Cyclodextrin - a Carrieir of Synthon? Synthesis of per-O-Methyl-β-cyclodextrin-GM3

Article abstract of DOI:10.1081/CAR-100102540

Cyclodextrins are currently under imnvestigation for their utility as drug delivery agents. One property of the parent cyclic oligosaccharides which requires further modification for advanced drug delivery applications is that of site-directing capability

Full text of DOI:10.1081/CAR-100102540

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CYCLODEXTRIN—A CARRIER OR SYNTHON? SYNTHESIS
OF PER-O-METHYL-β- CYCLODEXTRIN-GM3
W. Haque a & J. Diakur b
a Medicure , 24-62 Scurfield Blvd., Winnipeg, Manitoba, R3Y 1M5, Canada
b Faculty of Pharmacy and Pharmaceutical Sciences , University of Alberta , Edmonton,
Alberta, T6G 2N8, Canada
Published online: 11 Dec 2006.
To cite this article: W. Haque & J. Diakur (2001) CYCLODEXTRIN—A CARRIER OR SYNTHON? SYNTHESIS OF PER-O-
METHYL-β- CYCLODEXTRIN-GM3 , Journal of Carbohydrate Chemistry, 20:1, 17-29, DOI: 10.1081/CAR-100102540
To link to this article: http://dx.doi.org/10.1081/CAR-100102540
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J. CARBOHYDRATE CHEMISTRY, 20(1), 17–29 (2001)
CYCLODEXTRIN—A CARRIER OR SYNTHON?
SYNTHESIS OF PER-O-METHYL-ꢀ-
CYCLODEXTRIN-GM3
W. Haque1 and J. Diakur2,*
1Medicure, 24-62 Scurfield Blvd., Winnipeg, Manitoba, Canada R3Y 1M5,
2Faculty of Pharmacy and Pharmaceutical Sciences, University of
Alberta, Edmonton, Alberta, Canada T6G 2N8
ABSTRACT
Cyclodextrins are currently under investigation for their utility as drug
delivery agents. One property of the parent cyclic oligosaccharides which re-
quires further modification for advanced drug delivery applications is that of
site-directing capability. We have utilized ꢀ-cyclodextrin as a starting material
for the synthesis of the GM3 trisaccharide and have conjugated this targeting
ligand to a derivative of the parent ꢀ-cyclodextrin. Synthesis of this cyclodex-
trin derivative, namely 18, is presented.
INTRODUCTION
Pharmaceutical applications for the family of cyclic oligosaccharides collec-
tively knowns as the cyclodextrins (CyDs) have been contemplated for some time.1
Their ability to form inclusion complexes has been exploited for altering the chem-
ical and physical properties of guest (drug) molecules including solubility, in vivo
stability, reduction of toxicity and irritancy, etc. In order for these drug carriers to
function as advanced drug delivery systems, their ability to deliver drugs to a tar-
geted site requires optimization.2
Along with a relatively hydrophobic interior cavity for the molecular en-
capsulation of guest molecules, the architecture of CyDs is such that two faces
are apparent. The wider opening of the cyclic oligosaccharide is populated with
secondary hydroxyl groups while the opposite face houses an array of primary
17
Copyright © 2001 by Marcel Dekker, Inc.
www.dekker.com

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18
HAQUE AND DIAKUR
hydroxyls. These faces are separated by a distance of at least 7.9 Å.3 An imme-
diate consequence of this spatial arrangement is that the two faces can be ex-
ploited in a regioselective manner to provide both a region for drug complexa-
tion/conjugation and a region for arming with targeting ligands. Furthermore, the
symmetric nature of the CyD skeleton allows for the installation of the latter in
a multivalent fashion.4
The ganglioside GM3 is present on normal melanocytes. However, due to al-
tered levels of expression in melanoma, it can be recognized by murine cytotoxic
T cells and suppressor T cells as a tumor-associated antigen.5 The synthesis of this
antigen has been reported by several groups.6 We report herein, the synthesis of
this epitope from cyclodextrin, and the conjugation of this trisaccharide as a po-
tential targeting ligand, to an amino-activated ꢀ-CyD derivative.
RESULTS AND DISCUSSION
The cyclodextrin skeleton is well suited for providing rapid access to inter-
mediates for further elaboration into the lacto-series backbone in the sense that the
glycosidic linkage at the 4-hydroxyl position can be viewed as a protecting group.
Thus, as previously reported by Sato et al.,7 glucose synthon 1 can be readily ob-
tained by controlled acid hydrolysis of the fully benzylated ꢀ-CyD followed by
subsequent acetylation of the anomeric and 4-hydroxyl moieties as outlined in
Scheme 1. Anomeric activation as the chloride 2 is then accomplished by treatment
with HCl saturated ether.8 Reaction of 2 with p-nitrophenethyl alcohol using
freshly prepared silver-sieves as the promoter9 provided PNPE-glucose derivative
3 in 84 % yield as the ꢀ-glycoside (J1,2 ꢁ 7.5 Hz). De-O-acetylation of 3 afforded
glycosyl acceptor 4.
Scheme 1.

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19
Scheme 2.
Glycosidation of 4 with acetobromogalactose in the presence of silver salts
(Scheme 2) yielded the fully blocked lactose-PNPE derivative 5 in good yield
(75%). Compound 5 was subjected to de-O-esterification to give tetrol 6 (83%).
Simultaneous protection of the 4′- and 6′-hydroxy groups was subsequently real-
ized by reaction with ꢂ,ꢂ′-dimethoxytoluene and catalytic p-toluenesulfonic acid
to provide the desired sialyl acceptor as the 2′,3′-diol 7 (90% yield). Introduction
of this diol to sialic acid donor 1510 (1.68 equiv) in the presence of silver salts
(Scheme 3) led to a crude mixture containing starting diol 7 along with sialic acid
Scheme 3.

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HAQUE AND DIAKUR
by-products (Neu5Ac-2-OH and 2,3-dehydro-Neu5Ac) and the desired sialoside
8. Unfortunately, trisaccharide 8 could not be cleanly separated from this com-
plex mixture, and column chromatography over silica gel at this stage only al-
lowed for the separation of the Neu5Ac-2-OH derivative from the remainder of
the mixture. Partial separation of diol 7 from the remaining mixture was accom-
plished by crystallization from chloroform leaving a mixture consisting mainly of
the 2,3-dehydro-NeuNAc and 8. This mixture was then acetylated using 2:1 pyri-
dine-acetic anhydride, and the resulting product was readily separated to furnish
the desired sialoside 9 (29% overall yield from 7). Homonuclear decoupling ex-
periments provided confirmation of several of the proton NMR resonances in the
spectrum of this trisaccharide, and in particular, the downfield shift of H-2′ to ꢃ
5.15 ppm which confirmed the regioselectivity of the glycosidation reaction.
Next, the nitro moiety of 9 was converted into the p-NTFA derivative by the two
step sequence involving initial treatment with Zn/CuSO4 in tetrahydrofuran-
acetic acid-water,11 to give the free amine 10. Protection of this functionality as
the p-NTFA derivative was accomplished with pyridine-trifluoracetic anhydride
to provide the desired trisaccharide 11 (90% yield from the fully protected nitro
1
derivative 9). Although the H NMR spectrum of 11 was nearly identical to that
of the nitro derivative 9, the two proton doublet at ꢃ 8.0 ppm normally associated
with the 2-(p-nitrophenyl)ethyl derivatives had now shifted upfield. As well, the
19F NMR spectrum of 11 displayed a fluorine singlet at ꢃ -75.84 ppm.
Sequential deprotection of 11 was initiated by hydrogenation over wet 5%
palladium-on-carbon containing a small amount of acetic acid to give intermedi-
ate 12. Subsequent reaction of 12 with sodium methoxide in methanol revealed
that de-O-acetylation of the 2′ hydroxyl was somewhat sluggish, however,
prolonged treatment provided two lower Rf compounds. Neutralization of this re-
action mixture followed by subsequent separation by column chromatography
over silica gel gave the totally deblocked trisaccharide 13 (62%) followed by the
ninhydrin positive amino compound 14 (12%). From the observed sluggish
de-O-esterification of the acetylated hydroxyl group at C-2 of the galactose
moiety, it appears that the topology of the trisaccharide is such that the sialic acid
moiety has reduced steric accessibility to this region. The ꢂ-stereochemistry of
the glycosylation reaction was firmly established by the fact that the signal for
H-3′′e of the sialic acid residue was located at ꢃ 2.74 ppm,12 and the remaining
spectral data was consistent with the proposed structure. The amino derivative 14
was also available by treatment of 13 with ammonium hydroxide. The conjuga-
tion of a galactose ligand to mono-6-amino-6-deoxy-ꢀ-CyD using the isothio-
cyanate coupling strategy has been reported to proceed in reasonable yield.13 In
order to investigate the efficiency of conjugation of GM3 analog 14 with a CyD
carrier, the known mono-6-amino-per-O-methyl CyD 1614 was synthesized. Re-
action with thiophosgene provided isothiocyanate 17 in quantitative yield
(Scheme 4). Finally, conjugation of 17 with 14 in pyridine provided 18 in low
(23%) yield.

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21
Scheme 4.
CONCLUSION
The synthesis of a cyclodextrin derivative armed with a targeting ligand,
namely 18, has been accomplished, albeit in low yield. The parent cyclic oligosac-
charide has served as a convenient starting material to gain entry into the lactose

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HAQUE AND DIAKUR
backbone ganglioside series. Work is currently underway to prepare the heptava-
lent GM3-CyD.
EXPERIMENTAL
General Methods. NMR spectra were recorded on a Bruker AM 300 spec-
trometer (1H: 300 MHz; 19F: 282 MHz; 13C: 75 MHz) or Varian Unity 500 spec-
trometer in CDCl3 or D2O solution unless otherwise stated. Chemical shifts in
CDCl3 solutions are reported in parts per million downfield from TMS, or in the
case of D2O solutions, using HOD set at ꢃ 4.82 (25 °C) unless otherwise specified.
13C NMR spectral assignments were aided by the J-MOD technique.15 19F NMR
spectra are reported in parts per million using external C6F6 (set at 0.0 ppm). Fast
atom bombardment (FAB) mass spectra were obtained with a Kratos AE1 MS9
mass spectrometer while Electrospray (ES) mass spectra were obtained with a Mi-
cromass VG ZabSpec TOF instrument in the negative ion mode. Reactions were
monitored by thin-layer chromatography (TLC) on Kieselgel 60 F254 (Merck) and
visualization was accomplished by charring with 5% methanolic sulfuric acid. Col-
umn chromatography was performed using Merck 9385 silica gel (40 - 63ꢄ). Cy-
clodextrin was dried in vacuo over P2O5 at 60 °C prior to use. Chloroform was dis-
tilled from P2O5 and pyridine was distilled from CaH2 and stored over molecular
sieves 3 Å. Activated powdered molecular sieves 4 Å were obtained from Aldrich
Chemical Company and were dried in vacuo at 100 °C.
2-(p-Nitrophenyl)ethyl 2,3,6-Tri-O-benzyl-ꢀ-D-glucopyranoside (4). A solu-
tion of crude 2,3,6-tri-O-benzyl-ꢂ-D-glucopyranose7 (3.15 g, 7 mmol) in 2:1 pyri-
dine-acetic anhydride (75 mL) was stirred at room temperature for 48 h. The reac-
tion mixture was then concentrated and the residue was subjected to column
chromatography over silica gel using 7:1 hexane-ethyl acetate to provide 2.82 g (68
%) of 1,4-di-O-acetyl-2,3,6-tri-O-benzyl-ꢂ/ꢀ-D-glucopyranoside 1: Rf 0.29 (3:1
1
hexane-ethyl acetate); H NMR (CDCl3, 500 MHz) ꢃ 7.38 - 7.22 (m, 15H, aro-
matic), 6.34 (d, 0.5H, J1,2 ꢁ 3.9 Hz, H-1ꢃ), 5.63 (d, 0.5H, J1,2 ꢁ 7.8 Hz, H-1ꢀ),
5.11 (t, 0.5H, J ꢁ 10.3 Hz, H-4ꢂ), 5.10 (t, 0.5H, J ꢁ 10.3 Hz, H-4ꢀ), 4.87 (d, 0.5H,
J ꢁ 11.7 Hz, OCH2C6H5), 4.80 (d, 0.5H, J ꢁ 11.7 Hz, OCH2C6H5), 4.76 (d, 0.5H,
J ꢁ 11.2 Hz, OCH2C6H5), 4.72 (d, 0.5H, J ꢁ 11.7 Hz, OCH2C6H5), 4.68 (d, 0.5H,
J ꢁ 11.2 Hz, OCH2C6H5), 4.63 (m, 1.5H, OCH2C6H5), 4.51 (d, 0.5H, J ꢁ 11.7 Hz,
OCH2C6H5), 4.50 (d, 0.5H, J ꢁ 12.2 Hz, OCH2C6H5), 4.47 (d, 0.5H, J ꢁ 12.2 Hz,
OCH2C6H5), 4.46 (d, 0.5H, J ꢁ 12.2 Hz, OCH2C6H5), 3.93 (dt, 0.5H, J5,6 ꢁ 4.4
Hz, H-5), 3.86 (t, 0.5H, J ꢁ 9.5 Hz, H-3ꢂ), 3.71 (dd, 1H, J2,3 ꢁ 9.5 Hz, H-2ꢂ), 3.66
(t, 0.5H, J ꢁ 9.0 Hz, H-3ꢀ), 3.65 - 3.60 (m, 1H), 3.56 – 3.43 (m, 2H), 2.17 (s, 1.5H,
COCH3), 2.05 (s, 1.5H, COCH3), 1.84 (s, 1.5H, COCH3), 1.82 (s, 1.5H, COCH3).
Compound 1 (1.5 g, 2.8 mmol) was dissolved in hydrogen chloride saturated
ethyl ether, and the reaction was stirred at room temperature and monitored by
TLC (3:1 hexane-ethyl acetate). Upon completion of the reaction, argon was
passed through the solution for 30 min. The organic solution was then diluted with
ether and washed with water, saturated aqueous sodium hydrogen carbonate, brine,

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CYCLODEXTRIN
23
dried (Na2SO4), filtered and concentrated. Column chromatography over silica gel
using 4:1 hexane-ethyl acetate gave 860 mg (60%) of 4-O-acetyl-2,3,6-tri-O-ben-
1
zyl-ꢂ-D-glucopyranosyl chloride 2: Rf 0.37 (3:1 hexane-ethyl acetate); H NMR
(CDCl3, 500 MHz)ꢃ 7.38 - 7.22 (m, 15H, aromatic), 6.03 (d, 1H, J1,2 ꢁ 3.5 Hz, H-
1), 5.13 (t, 1H, J ꢁ 10.3 Hz, H-4ꢂ or ꢀ), 5.11 (t, 1H, J ꢁ 10.3 Hz, H-4ꢂ or ꢀ), 4.88
(d, 1H, J ꢁ 11.7 Hz, OCH2C6H5), 4.73 (d, 1H, J ꢁ 11.7 Hz, OCH2C6H5), 4.68 (d,
1H, J ꢁ 11.7 Hz, OCH2C6H5), 4.65 (d, 1H, J ꢁ 11.7 Hz, OCH2C6H5), 4.51 (d, 1H,
J ꢁ 11.7 Hz, OCH2C6H5), 4.47 (d, 1H, J ꢁ 11.7 Hz, OCH2C6H5), 3.93 (dt, 1H, J5,6
ꢁ 3.9 Hz, H-5), 3.96 (t, 1H, J ꢁ 9.5 Hz, H-3), 3.76 (dd, 1H, H-2), 3.51 (dd, 1H,
J
6a,6b ꢁ 11.0 Hz, H-6a), 3.46 (dd, 1H, H-6b), 1.84 (s, 3H, COCH3).
To a stirred suspension of dry powdered activated molecular sieves 4 Å (3.1
g) and toluene was added dropwise, a solution of silver trifluoromethanesulfonate
(2.6 g, 10 mmol) in toluene (25 mL), and the mixture was stirred in the dark at am-
bient temperature for 5 h. The solid was allowed to settle and then decanted. To the
suspension of Ag-sieves was added p-nitrophenethyl alcohol (1.1 g, 6.6 mmol) and
the reaction stirred for an additional 30 min. At this time, the suspension was
cooled to 0 °C under argon, and a solution of chloride 2 (850 mg, 1.7 mmol) in dry
chloroform (17 mL) was added dropwise. The mixture was allowed to warm to am-
bient temperature and the reaction progress was monitored by TLC (3:1 hexane-
ethyl acetate). After 20 h, the mixture was filtered through Celite and the pad
washed several times with chloroform. The combined organic filtrate was washed
with saturated aqueous sodium bicarbonate, brine, dried (Na2SO4), filtered and
concentrated. Column chromatography over silica gel using 4:1 hexane-ethyl ac-
etate gave 900 mg (84%) of 2-(p-nitrophenyl)ethyl 4-O-acetyl-2,3,6-tri-O-benzyl-
ꢀ-D-glucopyranoside 3: Rf 0.14 (4:1 hexane-ethyl acetate); [ꢂ]D -4.5° (c 0.25, chlo-
roform); 1H NMR (CDCl3, 500 MHz) ꢃ 8.05 (d, 2H, J ꢁ 9.0 Hz, aromatic), 7.4 -
7.1 (m, 17H, aromatic), 4.96 (bt, 1H, J ꢁ 9.0 Hz, H-4), 4.78 (d, 1H, J ꢁ 12.0 Hz,
OCH2C6H5), 4.65 (d, 1H, J ꢁ 11.5 Hz, OCH2C6H5), 4.59 (bd, 2H, J ꢁ 12.0 Hz,
OCH2C6H5), 4.51 (s, 2H, OCH2C6H5), 4.42 (d, 1H, J1,2 ꢁ 7.5 Hz, H-1), 4.24 (dt,
1H, Jd ꢁ 9.5 Hz, Jt ꢁ 6.0 Hz, OCH2), 3.80 (dt, 1H, Jt ꢁ 7.0 Hz, OCH2), 3.58 (t,
1H, J ꢁ 9.0 Hz, H-3), 3.56 - 3.52 (m, 3H, H-5, H-6a, H-6b), 3.44 (dd, 1H, J2,3 ꢁ
9.0 Hz, H-2), 3.04 (bt, 2H, OCH2CH2), 1.83 (s, 3H, COCH3).
A solution of 3 (890 mg, 1.39 mmol) in methanolic sodium methoxide was
stirred at room temperature overnight. The reaction mixture was then deionized
with IR 120 (Hꢅ) resin, filtered and concentrated. The residue was taken up in
chloroform and washed with aqueous saturated sodium bicarbonate solution, brine,
dried (Na2SO4), filtered and concentrated to give 830 mg (quantitative) of 4 as a
foam: Rf 0.37 (20:10:1 hexane-ethyl acetate-ethanol); [ꢂ]D -9.5° (c 0.5, chloro-
form); 1H NMR (CDCl3, 500 MHz) ꢃ 8.05 (d, 2H, J ꢁ 9.0 Hz, aromatic), 7.4 - 7.1
(m, 17H, aromatic), 4.90 (d, 1H, J ꢁ 11.5 Hz, OCH2C6H5), 4.70 (d, 1H, J ꢁ 11.5
Hz, OCH2C6H5), 4.66 (d, 1H, J ꢁ 12.0 Hz, OCH2C6H5), 4.60 (d, 1H, J ꢁ 11.5 Hz,
OCH2C6H5), 4.59 (d, 1H, J ꢁ 12.0 Hz, OCH2C6H5), 4.55 (d, 1H, J ꢁ 11.5 Hz,
OCH2C6H5), 4.41 (d, 1H, J1,2 ꢁ 7.5 Hz, H-1), 4.23 (dt, 1H, Jd ꢁ 9.5 Hz, Jt ꢁ 6.0
Hz, OCH2), 3.79 (dt, 1H, Jt ꢁ 6.5 Hz, OCH2), 3.77 (dd, 1H, J6a,6b ꢁ 10.5 Hz, J5,6a
ꢁ 3.5 Hz, H-6a), 3.65 (dd, 1H, J5,6b ꢁ 5.0 Hz, H-6b), 3.58 (td, 1H, Jt ꢁ 9.0 Hz,

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HAQUE AND DIAKUR
J
4,OH ꢁ 2.0 Hz, H-4), 3.46 (ddd, 1H, H-5), 3.40 (dd, 1H, J2,3 ꢁ 9.0 Hz, H-2), 3.38
(t, 1H, Jt ꢁ 9.0 Hz, H-3), 3.04 (bt, 2H, OCH2CH2).
Anal. Calcd for C35H37O8N (599.66): C, 70.10; H, 6.22; N, 2.34. Found: C,
69.79; H, 6.11; N, 2.33.
2-( p-Nitrophenyl)ethyl O-(2,3,4,6-Tetra-O-acetyl-ꢀ-D-galactopyranosyl)-
(1→4)-2,3,6-tri-O-benzyl-ꢀ-D-glucopyranoside (5). A mixture of 4 (2.38 g, 3.97
mmol), silver carbonate (5.47 g, 19.9 mmol), catalytic silver trifluoromethanesul-
fonate (51 mg, 0.2 mmol), and powdered Drierite (10 g) in dry dichloromethane
(25 mL) was cooled to -70 °C under argon. To the reaction mixture was added
dropwise, a solution of acetobromogalactose (3.27 g, 7.9 mmol) in dry toluene (10
mL), and the mixture was allowed to slowly warm to -10 °C and stirred overnight
at this temperature. The reaction mixture was then filtered through Celite and con-
centrated. Column chromatography over silica gel using 2:1 hexane-ethyl acetate
yielded 2.77 g (75%) of 5: Rf 0.11 (2:1 hexane-ethyl acetate); [ꢂ]D -1.3° (c 0.25,
chloroform); 1H NMR ꢃ 8.05 (d, 2H, J ꢁ 9.0 Hz, aromatic), 7.40 - 7.10 (m, 15H,
aromatic), 7.10 (m, 2H, aromatic), 5.25 (dd, 1H, J3′,4′ ꢁ 3.5 Hz, J4′,5′ ꢁ 1.0 Hz, H-
4′), 5.10 (dd, 1H, J2′,3′ ꢁ 10.5 Hz, J1′,2′ ꢁ 8.0 Hz, H-2’), 4.91 (d, 1H, J ꢁ 11.0 Hz,
OCH2C6H5), 4.81 (dd, 1H, H-3’), 4.78 (d, 1H, J ꢁ 11.0 Hz, OCH2C6H5), 4.74 (d,
1H, J ꢁ 12.0 Hz, OCH2C6H5), 4.61 (s, 2H, OCH2C6H5), 4.60 (d, 1H, J1′,2′ or J1,2
ꢁ 8.0 Hz, H-1′ or H-1), 4.47 (d, 1H, J ꢁ 12.0 Hz, OCH2C6H5), 4.38 (d, 1H, J1,2 or
′
J
1′,2′ ꢁ 8.0 Hz, H-1 or H-1′), 4.21 (dt, 1H, Jd ꢁ 9.5 Hz, Jt ꢁ 6.0 Hz, OCH2), 3.99
(dd, 1H, J6′a,6′bꢁ 11.0 Hz, J5′,6a′ ꢁ 7.5 Hz, H-6’a), 3.93 (t, 1H, J ꢁ 9.5 Hz, H-4),
3.84 (dd, 1H, J5′,6b′ ꢁ 6.0 Hz, H-6’b), 3.77 (dt, 1H, Jt ꢁ 6.0 Hz, OCH2), 3.72 - 3.69
(m, 2H, H-6a and H-6b), 3.56 (t, 1H, Jt ꢁ 9.0 Hz, H-3), 3.52 (bdt, 1H, J4′,5′ ꢁ 1.0
Hz, H-5’), 3.38 (dd, 1H, H-2), 3.37 (dt, 1H, Jd ꢁ 9.0 Hz, Jt ꢁ 2.5 Hz, H-5), 3.07 -
3.01 (m, 2H, OCH2CH2), 2.09 (s, 3H, COCH3), 1.98 (s, 3H, COCH3), 1.96 (s, 3H,
COCH3), 1.95 (s, 3H, COCH3).
Anal. Calcd for C49H55O17N (929.35): C, 63.27; H, 5.96; N, 1.51. Found: C,
62.88; H, 5.90; N, 1.48.
2-(p-Nitrophenyl)ethyl (4,6-O-Benzylidene-ꢀ-D-galactopyranosyl)-(1→4)-
2,3,6-tri-O-benzyl-ꢀ-D-glucopyranoside (7). To a solution of 5 (2.65 g, 2.85 mmol)
in dry methanol (20 mL) was added a solution of sodium metal (61 mg, 2.6 mmol)
in dry methanol (7 mL) and the solution was stirred for 18 h at room temperature.
The mixture was then neutralized with IR 120 (Hꢅ) resin, filtered and the solvents
were removed to give 2.1 g (quantitative) of 2-(p-nitrophenyl)ethyl (ꢀ-D-galac-
topyranosyl)-(1→4)-2,3,6-tri-O-benzyl-ꢀ-D-glucopyranoside 6 which was used
directly in the next step without further purification or characterization: Rf 0.18
(5:5:1 hexane-ethyl acetate-ethanol).
A solution of tetrol 6 (2.1 g, 2.78 mmol), benzaldehyde dimethyl acetal (740
mg, 4.87 mmol), and p-toluenesulphonic acid monohydrate (27 mg, 0.14 mmol) in
dry acetonitrile (30 mL) was stirred at room temperature overnight. The reaction
was quenched with an excess of triethylamine, and the reaction mixture was then
concentrated. Column chromatography over silica gel using chloroform then 100:1
chloroform-methanol as eluant gave 1.96 g (83%) of 7: Rf 0.22 (30:1 chloroform-

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CYCLODEXTRIN
25
methanol); [ꢂ]D ꢅ1° (c 1, chloroform); 1H NMR ꢃ 8.05 (d, 2H, J ꢁ 9.0 Hz, aro-
matic), 7.50 - 7.30 (m, 20H, aromatic), 7.10 (m, 2H, aromatic), 5.45 (s, 1H, ben-
zylidene), 4.99 (d, 1H, J ꢁ 11.0 Hz, OCH2C6H5), 4.90 (d, 1H, J ꢁ 11.0 Hz,
OCH2C6H5), 4.71 (d, 1H, J ꢁ 12.0 Hz, OCH2C6H5), 4.65 (d, 1H, J ꢁ 12.0 Hz,
OCH2C6H5), 4.61 (d, 1H, J ꢁ 12.0 Hz, OCH2C6H5), 4.58 (d, 1H, J ꢁ 12.0 Hz,
OCH2C6H5), 4.55 (d, 1H, J1′,2′ or J1,2 ꢁ 7.5 Hz, H-1′ or H-1), 4.40 (d, 1H, J1,2 or
J
1′,2′ ꢁ 7.5 Hz, H-1 or H-1′), 4.22 (dt, 1H, Jd ꢁ 9.5 Hz, Jt ꢁ 6.0 Hz, OCH2), 4.05
(dd, 1H, J6′a,6′b ꢁ 12.0 Hz, J5′,6a′ ꢁ 1.0 Hz, H-6′a), 4.04 - 4.01 (m, 2H), 4.02 (t, 1H,
J ꢁ 9.0 Hz, H-4), 3.79 (dt, 1H, Jt ꢁ 6.0 Hz, OCH2), 3.78 (dd, 1H, J5′,6b′ ꢁ 2.0 Hz,
H-6′b), 3.76 (m, 1H), 3.68 (t, 1H, J ꢁ 9.0 Hz, H-3), 3.62 (bm, 1H), 3.50-3.44 (m,
3H), 3.42 (dd, 1H, H-2), 3.04 (m, 2H, OCH2CH2), 2.88 (bs, 1H, OH), 2.41 (d, 1H,
Jd ꢁ 9.0 Hz, OH).
Anal. Calcd for C48H51O13N (849.34): C, 67.82; H, 6.05; N, 1.65. Found: C,
67.58; H, 5.93; N, 1.68.
2-(p-Nitrophenyl)ethyl (Benzyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero-ꢂ-D-galacto-2-nonulopyranosylonate)-(2→3)-(2-O-acetyl-
4,6-O-benzylidene-ꢀ-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-ꢀ-D-glu-
copyranoside (9). A mixture of diol 7 (1.9 g, 2.24 mmol), silver
trifluoromethanesulfonate (1.1 g 4.28 mmol), 2,6-di-t-butylpyridine (945 mg, 4.94
mmol), powdered Drierite (3 g), and anhydrous tetrahydrofuran (12 mL) was
strirred at room temperature under argon atmosphere for 8 h to ensure dryness. The
reaction mixture was then cooled to -35 °C under argon, and a solution of chloride
1510 (2.2 g, 3.37 mmol) in dry toluene (4 mL) was then added dropwise. Upon
completion of the addition, the reaction mixture was stirred for an additional 30
min at this temperature and then at 0 °C overnight. The reaction mixture was then
diluted with chloroform and filtered through Celite. The organic filtrate was
washed with saturated aqueous sodium bicarbonate, water, dried (Na2SO4), fil-
tered, and concentrated. The resulting yellowish foam was then purified by column
chromatography over silica gel using 20:10:1 hexane-ethyl acetate-ethanol as elu-
ant to give crude 2-(p-nitrophenyl)ethyl (benzyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-D-glycero-ꢂ-D-galacto-2-nonulopyranosylonate)-(2→3)-(4,6-
O-benzylidene-ꢀ-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-ꢀ-D-glucopyran
oside 8, contaminated with diol 7 and unsaturated Neu5NAc.
The crude 8 (1.2 g) was treated with 2:1 pyridine-acetic anhydride (24 mL) at
room temperature for 48 h and the mixture was then concentrated. After coevapo-
ration with toluene, the resulting yellowish foam was purified by column chro-
matography over silica gel using 95:5 chloroform-methanol to provide 940 mg
(29% overall yield from 7) as a white solid after lyophilization from benzene: Rf
0.36 (20:1 chloroform-methanol); [ꢂ]D ꢅ15.8° (c 0.5, chloroform); 1H NMR ꢃ 8.00
(d, 2H, J ꢁ 9.0 Hz, aromatic), 7.40 - 7.05 (m, 27H, aromatic), 5.58 (ddd, 1H, J7′′,8′′
ꢁ 9.0 Hz, J8′′,9′′b ꢁ 6.0 Hz, J8′′,9′′a ꢁ 2.5 Hz, H-8′′), 5.36 (dd, 1H, J6′′,7′′ ꢁ 2.5 Hz,
H-7′′), 5.15 (dd, 1H, J2′,3′ ꢁ 10.0 Hz, J1′,2′ ꢁ 8.0 Hz, H-2′), 5.13 (d, 1H, J ꢁ 12.0
Hz, OCH2C6H5), 5.05 (d, 1H, J ꢁ 12.0 Hz, OCH2C6H5), 5.04 (d, 1H, J ꢁ 12.0 Hz,
OCH2C6H5), 5.03 (d, 1H, J ꢁ 10.5 Hz, NH), 4.82 (s, 1H, benzylidene), 4.80 (d, 1H,

ORDER
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26
HAQUE AND DIAKUR
H-1′), 4.80 (m, 1H, H-4′′), 4.65-4.55 (m, 4H, OCH2C6H5), 4.38 (d, 1H, J1,2 ꢁ 8.0
Hz, H-1), 4.35 (dd, 1H, J9′′a,9′′b ꢁ 12.5 Hz, H-9˝a), 4.29 (dd, 1H, J3′,4′ ꢁ 3.5 Hz, H-
3′), 4.19 (dt, 1H, Jd ꢁ 9.5 Hz, Jt ꢁ 6.0 Hz, OCH2), 4.07 (q, 1H, J ꢁ 10.5 Hz, H-5˝),
3.97 (dd, 1H, H-9′′b), 3.90 (dd, 1H, J6′a,6′b ꢁ 12.5 Hz, J5′,6a′ ꢆ 1.0 Hz, H-6′a), 3.87
(dd, 1H, J6a,6b ꢁ 11.0 Hz, J5,6a ꢁ 1.5 Hz, H-6a), 3.84 (dd, 1H, H-6˝), 3.83 (t, 1H, J
ꢁ 9.5 Hz, H-4), 3.78 (dt, 1H, Jt ꢁ 7.0 Hz, OCH2), 3.72 (dd, 1H, J5,6b ꢁ 5.5 Hz, H-
6b), 3.63 (t, 1H, J ꢁ 9.0 Hz, H-3), 3.52 (dd, 1H, J5′,6b′ ꢁ 1.0 Hz, H-6′b), 3.49 (ddd,
1H, H-5), 3.40 (d, 1H, H-4′), 3.36 (dd, 1H, H-2), 3.05 (bs, 1H, H-5′), 3.04-3.00 (m,
2H, OCH2CH2), 2.73 (dd, 1H, J3′′e,3′′a ꢁ 12.5 Hz, J3′′e,4′′ ꢁ 4.5 Hz, H-3′′e), 2.20 (s,
3H, COCH3), 2.12 (s, 3H, COCH3), 2.06 (s, 3H, COCH3), 2.01 (s, 3H, COCH3),
2.00 (s, 3H, COCH3), 1.88 (s, 3H, COCH3), 1.80 (t, 1H, H-3′′a).
Anal. Calcd for C76H84O26N2 (1440.53): C, 63.31; H, 5.88; N, 1.94. Found:
C, 63.00; H, 5.77; N, 1.94.
2-(p-Trifluoroacetamidophenyl)ethyl (Benzyl 5-Acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-D-glycero-ꢂ-D-galacto-2-nonulopyranosylonate)-(2→3)-(2-
O-acetyl-4,6-O-benzylidene-ꢀ-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-ꢀ-
D-glucopyranoside (11). A solution of 9 (930 mg, 0.65 mmol) in 15:2:0.6
tetrahydrofuran-acetic acid-water was cooled to 0 °C and zinc powder (2 g) was
then added in one portion. To the rapidly stirred mixture was added 4 mL of aque-
ous copper sulfate solution (100 mg CuSO4·5H2O per 1 mL H2O) and the mixture
was stirred at room temperature for 1 h. After dilution with chloroform, the reac-
tion was filtered through Celite and the organic filtrate was washed with saturated
aqueous sodium bicarbonate, dried (Na2SO4), filtered, and concentrated. Finally,
the residue was coevaporated with toluene to give crude 2-(p-aminophenyl)ethyl
(benzyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-ꢂ-D-galacto-
2-nonulopyranosylonate)-(2→3)-(2-O-acetyl-4,6-O-benzylidene-ꢀ-D-galactopy-
ranosyl)-(1→4)-2,3,6-tri-O-benzyl-ꢀ-D-glucopyranoside 10: Rf 0.26 (20:1 chloro-
form-methanol, visualization with ninhydrin spray).
The crude amine 10 was dissolved in anhydrous dichloromethane (10 mL)
containing anhydrous pyridine (525 ꢄL, 6.5 mmol) and the reaction mixture was
cooled to -20 °C under argon. To the reaction was added trifluoroacetic anhydride
(305 ꢄL, 2.17 mmol) and the reaction mixture was stirred at this temperature for
an additional 10 min. The mixture was diluted with dichloromethane and then
washed with saturated aqueous sodium bicarbonate solution, dried (Na2SO4), fil-
tered, and concentrated. The resulting compound was purified by column chro-
matography over silica gel using 20:10:1 then 15:10:1 hexane-ethyl acetate-
ethanol as eluant to give 880 mg (90%) of 11 as a white solid after lyophilization
from benzene: Rf 0.1 15:10:1 (hexane-ethyl acetate-ethanol); [ꢂ]D ꢅ1.7° (c 0.4,
1
chloroform); H NMR ꢃ 7.70 (bs, 1H, NHTFA), 7.40 - 7.10 (m, 29H, aromatic),
5.57 (ddd, 1H, J7′′,8′′ ꢁ 9.0 Hz, J8′′,9′′b ꢁ 6.0 Hz, J8′′,9′′a ꢁ 2.5 Hz, H-8˝), 5.35 (dd,
1H, J6′′,7′′ ꢁ 2.5 Hz, H-7˝), 5.14 (dd, 1H, J2′,3′ ꢁ 10.5 Hz, J1′,2′ ꢁ 8.0 Hz, H-2′), 5.11
(d, 1H, J ꢁ 11.5 Hz, OCH2C6H5), 5.04 (d, 1H, J ꢁ 11.5 Hz, OCH2C6H5), 5.02 (d,
1H, J ꢁ 11.5 Hz, OCH2C6H5), 5.02 (d, 1H, J ꢁ 10.5 Hz, NH), 4.82 (s, 1H, ben-
zylidene), 4.80 (d, 1H, J ꢁ 11.5 Hz, OCH2C6H5), 4.79 (d, 1H, H-1′), 4.79 (m, 1H,

ORDER
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CYCLODEXTRIN
27
H-4′′), 4.62 (d, 1H, J ꢁ 12.0 Hz, OCH2C6H5), 4.61 (d, 1H, J ꢁ 11.5 Hz,
OCH2C6H5), 4.59 (d, 1H, J ꢁ 12.0 Hz, OCH2C6H5), 4.53 (d, 1H, J ꢁ 11.5 Hz,
OCH2C6H5), 4.37 (d, 1H, J1,2 ꢁ 8.0 Hz, H-1), 4.36 (dd, 1H, J9′′a,9′′b ꢁ 12.5 Hz, H-
9′′a), 4.29 (dd, 1H, J3′,4′ ꢁ 3.5 Hz, H-3′), 4.16 (dt, 1H, Jd ꢁ 9.5 Hz, Jt ꢁ 6.0 Hz,
OCH2), 4.06 (q, 1H, J ꢁ 10.5 Hz, H-5′′), 3.97 (dd, 1H, H-9˝b), 3.90 (dd, 1H, J6′a,6′b
ꢁ 12.0 Hz, J5′,6′a′ ꢆ 1.0 Hz, H-6′a), 3.87 (dd, 1H, J6a,6b ꢁ 11.0 Hz, J5,6a ꢁ 1.5 Hz,
H-6a), 3.83 (dd, 1H, H-6′′), 3.82 (t, 1H, J ꢁ 9.5 Hz, H-4), 3.73 (dt, 1H, Jt ꢁ 7.0 Hz,
OCH2), 3.72 (dd, 1H, J5,6b ꢁ 5.5 Hz, H-6b), 3.62 (t, 1H, J ꢁ 9.0 Hz, H-3), 3.52 (dd,
1H, J6′b,6′a ꢁ 12.0 Hz, J5′,6b′ ꢁ 1.0 Hz, H-6′b), 3.46 (ddd, 1H, H-5), 3.41 (d, 1H, H-
4′), 3.35 (dd, 1H, H-2), 3.05 (bs, 1H, H-5′), 2.92 (t, 2H, OCH2CH2), 2.73 (dd, 1H,
J3′′e,3′′a ꢁ 12.5 Hz, J3′′e,4′′ ꢁ 4.5 Hz, H-3′′e), 2.20 (s, 3H, COCH3), 2.11 (s, 3H,
COCH3), 2.05 (s, 3H, COCH3), 2.00 (s, 6H, COCH3), 1.87 (s, 3H, COCH3), 1.79
(t, 1H, H-3˝a); 19F NMR -75.84 (s).
Anal. Calcd for C78H85O25N2F3 (1506.54): C, 62.13; H, 5.69; N, 1.86. Found:
C, 61.78; H, 5.53; N, 1.88.
2-(p-Trifluoroacetamidophenyl)ethyl (5-Acetamido-3,5-dideoxy-D-glycero-
ꢂ-D-galacto-2-nonulopyranosylonic)-(2→3)-(ꢀ-D-galactopyranosyl)-(1→4)-ꢀ-D-
gluco-pyranoside (13). To a mixture of 11 (840 mg, 0.58 mmol), methanol (50
mL), and 5% palladium-on-carbon was added acetic acid (500 ꢄL), and the mix-
ture was hydrogenated at 5 psi, room temperature for 20 h. The reaction mixture
was then filtered through Celite and concentrated to give 538 mg of 2-(p-trifluo-
roacetamidophenyl)ethyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-
glycero-ꢂ-D-galacto-2-nonulopyranosylonate)-(2→3)-(2-O-acetyl-4,6-ꢀ-D-galac-
topyranosyl)-(1→4)-ꢀ-D-glucopyranoside 12 which was homogeneous by TLC, Rf
0.39 (65:35:3 chloroform-methanol-water).
A solution of 12 (538 mg) in dry methanol containing sodium metal (46
mg, 2 mmol) was stirred at room temperature and the reaction was monitored by
TLC (Rf 0.15 and 0.12 (6:1.2:1 ethyl acetate-methanol-water). The reaction mix-
ture was neutralized with acetic acid, filtered and concentrated. Column chro-
matography over silica gel using first 10:2:1 then 6:1.2:1 ethyl acetate-methanol-
water as solvent gave pure 13 (305 mg, 62%) followed by 14 (51 mg, 12%). Data
for 13: Rf 0.15 (6:1.2:1 ethyl acetate-methanol-water); [ꢂ]D ꢅ0.9° (c 1,
1
methanol); H NMR (D2O, HOD) ꢃ 7.45 - 7.35 (m, 4H, aromatic), 4.50 (d, 1H,
J1′,2′ ꢁ 8.0 Hz, H-1′), 4.46 (d, 1H, J1,2 ꢁ 8.0 Hz, H-1), 4.12 (dt, 1H, Jd ꢁ 10.0
Hz, Jt ꢁ 6.5 Hz, OCH2), 4.09 (dd, 1H, J2′,3′ ꢁ 10.0 Hz, J3′,4′ ꢁ 3.5 Hz, H-3′),
3.97 - 3.51 (m), 3.53 (dd, 1H, H-2′), 3.27 (t, 1H, J ꢁ 9.0 Hz, H-2), 2.95 (t, 2H,
OCH2CH2), 2.74 (dd, 1H, J3′′e,3′′a ꢁ 12.5 Hz, J3′′e,4′′ ꢁ 4.5 Hz, H-3′′e), 2.00 (s,
3H, NHCOCH3), 1.78 (t, 1H, H-3˝a); 19F NMR ꢃ -75.70 (s); 13C NMR ꢃ 175.72
(NHCOCH3), 174.57 (C-1), 138.18 (aromatic C), 133.64 (aromatic C), 130.46 (2
ꢇ aromatic C), 123.12 (2 ꢇ aromatic C), 103.35 (C-1), 102.78 (C-1′), 100.51 (C-
2′′), 78.76 (C-4), 76.18 (C-3′), 75.84 (C-5′), 75.43 (C-5), 75.04 (C-3), 73.57 (C-
6′′), 73.46 (C-2), 72.45 (C-8′′), 71.27 (OCH2), 70.05 (C-2′), 69.02 (C-4′′), 68.81
(C-7′′), 68.17 (C-4′), 63.28 (C-9′′), 61.69 (C-6′), 60.75 (C-6), 52.39 (C-5′′), 40.33
(C-3′′), 35.41 (CH2C6H4), 22.75 (NHCOCH3). Some of the 13C assignments re-

ORDER
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28
HAQUE AND DIAKUR
main tentative. FAB-MS, Calcd for C33H47O20N2F3: 848.73, (negative ion, TEA
matrix). Found: 847.4 (M-1).
N-{4-[2-{(Ammonium 5-Acetamido-3,5-dideoxy-D-glycero-ꢂ-D-galacto-2-
nonulopyranosylonate)-(2→3)-(ꢀ-D-galactopyranosyl)-(1→4)-(ꢀ-D-glucopyra-
nosyloxy)}ethyl]phenyl}-N′-[6A-deoxyheptakis(2,3-di-O-methyl)-
6B,6C,6D,6E,6F,6G-hexa-O-methyl-ꢀ-cyclodextrin-6A-yl]thiourea (18). Compound
13 (24 mg) was dissolved in ammonium hydroxide solution (5 mL) and the reac-
tion was stirred at room temperature overnight. Removal of solvents and column
chromatography over silica gel using 65:35:4.5 chloroform-methanol-water fur-
nished 2-(p-aminophenyl)ethyl (5-acetamido-3,5-dideoxy-D-glycero-ꢂ-D-galacto-
2-nonulopyranosylonic acid)-(2→3)-(ꢀ-D-galacto-pyranosyl)-(1→4)-ꢀ-D-glu-
copyranoside 14 (16 mg, 76%): Rf 0.06 (65:35:4.5 chloroform-methanol-water,
ninhydrin positive); 1H NMR (D2O, HOD) ꢃ 7.35 - 6.85 (m, 4H, aromatic), 4.52
(d, 1H, J ꢁ 8.0 Hz, H-1′ or H-1), 4.48 (d, 1H, J ꢁ 8.0 Hz, H-1 or H-1′), 4.11 (dt,
1H, obscured by H-3′, OCH2), 4.11 (dd, 1H, J2′,3′ ꢁ 10.0 Hz, J3′,4′ ꢁ 3.5 Hz, H-3′),
3.99 - 3.53 (m), 3.27 (t, 2H, J ꢁ 9.0 Hz), 2.85 (t, 2H, J ꢁ 7.0 Hz, OCH2CH2), 2.75
(dd, 1H, J3′′e,3′′a ꢁ 12.5 Hz, J3′′e,4′′ ꢁ 4.5 Hz, H-3˝e), 2.02 (s, 3H, NHCOCH3), 1.78
(t, 1H, H-3˝a).
To a stirred mixture of 1613 (21 mg, 15 ꢄmol) in chloroform (1 mL) and sat-
urated aqueous hydrogen carbonate (1 mL), was added thiophosgene (7 mg, 60
ꢄmol) in chloroform (0.6 mL) and the reaction stirred at room temperature for 30
min. The layers were then separated and the organic solution was dried (Na2SO4),
filtered, and concentrated to give crude 6A-isothiocyanato-6A-deoxyheptakis(2,3-
di-O-methyl)-6B,6C,6D,6E,6F,6G-hexa-O-methyl-ꢀ-cyclodextrin 17: Rf 16 0.5; Rf
17 0.72 (9:1 chloroform-methanol).
To a mixture of crude 17 (ꢀ21 mg) and 14 (9 mg, 12 ꢄmol) in methanol (750
ꢄL) was added pyridine (500 ꢄL) and the reaction was stirred at room temperature
for 48 h. The reaction mixture was concentrated and purified by column chro-
matography using first 65:35 chloroform-methanol to elute unreacted 17 followed
by 65:35:6 chloroform-methanol water to give 6 mg (23%) 18 and finally unre-
acted 14. Lyophilization of 18 from water gave a solid: Rf 0.46 (5:4:1 chloroform-
methanol-0.2% aqueous calcium chloride); 1H NMR (D2O, HOD) ꢃ 7.51 (br s, 2H,
aromatic), 7.40 (br d, 2H, aromatic), 5.67 (br s, 2H), 5.5 – 5.25 (m, 7H, H-1 cy-
clodextrin), 4.60 (d, 1H, J1,2 or J1′,2′ ꢁ 7.8 Hz, H-1 or H-1′ GM3), 4.56 (dd, 1H, J
ꢁ 8.0 Hz, J ꢁ 3.9 Hz), 4.3 – 3.3 (m, 132 H), 3.08 (t, 2H, J ꢁ 7.0 Hz), 3.02 (t, 2H,
J ꢁ 7.0 Hz), 2.84 (dd, 1H, J3e′′,3a′′ ꢁ 12.5 Hz, J3e′′,4′′ ꢁ 4.9 Hz, H-3e NeuNAc), 2.11
(s, 3H, COCH3), 1.88 (t, 1H, H-3a NeuNAc). ES-MS (negative ion), Calcd for
C94H154O52N3S ((M-1)-18): 2190.3. Found: 2189.9.
ACKNOWLEDGMENTS
We are grateful to Dr. A. Morales, Mass Spectrometer Service Laboratory,
Department of Chemistry, University of Alberta for assistance in obtaining ES
spectra.

ORDER
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CYCLODEXTRIN
29
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Received January 18, 2000
Accepted September 12, 2000

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