
1H NMR: ꢂH 50.91 (dd, 1 H, J2 ,3 =11.4 Hz,
chromatographed on a C-18 reverse phase HPLC
column using a step gradient of H2O±MeOH
(50:50±0:100) to yield the faster moving 18 (3.7 mg,
0
0
0
0
0
J3 ,4 =3.4 Hz, H-3 ), 5.68 (d, 1 H, JNH,2 =6.9 Hz,
NHAc), 5.36 (d, 1 H, H-40), 5.22 (dd, 1 H,
J1,2=7.9 Hz, J2,3=10.5 Hz, H-2), 5.11 (d, 1 H,
32%), [ꢁ]d 8.0ꢀ (c. 0.3; H2O). 1H NMR: ꢂH
0
0
0
0
0
0
J1 ,2 =8.2 Hz, H-1 ), 4.91 (dd, 1 H, J3,4=2.8 Hz, H-
3), 4.39 (d, 1 H, H-1), 4.28 (dd, 1 H, J5,6a=5.6 Hz,
J6a,6b=11.7 Hz, H-6a), 4.23 (dd, 1 H, J5,6b=
(CD3OD): 4.63 (d, 1 H, J1 ,2 =8.5 Hz, H-1 ), 4.18
(d, 1 H, J1,2=7.7 Hz, H-1), 4.01 (dd, 1 H,
J3,4=3.1 Hz, J4,5=<1 Hz, H-4), 3.91±3.78 (m, 4
6.4 Hz, H-6b), 4.1 (d, 1 H, H-4), 4.02 (d, 2 H,
J5 ,6ab=6.6 Hz, H-60a, H-60b), 3.90±3.82 (m, 2 H,
H, H-6a, H-2, H-60a, CH2O), 3.74 (broad d, 1 H,
J3 ,4 =3.2 Hz, H-4 ), 3.70 (dd, 1 H, J5 ,6 b=4.2 Hz,
0
0
0
0
0
0
H-50, CH2O), 3.70 (t, 1 H, H-5), 3.43 (dt, 1 H,
J6 a,6 b=11.2 Hz, H-60b), 3.63±3.55 (m, 3 H, H-3,
H-30, H-6b), 3.53±3.48 (m, 3 H, H-5, H-50, CH2O),
3.43 (dd, 1 H, J2,3=9.7 Hz, H-2), 2.48 (t, 2 H,
3J=7.2 Hz, CH2S), 2.02 (s, 3 H, Ac), 1.65±1.53 (m,
4 H, CH2CH2S, CH2CH2O), 1.42±1.28 (m, 24 H,
12 CH2). Electrospray MS: 662.3553 (calcd for
C30H57NSO11Na 662.3550). A slower moving
component, the disul®de 17 (7.9 mg, 68%) was
recovered.
0
0
3J=7.0 Hz, 2J=9.7 Hz, CH2O), 3.31 (ddd, 1 H, H-
0
3
2 ), 2.92 (t, 2 H, J=7.3 Hz, CH2SCN), 2.11, 2.08,
2.04, 2.01, 2.008, 1.97, 1.96 (7s, 21 H, 7 Ac), 1.77
(p, 2 H, 3J=7.2 Hz, 2CH2CH2SCN), 1.56±1.20 (m,
26 H, 13 CH2). Anal. calcd for C43H68SN2O17
(917.07): C, 56.32; H, 7.47; N, 3.05; S, 3.50. Found:
C, 56.40; H, 7.59; N, 2.84; S, 3.51.
Bis{16-[4-O-(2-acetamido-2-deoxy-b-d-galacto-
pyranosyl)-b-d-galactopyranosyloxy]hexadecanyl}
disul®de (17).ÐA solution of 16 (373.9 mg,
0.374 mmol) and sodium borohydride (280 mg,
3.74 mmol) in dry MeOH (3 mL) was stirred under
an argon atmosphere for 20 h, then the mixture
was neutralized with AcOH, the solution was con-
centrated, taken up in water, and applied onto a
Sep-Pak (C-18) cartridge (Waters). The cartridge
was washed with 20, 40, 60, and 80% solution of
MeOH in water, then with pure MeOH. The
methanol fraction containing sugar was con-
centrated to give 17 (189.4 mg, 79.2%), [ꢁ]d 8.3ꢀ
Acknowledgements
This work was supported by the Protein Engi-
neering (PENCE) and Canadian Bacterial Diseases
(CBDN) Network Centers of Excellence. The
authors thank Dr Elena Kitova for assistance in
the analysis and puri®cation of thiol and disul®de
products.
ꢀ
1
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J4,5=0.8 Hz, H-4), 3.88 (dd, 1 H, J5,6a=7.6 Hz,
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=
J6a,6b=11.1 Hz, H-6a), 3.85 (dd, 1 H, J2 ,3
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