Synthesis of three tethered trisaccharides to probe entropy contributions in carbohydrate-protein interactions

Article abstract of DOI:10.1560/YXQT-2JJU-YLAA-L04Q

Crystal structure data show that the branched trisaccharide 1 constitutes the complete antigenic determinant of the Salmonella serogroup B antigen that is recognized by monoclonal antibody SE155.4. In an effort to characterize the entropic costs associated with immobilization of glycosidic torsional angles in the bound state, three distinct intramolecularly tethered analogues of this trisaccharide 2-4 have been synthesized. Two trisaccharides are tethered by a methylene acetal via the O-2 position of the 3,6-dideoxy-hexose, abequose to either O-2 of galactose (2) or O-4 of mannose (3). The third tether, α,α'-di-thio-p-xylene, spans the C-6 atoms of the mannose and galactose residues to create trisaccharide 4. The acetal tethers of 2 and 3 span hydroxyl centers that are known to be involved in intramolecular sugar-sugar hydrogens bonds, but both trisaccharides are biologically inactive due to distorted conformations that cannot be accommodated in the antibody binding site. Trisaccharide 4 is active since both hydroxymethyl groups of galactose and mannose are solvent exposed in the bound state and the constrained conformation of 4 is virtually superimposable on the bound conformation of 1. Despite the retained complimentary and significant reduction of torsional flexibility, trisaccharide 4 exhibits a ΔG° = -7.6 kcal mol^-1 compared to ΔG° = -7.1 kcal mol^-1 for 1. The modest free energy gain for the tethered trisaccharide 4 arises from a small entropy gain (TΔΔS = 0.3 kcal mol^-1) and an even smaller enthalpic change (ΔΔH = -0.2 kcal mol^-1).

Full text of DOI:10.1560/YXQT-2JJU-YLAA-L04Q

Synthesis of Three Tethered Trisaccharides to Probe Entropy Contributions in
Carbohydrate–Protein Interactions
PING ZHANG AND DAVID R. BUNDLE*
Department of Chemistry, The University of Alberta, Edmonton, Alberta, T6G 2G2, Canada
(Received 4 September 2000 and in revised form 13 November 2000)
Abstract. Crystal structure data show that the branched trisaccharide 1 consti-
tutes the complete antigenic determinant of the Salmonella serogroup B antigen that
is recognized by monoclonal antibody SE155.4. In an effort to characterize the
entropic costs associated with immobilization of glycosidic torsional angles in the
bound state, three distinct intramolecularly tethered analogues of this trisaccharide
2–4 have been synthesized. Two trisaccharides are tethered by a methylene acetal
via the O-2 position of the 3,6-dideoxy-hexose, abequose to either O-2 of galactose
(2) or O-4 of mannose (3). The third tether, α,α′-di-thio-p-xylene, spans the C-6
atoms of the mannose and galactose residues to create trisaccharide 4. The acetal
tethers of 2 and 3 span hydroxyl centers that are known to be involved in intramo-
lecular sugar–sugar hydrogens bonds, but both trisaccharides are biologically inac-
tive due to distorted conformations that cannot be accommodated in the antibody
binding site. Trisaccharide 4 is active since both hydroxymethyl groups of galactose
and mannose are solvent exposed in the bound state and the constrained conforma-
tion of 4 is virtually superimposable on the bound conformation of 1. Despite the
retained complimentary and significant reduction of torsional flexibility, trisaccha-
ride 4 exhibits a ∆G° = –7.6 kcal mol–1 compared to ∆G° = –7.1 kcal mol–1 for 1.
The modest free energy gain for the tethered trisaccharide 4 arises from a small
entropy gain (T∆∆S = 0.3 kcal mol–1) and an even smaller enthalpic change (∆∆H =
–0.2 kcal mol–1).
INTRODUCTION
is dominated by aromatic amino acid residues and bur-
The crystal structure of the monoclonal antibody ies the 3,6-dideoxy-D-xylo-hexose (abequose, Abe) of
SE155.4 has been solved as both its Fab and single chain the trisaccharide epitope 1. The mannose and galactose
forms with various ligands at high resolution.1–4 These residues are partially solvent exposed, although both are
data provide the atomic detail that is needed to address postulated to make hydrogen bonds to the antibody
several issues related to the energetics of protein–sugar (Fig. 1).1–3 To date, in all crystal structures,1–3 the Abe–
interactions. The principle objective of the syntheses Man glycosidic linkage adopts a conformation close to
reported here was an investigation of binding energy that predicted by potential energy calculations.2 De-
changes that result when trisaccharide 1 is pre-orga- pending on the complex, the Gal–Man glycosidic link-
nized for binding by restriction of flexibility about the age exists in one of two conformations, I and II (Fig. 2),
glycosidic linkages, i.e., the entropic loss when a protein which are related by a shift in the Φ/Ψ glycosidic tor-
binding site selects a single bound conformation. The sional angles.1,3,4 In the complex a hydrogen bond be-
resulting freezing or restriction of inter-saccharide bond tween Abe O-2 and Gal O-2 is either direct (conforma-
rotamers that accompanies binding of unconstrained oli- tion I)1 or mediated by a water molecule (conformation
gosaccharides has been estimated to carry a conforma- II),3 while the hydrogen bonding pattern to the galactose
tional entropy penalty as large as 1–2 kcal mol–1 per residue is also modified.4
rotor.5 Other estimates for freezing single bond rotamers
are more conservative at ~0.6 kcal mol–1 per torsion.6,7
The binding site of the monoclonal antibody SE155.4
In order to facilitate a comparison of the energetics of
*Authortowhomcorrespondenceshouldbeaddressed. E-mail:
dave.bundle@ualberta.ca
Israel Journal of Chemistry
Vol. 40
2000 pp. 189–208

190
binding of tethered and native trisaccharides using titra- as base, the reaction proceeded smoothly at 0 °C. After
tion microcalorimetry,8 tethered trisaccharides 2–4 were chromatography on silica gel, the 2-monobenzylated
synthesized. In the bound state or in solution, trisaccha- product 8 was isolated in 29% yield, along with the 2,4-
ride 1 has been shown to have hydrogen bonds between dibenzylated compound 6 (17%) and 4-monobenzylated
Abe O-2 and Gal O-2, and between Abe O-2 and Man product 7 (6%) together with unreacted starting material
O-4.2 Trisaccharides 2 and 3 are each tethered by a 5 (25%).
methylene acetal in an attempt to simulate these confor-
To obtain the target abequose donor 12 in which the
mations. Trisaccharide 4, by contrast, is tethered be- OH-2 was selectively protected by a p-methoxybenzyl
tween Gal C-6 and Man C-6, two relatively close and group, we applied reaction conditions similar to those
solvent-exposed atoms that are well removed from the above with p-methoxybenzyl chloride. Similar results
protein surface, and hence reduce the possibility of pro- were observed, and the 2-O-p-methoxybenzylated prod-
tein–tether interactions. The activities of these and other uct 11 was isolated in 33% yield, along with the 2,4-di-
tethered derivatives9 have been assayed by solid phase O-p-methoxybenzylated derivative 9 (26%) and 4-
binding and titration microcalorimetry.8
mono-p-methoxybenzylated 10 (14%). However, in this
reaction no unreacted starting material 5 was recovered.
Our previous work10 indicated that both α- and β-
isomers of ethyl 3,6-dideoxy-1-thio-D-xylo-hexopyr-
anoside react equally well during glycosylation. Here
we used the α-isomer 17 as the glycosyl donor. Alkyla-
tion of 13 under conditions similar to those above gave
the 2-O-p-methoxybenzylated product 16, isolated in
27% yield, along with the 2,4-di-O-p-methoxy-
benzylated derivative 14 (23%) and 4-O-p-methoxy-
benzylated compound 15 (11%). The OH-4 hydroxyl
groups in both the anomeric isomers 11 and 16 were
subsequently benzylated, and the desired donors 12
(90%) and 17 (94%) were obtained in excellent yields.
The selective protection of the of ethyl 1-thiogalacto-
pyranoside with a 2-O-p-methoxybenzyl group started
with the acetal 18.12 Using aqueous fluoroboric acid in
methanol at 0 °C,12 the acetal groups were selectively
removed in the presence of the acid-sensitive p-meth-
oxybenzyl group. The triol 19 was isolated in 91% yield
and perbenzylation afforded the desired donor 20 in
85% yield.
SYNTHESIS AND DISCUSSION
The design of the required synthons for the preparation
of the tethered trisaccharides relied on thioglycosides as
glycosylation donors since our previous experience sug-
gested that this approach would give straightforward
results.10 In addition, the preparation of the desired teth-
ered trisaccharides 2 and 3 required the synthesis of a
common derivative of abequose 12 or 17 in which the
OH-2 was selectively protected by a p-methoxybenzyl
group, which could be selectively removed in order to
introduce the methylene acetal tether. The synthesis of 2
and 3 required two other intermediates, 20 and 32, in
which the OH-2 of the galactose unit and OH-4 of the
mannose residue were also selectively protected with
the p-methoxybenzyl group. In the case of the tethered
trisaccharide 4, both the OH-6 groups of the mannose
and galactose residues needed to be selectively pro-
tected and activated.
SYNTHESIS OF THE TETHERED
TRISACCHARIDE 2
Glycosylation of the known mannopyranosyl accep-
tor 2113 by the abequose donor 12 proceeded smoothly
The presence of axial and equatorial hydroxyl groups in in anhydrous dichloromethane using N-iodosuccin-
the 3,6-dideoxyhexosides 5 and 1310 suggests that there imide, and silver trifluoromethanesulfonate to give the
should be a significant difference in reactivity between desired disaccharide 22 in 64% yield. A similar yield
these O-2 and O-4 positions. The axial hydroxyl group was obtained with the corresponding α-isomer donor
OH-2 of a 4,6-protected mannopyranoside is reported to 17. The 2-O-benzoate was then removed by transester-
undergo benzoylation or benzylation reactions more ification (94%). Alcohol 23 was glycosylated with ethyl
readily than the equatorial O-3, so that the O-2 benzoy- 1-thiogalactopyranoside 20 using methyl triflate as pro-
lated or benzylated derivatives were obtained as major moter in anhydrous dichloromethane, and the desired
products.11
trisaccharide 24 was obtained in 94% yield. Subse-
Selective benzylation of the ethyl 1-thioglycoside of quently, both the p-methoxybenzyl protecting groups
abequose was primarily carried out with the β-isomer 5. were successfully removed by cerium ammonium nitrate
Under mild conditions, using barium oxide-barium hy- in acetonitrile–water. The reaction proceeded rapidly at
droxide octahydrate as base and benzyl bromide (1.1 room temperature; and after chromatography on silica
equivalent) as reagent in an aprotic solvent, the reaction gel, the diol 25 was obtained in 73% yield.
proceeded very slowly at 0 → 50 °C, and this approach
Formation of the formaldehyde acetal and closure of
was not pursued further. However, with sodium hydride the macrocyclic ring were completed in a one-pot reac-
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Fig. 1. Trisaccharide 1 complexed with antibody Se155.4. The abequose residue is buried in the binding site and the surfaces of
both mannose and galactose make contact with the protein, while parts of mannose and most of the galactose residues are
solvent-exposed. The interatomic distances for the two intramolecular sugar–sugar hydrogen bonds between Abe O-2 and Gal
O-2 and Man O-4 are shown. Also shown is the interatomic distance between the 06 atoms of the Gal and Man hydroxymethyl
groups.
tion.14 In the presence of excess of sodium hydride, the impurity (~30%) together with the major compound,
diol 25 was first converted to the disodium salt and chromatography on silica gel using different eluents
followed by addition of dibromomethane. Presumably could not resolve the mixture. The pattern of the methyl-
the intermediate bromomethyl derivative may be at- ene acetal signals could not be identified since they were
tacked by either another oxygen anion in the same mol- overlapped by the signals of benzyl protecting groups.
ecule or by one from another molecule. Intramolecular However, comparison with the NMR spectrum of 25
attack would lead to the desired tethered product 26, suggested the formation of the desired product. The
while the intermolecular attack would lead to undesired chemical shifts for the H-1 of the galactose unit shifted
oligomeric byproducts. As the two hydroxyl groups in significantly downfield from 5.10 to 5.91 ppm, while
the same molecule are close to each other in low energy the H-1s of abequose and mannose residues remained
conformations, the desired tether should be the major roughly the same. The H-3e resonance of the abequose
component in the reaction mixture. The reaction was unit shifted upfield from 2.16 to 1.93 ppm, while the H-3a
carried out by treating the diol 25 with 10 equiv of resonance shifted downfield from 1.60 to 2.03 ppm,
sodium hydride in anhydrous N,N-dimethylformamide respectively. The H-6 of the abequose unit shifted
and using only 1.3 equiv of dibromomethane. After 24 upfield from 1.07 ppm to 0.86 ppm. These are strong
h, TLC indicated the presence of one major spot. Al- indications that the methylene group was successfully
though NMR showed the presence of an unidentified incorporated at the two hydroxyl positions. The pres-
Zhang and Bundle / Synthesis of Tethered Trisaccharides to Probe Carbohydrate–Protein Interactions

192
Fig. 2. Hydrogen bonding maps for the solved crystal structures of trisaccharide 1 complexed with Fab2 and with single chain
Fv3. Conformation I is observed when 1 is complexed with Fab and has a directed Abe O-2 to Gal O-2 hydrogen bond. Bound
conformation II is observed in the single chain Fv crystal structure and shows the same hydrogen bond, but in this case mediated
via a bound water molecule.
Scheme 1
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193
Scheme 2
ence of such a short methylene group in the center of the spond to the two protons of the methylene tether. This
molecule alters significantly the conformation of the was confirmed by a two-dimensional experiment
glycosidic linkages, leading to substantial chemical (T-ROESY) that measured the through-space interac-
shift changes. The success in introducing the methylene tion and which clearly showed that one of these two
group was finally proved after removing all the protect- protons interacts with H-2 of the galactose unit, while
ing groups. Hydrogenation in methanol solution over the other interacts with H-2 of the abequose unit.
5% palladium on charcoal gave the target tethered
trisaccharide after purification by chromatography on
silica gel, followed by reverse phase chromatography on
SYNTHESIS OF THE TETHERED
TRISACCHARIDE 3
a C18 column. The overall yield for the two steps was The synthesis of tethered trisaccharide 3 started with the
15%. In addition to the three anomeric protons, the preparation of the mannose acceptor 32 selectively pro-
NMR spectrum showed clearly in the region 4.5 ppm to tected at the O-4 position. Using a known 2,3-O-
5.5 ppm, two sets of doublets resonating at 5.12 ppm isopropylidene mannopyranoside 2715 as starting mate-
(J = 8.4 Hz) and 4.93 ppm (J = 8.4 Hz), which corre- rial, the O-6 position was selectively benzylated using
Zhang and Bundle / Synthesis of Tethered Trisaccharides to Probe Carbohydrate–Protein Interactions

194
Scheme 3
di-n-butyltin oxide as activator and benzyl bromide as to afford 30 in 91% yield. In this case, the removal of the
reagent.16,17 The 6-O-monobenzylated product 29 was iso- isopropylidene acetal in the presence of an acid-sensi-
lated in 54% yield. Interestingly, no 4-O-monobenzylated tive p-methoxybenzyl group proceeded more slowly
product was observed. Instead, a 3,6-di-O-benzylated de- than for the galactose derivative 19. After 48 h reaction
rivative 2818 was obtained as the major side product in with aqueous fluoroboric acid in methanol, the 2,3-diol
17% yield. A possible mechanism to account for this 31 was isolated in 81% yield. The diol was selectively
observation involves reaction of a second molecule of benzoylated at O-2 through a 2-step reaction sequence
dibutyltin oxide with the initially formed 4,6-stannylene using trimethyl orthobenzoate to form first the 2,3-cy-
acetal via coordination to O-3 of compound 27, followed clic orthoester, followed by regioselective opening in
by hydrolysis of the activated acetonide to a transiently 80% aqueous acetic acid to give the desired acceptor 32
formed 2,3:4,6-di-O-stannylene acetal (Scheme 5). Selec- in 88% yield.
tive benzylation at the 3 and 6 positions gives 28.
Glycosylation of the acceptor 32 by donors 12 or 17,
The alcohol 29 was p-methoxybenzylated as above using methyl triflate as promoter, gave the disaccharide
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Scheme 4
34 in 83% yield after chromatography. The benzoate of galactose unit and abequose unit experience downfield
disaccharide 34 was first removed (→ 35, 93%), and the shifts from δH 5.17 and 5.05 ppm to 5.55 and 5.44 ppm.
resulting disaccharide acceptor 35 was glycosylated The final tethered trisaccharide 3 was obtained in 90%
with the thiogalactoside 33, using methyl triflate as yield after the removal of the benzyl groups.
activator. The target trisaccharide 36 was obtained in
The NMR spectrum of the final trisaccharide 3
83% yield. Both p-methoxybenzyl groups were success- showed beside the three anomeric protons, two sets of
fully removed using the cerium ammonium nitrate doublets resonating at 4.93 ppm (J = 6.6 Hz) and
method (→ 37, 79%). By analogy with 25, after reacting 4.85 ppm (J = 6.6 Hz), which correspond to the two
the disodium salt of 37 with dibromomethane, the teth- protons of the methylene tether. Two dimensional
ered trisaccharide 38 was isolated in pure form in 27% T-ROESY demonstrated that one of the tether protons
yield. The NMR spectrum of the tethered trisaccharide correlates with the H-4, H-6a, and H-6b protons of the
38 compared to the precursor 37, exhibited marked mannose unit, while the other tether proton correlates
changes in chemical shifts. The anomeric protons of the only with H-2 of the abequose unit.
Zhang and Bundle / Synthesis of Tethered Trisaccharides to Probe Carbohydrate–Protein Interactions

196
A
B
Scheme 5
SYNTHESIS OF THE TETHERED
TRISACCHARIDE 4
lective. Two major products were present in the reaction
mixture, and this strategy was abandoned.
The synthesis of the tethered trisaccharide 4 required acti-
The second strategy involved the use of two ben-
vation at the O-6-positions of both the galactose and man- zylidene groups to protect the 4- and 6-positions of both
nose units. As there are only two primary positions in this the mannose and galactose units. The remaining hydroxyl
trisaccharide structure, we chose to activate both O-6 groups could be protected using acetate esters. NBS open-
positions in a single-pot reaction at the trisaccharide stage. ing of both the 4,6-benzylidene acetal19,20 would lead si-
Using trisaccharide 113 as starting material, two strat- multaneously to an intermediate activated in both the
egies were considered. The first concerned the use of a mannose and galactose units. Thus, reaction with α,α-
bulky trityl protecting group to selectively protect the dimethoxytoluene gave the diacetal 42 in 54% yield, and
primary positions, followed by protection of the remain- the remaining hydroxyl groups were then acetylated (→
ing hydroxyl groups and selective removal of the trityl 43, 87%). Selective opening of the acetals was achieved
groups. The primary hydroxyl groups could then be by reacting 43 with N-bromosuccinimide (3 equiv) in
selectively activated by sulfonates. However, our inves- anhydrous carbon tetrachloride, and the dibromide 44
tigation showed that tritylation was not completely se- was obtained in 63% yield after chromatography.
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Table 1
500 MHz 1H NMR of the tethered trisaccharidesa 2, 3, and 4
H1
H2
H3
H4
H5
H6
H6′
(J12)
(J12, J23)
(J23, J34)
(J34, J45)
(J56)
(J56′, J66′)
2 Abe 5.90
3.94
2.12/1.95
3.90
4.19
1.13
(3.5) (13.1,3.0)
(4.0)
4.18
(10.5)
3.99
(1.4)
4.24
(9.2)
3.98
(1.0)
(6.6)
3.94
(2.2)
3.76
(6.8)
Man 5.08
3.98
(3.5)
3.76
(9.1)
3.66
4.05
3.83
(6.0)
3.73
(4.9)
(1.4)
Gal 5.23
(4.0)
(3.4)
Tether: 5.12 (8.4), 4.93 (8.4); OMe: 3.40
3 Abe 5.52
3.96
2.12/1.97
(3.0,3.7)
3.98
3.88
(0.9)
4.06
(9.7)
3.98
(1.1)
4.06
3.73
4.01
1.15
(6.6)
3.86
(2.0)
3.77
(7.4)
(4.0) (12.1,5.6)
Man 4.96
(1.8)
Gal 5.28
(4.0)
4.24
(3.0)
3.77
3.77
(6.0)
3.76
(4.8)
(9.7)
3.92
(3.3)
(10.3)
Tether: 4.93 (6.6), 4.85 (6.6); OMe: 3.41
4 Abe 5.30
(3.8)
3.96
1.95/1.93
3.82
3.98
3.36
3.46
1.13
(6.6)
3.23
Man 4.14
(1.7)
Gal 4.84
3.64
(3.5)
3.68
3.71
(3.1)
3.65
3.58
(9.8)
4.20
2.72
(2.1,15.2) (7.5,15.2)
2.95 2.83
(6.7,15.0) (7.6,15.0)
(3.8) (3.9,10.6) (3.7,10.6)
(1.1,3.3)
Tether: 7.49, 7.36, 7.24, 7.22, 3.87 (14.8), 3.81 (14.8), 3.79 (14.5), 3.76 (14.5); OMe: 3.39
a The chemical shifts for trisaccharide 1 in D2O are reported in ref 2.
Table 2
500 MHz 13C NMR of the tethered trisaccharidesa 2, 3, and 4
C1
C2
C3
C4
C5
C6
JC1,H1
Abe
Man
Gal
95.0
100.6
102.6
75.8
83.3
79.8
32.4
77.6
68.5
69.6
64.3
70.4
67.4
73.4
72.2
16.5
61.9
62.6
174.5
173.8
171.6
2
3
4
Tether:100.4; OMe: 55.8
Abe
Man
Gal
100.1
100.8
101.5
71.5
77.7
69.9
32.7
80.0
70.5
68.8
73.0
70.4
68.5
72.2
72.7
16.8
61.0
62.5
169.4
173.3
173.3
Tether: 94.7; OMe: 55.9
Abe
Man
Gal
101.0
99.9
103.4
64.2
81.8
69.2
33.5
78.2
69.9
69.3
70.2
70.6
67.6
72.6
72.2
15.9
34.0
34.8
171.8
173.1
171.6
Tether: 39.4, 39.0; OMe: 55.8
a The chemical shifts for trisaccharide 1 in D2O are reported in ref 2.
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198
Fig. 3. A comparison of the two bound conformations
of trisaccharide 1, conformations I and II with the
conformations adopted by the acetal-tethered trisac-
charides 2 and 3. In each case, the coordinates of the
abequose residue were superimposed to display the
relative orientations of the mannose and galactose
residues in the tethered trisaccharides. (A) Conforma-
tion II of trisaccharide 1 (blue), superimposed on
trisaccharide 2 (white). The bound water that mediates
the hydrogen bond present in the bound conformation
II is shown. (B) Conformation I of trisaccharide 1
(red), superimposed on trisaccharide 2 (white). (C)
Conformation II of trisaccharide 1 (blue), superim-
posed on trisaccharide 3 (white). The bound water that
mediates the hydrogen bond in the bound conforma-
tion II is shown. (D) Conformation I of trisaccharide 1
(red), superimposed on trisaccharide 3 (white).
Fig. 4. The tethered trisaccharide 4 (yellow) superim-
posed on A, bound conformation II (blue) and B,
bound conformation I (red) of trisaccharide 1. The
coordinates of the abequose residue were superim-
posed to display the relative orientations of the man-
nose and galactose residues in the tethered and non-
tethered trisaccharides.
Fig. 5. The proximity of the Man H-1 proton to the
center of the aromatic ring of the tether in trisaccha-
ride 4 is illustrated and this shielding by the ring
current accounts for the substantial upfield shift in the
resonance of this proton.
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The α,α′-di-thio-p-xylylene tether 41 was prepared permit free rotation about either the S–CH2 or CH2–
by reacting the commercial α,α′-dibromo-p-xylene 39 C6H4 bonds of the tether, the lines of the latter are
with thiourea, followed by hydrolysis of the intermediate broadened, while the motions of protons in other parts
thiourea salts 40. The dimercaptane 41 was converted to of the molecule are not constrained and show little if any
the dipotassium salt before use. The introduction of the linebroadening. The four benzyl protons of the tether
α,α′-di-thio-p-xylylene tether and closure of the macro- resonate at 3.87 ppm, 3.81 ppm, 3.79 ppm, and 3.76
cyclic ring was carried out between the dipotassium salt ppm, all of which appear as sharp doublets with J around
of 41 and the dibromide 44. The reaction proceeded in 14.5 Hz (Table 1).
very low yield, presumably due to the large average
Introduction of acetals between Abe O-2 and Gal
distance between the two reaction sites. Consequently, O-2 or between Abe O-2 and Man O-4 to produce
the intermolecular reaction competes with the intramo- trisaccharides 2 or 3 is intended to simulate constraints
lecular ring closure. Nevertheless, this reaction scheme that result from intramolecular hydrogen bonds in the
13
represents a short, direct route to the tethered product 45 bound state or DMSO solution.2 The C NMR spectra
(17%). The most significant NMR evidence for the of 2 or 3 (Table 2) confirm the presence of acetals linked
formation of 45 was the presence of 4 sets of doublets at to the atoms in question, since both C-2 atoms of Abe
2.93 ppm (J = 2.2 Hz, 15.0 Hz), 2.76 ppm (J = 8.1 Hz, and Gal show downfield shifts of ~10 ppm relative to 1.2
15.0 Hz), 2.67 ppm (J = 6.2 Hz, 15.2 Hz), and 2.49 ppm Similar shifts of slightly smaller magnitude ~7–8 ppm
(J = 9.3 Hz, 15.0 Hz), corresponding to the H-6a (Man), are observed when the acetal spans C-2 Abe and C-4
H-6a (Gal), H-6b (Gal), and H-6b (Man) protons. The Man. Altered glycosylation shifts21,22 (~5 ppm) are also
H-4 of the galactose unit shifted significantly downfield observed for C-2 but not C-3 of mannose in compound
from 5.81 ppm to 6.23 ppm. The H-1 of the mannose 2, while the anomeric carbon of Abe is shifted upfield
unit shifted upfield by 1.85 ppm from 5.67 ppm to by ~5 ppm. Based on published correlations of anomeric
3.82 ppm. This was considered as direct evidence of the shift changes vs. torsional angle changes,23 these obser-
presence of the tether in the molecule, as the anomeric vations would suggest that the Abe–Gal and the Gal–
proton should be located in the face of the aromatic ring Man glycosidic linkages adopt unusual torsional angles.
of the tether. The resulting anisotropic effect of the On the basis of the 13C chemical shifts of 2 in D2O, we
aromatic ring causes an upfield chemical shift of this would expect this tethered trisaccharide to be signifi-
proton. After subjecting 45 to Zemplén transesterification cantly distorted from the solution and bound conforma-
conditions, all the protecting groups were removed, and tions of 1. Molecular modeling confirms this prediction
the final tethered trisaccharide 4 was obtained by pre- (Fig. 3A,B). Large changes in chemical shifts are not
13
parative HPLC on a reverse-phase C18 column.
seen when the C shifts of 3 and 1 are compared, and
The NMR spectrum of the final product 4 recorded at molecular modeling confirms that the tethered trisac-
600 MHz NMR was particularly interesting. Four sets of charide 3 is less distorted than compound 2 (Fig. 3C,D).
doublet or doublets corresponding to the H-6a (Man), Since compound 4 contains thioether groups at C-6 of
H-6a (Gal), H-6b (Gal), and H-6b (Man) appear at 3.23, both mannose and galactose residues, the chemical shift
2.95, 2.83, and 2.72 ppm, respectively. The anomeric changes in this compound relative to 1 must be treated
proton, H-1, of the α-mannose unit resonates at unusu- more cautiously. However, a significant glycosylation
ally high field, 4.14 ppm, and the line shapes of the four shift of ~3 ppm can be seen on C-2 of mannose for
aromatic protons are of special interest since all appear compound 4, implying that the ψ angle of the Gal–Man
as broad resonances at 7.49, 7.36, 7.24, and 7.22 ppm. bond has shifted somewhat relative to the solution struc-
This is probably due to the slow rotation of the tether’s ture of 1. In fact, molecular modeling shows that the
aromatic ring. Since the aromatic ring is likely too close conformation of 4 correlates well with bound conformer
to the van der Waals surface of the trisaccharide to II (Fig. 4A), and the Gal–Man linkage is shifted some-
Table 3
Thermodynamics of antibody–oligosaccharide interaction (kcal mol–1) for native 1 and tethered
trisaccharide 4 at 25 0.2 °Ca
Ligand
KA (M–1)
∆G°
–7.10 0.10
–7.60 0.01
∆H°
–6.75 0.38
–6.95 0.05
T∆S°
0.34 0.48
0.65 0.06
1
4
1.60 0.28 × 105
3.85 0.05 × 105
a taken from ref 8. Reproduced with permission from J. Am. Chem. Soc. 1998, 120, 5317–5318.
Copyright 1998 Am. Chem. Soc.
Zhang and Bundle / Synthesis of Tethered Trisaccharides to Probe Carbohydrate–Protein Interactions

200
Ethyl 2,4-di-O-benzyl-3,6-dideoxy-1-thio-β-D-xylo-
hexopyranoside (6)10, ethyl 4-O-benzyl-3,6-dideoxy-1-thio-β-
D-xylo-hexopyranoside (7), ethyl 2-O-benzyl-3,6-dideoxy-1-
thio-β-D-xylo-hexopyranoside (8). A solution of ethyl 3,6-
dideoxy-1-thio-β-D-xylo-hexopyranoside (5) (53.2 mg,
0.28 mmol) in anhydrous DMF (2 mL) was cooled to 0 °C,
sodium hydride (80% dispersion in mineral oil, 17.4 mg,
0.58 mmol) was added, and the solution was stirred for 10 min.
Benzyl bromide (35.5 µL, 0.31 mmol) was added and the
solution was stirred for 1 h at 0 °C. The reaction was quenched
by addition of MeOH (0.5 mL), and the reaction mixture was
diluted with ether (30 mL), washed with 5% brine (2 × 15 mL),
dried over anhydrous Na2SO4, and evaporated under high
vacuum. Chromatography on silica gel using 20% → 30%
(v/v) EtOAc/pentane gave first the 2,4-dibenzylated com-
pound 6 (18.0 mg, 17%), followed by the 4-benzylated com-
pound 7 (4.8 mg, 6%), the 2-benzylated compound 8 (23.0 mg,
29%); and finally, unreacted starting material was recovered
(13.2 mg, 25%).
what relative to the bound conformer I (Fig. 4B). Both
depictions show that the constrained trisaccharide
4 closely resembles both bound conformations of 1.
This model is supported by the NMR data showing
linebroadening and the aromatic-ring-induced proton
shift on H-1 of mannose, which is explained by the
proximity of this anomeric proton to the center of the
aromatic ring (Fig. 5).
The conformational models would predict that the
trisaccharides 2 and 3 should be inactive since tethering
has distorted the relative positions of both the mannose
and galactose residues. Short tethers composed of sp3
hybridized carbons are poorly disposed to simulate the
linear geometry of intramolecular sugar–sugar hydro-
gen bonds. Conversely, longer tethers that span solvent-
exposed groups of the epitope have allowed the bound
conformation to be well simulated, while avoiding unfa-
vorable protein–tether contacts. In this context, com-
pound 4 would be predicted to be as active as 1, and
accurate thermodynamic data confirm this expectation
(Table 3).8 However, the same data show that there are
only small differences for the entropy term when com-
pound 1 and compound 4 are bound by antibody
SE155.4. This implies that the entropic costs to binding
the unconstrained trisaccharide 1 are not as large as
would be estimated based on some literature predic-
tions.5 If this is so, our data lend support to the conten-
tion that the bound form of an oligosaccharide is se-
lected from the most heavily populated conformational
families, and as suggested by Lemieux24 and supported
by Chervenak and Toone,25 the origins of the large
enthalpy and entropy changes that occur when carbohy-
drates are bound by proteins originate from reordering
of water about the new complex.
22
1
Data for 7: [α]D –58.9° (c 0.4, CHCl3). H NMR (360
MHz, CDCl3): δ 7.24–7.35 (m, 5H, C6H5CH2), 4.70 (d, 1H, J =
12.2 Hz, C6H5CH2), 4.47 (d, 1H, J = 12.2 Hz, C6H5CH2), 4.27
(d, 1H, J = 9.5 Hz, H-1), 3.80 (m, 1H, H-2), 3.62 (dq, 1H, J =
1.5 Hz, 6.5 Hz, H-5), 3.43 (m, 1H, H-4), 2.75 (m, 2H, SEt),
2.56 (m, 1H, H-3e), 2.31 (b s, 1H, OH-2), 1.48 (m, 1H, H-3a),
1.33 (t, 3H, J = 7.5 Hz, SEt), 1.27 (d, 3H, J = 6.5 Hz, H-6).
High Res. ES-MS: 305.117887 (M + Na+). Anal. Calcd for
C15H22O3S: C: 63.80%, H: 7.85%. Found: C: 63.44%, H:
7.90%.
22
1
Data for 8: [α]D –54.7° (c 0.3, CHCl3). H NMR (360
MHz, CDCl3): δ 7.23–7.37 (m, 5H, C6H5CH2), 4.70 (d, 1H, J =
11.5 Hz, C6H5CH2), 4.58 (d, 1H, J = 11.5 Hz, C6H5CH2), 4.48
(d, 1H, J = 9.6 Hz, H-1), 3.71 (m, 1H, H-4), 3.63 (dq, 1H, H-
5), 3.56 (m, 1H, H-2), 2.73 (m, 2H, SCH2CH3), 2.45 (m, 1H,
H-3e), 1.62 (m, 1H, H-3a), 1.31 (t, 3H, J = 7.4 Hz, SCH2CH3),
1.23 (d, 3H, J = 6.5 Hz, H-6). High Res. ES-MS: 305.117846
(M + Na+). Anal. Calcd for C25H22O3S: C: 63.80%. H: 7.85%.
Found: C: 63.96%, H: 8.07%.
EXPERIMENTAL
Optical rotations were performed on a Perkin-Elmer 241 pola-
Ethyl 2,4-di-O-(p-methoxybenzyl)-3,6-dideoxy-1-thio-β-D-
rimeter at 22 2 °C. Analytical TLC was employed on silica xylo-hexopyranoside (9), ethyl 4-O-(p-methoxybenzyl)-3,6-
gel 60-F254 (E. Merck, Darmstadt) with detection by quenching dideoxy-1-thio-β-D-xylo-hexopyranoside (10), ethyl 2-O-(p-
of fluorescence and/or by charring with sulfuric acid. methoxybenzyl)-3,6-dideoxy-1-thio-β-D-xylo-hexopyranoside
Iatrobeads refer to a beaded silica gel 6RS-8060 manufactured (11). A solution of ethyl 3,6-dideoxy-1-thio-β-D-xylo-
by Iatron Laboratories (Tokyo). All commercial reagents were hexopyranoside (5) (442.0 mg 2.30 mmol) in anhydrous DMF
used as supplied, and chromatography solvents were distilled (7 mL) was cooled to 0 °C, sodium hydride (80% dispersion in
prior to use. Column chromatography was performed on silica mineral oil, 145.0 mg, 4.80 mmol) was added, and the solution
1
gel 60 (40–60 mesh). H NMR spectra were recorded at 360 was stirred for 10 min. p-Methoxybenzyl chloride (370 µL,
MHz (Brüker WM-360) and first-order proton chemical shifts 2.73 mmol) was added, and the solution was stirred for 1 h at 0
δH are referenced to either internal CHCl3 (δH 7.24, CDCl3) or °C. The reaction was quenched by addition of MeOH (2 mL),
0.1% (v/v) internal acetone (δH 2.225, D2O). 13C NMR spectra and the reaction mixture was diluted with CH2Cl2, washed
were recorded at 75.5 MHz (Brüker AM-300) and 13C chemi- with H2O, dried over anhydrous Na2SO4, and evaporated under
cal shifts δC are referenced to internal CHCl3 (δC 77.00, high vacuum. Chromatography on silica gel using 30% (v/v)
CDCl3). Organic solutions were dried prior to concentration EtOAc/pentane gave first the di-p-methoxybenzylated com-
under vacuum at <40 °C (bath). Microanalyses and pound 9 (256.8 mg, 26%), followed by the 4-p-
electrospray mass spectra were carried out by the analytical methoxybenzylated compound 10 (97.4 mg, 14%), and 2-p-
services of this Department.
methoxybenzylated compound 11 (233.6 mg, 33%).
Israel Journal of Chemistry
40
2001

201
Data for 9: [α]D –53.2° (c 1.4, CHCl3). H NMR (360 Res. ES-MS: 425.17656 (M + Na+). Anal. Calcd for C23H30O4S:
MHz, CDCl3): δ 7.28 (d, 2H, J = 8.7 Hz, o-H-MeOC6H5CH2), C: 68.60%, H: 7.51%. Found: C: 68.55%, H: 7.64%.
22
1
7.18 (d, 2H, J = 8.7 Hz, o-H-MeOC6H5CH2), 6.81–6.88 (m,
Ethyl 2,4-di-O-(p-methoxybenzyl)-3,6-dideoxy-1-thio-α-D-
4H, m-H-MeOC6H5CH2), 4.63 (d, 1H, J = 11.3 Hz,
xylo-hexopyranoside (14), ethyl 4-O-(p-methoxybenzyl)-3,6-
MeOC6H5CH2), 4.48 (d, 1H, J = 12.0 Hz, MeOC6H5CH2), 4.47
dideoxy-1-thio-α-D-xylo-hexopyranoside (15), ethyl 2-O-(p-
(d, 1H, J = 11.9 Hz, MeOC6H5CH2), 4.42 (d, 1H, J = 9.4 Hz,
methoxybenzyl)-3,6-dideoxy-1-thio-α-D-xylo-hexopyranoside
H-1), 4.30 (d, 1H, J = 12.0 Hz, MeOC6H5CH2), 3.79 (s, 3H,
(16). A solution of ethyl 2-O-3,6-dideoxy-1-thio-α-D-xylo-
OMe), 3.78 (s, 3H, OMe), 3.50–3.60 (m, 2H, H-2 + H-5), 3.34
hexopyranoside (13) (862.3 mg 4.49 mmol) in anhydrous
(m, 1H, H-4), 2.70 (m, 2H, SCH2CH3), 2.34 (m, 1H, H-3e),
DMF (15 mL) was cooled to 0 °C, sodium hydride (80%
1.40 (m, 1H, H-3a), 1.27 (t, 3H, J = 7.5 Hz, SCH2CH3), 1.19
dispersion in mineral oil, 282.9 mg, 9.43 mmol) was added,
(d, 3H, J = 6.5 Hz, H-6). High Res. ES-MS: 455.186155 (M +
and the solution was stirred for 10 min. p-Methoxybenzyl chlo-
Na+). Anal. Calcd for C24H32O5S: C: 66.64%, H: 7.46%.
ride (683.0 µL, 4.94 mmol) was added and the solution was
Found: C: 66.74%, H: 7.53%.
stirred for 1 h at 0 °C. The reaction was quenched by addition of
22
1
MeOH (3 mL), the reaction mixture was diluted with CH2Cl2,
washed with H2O, dried over anhydrous Na2SO4, and evapo-
rated under high vacuum. Chromatography on silica gel using
30% (v/v) EtOAc/pentane gave first the di-p-methoxy-
benzylated compound 14 (450.0 mg, 23%), followed by the 4-p-
methoxybenzylated compound 15 (150.0 mg, 11%), and 2-p-
methoxybenzylated compound 16 (377.8 mg, 27%). Unreacted
starting material (210.0 mg, 24%) was also recovered.
Data for 10: [α]D –48.9° (c 0.5, CHCl3). H NMR (360
MHz, CDCl3): δ 7.25 (d, 2H, J = 8.7 Hz, o-H-MeOC6H5CH2),
6.86 (d, 2H, J = 8.7 Hz, m-H-MeOC6H5CH2), 4.64 (d, 1H, J =
11.9 Hz, MeOC6H5CH2), 4.39 (d, 1H, J = 12.1 Hz,
MeOC6H5CH2), 4.24 (d, 1H, J = 9.5 Hz, H-1), 3.80 (s, 3H,
OMe), 3.76 (m, 1H, H-2), 3.60 (dq, 1H, H-5), 3.41 (m, 1H, H-
4), 2.74 (m, 2H, SCH2CH3), 2.53 (m, 1H, H-3e), 2.32 (d, 1H,
J = 1.7 Hz, OH-2), 1.44 (m, 1H, H-3a), 1.30 (t, 3H, J = 7.4 Hz,
SCH2CH3), 1.26 (d, 3H, J = 6.6 Hz, H-6). High Res. ES-MS:
335.128655 (M + Na+). Anal. Calcd for C16H24O4S: C:
61.51%, H: 7.74%. Found: C: 61.84%, H: 7.85%.
Data for 14: [α]D22 +83.5° (c 0.34, CHCl3). 1H NMR (360
MHz, CDCl3): δ 7.30 (d, 2H, J = 8.6 Hz, o-H-MeOC6H5CH2),
7.21 (d, 2H, J = 8.6 Hz, o-H-MeOC6H5CH2), 6.85 (m, 4H, m-
H-MeOC6H5CH2), 5.47 (d, 1H, J = 5.0 Hz, H-1), 4.58 (d, 1H,
J = 11.4 Hz, MeOC6H5CH2), 4.50 (d, 1H, J = 11.8 Hz,
MeOC6H5CH2), 4.40 (d, 1H, J = 11.4 Hz, MeOC6H5CH2), 4.32
(d, 1H, J = 11.8 Hz, MeOC6H5CH2), 4.20 (dq, 1H, H-5), 4.03
(m, 1H, H-2), 3.78 (s, 3H, OMe), 3.73 (m, 1H, H-4), 2.57 (m,
2H, SCH2CH3), 2.04 (m, 1H, H-3e), 1.86 (m, 1H, H-3a), 1.62
(d, 1H, J = 6.8 Hz, OH-4), 1.29 (t, 3H, J = 7.4 Hz, SCH2CH3),
1.17 (d, 3H, J = 6.6 Hz, H-6). High Res. ES-MS: 455.185844
(M + Na+). Anal. Calcd for C24H32O5S: C: 66.64%, H: 7.46%.
Found: C: 66.27%, H: 7.46%.
22
1
Data for 11: [α]D –46.3° (c 1.1, CHCl3). H NMR (360
MHz, CDCl3): δ 7.27 (d, 2H, J = 8.6 Hz, o-H-MeOC6H5CH2),
6.82 (d, 2H, J = 8.6 Hz, m-H-MeOC6H5CH2), 4.62 (d, 1H, J =
11.1 Hz, MeOC6H5CH2), 4.51 (d, 1H, J = 11.1 Hz,
MeOC6H5CH2), 4.46 (d, 1H, J = 9.6 Hz, H-1), 3.78 (s, 3H,
OMe), 3.69 (m, 1H, H-4), 3.62 (m, 1H, H-5), 3.55 (m, 1H, H-
2), 2.70 (m, 2H, SCH2CH3), 2.40 (m, 1H, H-3e), 1.96 (b s, 1H,
OH-4), 1.60 (m, 1H, H-3a), 1.30 (t, 3H, J = 7.5 Hz, SCH2CH3),
1.24 (d, 3H, J = 6.5 Hz, H-6). High Res. ES-MS: 335.12957
(M + Na+). Anal. Calcd for C16H24O4S: C: 61.51%, H: 7.74%.
Found: C: 61.74%, H: 7.98%.
22
1
Data for 15: [α]D +117° (c 0.5, CHCl3). H NMR (360
MHz, CDCl3): δ 7.26 (d, 2H, J = 8.7 Hz, o-H-MeOC6H5CH2),
6.86 (d, 2H, J = 8.6 Hz, m-H-MeOC6H5CH2), 5.28 (d, 1H, J =
5.0 Hz, H-1), 4.61 (d, 1H, J = 11.7 Hz, MeOC6H5CH2), 4.37 (d,
1H, J = 11.8 Hz, MeOC6H5CH2), 4.24 (m, 1H, H-2), 4.11 (dq,
1H, H-5), 3.79 (s, 3H, OMe), 3.38 (m, 1H, H-4), 2.65 (m, 2H,
SCH2CH3), 2.19 (m, 1H, H-3e), 1.45 (m, 1H, H-3a), 1.29 (t,
3H, J = 7.4 Hz, SCH2CH3), 1.17 (d, 3H, J = 6.6 Hz, H-6). High
Res. ES-MS: 335.12486 (M + Na+). Anal. Calcd for
C16H24O4S: C: 61.51%, H: 7.74%. Found: C: 61.78%, H:
7.89%.
Ethyl 4-O-benzyl-2-O-(p-methoxybenzyl)-3,6-dideoxy-1-
thio-β-D-xylo-hexopyranoside (12). Sodium hydride (80%
dispersion in mineral oil, 119.7 mg, 3.99 mmol) was added by
portions to a solution containing compound 11 (417.0 mg,
1.33 mmol) and benzyl bromide (485 µL, 3.99 mmol) in
anhydrous DMF (10 mL). The reaction was continued at room
temperature for 1 h and quenched by addition of methanol
(1.0 mL). The reaction mixture was diluted with EtOAc
(75 mL), and washed with 5% brine (2 × 30 mL), dried over
anhydrous Na2SO4, filtered, and evaporated. The pure com-
pound 12 (483.0 mg, 90%) was isolated by flash chromatogra-
Data for 16: [α]D22 +190.5° (c 0.8, CHCl3). 1H NMR (360
MHz, CDCl3): δ 7.26 (d, 2H, J = 8.5 Hz, o-H-MeOC6H5CH2),
22
phy on silica gel using 8% (v/v) EtOAc/pentane. [α]D
+119.3° (c 1.7, CHCl3). 1H NMR (360 MHz, CDCl3): δ 7.23– 6.85 (d, 2H, J = 8.6 Hz, m-H-MeOC6H5CH2), 5.42 (d, 1H, J =
7.34 (m, 7H, C6H5CH2 + o-H-MeOC6H5CH2), 6.85 (d, 2H, J = 5.1 Hz, H-1), 4.56 (d, 1H, J = 11.4 Hz, MeOC6H5CH2), 4.41 (d,
8.7 Hz, m-H-MeOC6H5CH2), 4.62 (d, 1H, J = 11.2 Hz, 1H, J = 11.4 Hz, MeOC6H5CH2), 4.29 (dq, 1H, J = 1.1 Hz, 6.6
MeOC6H5CH2), 4.52 (d, 1H, J = 12.2 Hz, C6H5CH2), 4.47 (d, Hz, H-5), 4.03 (m, 1H, H-2), 3.78 (s, 3H, OMe), 3.73 (m, 1H,
1H, J = 11.3 Hz, MeOC6H5CH2), 4.43 (d, 1H, J = 9.5 Hz, H-1), H-4), 2.57 (m, 2H, SCH2CH3), 2.04 (m, 1H, H-3e), 1.86 (m,
4.37 (d, 1H, J = 12.2 Hz, C6H5CH2), 3.77 (s, 3H, OMe), 3.51– 1H, H-3a), 1.62 (d, 1H, J = 6.8 Hz, OH-4), 1.29 (t, 3H, J = 7.4
3.62 (m, 2H, H-2 + H-5), 3.38 (m, 1H, H-4), 2.71 (m, 2H, Hz, SCH2CH3), 1.17 (d, 3H, J = 6.6 Hz, H-6). High Res. ES-
SCH2CH3), 2.38 (m, 1H, H-3e), 1.42 (m, 1H, H-3a), 1.28 (t, MS: 335.12944 (M + Na+). Anal. Calcd for C16H24O4S: C:
3H, J = 7.5 Hz, SCH2CH3), 1.24 (d, 3H, J = 6.4 Hz, H-6). High 61.51%, H: 7.74%. Found: C: 61.70%, H: 7.79%.
Zhang and Bundle / Synthesis of Tethered Trisaccharides to Probe Carbohydrate–Protein Interactions

202
Ethyl 4-O-benzyl-2-O-(p-methoxybenzyl)-3,6-dideoxy-1- 2H, J = 8.6 Hz, o-H-MeOC6H5CH2), 6.88 (d, 2H, J = 8.7 Hz,
thio-α-D-xylo-hexopyranoside (17). Sodium hydride (80% m-H-MeOC6H5CH2), 4.88 (d, 1H, J = 10.8 Hz, MeOC6H5CH2),
dispersion in mineral oil, 154.0 mg, 5.10 mmol) was added by 4.59 (d, 1H, J = 10.8 Hz, MeOC6H5CH2), 4.41 (d, 1H, J = 9.5
portions to a solution containing compound 16 (320.7 mg, Hz, H-1), 4.00 (dd, 1H, J = 0.9 Hz, 3.2Hz, H-4), 3.92 (dd, 1H,
1.03 mmol) and benzyl bromide (375 µL, 3.09 mmol) in J = 6.1 Hz, 11.9 Hz, H-6a), 3.82 (dd, 1H, J = 4.3 Hz, 11.9 Hz,
anhydrous DMF (12 mL). The reaction was continued at room H-6b), 3.79 (s, 3H, OMe), 3.58 (dd, 1H, J = 3.3 Hz, 8.9 Hz,
temperature for 1 h and quenched by addition of methanol H-3), 3.49 (m, 2H, H-2 + H-5), 2.77 (m, 2H, SCH2CH3), 1.32
(0.5 mL). The reaction mixture was diluted with EtOAc (t, 3H, J = 7.4 Hz, SCH2CH3). High Res. ES-MS: 367.119072
(75 mL), and washed with 5% brine (2 × 30 mL), dried over (M + Na+). Anal. Calcd for C16H24O6S: C: 55.80%, H: 7.02%.
anhydrous Na2SO4, filtered, and evaporated. The pure com- Found: C: 55.59%, H: 7.06%.
pound 17 (388.0 mg, 94%) was isolated by flash chromatogra-
Ethyl 3,4,6-tri-O-benzyl-2-O-(p-methoxybenzyl)-1-thio-β-
22
phy on silica gel using 8% (v/v) EtOAc/pentane. [α]D
D-galactopyranoside (20). Sodium hydride (80% dispersion in
+119.3° (c 1.7, CHCl3). 1H NMR (360 MHz, CDCl3): δ 7.24–
mineral oil, 435 mg 14.5 mmol) was added by portions to a
7.36 (m, 7H, C6H5CH2 + o-H-MeOC6H5CH2), 6.85 (d, 2H, J =
cold solution of compound 19 (1.0 g 2.9 mmol) and benzyl
8.6 Hz, m-H-MeOC6H5CH2), 5.46 (d, 1H, J = 5.0 Hz, H-1),
bromide (3.17 mL, 8.7 mmol) in anhydrous DMF (12 mL) at
4.59 (d, 1H, J = 12.1 Hz, MeOC6H5CH2), 4.58 (d, 1H, J = 11.4
0 °C. The reaction was continued for 4 h while the temperature
Hz, C6H5CH2), 4.39 (d, 2H, J ≈ 11.5 Hz, ArCH2–), 4.21 (dq,
was allowed to rise to room temperature. The reaction was
1H, H-5), 4.09 (m, 1H, H-2), 3.78 (s, 3H, OMe), 3.43 (m, 1H,
quenched by addition of methanol (1.0 mL). The solution was
H-4), 2.56 (m, 2H, SCH2CH3), 2.12 (m, 1H, H-3e), 1.73 (m,
diluted with EtOAc (80 mL), washed with 5% (2 × 30 mL),
1H, H-3a), 1.28 (t, 3H, J = 7.4 Hz, SCH2CH3), 1.17 (d, 3H, J =
dried over anhydrous Na2SO4, and concentrated. The pure
6.6 Hz, H-6). High Res. ES-MS: 425.17696 (M + Na+). Anal.
compound 20 (1.52 g, 85%) was obtained by flash chromatog-
Calcd for C23H30O4S: C: 68.60%, H: 7.51%. Found: C:
raphy on silica gel using 5% (v/v) EtOAc/toluene as eluent.
68.20%, H: 7.57%.
22
1
[α]D + 2.4° (c 1.0, CHCl3). H NMR (360 MHz, CDCl3): δ
Ethyl 3,4-di-O-isopropylidene-2-O-(p-methoxybenzyl)-6- 7.24–7.38 (m, 17H, C6H5CH2 + o-H-MeOC6H5CH2), 6.85 (d,
O-(2-methoxy-2-propyl)-1-thio-β-D-galactopyranoside (18). 1H, J = 8.6 Hz, m-H-MeOC6H5CH2), 4.97 (d, 1H, J = 11.7 Hz,
A solution of ethyl 3,4-di-O-isopropylidene-6-O-(2-methoxy- ArCH2–), 4.40–4.86 (m, 8H, H-1 + ArCH2–), 3.95 (b d, 1H, J =
2-propyl)-1-thio-β-D-galactopyranoside (5.55 g 16.5 mmol) in 2.5 Hz, H-4), 3.83 (t, 1H, J = 9.5 Hz, H-2), 3.79 (s, 3H, OMe),
anhydrous DMF (20 mL) was treated with NaH (80% disper- 3.55–3.61 (m, 4H, H-3 + H-5 + H-6a + H-6b), 2.74 (m, 2H,
sion in mineral oil, 2.48 g, 82.7 mmol) at 0 °C for 20 min, p- SCH2CH3), 1.30 (t, 3H, J = 7.4 Hz, SCH2CH3). High Res. ES-
methoxybenzyl chloride (5.0 mL, 36.9 mmol) was added MS: 637.26022 (M + Na+). Anal. Calcd for C37H42O6S: C:
dropwise and the reaction was continued for 5 h while the 72.28%, H: 6.89%. Found: C: 72.22%, H: 7.13%.
temperature was allowed to rise slowly to room temperature.
Methyl 2-O-benzoyl-4,6-di-O-benzyl-3-O-(2′-O-(p-
The reaction was quenched by addition of methanol (5.0 mL),
methoxybenzyl)-4′-O-benzyl-3′,6′-dideoxy-α-D-xylo
the reaction mixture was diluted with CH2Cl2, washed with
hexopyranosyl)-α-D-mannopyranoside (22). A mixture con-
H2O, and concentrated. Chromatography of the residue on
taining acceptor 21 (200.8 mg 0.42 mmol), donor 12 or 17
silica gel using 20/80/1 EtOAc/pentane/Et3N as eluent af-
(337 mg, 0.84 mmol), and molecular sieve 4Å (1.0 g) was
22
forded pure compound 18 (3.43 g) in 46% yield. [α]D
dried under high vacuum for 2 h, CH2Cl2 (10 mL) was added,
–4.8° (c 0.5, CHCl3). 1H NMR (360 MHz, CDCl3): δ 7.33 (d,
the mixture was protected under argon, and stirred for 1 h at
2H, J = 8.7 Hz, o-H-MeOC6H5CH2), 6.88 (d, 1H, J = 8.7 Hz,
0 °C. N-iodosuccinimide (248.7 mg,1.05 mmol) was added, a
m-H-MeOC6H5CH2), 4.74 (d, 1H, J = 11.2 Hz, MeOC6H5CH2),
few crystals of silver trifluoromethanesulfonate were added,
4.64 (d, 1H, J = 11.2 Hz, MeOC6H5CH2), 4.46 (d, 1H, J = 9.7
and the reaction was continued for 15 min. Et3N (1 mL) was
Hz, H-1), 4.28 (dd, 1H, J = 2.0 Hz, 5.6 Hz, H-4), 4.22 (t, 1H,
added to quench the reaction, the solids were filtered off
J = 5.7 Hz, H-3), 3.92 (m, 1H, H-5), 3.77 (s, 3H, OMe), 3.58
through a thin bed of Celite, and the residues washed with
(m, 2H, H-6a + H-6b), 3.37 (dd, 1H, J = 6.3 Hz, 9.7 Hz, H-2),
more CH2Cl2. The combined organic solution was washed
3.17 (s, 3H, Me2C(OMe)-), 2.66 (m, 2H, SCH2CH3), 1.41 (s,
with a 1:1 mixture of saturated aqueous NaHCO3 and 10%
3H, Me), 1.30 (s, 3H, Me), 1.28 (s, 6H, Me2C(OMe)-), 1.24 (t,
aqueous Na2S2O3, dried over anhydrous Na2SO4, and concen-
3H, J = 7.5 Hz, SCH2CH3). High Res. ES-MS: 479.207941 (M
trated. Chromatography on silica gel using 8% (v/v) EtOAc/
+ Na+). Anal. Calcd for C23H36O7S: C: 60.50%, H: 7.95%.
Found: C: 60.85%, H: 7.72%.
22
toluene gave pure disaccharide 22 (220 mg, 64%). [α]D
+
17.1° (c 0.6, CHCl3). 1H NMR (360 MHz, CDCl3): δ 8.04 (m,
Ethyl 2-O-(p-methoxybenzyl)-1-thio-β-D-galactopyrano- 2H, o-H-C6H5CO), 7.54 (m, 1H, p-H-C6H5CO), 7.11–7.39 (m,
side (19). A solution of 18 (1.34 g, 2.90 mmol) in MeOH 17H, m-H-C6H5CO + MeOC6H5CH2), 7.05 (d, 1H, J = 8.6 Hz,
(15 mL) was treated with a solution of HBF4 (48% aqueous, m-H-MeOC6H5CH2), 6.70 (d, 1H, J = 8.7 Hz, o-H-
134 µL) at 0 °C overnight, NEt3 (0.5 mL) was added and the MeOC6H5CH2), 5.38 (dd, 1H, J = 2.0 Hz, 3.1 Hz, H-2), 5.12–
solution was concentrated under high vacuum. The pure com- 5.18 (m, 2H, H-1′ + ArCH2–), 4.85 (d, 1H, J = 1.8 Hz, H-1),
pound 19 (0.92 g, 91%) was isolated by chromatography on 4.66 (d, 1H, J = 12.0 Hz, ArCH2–), 4.56 (d, 1H, J = 11.3 Hz,
22
silica gel using 3% (v/v) MeOH/CH2Cl2 as eluent. [α]D
+
ArCH2–), 4.50 (d, 1H, J = 12.1 Hz, ArCH2–), 4.46 (d, 1H, J =
12.7° (c 1.1, CHCl3). 1H NMR (360 MHz, CDCl3): δ 7.32 (d, 12.4 Hz, ArCH2–), 4.28 – 4.38 (m, 4H, H-3 + ArCH2–), 4.14 (t,
Israel Journal of Chemistry 40 2001

203
3H, J = 9.6 Hz, H-4), 3.91 (dq, 1H, H-5′), 3.71–3.88 (m, 7H, ArCH2–), 4.57 (d, 1H, J = 11.8 Hz, ArCH2–), 4.54 (d, 1H, J =
OMe + MeOC6H5CH2 + H-2′ +H-5 + H-6a + H-6b + OMe + 11.4 Hz, ArCH2–), 4.47 (d, 1H, J = 11.9 Hz, ArCH2–), 4.45 (d,
MeOC6H5CH2), 3.40 (s, 3H, OMe), 3.30 (br, 1H, H-4′), 1.98 1H, J = 12.0 Hz, ArCH2–), 4.40 (d, 2H, J = 11.2 Hz, ArCH2–),
(m, 1H, H-3e′), 1.76 (m, 1H, H-3a′), 1.05 (d, 3H, J = 6.6 Hz, H- 4.35 (d, 1H, J = 11.7 Hz, ArCH2–), 3.91 – 4.30 (m, 10 H, 3 ×
6′). High Res. ES-MS: 841.356686 (M + Na+). Anal. Calcd for ArCH2– + H-3 + H-2 + H-5″ + H-5′ + H-4 + H-3″ + H-2″),
C49H54O11: C: 71.86%, H: 6.65%. Found: C: 71.84%, H: 6.83 %. 3.87 (d, 1H, J = 2.0 Hz, H-4″), 3.62–3.80 (m, 10H, H-5 +H-2′
+ H-6a + H-6b + 2 × OMe+MeOC6H5CH2), 3.53 (dd, 1H, J =
Methyl 4,6-di-O-benzyl-3-O-(2′-O-(p-methoxybenzyl)-4′-
6.5 Hz, 9.8 Hz, H-6a″), 3.46 (dd, 1H, J = 6.1 Hz, 9.8 Hz, H-
O-benzyl-3′,6′-dideoxy-α-D-xylo-hexopyranosyl)-α-D-
6b″), 3.24 (s, 3H, OMe), 3.14 (br, 1H, H-4′), 1.94 (m, 1H, H-
mannopyranoside (23). Disaccharide 22 (170 mg, 0.208
3e′), 1.70 (m, 1H, H-3a′), 1.07 (d, 3H, J = 6.5 Hz, H-6′). High
mmol) was dissolved in anhydrous methanol (5 mL), a solu-
Res. ES-MS: 1289.581045 (M + Na+).
tion of sodium methoxide in anhydrous methanol (200 µL,
1.27 M) was added, and the mixture was stirred for 2 h at room
Methyl 4,6-di-O-benzyl-2-O-(3″,4″,6″-tri-O-benzyl-α-D-
temperature. When the starting material was consumed, the galactopyranosyl)-3-O-(4′-O-benzyl-3′,6′-dideoxy-α-D-xylo-
solution was neutralized with Amberlite IR-120 (H+). The hexopyranosyl)-α-D-mannopyranoside (25). A solution of
resin was filtered off and the solution was concentrated under trisaccharide 24 (346.5 mg, 0.273 mmol) in CH3CN (15 mL)
high vacuum. The alcohol 23 was purified by flash chromatog- was treated with water (1.0 mL) and ceric ammonium nitrate
raphy using 25% (v/v) EtOAc/toluene as eluent (139 mg, (750 mg 1.36 mmol) for 1.5 h at room temperature. The
22
1
94%). [α]D + 66.8° (c 0.5, CHCl3). H NMR (360 MHz, reaction mixture was diluted with CH2Cl2 (50 mL), washed
CDCl3): δ 7.10–7.34 (m, 15H, C6H5CH2), 7.08 (d, 2H, J = 8.6 with water (3 × 30 mL), dried over anhydrous Na2SO4, and
Hz, o-H-MeOC6H5CH2), 6.72 (d, 2H, J = 8.7 Hz, m-H- evaporated. The crude material was chromatographed on
C6H5CH2), 5.16 (d, 1H, J = 3.4 Hz, H-1′), 5.04 (d, 1H, J = 11.3 silica gel using 25% (v/v) EtOAc/toluene to give 25
Hz, ArCH2–), 4.74 (d, 1H, J = 1.6 Hz, H-1), 4.60 (d, 1H, J = (203.6 mg, 73%). [α]D22 + 57.6° (c 0.8, CHCl3). 1H NMR (360
12.2 Hz, ArCH2–), 4.53 (d, 1H, J = 12.0 Hz, ArCH2–), 4.50 (d, MHz, CDCl3): δ 7.17–7.36 (m, 30H, C6H5CH2–), 5.16 (d, 1H,
1H, J = 12.1 Hz, ArCH2–), 4.44 (d, 1H, J = 11.2 Hz, ArCH2–), J = 3.6 Hz, H-1′), 5.10 (d, 1H, J = 4.0 Hz, H-1″), 4.86 (d, 1H,
4.39 (d, 1H, J = 11.9 Hz, ArCH2–), 4.36 (d, 1H, J = 12.1 Hz, J = 11.4 Hz, ArCH2–), 4.77 (d, 1H, J = 1.4 Hz, H-1), 4.74 (d,
ArCH2–), 4.32 (d, 1H, J = 12.1 Hz, ArCH2–), 4.03–4.11 (m, 1H, J = 10.6 Hz, ArCH2–), 4.38–4.70 (m, 10H, ArCH2–),
2H, H-3 + H-5′), 3.98 (dd, 1H, J = 1.8 Hz, 3.34 Hz, H-2), 3.86 4.09–4.18 (m, 3H, H-3 + H-2″ + H-5″), 3.95 (m, 1H, H-2′),
(t, 1H, J = 9.3 Hz, H-4), 3.64–3.85 (m, 8H, H-2′ + H-5 + H-6a 3.77–3.91 (m, 5H, H-2 + H-4 + H-5′ + H-3″ + H-4″), 3.66–
+ H-6b + OMe+MeOC6H5CH2), 3.45 (br. 1H, H-4′), 3.36 (s, 3.74 (m, 2H, H-5 + H-6a), 3.60 (dd, 1H, J = 2.6 Hz, 10 Hz, H-
3H, OMe), 2.12 (m, 1H, H-3e′), 1.87 (m, 1H, H-3a′), 1.15 (d, 6b), 3.57 (dd, 1H, J = 6.5 Hz, 9.5 Hz, H-6a″), 3.49 (dd, 1H, J =
3H, J = 6.6 Hz, H-6′). High Res. ES-MS: 737.331267 (M + 6.1 Hz, 9.6 Hz, H-6b″), 3.39 (br, 1H, H-4′), 3.21 (s, 3H, OMe),
Na+). Anal. Calcd for C42H50O10: C: 70.57%, H: 7.05%. Found: 2.16 (m, 1H, H-3e′), 1.60 (m, 1H, H-3a′), 1.13 (d, 3H, J = 6.5
C: 70.39%, H: 7.09%.
Hz, H-6′). High Res. ES-MS: 1049.467115 (M + Na+). Anal.
Calcd for C61H69O14: C: 71.33%, H: 6.87%. Found: C: 70.68%,
H: 6.86%.
Methyl 4,6-di-O-benzyl-2-O-(3″,4″,6″-tri-O-benzyl-2″-O-
(p-methoxybenzyl)-α-D-galactopyranosyl)-3-O-(2′-O-(p-
methoxybenzyl)-4′-O-benzyl-3′,6′-dideoxy-α-D-xylo-
hexopyranosyl)-α-D-mannopyranoside (24). A mixture of dis-
accharide 23 (226.6 mg, 0.317 mmol), donor 20 (487.9 mg,
0.79 mmol), and molecular sieve 4Å (1.0 g) was dried under
high vacuum for 2 h, CH2Cl2 (10 mL) was added, and the
mixture was stirred for 1 h at 0 °C under argon. 2,6-Di-tert-
butyl-4-methylpyridine (200.0 mg, 0.95 mmol) and methyl
trifluoromethanesulfonate (101 µL, 0.857 mmol) were added
at 0 °C. After 30 min, the solid was filtered off through a thin
bed of Celite, and washed with more CH2Cl2 (80 mL). The
combined organic solution was washed with H2O (1 × 20 mL),
dried over anhydrous Na2SO4, filtered, and concentrated. The
trisaccharide 24 was obtained by chromatography using 5%
(v/v) EtOAc/toluene as eluent (378.6 mg, 94%). [α]D22 + 47° (c
Methyl 4,6-di-O-benzyl-2-O-(3″,4″,6″-tri-O-benzyl-α-D-
galactopyranosyl)-3-O-(4′-O-benzyl-3′,6′-dideoxy-α-D-xylo-
hexopyranosyl)-2′,2″-di-O-methylene-α-D-mannopyranoside
(26). A solution of the trisaccharide diol 25 (107 mg,
0.104 mmol) in anhydrous DMF (3 mL) was treated with
sodium hydride (80% dispersion in mineral oil, 25 mg,
1.04 mmol) for 10 min., and dibromomethane (10 µl,
0.142 mmol) was added dropwise. The reaction was stirred at
room temperature for 24 hr, methanol (0.5 mL) was added to
quench the reaction, and the reaction mixture was concen-
trated under high vacuum. Chromatography using 5% (v/v)
EtOAc/toluene as eluent could not purify the trisaccharide 26,
the impure fractions (40.0 mg, containing ~30% of another
unidentified product) of the compound was collected and
deprotected. 1H NMR (360 MHz, CDCl3): selected data: δ 5.91
(d, 1H, J = 2.6 Hz, H-1″), 5.16 (d, 1H, J = 3.7 Hz, H-1′), 4.94
(d, 1H, J ª 1 Hz, H-1), 3.10 (s, 3H, OMe), 2.03 (m, 1H, H-3a′),
1.93 (m, 1H, H-3e′), 0.86 (d, 3H, J = 6.5 Hz, H-6′).
1
0.5, CHCl3). H NMR (360 MHz, CDCl3): δ 7.08–7.34 (m,
32H, o-H-MeOC6H5CH2 + C6H5), 6.91 (d, 2H, J = 8.6 Hz, o-H-
MeOC6H5CH2), 6.71 (d, 2H, J = 8.7 Hz, m-H-MeOC6H5CH2),
6.61 (d, 2H, J = 8.7 Hz, m-H-MeOC6H5CH2), 5.53 (d, 1H, J =
3.6 Hz, H-1″), 5.19 (d, 1H, J = 3.2 Hz, H-1′), 5.00 (d, 1H, J =
12.4 Hz, ArCH2–), 4.93 (d, 1H, J = 11.2 Hz, ArCH2–), 4.91 (d,
1H, J = 11.4 Hz, ArCH2–), 4.77 (d, 1H, J = 1.8 Hz, H-1), 4.73
(d, 1H, J = 11.8 Hz, ArCH2–), 4.63 (d, 2H, J = 11.7 Hz,
Methyl 2-O-(α-D-galactopyranosyl)-3-O-(3′,6′-dideoxy-α-
D-xylo-hexopyranosyl)-2′,2″-di-O-methylene-α-D-manno-
pyranoside (2). To a solution of the impure protected and
Zhang and Bundle / Synthesis of Tethered Trisaccharides to Probe Carbohydrate–Protein Interactions

204
tethered trisaccharide 26 (33 mg) and 5% Pd on charcoal (100 mL), and the organic solution was washed with 5% brine
(35 mg) in methanol (25 mL) was added 3 drops of acetic acid, (3 × 30 mL), dried over anhydrous Na2SO4, filtered, and
and the resulting mixture was hydrogenated overnight. The evaporated. Chromatography on silica gel using 7% (v/v)
22
catalyst was filtered off through a Millipore membrane filter EtOAc/toluene yielded compound 30 (2.0 g, 91%). [α]D
+
1
(0.25 micron), and the filtrate was concentrated under high 40.5° (c 1.1, CHCl3). H NMR (360 MHz, CDCl3): δ 7.24–
vacuum. The crude material was first purified by silica gel 7.34 (m, 5 H, C6H5CH2), 7.16 (d, 2H, J = 8.5 Hz, o-H-
chromatography using 10 → 20% (v/v) MeOH/CH2Cl2 as MeOC6H5CH2), 6.80 (d, 2H, J = 8.6 Hz, m-H-MeOC6H5CH2),
eluent, followed by reversed phase silica gel chromatography 4.92 (s, 1H, H-1), 4.76 (d, 1H, J = 11.1 Hz, ArCH2–), 4.61 (d,
(C18) using 10 → 15% MeOH/H2O as eluent to give the pure, 1H, J = 12.2 Hz, ArCH2–), 4.53 (d, 1H, J = 12.2 Hz, ArCH2–),
22
tethered trisaccharide 2 (8.0 mg) which was lyophilized. [α]D
4.45 (d, 1H, J = 11.1 Hz, ArCH2–), 4.27 (‘t’, 1H, J ª 6.4 Hz, H-
+ 25.3° (c 0.3, H2O). 1H and 13C NMR (500 MHz, D2O): See 3), 4.11 (d, 1H, J = 5.9 Hz, H-2), 3.77 (s, 3H,
Tables 1 and 2. High Res. ES-MS: 521.184860 (M + Na+).
MeOC6H5CH2+OMe), 3.61 – 3.75 (m, 3H, H-5 + H6a + H-6b),
3.52 (dd, 1H, J = 7.0 Hz, 9.9 Hz, H-4), 3.37 (s, 3H, OMe), 1.50
(s, 3H, Me), 1.35 (s, 3H, Me). High Res. ES-MS: 467.20449
(M + Na+). Anal. Calcd for C25H32O7: C: 67.55%, H: 7.26%.
Found: C: 67.35%, H: 7.24%.
Methyl 3,6-di-O-benzyl-α-D-mannopyranoside (28) and
Methyl 2,3-O-isopropylidene-6-O-benzyl-α-D-mannopyr-
anoside (29). A mixture of methyl 2,3-di-O-isopropylidene-α-
D-mannopyranoside (27) (5.00 g, 21.4 mmol) and di-n-
butyltin oxide (7.50 g, 30.1 mmol) in toluene (250 mL) was
refluxed for 1.5 h, with a Dean–Stark tube and the water
generated during the reaction was removed. The solution was
cooled and evaporated to dryness under reduced pressure. The
resulting syrupy material was dissolved in anhydrous DMF (60
mL), benzyl bromide (8.8 mL, 74.0 mmol) and cesium fluoride
(6.50 g, 42.8 mmol) were added, and the reaction was stirred at
room temperature for 3 h. The mixture was filtered through a
thin bed of silica gel using EtOAc as eluent, the clear organic
solution was concentrated, and the mixture was purified by
chromatography on silica gel using 25% (v/v) EtOAc/pentane to
afford first: 29 (3.70 g, 54%); followed by 28 (1.18 g, 17%).
Methyl 6-O-benzyl-4-O-(p-methoxybenzyl)-α-D-manno-
pyranoside (31). A solution of compound 30 (650 mg,
1.46 mmol) in methanol (8 mL) was cooled to 0 °C, HBF4
(48% aqueous, 65 µl) was added, the mixture was stirred for
24 h while it rose to ambient temperature. Another aliquot of
HBF4 (48% aqueous, 65 µL) was added, and the reaction was
continued for a further 24 h. NEt3 (2 mL) was added, the
solution was evaporated under high vacuum. Chromatography
on silica gel using 5% (v/v) MeOH/CH2Cl2 as eluent afforded
22
the desired diol 31 (479 mg, 81%). [α]D + 69.1° (c 0.8,
1
CHCl3). H NMR (360 MHz, CDCl3): δ 7.24–7.37 (m, 5H,
C6H5CH2), 7.14 (d, 1H, J = 8.7 Hz, o-H-MeOC6H5CH2), 6.81
(d, 1H, J = 8.7 Hz, m-H-MeOC6H5CH2), 4.71 (d, 1H, J = 0.7
22
1
Data for 28: [α]D + 26° (c 1.5, CHCl3). H NMR (360 Hz, H-1), 4.65 (d, 1H, J = 11.8 Hz, ArCH2–),), 4.62 (d, 1H, J =
MHz, CDCl3): δ 7.24–7.36 (m, 10H, C6H5CH2), 4.76 (d, 1H, 10.3 Hz, ArCH2–), 4.53 (d, 1H, J = 12.1 Hz, ArCH2–), 4.48 (d,
J = 1.6 Hz, H-1), 4.70 (d, 1H, J = 11.6 Hz, C6H5CH2), 4.63 (d, 1H, J = 11.0 Hz, ArCH2–), 3.66 – 3.87 (m, 9H, H-2 + H-3 + H-
1H, J = 11.6 Hz, C6H5CH2), 4.61 (d, 1H, J = 12.0 Hz, 4 + H-5 + H-6a + H-6b + OMe+MeOC6H5CH2), 3.33 (s, 3H,
C6H5CH2), 4.56 (d, 1H, J = 12.0 Hz, C6H5CH2), 3.98 (dd, 1H, OMe). High Res. ES-MS: 427.17303 (M + Na+). Anal. Calcd
J = 1.6 Hz, 3.3 Hz, H-2), 3.90 (t, 1H, J = 9.1 Hz, H-4), 3.68– for C22H28O7: C: 65.33%, H: 6.98%. Found: C: 65.00%, H:
3.77 (m, 3H, H-5 + H-6a + H-6b), 3.66 (dd, 1H, J = 3.3 Hz, 9.2 6.98%.
Hz, H-3), 3.36 (s, 3H, OMe). High Res. FAB MS: 397.0 (M +
Methyl 2-O-benzoyl-6-O-benzyl-4-O-(p-methoxybenzyl)-
Na+). Anal. Calcd for C21H26O6: C: 67.36%, H: 7.00%. Found:
α-D-mannopyranoside (32). A solution of diol 31 (631.4 mg,
C: 66.60%, H: 6.97%.
1.56 mmol), trimethyl orthobenzoate (821 µL, 4.86 mmol) and
22
1
camphorsulphonic acid (70 mg) in CHCl3 (50 mL) was con-
centrated on a Rotavap under reduced pressure to ~15 mL,
another portion of CHCl3 (50 mL) was added and the resulting
solution was concentrated to ~15 mL again. A third portion of
CHCl3 (50 mL) was added and the same process was repeated.
TLC exam showed that the starting material had disappeared.
Et3N (2.5 mL) was added and the mixture was evaporated to
dryness under high vacuum. The syrupy mixture was dis-
solved in 80% (v/v) HOAc/H2O (30 mL), and the reaction was
continued at room temperature for 1 h. The solution was
evaporated to dryness. After chromatography on silica gel
using 30% (v/v) EtOAc/pentane as eluent, the compound 32
was obtained as a colorless foam (702 mg, 88%). [α]D22 –2.0°
(c 1.8, CHCl3). 1H NMR (360 MHz, CDCl3): δ 8.03 (m, 2H, o-
H-C6H5CO), 7.55 (m, 1H, p-H-C6H5CO), 7.28–7.58 (m, 7H,
m-H-C6H5CO + C6H5CH2), 7.18 (d, 2H, J = 6.6 Hz, o-H-
C6H5CH2), 6.80 (d, 2H, J = 6.6 Hz, m-H-C6H5CH2), 5.34 (1H,
dd, J =1.8 Hz, 3.4 Hz, H-2), 4.84 (d, 1H, J = 1.7 Hz, H-1), 4.75
Data for 29: [α]D + 8.3° (c 1.9, CHCl3). H NMR (360
MHz, CDCl3): δ 7.17–7.25 (m, 5H, C6H5CH2), 4.84 (s, 1H,
H-1), 4.55 (d, 1H, J = 12.1 Hz, C6H5CH2), 4.48 (d, 1H, J = 12.1
Hz, C6H5CH2), 4.02 (br s, 2H, H-2 + H-3), 3.61–3.66 (m, 4H,
H-4 + H-5 + H-6 + H-6′), 3.30 (s, 3H, OMe), 1.42 (s, 3H, Me),
1.25 (s, 3H, Me). High Res. ES-MS: 347.147751 (M + Na+).
Anal. Calcd for C17H24O6: C: 62.95%, H: 7.46%. Found: C:
62.94%, H: 7.55%.
Methyl 2,3-O-isopropylidene-6-O-benzyl-4-O-(p-metho-
xybenzyl)-α-D-mannopyranoside (30). The alcohol (29) (1.60
g, 4.96 mmol) was dissolved in anhydrous DMF (15 mL) and
the solution was cooled in an ice bath. Sodium hydride (80%
dispersion in mineral oil, 445 mg, 14.8 mmol) was added by
portions, the mixture was stirred for 20 min. p-Methoxybenzyl
chloride (1.4 mL, 9.90 mmol) was added dropwise, the reac-
tion was continued at 0 °C for 2 h. MeOH (0.5 mL) was added
to quench the reaction. The mixture was diluted with EtOAc
Israel Journal of Chemistry
40
2001

205
(d, 1H, J = 12.0 Hz, ArCH2–), 4.71 (d, 1H, J = 10.9 Hz, OMe+MeOC6H5CH2), 3.73 (s, 3H, OMe-MeOC6H5CH2),
ArCH2–), 4.58 (d, 1H, J = 10.8 Hz, ArCH2–), 4.57 (d, 1H, J = 3.63–3.72 (m, 3H, H-5 + H-6a + H-6b), 3.45 (br., 1H, H-4′),
12.0 Hz, ArCH2–), 4.21 (dd, 1H, J = 3.4 Hz, 9.4 Hz, H-3), 3.97 3.35 (s, 3H, OMe), 2.12 (m, 1H, H-3e′), 1.87 (m, 1H, H-3a′),
(t, 1H, J = 9.4 Hz, H-4), 3.89 (dd, 1H, J = 4.1 Hz, 11.1 Hz, 1.16 (d, 3H, J = 6.6 Hz, H-6′). Anal. Calcd for C43H52O11: C:
H-6a), 3.77–3.81 (m, 2H, H-5 + H-6b), 3.75 (s, 3H, OMe + 69.33%, H: 7.04%. Found: C: 69.11%, H: 7.25%.
MeOC6H5CH2), 3.38 (s, 3H, OMe). High Res. ES-MS:
Methyl 6-O-benzyl-4-O-(p-methoxybenzyl)-2-O-(2″,3″,
531.19945 (M + Na+). Anal. Calcd for C29H32O8: C: 68.50%,
4″,6″-tetra-O-benzyl-α-D-galactopyranosyl)-3-O-(2′-O-(p-
H: 6.34%. Found: C: 68.85%, H: 6.16%.
methoxybenzyl)-4′-O-benzyl-3′,6′-dideoxy-α-D-xylo-
Methyl 2-O-benzoyl-6-O-benzyl-4-O-(p-methoxybenzyl)- hexopyranosyl)-α-D-mannopyranoside (36). A mixture of dis-
3-O-(2′-O-(p-methoxybenzyl)-4′-O-benzyl-3′,6′-dideoxy-α-D- accharide 35 (177 mg, 0.237 mmol), donor 33 (347 mg, 0.593
xylo-hexopyranosyl)-α-D-mannopyranoside (34). A mixture mmol), and molecular sieve 4Å (600 mg) was dried under high
containing acceptor 32 (223 mg, 0.438 mmol), donor 12 or 17 vacuum for 2 h, CH2Cl2 (8 mL) was added, and the mixture
(264 mg, 0.675 mmol), and molecular sieve 4Å (0.5 g) was was stirred for 1 h at 0 °C under argon, followed by the
dried under high vacuum for 2 h. CH2Cl2 (10 mL) was added, addition of 2,6 di-tert-butyl-4-methylpyridine (149 mg, 0.73
the mixture was protected under argon, and stirred for 1 h at mmol) and methyl trifluoromethanesulfonate (81.5 µL, 0.71
0 °C. 2,6 Di-tert-butyl-4-methylpyridine (273 mg, 1.30 mmol) mmol). After 30 min, the reaction was quenched by addition of
and methyl trifluoromethanesulfonate (137 µL, 1.17 mmol) Et3N (1 mL). The solid was filtered off through a thin bed of
were added, and the reaction was continued for 30 min. Et3N Celite, and washed with CH2Cl2. The combined organic solu-
(0.5 mL) was added to quench the reaction, the solids were tion was evaporated to dryness. The trisaccharide 36 was
filtered off through a thin bed of Celite, and washed with more obtained by chromatography using 5% (v/v) EtOAc/toluene as
CH2Cl2. The combined organic solution was evaporated, and eluent (249 mg, 83%). [α]D22 + 57.5° (c 0.9, CHCl3). 1H NMR
chromatography on silica gel using 8% (v/v) EtOAc/toluene (360 MHz, CDCl3): δ 7.14–7.45 (m, 3H, C6H5CH2), 7.04 (d,
gave pure disaccharide 34 (309 mg, 83%). [α]D22 + 22.4° (c 0.5, 2H, J = 8.6 Hz, MeOC6H5CH2), 7.00 (d, 2H, J = 8.6 Hz,
1
CHCl3). H NMR (360 MHz, CDCl3): δ 8.03 (m, 2H, o-H- MeOC6H5CH2), 6.75 (d, 2H, J = 8.6 Hz, MeOC6H5CH2), 6.67
C6H5CO), 7.54 (m, 1H, p-H-C6H5CO), 7.21–7.37 (m, 12 H, (d, 2H, J = 8.6 Hz, MeOC6H5CH2), 5.56 (d, 1H, J = 3.6 Hz, H-
C6H5CH2 + m-H-C6H5CO), 7.10 (m, 4H, o-H-MeOC6H5CH2), 1″), 5.20 (d, 1H, J = 3.3 Hz, H-1′), 5.01 (d, 1H, J = 12.0 Hz,
6.73 (m, 4H, m-H-MeOC6H5CH2), 5.37 (dd, 1H, J = 2.0 Hz, ArCH2–), 4.91–4.95 (m, 2H, ArCH2–), 4.80 (d, 1H, J = 1.8 Hz,
2.9 Hz, H-2), 5.13 (d, 1H, J = 3.1 Hz, H-1′), 5.04 (d, 1H, J = H-1′), 4.75 (d, 1H, J = 11.8 Hz, ArCH2–), 4.64 (d, 1H, J = 12.0
10.7 Hz, ArCH2–), 4.84 (d, 1H, J = 1.6 Hz, H-1), 4.65 (d, 1H, Hz, ArCH2–), 4.59 (d, 1H, J = 11.8 Hz, ArCH2–), 4.57 (d, 1H,
J = 12.0 Hz, ArCH2–), 4.27 – 4.51 (m, 7H, ArCH2– + H-3), J = 11.5 Hz, ArCH2–), 4.50 (d, 1H, J = 11.8 Hz, ArCH2–), 4.48
4.11 (t, 1H, J = 9.5 Hz, H-4), 3.90 (m, 1H, H-5′), 3.70–3.84 (m, (d, 1H, J = 12.0 Hz, ArCH2–), 4.42 (d, 2H, J = 11.8 Hz,
10H, 2 × OMe MeOC6H5CH2+ H-2′ + H-5 +H-6a + H-6b), 2.00 ArCH2–), 4.21 – 4.32 (m, 5H, H-3 + ArCH2–), 3.97–4.16 (m,
(m, 1H, H-3e′), 1.76 (m, 1H, H-3a′), 1.03 (d, 3H, J = 6.6 Hz, H- 6H, H-2 + H-5″ + H-2″ + H-5′ + H-4 + H-3″), 3.90 (d, 1H, J =
6′). High Res. ES-MS: 871.366398 (M + Na+). Anal. Calcd for 2.0 Hz, H-4″), 3.63–3.82 (m, 10 H, 2 × OMe + MeOC6H5CH2
C50H56O12: C: 70.74%, H: 6.65%. Found: C: 70.55%, H: 6.88%. + H-5 + H-6a + H-6b + H-2′), 3.55 (dd, 1H, J = 6.5 Hz, 9.8 Hz,
H-6a″), 3.49 (dd, 1H, J = 6.1 Hz, 9.7 Hz, H-6b″), 3.25 (s, 3H,
Methyl 6-O-benzyl-4-O-(p-methoxybenzyl)-3-O-(2′-O-(p-
OMe), 3.11 (br. 1H, H-4′), 1.95 (m, 1H, H-3e′), 1.71 (m, 1H,
methoxybenzyl)-4′-O-benzyl-3′,6′-dideoxy-α-D-xylo-
H-3a′), 1.09 (d, 3H, J = 6.5 Hz, H-6′). High Res. ES-MS:
hexopyranosyl)-α-D-mannopyranoside (35). Disaccharide 34
(200 mg, 0.24 mmol) was dissolved in anhydrous methanol
(10 mL), a solution of sodium methoxide in anhydrous metha-
nol (1 mL, 1.27 M) was added, and the mixture was stirred for
2 h at room temperature. When the starting material was
consumed, the solution was neutralized with Amberlite IR-
120 (H+). The resin was filtered off and the solution was
concentrated under high vacuum. The alcohol 23 was purified
by flash chromatography using 25% (v/v) EtOAc/toluene as
eluent (164 mg, 93%). [α]D22 + 59.9° (c 0.8, CHCl3). 1H NMR
(360 MHz, CDCl3): δ 7.25–7.33 (m, 10H, C6H5CH2), 7.12 (d,
2H, J = 8.6 Hz, m-H-MeOC6H5CH2), 7.02 (d, 2H, J = 8.6 Hz,
m-H-MeOC6H5CH2), 6.70 – 6.75 (m, 4H, o-H-MeOC6H5CH2),
5.15 (d, 1H, J = 3.4 Hz, H-1′), 4.93 (d, 1H, J = 10.6 Hz,
ArCH2–), 4.73 (d, 1H, J = 1.5 Hz, H-1), 4.60 (d, 1H, J = 12.2
Hz, ArCH2–), 4.53 (d, 1H, J = 12.0 Hz, ArCH2–), 4.49 (d, 1H,
J = 12.3 Hz, ArCH2–), 4.43 (d, 1H, J = 11.8 Hz, ArCH2–), 4.37
(d, 2H, J ≈ 11.0 Hz, ArCH2–), 4.36 (d, 1H, J = 10.6 Hz,
ArCH2–), 4.02–4.09 (m, 2H, H-3 + H-5′), 3.97 (dd, 1H, J = 1.7
Hz, 3.3 Hz, H-2), 3.80–3.88 (m, 2H, H-2′ + H-4), 3.74 (s, 3H,
1289.57662 (M + Na+).
Methyl 6-O-benzyl-2-O-(2″,3″,4″,6″-tetra-O-benzyl-α-D-
galactopyranosyl)-3-O-(4′-O-benzyl-3′,6′-dideoxy-α-D-xylo-
hexopyranosyl)-α-D-mannopyranoside (37). A solution of
trisaccharide 36 (136 mg, 0.107 mmol) in CH3CN (5 mL) was
treated with water (400 µL) and ceric ammonium nitrate
(300 mg, 0.549 mmol) for 1.5 h at room temperature. The
reaction mixture was diluted with CH2Cl2 (50 mL), washed
with water (2 × 20 mL), dried over anhydrous Na2SO4, and
evaporated. The crude material was chromatographed on
silica gel using 25% (v/v) EtOAc/toluene to give 37 (86.9 mg,
22
1
79%). [α]D + 40.3° (c 0.6, CHCl3). H NMR (360 MHz,
CDCl3): δ 7.17–7.34 (m, 30H, C6H5CH2), 5.17 (d, 1H, J = 3.6
Hz, H-1″), 5.05 (d, 1H, J = 3.6 Hz, H-1′), 4.89 (d, 1H, J = 11.5
Hz, PhCH2–), 4.84 (d, 1H, J = 11.8 Hz, PhCH2–), 4.82 (d, 1H,
J ≈ 1 Hz, H-1), 4.70 (d, J = 12.0 Hz, PhCH2–), 4.49–4.67 (m,
6H, PhCH2–), 4.47 (d, 1H, J = 11.8 Hz, PhCH2–), 4.40 (d, 1H,
J = 11.9 Hz, PhCH2–), 4.21 (d, 1H, J = 12.1 Hz, PhCH2–), 4.15
(t, 1H, J = 9.5 Hz, H-4), 4.03–4.10 (m, 2H, H-2″ + H-5″),
Zhang and Bundle / Synthesis of Tethered Trisaccharides to Probe Carbohydrate–Protein Interactions

206
3.88–3.99 (m, 6H, H-2 + H-3 + H-2′ + H-5′ + H-3″ + H-4″), tion was washed with H2O (2 × 25 mL), dried over anhydrous
3.67–3.83 (m, 3H, H-5 + H-6a + H-6b), 3.52 (dd, 1H, J = 6.5 Na2SO4, and evaporated. The residue (540 mg, 84%) was
Hz, 9.6 Hz, H-6a″), 3.45 (dd, 1H, J = 6.1 Hz, 9.6 Hz, H-6b″), sufficiently pure and used without any further purification.
3.19 (s, 3H, OMe), 2.94 (br, 1H, H-4′), 1.95 (m, 1H, H-3e′), 1H NMR (360 MHz, CDCl3): δ 7.26 (s, 4H, –C6H4–), 3.71 (d,
1.50 (m, 1H, H-3a′), 1.07 (d, 1H, J = 6.5 Hz, H-6′). High Res. 4H, J = 7.5 Hz, –CH2–), 1.74 (t, 2H, J = 7.5 Hz, –SH). High
ES-MS: 1049.467065 (M + Na+). Anal. Calcd for C61H69O14: RES. ES-MS: 170.02221 (M + Na+). Anal. Calcd for C8H10S2:
C: 71.39%, H: 6.78%. Found: C: 70.94%, H: 6.81%.
C: 56.43%, H: 5.92%. Found: C: 56.57%, H: 5.85%.
Methyl 6-O-benzyl-2-O-(2″,3″,4″,6″-tetra-O-benzyl-α-D-
Methyl 4,6-O-benzylidene-2-O-(4″,6″-O-benzylidene-α-D-
galactopyranosyl)-3-O-(4′-O-benzyl-3′,6′-dideoxy-α-D-xylo- galactopyranosyl)-3-O-(3′,6′-dideoxy-α-D-xylo-hexo-
hexopyranosyl)-4,2′-di-O-methylene-α-D-mannopyranoside pyranosyl)-α-D-mannopyranoside (42). Benzaldehyde dim-
(38). A solution of trisaccharide diol 37 (132 mg, 0.129 mmol) ethyl acetal (842 µL, 5.50 mmol) and camphorsulphonic acid
in anhydrous DMF (3 mL) was treated with sodium hydride (500 mg, 2.15 mmol) was added to a mixture of trisaccharide 1
(80% dispersion in mineral oil, 15 mg, 0.625 mmol) for 10 (372 mg, 0.77 mmol) in anhydrous DMF (6 mL). The mixture
min, dibromomethane (18 µL, 0.258 mmol) was added was stirred at room temperature for 18 h, then Et3N (1 mL)
dropwise, and the reaction was stirred at room temperature for was added to quench the reaction, and the mixture was evapo-
18 h. Methanol (1 mL) was added to quench the reaction. The rated to dryness under reduced pressure. Compound 42 was
reaction mixture was concentrated under high vacuum and obtained as colorless foam by chromatography on silica gel
22
purified by chromatography on silica gel using 5% (v/v) using 4% (v/v) MeOH/CH2Cl2 as eluent (275 mg, 54%). [α]D
EtOAc/toluene to give the tethered trisaccharide 38 (37 mg, + 52.5° (c 1.3, CHCl3). 1H NMR (360 MHz, CDCl3): δ 7.29–
27% yield). [α]D22 + 31.1° (c 1.0, CHCl3). 1H NMR (360 MHz, 7.49 (m, 10H, C6H5), 5.59 (s, 1H, C6H5CH), 5.54 (s, 1H,
CDCl3): δ 712–7.40 (m, 30H, C6H5CH2), 5.55 (d, 1H, J = 3.6 C6H5CH), 5.28 (d, 1H, J = 2.0 Hz, H-1″), 5.26 (d, 1H, J = 3.7
Hz, H-1″), 5.44 (d, 1H, J = 3.6 Hz, H-1′), 4.93 (d, 1H, J = 11.8 Hz, H-1′), 4.72 (d, 1H, J = 1.5 Hz, H-1), 4.31 (dd, 1H, J = 3.3
Hz, PhCH2–), 4.92 (d, 1H, J = 11.4 Hz, PhCH2–), 4.79 (d, 1H, Hz, 9.9 Hz, H-3), 4.20–4.29 (m, 3H, H-4″ + H-6a + H-6a″),
J ≈ 1 Hz, H-1), 4.77 (d, 1H, J = 12.1 Hz, PhCH2–), 4.34–4.66 4.14 (t, 1H, J = 9.7 Hz, H-6b), 4.08 (dd, 1H, J = 1.6 Hz, 12.6 Hz,
(m, 11H, –CHaHb– + PhCH2–), 4.17–4.23 (m, 2H, H-2 + H- H-6b″), 3.98–4.02 (m, 3H, H-2″ + H-3″ + H-2), 3.87–3.96 (m,
4), 4.09 (dd, 1H, J = 3.6 Hz, 10.6 Hz, H-3″), 3.94–4.02 (m, 3H, 2H, H-2′ + H-5′), 3.83 (br, 1H, H-5″), 3.73–3.82 (m, 2H, H-4 +
H-2 + H-3″ + H-5″), 3.87 (br d, 1H, J ≈ 1.7 Hz, H-4″), 3.81 H-5), 3.70 (br, 1H, H-4′), 3.38 (s, 3H, OMe), 2.04 (m, 1H, H-
(dq, 1H, H-2′), 3.67–3.80 (m, 3H, H-2′ + H-5 + H-6a), 3.54– 3e′), 1.76 (m, 1H, H-3a′), 1.17 (d, 3H, J = 6.6 Hz, H-6′). High
3.62 (m, 2H, H-6b + H-6a″), 3.47 (dd, 1H, J = 6.6 Hz, 9.6 Hz, Res. ES-MS: 685.24310 (M + Na+). Anal. Calcd for C33H42O14:
H-6b″), 3.24 (s, 3H, OMe), 3.05 (br, 1H, H-4′), 2.06 (m, 1H, C: 59.81%, H: 6.39%. Found: C: 58.64%, H: 6.61%.
H-3e′), 1.69 (m, 1H, H-3a′), 1.18 (d, 1H, J = 6.5 Hz, H-6′).
Methyl 4,6-O-benzylidene-2-O-(2″,3″-di-O-acetyl-4″,6″-
O-benzylidene-α-D-galactopyranosyl)-3-O-(2′,4′-di-O-
FAB MS: 1061.3 (M + Na+).
Methyl 2-O-(α-D-galactopyranosyl)-3-O-(3′,6′-dideoxy-α- acetyl-3′,6′-dideoxy-α-D-xylo-hexopyranosyl)-a-D-
D-xylo-hexopyranosyl)-4,2′-di-O-methylene-α-D-manno- mannopyranoside (43). Trisaccharide 42 (178 mg, 0.27 mmol)
pyranoside (3). To a solution of the tethered trisaccharide 38 was treated with a 1:1 mixture of Ac2O/pyridine (6 mL) at
(44 mg, 0.042 mmol) and 5% Pd on charcoal (53 mg) in room temperature for 18 h, and the mixture was evaporated to
methanol (30 mL) was added acetic acid (10 mL), the resulting dryness under reduced pressure. The acetylated trisaccharide
mixture was hydrogenated overnight. The mixture was filtered 43 was purified by chromatography on silica gel using 25% →
22
through a Millipore membrane filter (0.25 micron), and the 30% (v/v) EtOAc/toluene as eluent (195 mg, 87%). [α]D
+
organic solution was concentrated under high vacuum. The 102.6° (c 1.0, CHCl3). 1H NMR (360 MHz, CDCl3): δ 7.50 (m,
crude material was first purified by silica gel chromatography 2H, C6H5), 7.31–7.41 (m, 8H, C6H5), 5.25 (m, 3H, H-1″ +
using 10 → 20% (v/v) MeOH/CH2Cl2 as eluent, followed by PhCH), 5.42 (dd, 1H, J = 3.4 Hz, 10.9 Hz, H-2″), 5.36 (dd, 1H,
reversed phase silica gel chromatography (C18) using 10 → J = 3.1 Hz, 10.9 Hz, H-3″), 5.04–5.11 (m, 2H, H-1′ + H-2′),
15% MeOH/H2O as eluent to give pure tethered trisaccharide 5.00 (br, 1H, H-4′), 4.64 (d, 1H, J = 1.6 Hz, H-1), 4.52 (1H, dd,
3 (19 mg, 90 %) which was lyophilized. [α]D22 + 78.3° (c 0.3, J = 0.9 Hz, 3.1 Hz, H-4″), 4.27 (dd, 1H, J = 1.3 Hz, 12.6 Hz,
H2O). 1H and 13C NMR (500 MHz, D2O): See Tables 1 and 2. H-6a), 4.25 (dd, 1H, J = 2.7Hz, 10.3 Hz, H-3), 4.22 (dd, 1H,
ES-MS: 521.18665 (M + Na+). Anal. Calcd for C20H34O14: C: J = 4.4 Hz, 9.9 Hz, H-6a″), 4.05–4.13 (m, 2H, H-6b + H-6b″),
67.55%, H: 7.26%; Found: C: 67.35%, H: 7.24%.
4.00 (‘t’, 1H, J ≈ 2.2 Hz, H-2), 3.94 (dq, 1H, H-5′), 3.87 (t, 1H,
J = 10.3 Hz, H-4), 3.73–3.83 (m, 2H, H-5 + H-5″), 3.38 (s, 3H,
OMe), 2.11 (m, 1H, H-3e′), 2.10 (s, 3H, OAc), 2.09 (s, 3H,
OAc), 2.08 (s, 3H, OAc), 1.88 (m, 1H, H-3a′), 1.44 (s, 3H,
OAc), 1.10 (d, 1H, J = 6.5 Hz, H-6′). High Res. ES-MS:
853.28975 (M + Na+). Anal. Calcd for C41H50O18: C: 59.27%,
H: 6.07%. Found: C: 59.14%, H: 6.24%.
1,4-benzene-di-methanethiol (41). Thiourea (634 mg,
8.3 mmol) was added to a solution of α,α′-dibromo-p-xylene
(1.0 g, 3.8 mmol) in acetone (20 mL). The mixture was heated
to reflux for 1 h, and then cooled to room temperature. A
precipitate of 40 was filtered off, and dried under high
vacuum. The solid was dissolved in 2M NaOH (20 mL), the
mixture was heated to reflux under argon for 2 h, and the
Methyl 4-O-benzoyl-6-bromo-6-deoxy-2-O-(2″,3″-di-O-
solution was neutralized to pH ~2 with 2M HCl. The acetyl-4″-O-benzoyl-6″-bromo-6″-deoxy-α-D-galacto-
dimercaptane 41 was extracted into CH2Cl2, the organic solu- pyranosyl)-3-O-(2′,4′-di-O-acetyl-3′,6′-dideoxy-α-D-xylo-
Israel Journal of Chemistry
40
2001

207
hexopyranosyl)-α-D-mannopyranoside (44). A mixture con- Hz, 15.2 Hz, H-6b″), 2.49 (dd, 1H, J = 9.3 Hz, 15.0 Hz, H-6b),
taining compound 43 (275 mg, 0.33 mmol), N-bromosuccin- 2.03–2.10 (m, 10H, OAc + H-3a′), 1.83 (m, 1H, H-3e′), 1.35
imide (176 mg, 0.59 mmol) and barium carbonate (262 mg, (s, 3H, OAc), 1.08 (d, 3H, J = 6.6 Hz, H-6′). High Res. ES-
1.32 mmol) in anhydrous carbon tetrachloride (10 mL) was MS: 1019.28002 (M + Na+).
heated to reflux for 3 h under the an atmosphere of argon. The
Methyl 6-thio-6-deoxy-2-O-(6″-thio-6″-deoxy-α-D-gal-
actopyranosyl)-3-O-(3′,6′-dideoxy-α-D-xylo-hexopyranosyl)-
6,6″-(1,4-benzene di methylene)-α-D-mannopyranoside (4).
The protected trisaccharide 45 (14.0 mg) was dissolved in
anhydrous MeOH (3 mL), a solution of NaOMe in anhydrous
MeOH (1.27 M, 0.5 mL) was added, and the reaction was
continued for 18 h. The mixture was neutralized with a few drop
of HOAc, and the solution was concentrated to dryness under
reduced pressure. The crude material was purified by prepara-
tive HPLC on reversed phase silica gel (C18) using 0% → 80%
(v/v) MeOH/H2O as eluent to afforded the tethered trisaccha-
ride 4 in pure form (7.0 mg, 80%) which was lyophilized.
[α]D22 + 10.8° (c 0.2, H2O). 1H and 13C NMR (500 MHz, D2O):
See Tables 1 and 2. High Res. ES-MS: 643.18556 (M + Na+).
solid was filtered off and washed with more EtOAc. The
organic solution was evaporated to dryness under reduced
pressure and the crude material was purified by chromatogra-
phy on silica gel using 15% (v/v) EtOAc/toluene to afford the
dibromido derivative of trisaccharide 44 (205 mg, 63%) as
colorless foam. [α]D22 + 122.5° (c 0.4, CHCl3). 1H NMR (360
MHz, CDCl3): δ 8.09 (m, 2H, o-H-C6H5CO), 7.95 (m, 2H,
o-H-C6H5CO), 7.56–7.65 (m, 2H, p-H-C6H5CO), 7.41–7.53
(m, 4H, m-H-C6H5CO), 5.81 (‘d’, 1H, J ≈ 2.9 Hz, H-4″), 5.67
(d, 1H, J = 3.8 Hz, H-1″), 5.53 (t, 1H, J = 9.9 Hz, H-4), 5.49
(dd, 1H, J = 3.3 Hz, 10.9 Hz, H-3″), 5.21 (dd, 1G, J = 3.8 Hz,
10.9 Hz, H-2″), 5.05 (br, 1H, H-4′), 5.01 (d, 1H, J = 3.5 Hz,
H-1′), 4.94 (m, 2H, H-1 + H-2′), 4.42 (dd, 1H, J = 2.4 Hz, 9.9
Hz, H-3), 4.38 (m, 1H, H-5″), 4.13 (‘t’, 1H, J ≈ 2.1 Hz, H-2),
4.01 (dq, 1H, H-5′), 3.95 (m, 1H, H-5), 3.49 (s, 3H, OMe),
3.38–3.48 (m, 4H, H-6a + H-6b + H-6a″ + H-6b″), 2.18 (s, 3H,
OAc), 2.08 (m, 4H, OAc + H-3e′), 1.97 (s, 3H, OAc), 1.89 (m,
1H, H-3a′), 1.50 (s, 3H, OAc), 1.15 (d, 3H, J = 6.4 Hz, H-6′).
High Res. ES-MS: 1011.10930 (M + Na+). Anal. Calcd for
C41H48O18Br2: C: 49.81%, H: 4.89%. Found: C: 50.44%, H:
4.97%.
Acknowledgments. We thank Dr. C.C. Ling for valuable discus-
sions and for assistance with the production of the molecular
graphics. Financial support for the research was provided by the
University of Alberta and a research operating grant from the
Natural Science and Engineering Research Council of Canada.
REFERENCES AND NOTES
(1) Cygler, M.; Rose, D.R.; Bundle, D.R. Science 1991,
253, 442–446.
(2) Bundle, D.R.; Baumann, H.; Brisson, J.-R.; Gagne,
S.M.; Zdanov, A.; Cygler, M. Biochemistry 1994, 33,
5183–5192.
(3) Zdanov, A.; Li, Y.; Bundle, D.R.; Deng, S.-J.;
MacKenzie, C.R.; Narang S.A.; Young, N.M.; Cygler,
M. Proc. Natl. Acad. Sci. U.S.A., 1994, 91, 6423–6427.
(4) Bundle, D.R. In Carbohydrates; Hecht, S., Ed.; Oxford
University Press: Oxford, 1998; pp 370–440.
(5) (a) Carver, J.P.; Pure Appl. Chem. 1993, 65, 763–770. (b)
Carver, J.P.; Michnick, S.W.; Imberty, A.; Cumming,
D.A. In Carbohydrate Recognition in Cellular Function;
Bock, G. and Harnett, S., Eds.; Ciba Foundation Sympo-
sium; Wiley: Chichester, 1989; Vol. 145, pp 6–26.
(6) Searle, M.S.; Williams, D.H. J. Am. Chem. Soc. 1992,
114, 10690–10697.
Methyl 4-O-benzoyl-6-thio-6-deoxy-2-O-(2″,3″-di-O-
acetyl-4″-O-benzoyl-6″-thio-6″-deoxy-α-D-galacto-
pyranosyl)-3-O-(2′,4′-di-O-acetyl-3′,6′-dideoxy-α-D-xylo-
hexopyranosyl)-6,6″-(1,4-benzene di methylene)-α-D-manno-
pyranoside (45). The dimercaptane 41 (482.1 mg, 2.83 mmol)
was dissolved in anhydrous THF (8 mL) under argon, potas-
sium tert-butoxide (668.8 mg, 5.66 mmol) was added, and the
mixture was stirred for 30 min. Anhydrous ether (20 mL) was
added, the mixture was stirred for 10 min, the yellowish
precipitate was filtered, washed with more anhydrous ether
(30 mL), and dried under high vacuum (650 mg).
The trisaccharide 44 (18.0 mg, 0.018 mmol) was dissolved
in anhydrous DMF (1.0 mL) under argon, the dipotassium salt
of 41 (21.0 mg, 0.085 mmol) and potassium iodide (5 mg,
0.030 mmol) were added, and the mixture was heated to 50 °C
for 2 h. More dipotassium salt of 41 (27.4 mg, 0.11 mmol) was
added, and the reaction was continued overnight at 50 °C. The
mixture was concentrated and purified by repeated chroma-
tography on silica gel using 15% (v/v) EtOAc/toluene as
eluent. The product which has a Rf of 0.49 (30% AcOEt/
(7) Mammen, M.; Shakhnovich, E.I.; Whitesides, G.M. J.
Org. Chem. 1998, 63, 3168–3175.
(8) Bundle, D.R.; Alibes, R.; Nilar, S.; Otter, A., Warwas;
M.; Zhang, P. J. Am. Chem. Soc. 1998, 120, 5317–
5318.
22
toluene) was isolated (3.0 mg, 17%). [α]D + 81.4° (c 0.2,
1
CHCl3). H NMR (360 MHz, CDCl3): δ 8.08 (m, 2H, o-H-
C6H5CO), 7.91 (m, 2H, o-H-C6H5CO), 7.05–7.65 (m, 10H, m-
H-C6H5CO + p-H-C6H5CO + –CH2C6H4CH2–), 6.23 (‘d’, 1H,
J ≈ 2.9 Hz, H-4″), 5.32 (t, 1H, J = 9.9 Hz, H-4), 5.23 (d, 1H, J
= 4.0 Hz, H-1″), 5.15 (dd, 1H, J = 2.9 Hz, 10.9 Hz, H-3″), 5.00
(dd, 1H, J = 3.9 Hz, 11.0 Hz, H-2″), 4.86–4.97 (m, 3H, H-1′ +
H-2′ + H-4′), 4.18 (dd, 1H, J = 2.4 Hz, 10.0 Hz, H-3), 3.40–
3.89 (m, 9H, H-1 + H-2 + H-3 + H-5 + H-5″ + –CH2C6H4CH2–),
3.39 (s, 3H, OMe), 2.93 (dd, 1H, J = 2.2 Hz, 15.0 Hz, H-6a),
2.76 (dd, 1H, J = 8.1 Hz, 15.0 Hz, H-6a″), 2.67 (dd, 1H, J = 6.2
(9) Alibes, R.; Bundle, D.R. J. Org. Chem. 1998, 63, 6288–
6301.
(10) Lowary, T.L.; Eichler, E.; Bundle, D.R. J. Org. Chem.
1995, 60, 7316–7327.
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29–40. Kondo, Y; Noumi, K.; Kitagawa, S.; Hirano, S.
Carbohydr. Res. 1983, 123, 157–159. Szeja, W.
Carbohydr. Res. 1983, 115, 240–242.
(12) Pozsgay, V.; Glaudemans, C.P.J.; Robbins, J.B.;
Zhang and Bundle / Synthesis of Tethered Trisaccharides to Probe Carbohydrate–Protein Interactions

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