Studies on α(1→5) linked octyl arabinofuranosyl disaccharides for mycobacterial arabinosyl transferase activity

Article abstract of DOI:10.1016/S0968-0896(01)00180-8

The appearance multi-drug resistant Mycobacterium tuberculosis (MTB) throughout the world has prompted a search for new, safer and more active agents against tuberculosis. Based on studies of the biosynthesis of mycobacterial cell wall polysaccharides, octyl 5-O-(α-D-arabinofuranosyl)-α-D-arabinofuranoside analogues were synthesized and evaluated as inhibitors for M. tuberculosis and Mycobacterium avium. A cell free assay system has been used for the evaluation of these disaccharides as substrates for mycobacterial arabinosyltransferase activity.

Full text of DOI:10.1016/S0968-0896(01)00180-8

Bioorganic & Medicinal Chemistry 9 (2001) 3145–3151
Studies on ꢀ(1!5) Linked Octyl Arabinofuranosyl Disaccharides
for Mycobacterial Arabinosyl Transferase Activity
Ashish K. Pathak,a Vibha Pathak,a Joseph A. Maddry,a William J. Suling,b
Sudagar S. Gurcha,c Gurdyal S. Besrac and Robert C. Reynoldsa,*
aDepartment of Organic Chemistry, Southern Research Institute, PO Box 55305, Birmingham, AL 35255, USA
bDepartment of Microbiology, Southern Research Institute, PO Box 55305, Birmingham, AL 35255, USA
cThe School of Microbiological, Immunological & Virological Sciences, The University of Newcastle upon Tyne,
The Medical School, Framlington Place, Newcastle upon Tyne NE24HH, UK
Received 2 April 2001; accepted 10 May 2001
Abstract—The appearance multi-drug resistant Mycobacterium tuberculosis (MTB) throughout the world has prompted a search for
new, safer and more active agents against tuberculosis. Based on studies of the biosynthesis of mycobacterial cell wall poly-
saccharides, octyl 5-O-(a-d-arabinofuranosyl)-a-d-arabinofuranoside analogues were synthesized and evaluated as inhibitors for
M. tuberculosis and Mycobacterium avium. A cell free assay system has been used for the evaluation of these disaccharides as sub-
strates for mycobacterial arabinosyltransferase activity. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
of the more active anti-tubercular agents that are cur-
rently in use act on the cell wall of MTB.6 For example,
ethambutol (EMB) and isoniazid (INH) target two cri-
tical cell wall components, the arabinogalactan and the
mycolic acids, respectively.6 The arabinogalactan con-
tains both arabinofuranose and galactofuranose moi-
eties, and this cell wall component critically underpins
the cell wall infrastructure, maintaining cell wall integ-
rity. Neither of these sugar forms is found in humans,
thus making the biochemistry and attendant enzyme
pathways ideal targets for the development of new,
selective anti-tubercular agents. The arabinan portion of
the cell wall is composed of arabinofuranose homo-
polymers with different linkages, namely a(1!5),
a(1!2) and a(1!3), and most likely requires several
different sugar processing enzymes, or arabinosyl-
transferases, for its complete synthesis.7
In spite of the availability of highly active anti-tuber-
cular agents, tuberculosis remains one of the primary
causes of human death and suffering worldwide.1
Approximately 2–3 million people die annually from the
disease, and each year an additional 8 million people
become ill with tuberculosis. It is estimated that one
third of the world’s population is infected with the bac-
teria that causes tuberculosis. Of even greater concern is
the development of highly drug-resistant forms of the
disease, multiple drug-resistant tuberculosis (MDR-
TB), that raises the specter that tuberculosis may once
again become an incurable disease. Treatment of MDR-
TB is both difficult and expensive,2,3 and for more
intractable forms, few treatment options are available.4
Public health officials worldwide are now calling for
increased funding and research for the development of
newer, safer drugs and vaccines to combat this deadly
disease.5
A key mycobacterial arabinose donor b-d-arabinofur-
anosyl-1-monophosphoryldecaprenol has been identi-
fied within lipid extracts and is implicated in the
biogenesis of the two major cell wall polysaccharides
arabinogalactan (AG) and liporabinomannan (LAM).8
Several synthetic O- and S-alkyl arabinofuranoside
acceptors have been prepared as acceptors for the
development of an arabinosyltransferase assay.9
Recently, several workers have also synthesized the
arabinosyl oligosaccharides present in the cell wall of
The biosynthesis of the mycobacterial cell wall is an
excellent target for future drug development based on
recent structural studies as well as the fact that several
*Corresponding author. Tel.: +1-205-581-2454; fax.: +1-205-581-
2877; e-mail: reynolds@sri.org
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00180-8

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A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3145–3151
mycobacteria for further biological and structural stud-
ies.10,11 The highly branched arabinofuranosyl hexa-
saccharide (Fig. 1) found at the non-reducing termini of
mycobacterial AG and LAM was prepared as its methyl
glycoside via a route that is both highly efficient and
stereocontrolled.10 An alternative synthetic approach
has been reported starting from 1,2,5-orthoester of d-
arabinose to synthesize an octyl penta-arabinofurano-
side component of the mycobacterial cell wall.11 High-
resolution NMRstudies have been done on methyl
arabinofuranose polysaccharides to determine the iden-
tity and equilibrium populations of preferred con-
formers for each furanose ring.10d Additionally, gas-
phase computational methods have also been applied to
synthetic arabinofuranose saccharides in order to deter-
mine the energy profiles and structural parameters as a
function ring conformation.12 How these results relate
to the actual enzyme-bound intermediates or structure
of the arabinofuranose ring in AG are unclear.
Result and discussion
Synthesis
The synthesis of arabinosyl disaccharides is represented
in Scheme 1. Several approaches were used for the
synthesis of these arabinofuranosides10,11 in order to
prepare a(1!5) disaccharides having bulky and small
protecting groups at the reducing end, and to study the
effects of these substitutions on activity. The reaction of
1, 1,2,3,5-tetra-O-acetyl-d-arabinofuranoside,14 with n-
octanol in the presence of SnCl4 as Lewis acid gave 2 as
the pure a-isomer (75%) after column chromato-
graphy.13a The octyl compound 2 was then deacetyled
with 7 N NH3/MeOH to give 3 in an overall yield of
86% after purification. The 5-position of the sugar 3
was blocked with a trityl group to get compound 4 in
73% yield. Compound 4 was further blocked at both
the 2- and 3-positions with either benzyl or methyl
groups and then detritylated with 5% trifluoroacetic
acid in chloroform at room temperature to give com-
pound 5 or 6 as glycosyl acceptors for disaccharide
synthesis. The trichloroacetimidate donor15 and the
acceptors 5 or 6 were reacted for 2 h in the presence of
the promoter BF3.Et2O at 0 ꢀC under an inert atmo-
sphere in dry CH2Cl2 at room temperature over pow-
dered 4 A molecular seives. After workup and column
chromatography on silica gel G (70–230 mesh), the pure
disaccharides 8 and 9 were obtained in 88 and 85%
yield, respectively. The disaccharides were debenzoy-
lated with 7 N NH3/MeOH to yield the partially
blocked disaccharides 10 and 11 in 98 and 90% yield,
respectively. Lastly, the fully de-blocked disaccharide
12, which more closely resembles the natural substrate
for cell wall polysaccharide synthesis, was made by Pd/
C catalyzed hydrogenation of the disaccharide 10. All
the new compounds were characterized by CHN analy-
sis, FABMS and NMRspectroscopy. The anomeric
carbons of a-d-arabinofuranosides resonate in the range
of 108–105 ppm whereas in the proton NMRspectrum,
As a continuation of our work to design probes and
inhibitors of mycobacterial glycosyltransferases,13 we
report herein the synthesis of 1-O-octyl-arabinofur-
anosyl disaccharide derivatives that are similar to the
natural substrate (Fig. 2). Their anti-mycobacterial
activity and activity as arabinosyltransferase acceptors
are also reported.
3
the anomeric protons showed a coupling JH1,H2 in the
range of 0.8–1.2 Hz suggesting a-glycosylation.16 Fur-
ther, the NOE, decoupling, D2O exchanged and APT
experiments were performed as required in order to
confirm NMRassignments and stereochemistry at the
anomeric centers.
In-vitro assay
In-vitro assays17 of arabinofuranosyl disaccharides were
done on Mycobacterium tuberculosis (MTB H37Ra) and
Mycobacterium avium (NJ 211). The disaccharides 8, 9,
11, 12 have shown activity >128 mg/mL whereas the
disaccharide 10 have shown >12.8ꢁ128 mg/mL for
both strains.
Figure 1. Hexarabinoside found in mycobacterial arabinogalactan
(AG) and lipoarabinomannan (LAM).
Acceptor activity
Based on the previous use of specific arabinose based
neoglycolipid acceptors,9 compounds 10 and 11 were
synthesized and compared with the fully de-blocked
arabinose acceptor 12. Assays performed in the presence
of membranes resulted in [14C]Araf incorporation from
DP-[14C]A for both compounds 11 and 12 with no
Figure 2. Structure of a(1!5) linked arabinofuranosyl disaccharide
similar to natural substrate.

A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3145–3151
3147
Scheme 1. Synthesis of a(1!5) linked arabinofuranosyl disaccharides. Reagents & Conditions: (a) C8H17OH, SnCl4, CH3CN, 30 min, rt, 75%; (b)
7N NH3/MeOH, rt, 5 h, 86%; (c) TrCl, Py, rt, overnight, 73%; (d) BnBr/Mel, NaH, THF, rt, 3 h, 5% TFA/CHCl3, CHCl3, 1 h, rt, 5: 78%, 6:
66%; (e) BF3.Et2O, CH2Cl2, 0ꢀC, 30 min, 8: 88%, 9: 85%; (f) 7N NH3/MeOH, rt, overnight, 10: 98%, 11: 90%; (g) Pd/C, H2, MeOH, rt, over-
night, 99%
detectable activity for 10 (see Fig. 3). TLC/auto-
radiography (Fig. 3) demonstrated the enzymatic con-
version of the both disaccharide 11 and 12 to their
corresponding trisaccharide products, [14C]Araf to the
50-OH and 30-OH of 12 as reported previously9 and,
presumably, a similar linkage profile for 11. Compound
10 was not recognized as a substrate for the arabinosyl-
transferase enzyme(s), presumably due to the bulky
benzyl ether protecting groups on the reducing sugar. In
contrast, 11, which possessed methyl ether protecting
groups on the reducing sugar, was recognized as a sub-
strate, however, with a significant loss of acceptor
recognition (see Fig. 3). Further competition-based,
experiments established that 10 and 11 were effective
inhibitors of their native acceptors (12) in the arabino-
syltransferase assay resulting in IC50 values of 1.12 and
3.70 mM for 10 and 11, respectively.
Experimental
Synthesis
General procedure. All reactions were performed under
a dry argon atmosphere and reaction temperatures were
measured externally. Anhydrous solvents from Aldrich
were used in the reactions as such. Whenever necessary,
Figure 3. An autoradiogram of reactions products produced through
the inclusion of 10, 11, and 12, mycobacterial membranes and
DP[14C]A. Lane 1, no acceptor; lane 2, 10; lane 3, 11; and lane 4, 12.
The compounds were included at a final concentration of 2 mM and
TLC/autoradiography performed using chloroform/methanol/ammo-
nium hydroxide/water (65:25:0.4:3.6) and products revealed through
exposure to Kodak X-Omat film at À70 ꢀC for 3 days.
compounds and starting materials were dried by azeo-
tropic removal of water with toluene under reduced
pressure. Reactions were monitored by thin-layer chro-
matography (TLC) on precoated E. Merck silica gel
(60F254) plates (0.25 mm) and visualized using UV light
(254 nm) and/or heating after spray with (NH4)2SO4
solution (150 g ammonium sulfate, 30 mL H2SO4,
750 mL H2O). All solvents used for work up and chro-
matography were reagent grade from Fisher Scientific.
Flash chromatography was carried out on Fischer silica
gel 60 (230–400 mesh). Melting points were determined
with a Mel-Temp II capillary melting points apparatus
and is uncorrected. 1H and 13C NMRspectra were
recorded on Nicolet NT 300NB instrument at 300 and

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A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3145–3151
75 MHz, respectively. Coupling constants (J) are repor-
ted in Hz and chemical shifts are in ppm (d) relative to
residual solvent peak or internal standard. Microanalysis
was performed on a Perkin–Elmer 2400 CHN analyzer.
FABMS were recorded on a Varian/MAT 311A double-
focusing mass spectrometer either by adding NBA/LiCl.
98:2) gave compound 4 as pale yellow oil (7.16 g, 73%).
FAB-MS (LiCl) m/e 505 [M+Li]+. 1H NMR
(300 MHz, CDCl3): d 7.42–7.25 (15H, m, aromatic),
5.09 (1H, s, H-1), 4.10 (1H, dd, J=1.2, 2.0 Hz, H-4),
3.97 (1H, br d, J=11.2 Hz, H-2), 3.87 (1H, br d,
J=10.8 Hz, H-3), 3.83 (1H, d, J=11.2 Hz, 2-OH, D2O
exchangeable), 3.73 (1H, m, OCH2), 3.66 (1H, dd,
J=2.8, 10.5 Hz, H-5a), 3.46 (1H, m, OCH2), 3.24 (1H,
dd, J=2.0, 10.5 Hz, H-5b), 2.92 (1H, d, J=11.9 Hz, 3-
OH, D2O exchangeable), 1.55 (2H, m, CH2), 1.27 (10H,
m, 5ÂCH2), 0.87 (3H, m, CH3). 13C NMR(75 MHz,
CDCl3): d 142.80, 128.80, 128.04, 127.98, 127.44 (aro-
matic), 108.06 (C-1), 88.40 (C), 86.47 (C-4), 78.67 (C-2),
78.25 (C-3), 67.66 (OCH2), 63.47 (C-5), 31.77, 29.52,
29.30, 29.19, 26.12, 22.62 (6ÂCH2), 14.07 (CH3).
Octyl 2,3,5-tri-O-acetyl-ꢀ-D-arabinofuranoside (2). An
1
anomeric mixture of d-arabinofuranose tetraacetate14
(10.0 g, 31.45 mmol) was dissolved in dry CH3CN
(150 mL) was added SnCl4 (4.4 mL, 37.74 mmol) at 0 ꢀC
and the mixture was stirred for 30 min. To this cold
solution, n-octanol (5.94 mL, 37.74 mmol) was added
dropwise over a period of 30 min. It was again stirred
for 1 h at room temperature. Celite (2.0 g) was added
cooled in an ice-water bath and a saturated aqueous
NaHCO3 solution was added dropwise to precipitate tin
salts. After complete precipitation, the mixture was fil-
tered through Celite and washed with chloroform
(2Â10 mL), concentrated to syrup, and redissolved in
CHCl3 (400 mL). The organic solution was next washed
with water (2Â50 mL) and brine (2Â50 mL), dried over
Na2SO4 and concentrated in vacuo to give the crude oil.
Flash chromatography (cyclohexane/EtOAc, 5:1) gave 2
Octyl 2,3-di-O-benzyl-ꢀ-D-arabinofuranoside (5). Com-
pound 4 (4.0 g, 7.94 mmol) was dissolved in dry THF
(50 mL) and NaH (60% dispersion in mineral oil,
571 mg, 23.82 mmol) was added, and the mixture was
stirred at room temperature for 15 min. The reaction
was cooled to 0 ꢀC and benzyl bromide (2.83 mL,
23.82 mmol) was added dropwise, and the resulting
mixture was stirred for 3 h at room temperature.
Methanol (20 mL) was added, and the reaction was
concentrated to dryness, redissolved in CHCl3 (250 mL),
washed with water (2Â50 mL) and brine (50 mL), dried
over Na2SO4, and concentrated to a colorless oil. The
oil was dissolved in CHCl3 and cooled to 0 ꢀC, and 5%
trifluoroacetic acid in CHCl3 (10 mL) was added drop-
wise. The reaction mixture was stirred for 1 h, coevapo-
rated with ethanol (2Â20 mL), and concentrated to
syrup. Flash chromatography (cyclohexane/EtOAc
10:1) yielded compound 5 (2.25 g, 78%) as a colorless
as colorless oil (9.1 g, 75%). FAB-MS (LiCl) m/e 467
1
[M+Li]+. H NMR(300 MHz, CDCl ): d 5.07 (1H, d,
3
J=0.4, 1.5 Hz, H-2), 5.02 (1H, s, H-1), 4.96 (1H, ddd,
J=0.7, 1.5, 4.9 Hz, H-3), 4.44 (1H, m, H-5a), 4.22 (2H,
m, H-4, H-5b), 3.69 (1H, m, OCH2), 3.45 (1H, m,
OCH2), 2.109, 2.106, 2.103 (each 3H, s, 3ÂOAc), 1.57
(2H, m, CH2), 1.28 (10H, m, 5ÂCH2), 0.88 (3H, m,
CH3).
Octyl ꢀ-D-arabinofuranoside (3). To a solution of com-
pound 2 (9.00 g, 23.19 mmol) in dry methanol (50 mL)
was added a NaOMe solution in methanol (25% w/v,
9.0 mL) dropwise while cooling in an ice-bath. The
reaction mixture was allowed to stir at room tempera-
ture for 5 h. It was concentrated in vacuo to give a
syrup, which after flash chromatography (CHCl3/
MeOH 95:5) gave a viscous oil. Crystallization from
diethylether gave pure 3 as solid (5.22 g, 86%). Mp
1
syrup. FAB-MS (LiCl) m/e 449 [M+Li]+. H NMR
(300 MHz, CDCl3): d 7.38–7.28 (10H, m, aromatic),
5.02 (1H, d, H-1), 4.62–4.49 (4H, 4Âd, each J=11.8 Hz,
2ÂCH2–aromatic), 4.15–4.11 (1H, m, H-4), 4.03 (1H,
dd, J=1.1, 3.1 Hz, H-2), 3.98 (1H, dd, J=3.1, 6.6 Hz,
H-3), 3.84 (1H, dd, J=2.8, 12.1 Hz, H-5a), 3.70 (1H, m,
OCH2), 3.65 (1H, dd, J=3.9, 12.1 Hz, H-5b), 3.39 (1H,
m, OCH2), 1.59 (2H, m, CH2), 1.27 (10H, m, CH2), 0.86
49 ꢀC. FAB-MS (NBA) m/e 263 [M+H]+. H NMR
(3H, m, CH3). 13C NMR(75 MHz, CDCl ): d 137.84,
1
3
(300 MHz, CDCl3): d 5.01 (1H, s, H-1), 4.18 (1H, ddd,
J=1.5, 2.0, 2.5 Hz, H-4), 4.00 (2H, m, H-2, H-3), 3.90
(1H, dd, J=2.5, 11.7 Hz, H-5a), 3.82 (1H, dd, J=2.0,
11.7 Hz, H-5b), 3.69 (1H, m, OCH2), 3.45 (1H, m,
OCH2), 2.99 (1H, d, J=11.4 Hz, 3-OH, D2O exchange-
able), 2.40 (1H, m, 2-OH, D2O exchangeable), 1.58
(2H, m, CH2), 1.26 (10H, m, 5ÂCH2), 0.88 (3H, m,
137.40, 128.42, 128.36, 127.90, 127.83, 127.69 (aro-
matic), 106.21 (C-1), 88.10 (C-2), 82.66 (C-3), 81.68 (C-
4), 72.24, 71.92 (CH2-aromatic), 67.66 (OCH2), 62.18
(C-5), 31.80, 29.49, 29.34, 29.25, 26.11, 22.63 (6ÂCH2),
14.07 (CH3).
Octyl 2,3-di-O-methyl-ꢀ-D-arabinofuranoside (6). Com-
pound 4 (2.0 g, 3.97 mmol) was dissolved in dry THF
(20 mL) and NaH (60% dispersion in mineral oil,
286 mg, 11.91 mmol) was added. The mixture was stir-
red at room temperature for 15 min, next, the reaction
was cooled to 0 ꢀC, and iodomethane (0.7 mL,
9.93 mmol) was added dropwise followed by stirring for
3 h at room temperature. Methanol (10 mL) was added,
the solution was concentrated to dryness, the oil was
redissolved in CHCl3 (100 mL), and the organic solution
was washed with water (2Â20 mL) and brine (20 mL),
and dried over Na2SO4. The dry organic solution was
concentrated to a colorless oil that was dissolved in
CHCl3. The chloroform solution was cooled to 0 ꢀC,
CH3). 13C NMR(CDCl ): d 107.95 (C-1), 86.70 (C-4),
3
79.02 (C-2), 77.88 (C-3), 67.80 (OCH2), 61.78 (C-5),
31.77, 29.46, 29.29, 29.19, 26.08, 22.61 (6ÂCH2), 14.06
(CH3).
Octyl 5-O-trityl-ꢀ-D-arabinofuranoside (4). To a dry
pyridine (60 mL) solution of compound 3 (5.1 g,
19.46 mmol) was added trityl chloride (8.14 g,
29.19 mmol), and the reaction mixture was stirred at
50 ꢀC overnight. The reaction mixture was coevaporated
with toluene (2Â50 mL), redissolved in CHCl3 (150 mL),
washed with water (2Â20 mL), dried over Na2SO4 and
concentrated. Column chromatography (CHCl3/MeOH

A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3145–3151
3149
and 5% trifluoroacetic acid in CHCl3 (5 mL) was added
dropwise. The reaction mixture was then stirred for
30 min, coevaporated with ethanol (2Â10 mL), and
concentrated to syrup. After purification on silica gel
column using cyclohexane/ethylacetate (5:1) as eluant,
compound 6 was obtained as a colorless, viscous oil
(each 3H, s, OCH3), 1.56 (2H, m, CH2), 1.26 (10H, m,
CH2), 0.87 (3H, m, CH3). 13C NMR(75 MHz, CDCl ):
3
d 166.22, 165.80, 165.27 (3ÂC¼O), 133.47, 133.02,
130.03, 129.86, 129.74, 129.71, 128.50, 128.45, 128.28
(aromatic), 105.84 (C-10), 105.55 (C-1), 90.05 (C-2),
85.47 (C-3), 81.86 (C-20), 81.45 (C-4), 80.04 (C-40), 77.89
(C-30), 67.71 (OCH2), 66.71 (C-50), 63.81 (C-5), 31.82,
29.45, 29.36, 29.25, 26.08, 22.65 (6ÂCH2), 14.10 (CH3).
1
(66%). FABMS (LiCl) m/e 297 [M+Li]+. H NMR
(300 MHz, CDCl3): d 4.99 (1H, s, H-1), 4.06 (1H, m, H-
4), 3.89 (1H, m, H-5a), 3.73 (1H, dd, J=1.1, 2.6 Hz, H-
2), 3.68 (3H, m, H-3, H-5b, OCH2), 3.43 (1H, m,
OCH2), 3.42, 3.41 (each 3H, s, 2ÂOCH3), 1.87 (1H, dd,
J=4.4, 8.1 Hz, 5-OH, D2O exchangeable), 1.58 (2H, m,
CH2), 1.28 (10H, m, CH2), 0.88 (3H, m, CH3).
Octyl 5-O-(ꢀ-D-arabinofuranosyl)-2,3-di-O-benzyl-ꢀ-D-
arabinofuranoside (10). To a solution of disaccharide 8
(1.70 g, 1.92 mmol) in dry methanol (25 mL) was added
7 N NH3/MeOH (5 mL) dropwise, and the reaction
mixture was allowed to stir at room temperature over-
night. Concentration in vacuo to give a syrup, followed
by flash chromatography (CHCl3/MeOH 96:4) gave 10
as colorless oil (1.08 g, 98%). FAB-MS (NBA) m/e 575
[M+H]+. C32H46O9. 0.7 H2O (found: C, 65.30; H, 7.83.
requires C, 65.40; H, 7.99). 1H NMR(300 MHz,
CDCl3): d 7.38–7.26 (m, aromatic), 5.03 (1H, s, H-10),
5.01 (1H, s, H-1), 4.61–4.37 (4H, m, OCH2–aromatic),
4.16 (1H, ddd, J=3.1, 5.3, 5.9 Hz, H-40), 4.02 (1H, d,
J=9.7 Hz, H-30), 3.98 (1H, dd, J=0.9, 2.4 Hz, H-2),
3.94 (1H, m, H-20), 3.90 (1H, ddd, J=1.9, 2.3, 5.5 Hz,
H-4), 3.84–3.76 (4H, m, H-3, H-5a, H-5b, H-50a), 3.70–
3.62 (2H, m, H-50b, OCH2), 3.56 (1H, d, J=9.8 Hz, 30-
OH, D2O exchangeable), 3.38 (1H, m, OCH2), 3.22 (1H,
d, J=11.9 Hz, 20-OH, D2O exchangeable), 1.56 (2H, m,
CH2), 1.28 (10H, m, CH2), 0.88 (3H, m, CH3). 13C
NMR(75 MHz, CDCl 3): d 137.57, 137.19 (C, aromatic),
128.49, 128.41, 128.14, 128.04, 127.79 (CH, aromatic),
107.46 (C-10), 105.94 (C-1), 87.32 (C-2), 86.94 (C-40),
83.60 (C-3), 80.38 (C-4), 78.88 (C-20), 77.77 (C-30),
72.04, 71.90 (CH2–aromatic), 67.67 (OCH2), 65.89 (C-
5), 61.70 (C-50), 31.82, 29.43, 29.36, 29.26, 26.11, 22.65
(6ÂCH2), 14.11 (CH3).
Octyl 5-O-(2,3,5-tri-O-benzoyl-ꢀ-D-arabinofuranosyl)-
2,3-di-O-benzyl-ꢀ-D-arabinofuranoside (8). The tri-
chloroacetimidate donor 7 (2.46 g, 4.06 mmol) and the
acceptor 5 (1.12 g, 2.54 mmol) were dissolved in dry
CH2Cl2 (30 mL) and dry powdered 4 A molecular sieves
were added. The mixture was stirred for 15 min and
ꢀ
ꢂ
cooled to 0 C. The promoter BF3 Et2O (0.52 mL,
4.06 mmol) was dissolved in 2 mL of dry CH2Cl2 and
added to the reaction mixture dropwise. The reaction
mixture was stirred for an additional 30 min while
warming to ambient temperature and filtered. The fil-
trate was washed with cold saturated anhydrous
NaHCO3 followed by distilled water, and the organic
layer was dried over Na2SO4 and concentrated. Column
chromatography of the resulting oil on silica gel (cyclo-
hexane/EtOAc, 10:1) afforded the pure disaccharide 8
(1.96 g, 88%) as a colorless syrup. FAB-MS (LiCl) m/e
893 [M+Li]+. C53H58O12. 1.0 H2O (found: C, 70.35; H,
1
7.72, requires C, 70.3; H, 6.68). H NMR(300 MHz,
CDCl3): d 8.07–7.96, 7.57–7.19 (25H, each m, aromatic),
5.59 (1H, d, J=1.0 Hz, H-20), 5.53 (1H, d, J=4.7 Hz, H-
30), 5.36 (1H, s, H-10), 5.05 (1H, s, H-1), 4.78 (1H, dd,
J=3.2, 12.0 Hz, H-50a), 4.62 (1H, dd, J=4.8, 12.0 Hz, H-
50b), 4.50 (4H, m, 2ÂCH2-aromatic), 4.38 (1H, m, H-40),
4.20 (1H, m, H-4), 4.04 (1H, dd, J=3.5, 6.6 Hz, H-3),
4.03 (1H, d, J=1.0 Hz, H-2), 3.94 (1H, dd, J=4.4,
11.1 Hz, H-5a), 3.75 (1H, dd, J=3.4, 11.1 Hz, H-5b),
3.69 (1H, m, OCH2), 3.37 (1H, m, OCH2), 1.56 (2H, m,
CH2), 1.26 (10H, m, CH2), 0.87 (3H, m, CH3).
Octyl 5-O-(ꢀ-D-arabinofuranosyl)-2,3-di-O-methyl-ꢀ-D-
arabinofuranoside (11). From disaccharide 9 (190 mg,
0.26 mmol) by the method reported for the preparation
of 10, compound 11 was obtained as a syrup (98 mg,
90%) after column chromatography (CHCl3/MeOH,
95:5). FAB-MS (LiCl) m/e 429 [M+Li]+. C20H38O9. 0.2
H2O (found: C, 56.32; H, 8.99, requires C, 56.38; H,
9.08). 1H NMR(300 MHz, CDCl 3): d 5.07 (1H, s, H-10),
5.01 (1H, s, H-1), 4.19 (1H, ddd, J=2.2, 4.3, 4.4 Hz, H-
40), 4.11 (1H, ddd, J=3.6, 4.9, 5.8 Hz, H-4), 4.07 (1H,
m, H-20), 4.00 (1H, m, H-30), 3.90 (1H, dd, J=5.1,
11.8 Hz, H-50a), 3.89 (1H, dd, J=3.0, 10.4 Hz, H-5a),
3.81 (1H, dd, J=2.1, 11.8 Hz, H-50b), 3.73 (1H, dd,
J=3.6, 10.4 Hz, H-5b), 3.71 (1H, d, J=1.3 Hz, H-2),
3.67 (1H, m, OCH2), 3.54 (1H, dd, J=1.5, 5.8 Hz, H-3),
3.41 (2H, m, OCH2, 20-OH, D2O exchangeable), 3.40
(6H, s, 2ÂOCH3), 1.63 (1H, s, 30-OH, D2O exchange-
able), 1.57 (2H, m, CH2), 1.29 (10H, m, CH2), 0.88 (3H,
Octyl 5-O-(2,3,5-tri-O-benzoyl-ꢀ-D-arabinofuranosyl)-
2,3-di-O-methyl-ꢀ-D-arabinofuranoside (9). The accep-
tor 6 (123 mg, 0.42 mmol) was reacted with donor 7
ꢂ
(305 mg, 0.50 mmol) in the presence of BF3 Et2O
(0.06 mL, 0.5 mmol) as described for the preparation of
8. After purification on a silica gel column (cyclohex-
ane/EtOAc, 5:1) disaccharide 9 was obtained as a col-
orless syrup (264 mg, 85%). FAB-MS (LiCl) m/e 741
[M+Li]+. C41H50O12. 1.3 H2O (Found: C, 64.87; H,
1
6.74. requires C, 64.95; H, 6.99). H NMR(300 MHz,
CDCl3): d 8.12–7.98, 7.60–7.24 (15H, each m, aro-
matic), 5.61 (1H, d, J=1.1 Hz, H-20), 5.56 (1H, d,
J=4.6 Hz, H-30), 5.40 (1H, s, H-10), 4.99 (1H, s, H-1),
4.84 (1H, dd, J=3.3, 11.9 Hz, H-50a), 4.68 (1H, dd,
J=4.9, 11.9 Hz, H-50b), 4.60 (1H, ddd, J=3.3, 4.6,
4.9 Hz, H-40), 4.15–4.11 (1H, m, H-4), 3.97 (1H, dd,
J=4.7, 11.1 Hz, H-5a), 3.79 (1H, dd, J=3.7, 11.1 Hz, H-
5b), 3.73 (1H, m, H-3), 3.72 (1H, d, J=1.0 Hz, H-2),
3.68 (1H, m, OCH2), 3.39 (1H, m, OCH2), 3.38, 3.36
m, CH3). 13C NMR(75 MHz, CDCl ): d 107.80 (C-10),
3
105.48 (C-1), 89.37 (C-2), 87.11 (C-40), 86.43 (C-3),
80.55 (C-4), 78.94 (C-30), 77.81 (C-20), 67.71 (OCH2),
66.55 (C-5), 61.84 (C-50), 57.99, 57.52 (2ÂOCH3), 31.77,
29.37, 29.30, 29.20, 26.02, 22.61 (6ÂCH2), 14.06 (CH3).
Octyl 5-O-(ꢀ-D-arabinofuranosyl)-ꢀ-D-arabinofurano-
side (12). To a methanol solution (5 mL) of dis-

3150
A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3145–3151
accharide 10 (107 mg, 0.87 mmol) was added Pd/C
(10%, 50 mg), and the mixture was stirred at room
temperature under hydrogen (30 mL, 24 h). Filtration
through a Celite pad and concentration gave viscous,
colorless oil. Flash chromatography (CHCl3/MeOH
5:1) gave compound 12 as a colorless syrup (74 mg,
99%). FAB-MS (LiCl) m/e 401 [M+Li]+. C18H34O9.
on aluminum backed Silica Gel 60 F254 plates (E.
Merck, Darmstadt, Germany). Autoradiograms were
obtained by exposing TLC’s to X-ray film (Kodak X-
Omat) for 3 days. Competition based experiments were
performed by mixing compounds together at various
concentrations (12, 0.4 mM with 10 or 11 at 0.5, 1.0, 2.0,
4.0 and 6.0 mM) followed by thin-layer chromato-
graphy/autoradiography as described earlier to deter-
mine the extent of product formation.
0.5 H2O (found: C, 53.56; H, 8.72, requires C, 53.58; H,
1
8.74). H NMR(300 MHz, D O, acetone as reference):
2
d 4.96 (1H, d, J=1.2 Hz, H-1), 4.86 (1H, d, J=1.5 Hz, H-
10), 4.02 (1H, dd, J=1.4, 3.2 Hz, H-2), 3.94 (4H, m, H-4,
H-20, H-30, H-40), 3.85 (1H, dd, J=3.1, 5.9 Hz, H-3), 3.79
(1H, dd, J=4.7, 11.3 Hz, H-50a), 3.72 (1H, dd, J=3.4,
12.3 Hz, H-5a), 3.66–3.58 (3H, m, H-5b, H-50b, OCH2),
3.38 (1H, m, OCH2), 1.49 (2H, m, CH2), 1.20 (10H, m,
CH2), 0.79 (3H, m, CH3). 13C NMR(75 MHz, D 2O, ace-
tone as reference): d 107.79 (C-10), 107.64 (C-1), 84.27 (C-
40), 82.04 (C-4), 81.5 (C-2), 81.25 (C-20), 76.94 (C-3), 76.89
(C-30), 68.59 (OCH2), 66.72 (C-50), 61.46 (C-5), 31.86,
29.37, 29.34, 29.26, 25.95, 22.65 (6ÂCH2), 13.96 (CH3).
Acknowledgements
The authors are thankful to Dr. J. M. Riordan for
NMRspectra and Mr. M. D. Richardson for FABMS
data. This work was supported by NIH/NIAID grants
R01AI45317 and R01AI38667. GSB who is currently a
Lister Institute-Jenner Research Fellow acknowledges
support from The Medical Research Council (49343
and 49342).
Arabinosyltransferase assay
Compounds 10–12, at a range of concentrations from
0.25 to 6.0 mM (which were stored as 100 mM ethanol
stocks) and DP[14C]A [20,000 cpm, 9 mM, 10 mL (stored
in chloroform/methanol, 2:1)], were dried under a
stream of argon in a microcentrifuge tube (1.5 mL) and
placed in a vacuum desiccator for 15 min to remove any
residual solvent. The dried constituents of the assay
were then resuspended in 8 mL of a 1% aqueous solution
of Igepal. The remaining constituents of the arabino-
syltransferase assay containing 50 mM MOPS (adjusted
to pH 8.0 with KOH), 5 mM b-mercaptoethanol,
10 mM MgCl2, 1 mM ATP, membranes (250 mg) were
added to a final reaction volume of 80 mL. The reaction
mixtures were then incubated at 37 ꢀC for 1 h. A
CHCl3/CH3OH (1:1, 533 mL) solution was then added
to the incubation tubes and the entire contents cen-
trifuged at 18,000g. The supernatant was recovered and
dried under a stream of argon and re-suspended in
C2H5OH/H2O (1:1, 1 mL) and loaded onto a pre-equi-
librated [C2H5OH/H2O (1:1)] 1 mL Whatman strong
anion exchange (SAX) cartridge which was washed with
3 mL of ethanol. The eluate was dried and the resulting
products partitioned between the two phases arising
from a mixture of n-butanol (3 mL) and H2O (3 mL).
The resulting organic phase was recovered following
centrifugation at 3500g and the aqueous phase was
again extracted twice with 3 mL of n-butanol saturated
water, the pooled extracts were back-washed twice with
water saturated with n-butanol (3 mL). The n-butanol-
saturated water fraction was dried and re-suspended in
200 mL of n-butanol. The total cpm of radiolabeled
material extractable into the n-butanol phase was mea-
sured by scintillation counting using 10% of the labeled
material and 10 mL of EcoScintA (National Diag-
nostics, Atlanta, USA). The incorporation of [14C]Araf
was determined by subtracting counts present in control
assays (incubation of the reaction components in the
absence of the compounds). Another 10% of the labeled
material was subjected to thin-layer chromatography
(TLC) in CHCl3/CH3OH/NH4OH/H2O (65:25:0.5:3.6)
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