Chemical synthesis of cholesteryl β-D-galactofuranoside and -pyranoside

Article abstract of DOI:10.1016/j.carres.2005.06.015

Two isomeric cholesteryl galactosides, cholesteryl β-D- galactofuranoside and -pyranoside, have been synthesized by the Koenigs-Knorr reaction. Glycosylation of cholesterol with 2,3,5,6-tetra-O-benzoyl-D- galactofuranosyl bromide, followed by Zemplen saponification with sodium methoxide, gave cholesteryl β-D-galactofuranoside. By using 2,3,4,6-tetra-O-acetyl-D-galactopyranosyl bromide as the glycosyl donor, followed by alkaline hydrolysis, cholesteryl β-D-galactopyranoside was obtained. The title compounds were characterized by their IR spectra and by their ¹H and ¹³C NMR spectra. Structure considerations of the two cholesteryl galactosides correlated with data in the literature, thus confirming that cholesteryl β-D-galactopyranoside is an antigenic lipid of Lyme disease agent, Borrelia burgdorferi.

Full text of DOI:10.1016/j.carres.2005.06.015

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 2052–2054
Note
Chemical synthesis of cholesteryl b-D-galactofuranoside
and -pyranoside
Dumitru Petru Iga,a,* Silvia Iga,a Richard R. Schmidtb and Maria-Cristina Buzasc
aUniversity of Bucharest, Faculty for Biology, Splaiul Independentei 95, Bucuresti 5 R-76201, Romania
bFachbereich Chemie, Universita¨t Konstanz, Fach M 725, D-78457 Konstanz, Germany
cPolytechnic University of Bucharest, Faculty of Industrial Chemistry, Department of Organic Chemistry,
Spl. Independentei 313, Bucharest 6, Romania
Received 9 February 2005; accepted 14 June 2005
Available online 14 July 2005
Abstract—Two isomeric cholesteryl galactosides, cholesteryl b-D-galactofuranoside and -pyranoside, have been synthesized by the
Koenigs–Knorr reaction. Glycosylation of cholesterol with 2,3,5,6-tetra-O-benzoyl-D-galactofuranosyl bromide, followed by
´
Zemplen saponification with sodium methoxide, gave cholesteryl b-D-galactofuranoside. By using 2,3,4,6-tetra-O-acetyl-D-galacto-
pyranosyl bromide as the glycosyl donor, followed by alkaline hydrolysis, cholesteryl b-D-galactopyranoside was obtained. The title
compounds were characterized by their IR spectra and by their 1H and 13C NMR spectra. Structure considerations of the two cho-
lesteryl galactosides correlated with data in the literature, thus confirming that cholesteryl b-D-galactopyranoside is an antigenic
lipid of Lyme disease agent, Borrelia burgdorferi.
Ó 2005 Elsevier Ltd. All rights reserved.
Keywords: Cholesteryl b-D-galactofuranoside; Cholesteryl b-D-galactopyranoside; Sterylglycosides; Glycosylation
The isolation and/or synthesis of steryl glycosides
have been motivated by reasons related to either physio-
logical and biochemical phenomena or relative chemical
reactivity of different hydroxyl groups on the same
steroid.1 One of the first chemical preparations of cho-
lesteryl b-D-glucopyranoside, as well as of similar ste-
roids derived from cholesterol, was carried out to
explain structure–function relationships of saponins,
which are natural compounds having a similar structure
to that of the cholesteryl glucosides.2 In this way, choles-
terol became a model acceptor for glycosylation reac-
tions.3–5 About 45 glycosides, glucuronides, glucosides
and N-acetylglucosaminides of the most prominent five
naturally occurring bile acids, as well as a glucuronide
conjugate of pregnandiol,7 have been prepared by the
Koenigs–Knorr reaction.6 In the present paper the syn-
theses of cholesteryl b-D-galactofuranoside and -pyrano-
side are described, the latter compound being known as
a constituent of the cell-wall membrane of Borrelia burg-
dorferi, the etiologic agent of Lyme disease.8,9
For the synthesis of cholesteryl b-D-galactofuranoside
(3), a mixture of penta-O-benzoyl-a,b-D-galactofura-
noses (1, Scheme 1) was prepared by repeated crystalli-
zation of the benzoylation products of D-galactose in
hot pyridine, and the structure of each component was
1
confirmed by its H and 13C NMR spectra.10
Bromination of this mixture with hydrobromic acid in
glacial acetic acid11,12 gave tetra-O-benzoyl-a-D-galacto-
furanosyl bromide (2, Scheme 1), which was used for the
Koenigs–Knorr glycosylation of cholesterol in conjunc-
tion with cadmium carbonate.6 The glycosylation
mixture was submitted to alkaline hydrolysis, neutrali-
zation and Folch partition. The main reaction product
was recovered, together with unreacted cholesterol, in
the chloroform phase and the latter two compounds
were separated by column chromatography on silica
gel.13 Finally, a compound (3, Scheme 1) containing
equimolar amounts of cholesterol and galactose and
*
Corresponding author. Tel.: +40 21 411 5207; fax: +40 21 411
3933; e-mail: pdiga49@yahoo.com
0008-6215/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.06.015

D. P. Iga et al. / Carbohydrate Research 340 (2005) 2052–2054
2053
spectra were determined at 400 and 100 MHz, respec-
tively, in CDCl3, by using a Bruker ARX 400 spectro-
meter. For both cholesteryl galactosides synthesized in
this paper, per-O-acetylated derivatives were used for
O
OBz
HBr
OBz
C6H5COCl, Py
D-Gal
AcOH
BzO
BzO
OBz
100 °C
1
recording the H and 13C NMR spectra. All reagents
1
were purchased from E. Merck or Fluka Chemical Co.
O
OBz
Cholesterol, CdCO
NaOMe, MeOH
.
1
3
1.2. Cholesteryl b-D-galactofuranoside
Br
OBz
.
2
BzO
BzO
Penta-O-benzoyl-a,b-D-galactofuranoses (1) were pre-
pared by benzoylation of D-galactose in hot pyridine, fol-
lowed by repeated crystallization, and their structures
CH
H
3
2
CH3
CH3
H3C
H
CH3
H
1
were confirmed by H and 13C NMR spectroscopy.10
O
OH
O
H
Bromination of this mixture with HBr in glacial
HOAc11,12 gave tetra-O-benzoyl-a-D-galactofuranosyl
bromide (2) (TLC, solvent 1). A solution of HBr (33%)
in glacial HOAc (2.68 mL, 15 mmol) was cooled on ice
under exclusion of moisture, and then 2.1 g (3 mmol)
of penta-O-benzoyl-a,b-D-galactofuranoses and 10 mL
of 1,2-dichloroethane were added. Tetra-O-benzoyl-a-
D-galactofuranosyl bromide was extracted with cold
CHCl3 and processed in the usual manner12 to give
1.25 g (1.89 mmol, 90%) of 2. Product 2 was dissolved
in 10 mL of dry toluene and added to a suspension
consisting of 12 mL of dry toluene, 2 g of Drierite, 2 g
H
H
HO
HO
OH
3
Scheme 1.
having the H and 13C NMR characteristics of choles-
teryl b-D-galactofuranoside was obtained.
1
For the synthesis of cholesteryl b-D-galactopyranoside,
penta-O-acetyl-b-D-galactopyranose, obtained by acety-
lation of D-galactose in pyridine,14 followed by column
chromatography on silica gel, was converted to tetra-O-
acetyl-a-D-galactopyranosyl bromide by treating it with
hydrobromic acid. Koenigs–Knorr reaction of this com-
pound with cholesterol, followed by alkaline hydrolysis
with sodium methoxide, led to a compound containing
6
of CdCO3 and 0.608 g (1.58 mmol) of cholesterol and
refluxed for 7 h. The suspension was then diluted with
one volume of CHCl3, mixed with Celite and filtered.
The filtrate was concentrated to dryness in a vacuum,
and the residue was dissolved in 40 mL of 0.15 M
NaOMe and stirred overnight at room temperature.
The solution was neutralized with methanolic HCl,
and the product was partitioned as indicated by Folch.13
The organic phase was evaporated, and the residue was
submitted to column chromatography on silica gel in a
continuous gradient of MeOH in CHCl3. By mixing
the appropriate fractions, 0.54 g (0.97 mmol, 61.6%) of
cholesteryl b-D-galactofuranoside (3) was obtained.
1
equimolar amounts of cholesterol and galactose and H
and 13C NMR spectra characteristic of cholesteryl b-D-
galactopyranoside.8,9
Cholesteryl galactopyranosides have been characte-
rized in the cell-wall membrane of B. burgdorferi, the
agent of Lyme disease.8,9 On the other hand, steryl glu-
cosides present spectacular biochemical functions: sito-
steryl b-D-glucopyranoside is a primer for cellulose
synthesis in plants,15 and steryl glucosides are glucosyl
donors for ceramides that are involved in the biosynthe-
sis of glucocerebrosides.16
After crystallization from MeOH the product gave nee-
25
D
dles: mp 151–153 °C; ½aŠ À79 (c 1.035, 1:4 CHCl3–
MeOH); Rf 0.58, solvent 2; IR (KBr): m 3364, 2934.38,
1463.64, 1379.20, 1078.45, 650.88; 1H NM R:d 5.17
(J1 ,2 <1; H-10), 5.03 (J2 ,3 2.0; H-20), 5.00 (J3 ,4 6.2; H-
1. Experimental
0
0
0
0
0
0
30), 4.28 (J4 ,5 3.6; H-40), 5.37 (J5 ,6 a 3.2; H-50), 4.21
0
0
0
0
(J5 ,6 b 3.6; H-6a0), 4.31(J6 ,6 b 11.8; H-6b0) as well as val-
ues characteristic for the cholesteryl moiety;8,9 13C
NMR: d 103.95 (C-10), 81.93 (C-20), 77.01 (C-30), 79.43
(C-40), 69.18 (C-50), 62.70 (C-60) as well as values charac-
teristic of cholesterol.8,9 Anal. Calcd for C33H56O6: C,
72.26; H, 10.22. Found: C, 72.48; H, 10.47.
1.1. General methods
0
0
0
0
Thin-layer chromatography (TLC) was performed on
E. Merck Silica Gel 60 plastic plates with the following
mixtures: 7:1 toluene–MeOH (solvent 1) and 50:10:1
chloroform–MeOH–water (solvent 2). Visualization
was accomplished by dipping the plates in a solution of
ammonium molybdate, sulfuric acid and cerium(IV) sul-
fate, followed by heating. Column chromatography was
conducted with Silica Gel (E. Merck, 5–40 lm). IR spec-
tra (KBr) of the two cholesteryl galactosides were
Acidic hydrolysis of the product (10 mg) in a boiling
mixture of water–EtOH (10 mL) containing 1 MHCl
needed less than 5 min for total reaction (TLC, sol-
vent 2). The solution was concentrated to dryness
and the residue was partitioned between water and
CHCl3. The organic phase was used for cholesterol
recorded on deacylated compounds. H and 13C NM R
1

2054
D. P. Iga et al. / Carbohydrate Research 340 (2005) 2052–2054
determination (Liebermann–Burchardt), and the water
phase for galactose determination (anthrone). A molar
ratio of cholesterol–galactose of 0.98:1.01 was found.
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´
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Acknowledgements
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