Synthesis of deoxygenated α(1 → 5)-linked arabinofuranose disaccharides as substrates and inhibitors of arabinosyltransferases of Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmc.2008.11.027

Arabinosyltransferases (AraTs) play a critical role in mycobacterial cell wall biosynthesis and are potential drug targets for the treatment of tuberculosis, especially multi-drug resistant forms of M. tuberculosis (MTB). Herein, we report the synthesis a

Full text of DOI:10.1016/j.bmc.2008.11.027

Bioorganic & Medicinal Chemistry 17 (2009) 872–881
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of deoxygenated a(1 ? 5)-linked arabinofuranose disaccharides
as substrates and inhibitors of arabinosyltransferases of Mycobacterium
tuberculosis
Ashish K. Pathak a, Vibha Pathak a, William J. Suling a, James R. Riordan a, Sudagar S. Gurcha b,
Gurdyal S. Besra b, Robert C. Reynolds a,
*
a Drug Discovery Division, Southern Research Institute, PO Box 55305, Birmingham, AL 35255, USA
b School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Arabinosyltransferases (AraTs) play a critical role in mycobacterial cell wall biosynthesis and are poten-
Received 10 October 2008
Revised 10 November 2008
Accepted 12 November 2008
Available online 18 November 2008
tial drug targets for the treatment of tuberculosis, especially multi-drug resistant forms of M. tuberculosis
(MTB). Herein, we report the synthesis and acceptor/inhibitory activity of Araf
a(1 ? 5) Araf disaccha-
rides possessing deoxygenation at the reducing sugar of the disaccharide. Deoxygenation at either the
C-2 or C-3 position of Araf was achieved via a free radical procedure using xanthate derivatives of the
hydroxyl group. The a(1 ? 5)-linked disaccharides were produced by coupling n-octyl a-Araf 2-/3-deoxy,
2-fluoro glycosyl acceptors with an Araf thioglycosyl donor. The target disaccharides were tested in a cell
free mycobacterial AraTs assay as well as an in vitro assay against MTB H37Ra and M. avium complex
strains.
Keywords:
Arabinosyltransferases
Mycobacterium tuberculosis
Deoxyarabinofuranose
2-Fluoroarabinofuranose
Disaccharides
Ó 2008 Elsevier Ltd. All rights reserved.
Inhibitors
1. Introduction
acids.6 Among the front line drugs for treatment of TB, two drugs
isoniazid (INH) and ethambutol (EMB) target the mycobacterial
Tuberculosis (TB) is one of the primary killers worldwide in
spite of the availability of several potent anti-mycobacterial
agents.1 Millions die annually from this disease, and the problem
is amplified by the apparent synergism with HIV.2,3 Mycobacte-
rial diseases have attracted renewed attention in recent years
because of their increased incidence worldwide and the emer-
gence of multi-drug resistant (MDR) and extensively drug resis-
tant (XDR) strains.4 MDR-TB infections are significantly more
difficult to treat with second-line therapies that are typically
more expensive and have considerable side-effects. XDR-TB5
develops when these second-line drugs are also misused or mis-
managed and therefore become ineffective. Because XDR-TB is
resistant to first- and second-line drugs, treatment options are
seriously limited.
cell wall that is essential for the survival of pathogen.7 The struc-
ture of the cell wall has now been systematically elucidated in
terms of its component complex polysaccharides, the specific
chemical linkages therein, and the macromolecular structure of
the mycolylarabinogalactan complex.8 The two major oligosaccha-
ride portions, lipoarabinomannan (LAM) and arabinogalactan (AG),
contain arabinofuranose (Araf) units.
Chemical analysis of the fragments of LAM and AG have re-
vealed that both are composed of linear arabinan consisting of
a(1 ? 5) linked Araf units, and a branched Araf hexasaccharide at
the terminus with (1 ? 3) and b(1 ? 2) linked Araf units. The
a
assembly of the arabinan portions of cell wall polysaccharides in
mycobacteria involves a family of AraTs9 that promote the poly-
merization of Araf units using decaprenolphosphoarabinofuranose
(DPA) as the sugar donor. Mycobacterial viability requires an intact
arabinan, and thus compounds that inhibit these glycosyltransfer-
ases (GTs) are both useful biochemical tools as well as potential
lead compounds for new selective anti-tubercular agents as Araf
is not present in mammals.
During the last two decades, new programs have been initiated
to elucidate the systems biology of the tubercle bacillus with a fo-
cus on new, valid targets for novel anti-tubercular drug discovery.
Many unique metabolic processes occur during the biosynthesis of
cell wall components, including arabinogalactan and mycolic
At the inception of the mycobacterial GTs program, our inten-
tion was to prepare prototype disaccharides that would be sub-
strates for assay development and could probe the acceptor
activity of the various cell wall GTs.10,11 Neo-glycosides 1a, 1b
* Corresponding author. Tel.: +1 205 581 2454; fax: +1 205 581 2877.
E-mail address: Reynolds@sri.org (R.C. Reynolds).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.027

A. K. Pathak et al. / Bioorg. Med. Chem. 17 (2009) 872–881
873
and 1c (Chart 1) were previously synthesized and evaluated for
their potential as acceptors/inhibitors.11 As those efforts advanced,
our work turned to examining the various substitution patterns of
the acceptor disaccharides to study the acceptor tolerance for var-
ious alterations, and the ability of these substitutions to affect inhi-
bition relative to the standard acceptor disaccharides for each
transferase. Based on the substrate activity of a control acceptor,
ters, sulfonates, or even by direct reduction of hydroxyl groups.
Unsaturated sugars are also excellent sources for deoxygenated
derivatives via electrophilic addition of hydrogen halides, and gly-
cals through acid catalyzed addition of water or alcohols.20 Other
more direct, approaches have also been developed recently21 and
the syntheses of a small number of deoxy furanose derivatives,
especially 2-deoxy-glycosides, have also been reported.22 Specifi-
cally, 2-deoxy Araf di- and tri-saccharides were recently reported
through reductive desulfonylation.23
The most common preparative method used is radical chain
chemistry for the transformation of a secondary alcohol to the cor-
responding deoxy derivative. Firstly, alcohols are converted to a
thiocarbonyl derivative (thioxobenzoates, xanthates, or thiocarb-
onylimidazolides), and, on reduction with tributyltin hydride,
these derivatives afford deoxy compounds in good yields.24 Utiliz-
ing this approach, we report the synthesis of 1-O-octyl-Araf
n-octyl Araf a(1 ? 5) Araf (1a), several analogs possessing N-alkyl,
amide and sulphonamide functionalities at the C-5 position of the
non-reducing sugar were prepared and screened as competitive
inhibitors.12 Among a small set of disaccharides 2, N,N-dic-
yclohexylamino disaccharide analog 3 showed superior activity
as an inhibitor.12
Ideally, and with the growing body of SAR information, we
could begin to move from what might be considered routine accep-
tor-like and non-drug-like disaccharides to compounds that would
more closely fit drug-like molecules. Next, we prepared symmetri-
cal C-linked and pseudo-symmetrical O-linked disaccharides 4 and
5 possessing N,N-dicyclohexylamino moieties. Both C-sugars and
C-linked disaccharides may offer advantages as enzyme probes
and inhibitors, and most importantly, this linkage may prevent gly-
cosidase-mediated cleavage. However, both 4 and 5 showed only
modest inhibitory activity in the cell free assay system as well as
in vitro against MTB H37Ra and M. avium strains.13 In a parallel
study, the Lowary group has also synthesized Araf di- and tri-sac-
charide analogs possessing substitution at the C-5 position(s) of
the non-reducing sugars; activity was not reported for these
compounds.14
Our eventual goal was to move from prototype acceptor disac-
charides to potent drug-like GTs inhibitors. In this work, our goal
was to assess the requirement for typical saccharide-like OH sub-
stitutions (e.g., hydroxy to deoxy sugars), and some of these substi-
tutions are reported herein. Secondly, the 2-deoxy-2-fluoro-Araf
substitution is known to stabilize glycosidic linkages, and might
improve drug-like properties of even more saccharide-like
inhibitors.
a(1 ? 5) Araf disaccharides 6 and 7 possessing deoxygenation at
the 2- and 3-position of the reducing end, respectively, as shown
in Figure 1. Also, disaccharide 8 was synthesized possessing 2-
deoxy-2-fluoro at the reducing end of the disaccharide (Fig. 1)
starting from 2-fluoro-1,3,5-tri-O-benzoyl-a-D-arabinofuranose.
These analogs were prepared in order to further define the growing
SAR profile of the mycobacterial AraTs relative to the simple Araf
a(1 ? 5) Araf template acceptor.
2. Result and discussion
The synthesis of target 2-deoxy and 3-deoxy Araf disaccharides
6 and 7 were initiated from a common known Araf glycoside pre-
cursor 1125 (Schemes 1 and 2). The synthesis of precursor 11 began
with commercially available
formed to 1,2,3,5-tetra-O-acetyl-
9 was subsequently converted to n-octyl
D
-arabinose that efficiently trans-
-arabinofuranose 9.26 Glycoside
-arabinofuranoside
D
a-D
(10) as reported earlier.11 Reaction of 10 with 1,3-dichloro-
1,1,3,3-tetraisopropyl-disiloxane (TIPDSCl2) in pyridine and purifi-
cation by column chromatography on SiO2 afforded the 3,5-silyl
blocked glycoside 11 (Scheme 1).
Deoxy sugars as well as their fluoro counterparts are present in
many natural products and are a medicinally useful group of com-
pounds.15 Deoxy derivatives have been prepared as inhibitors of
glycosidases,16 GTs,17 and also to establish which hydroxyl groups
are involved in interaction with lectins.18 The preparation and bio-
logical activity of deoxy sugars and deoxy sugar oligosaccharides
have been reviewed.19 Some of the general methods for the prep-
aration of deoxy sugars are reductive methods, using such starting
materials as epoxides, thio sugars, C-halosugars, carboxylate es-
The synthesis of n-octyl 2-deoxy glycosyl acceptor 14 was
achieved in three steps starting from precursor glycoside 11. The
deoxygenation at the 2-position in glycoside 11 was carried out
in one pot by converting 11 to the xanthate intermediate through
reaction with CS2, MeI in the presence of NaH followed by radical
reduction with Bu3SnH and AIBN. After workup and purification,
2-deoxy glycoside 12 was obtained in 86% overall yield. Glycoside
12 was desilylated using Et4N+Fꢀ in THF to give 13 in 89% yield. The
final step in the synthesis of glycosyl acceptor 14 was carried out
without purification of the intermediates. The 5-OH group in 13
was selectively tritylated, and the product was subsequently ben-
zoylated and detritylated to give 14 in 65% overall yield after puri-
fication. The 1H NMR spectrum of glycosyl acceptor 14 in CDCl3
OH
HO
OH
R
O
O
OH
O
O
OR
O
O
OH
OH
OC8H17
OC8H17
2
1a: R = H
1b: R = Me
1c: R = Bn
OH
OR
HO
OH
HO
OH
R =
N-alkyl, amide, sulfonamide
O
O
OH
O
O
O
O
OH
OH
OC8H17
OC8H17
6
7
N
OH
OH
O
N
OH
O
HO
OH
X
OH
O
O
OH
O
OH
O
OH
F
N
O
O
4: X =O
5: X = CH2
OH
OH
OC8H17
3
OH
OC8H17
8
OH
Chart 1. Previously synthesized Araf
a
(1 ? 5) Araf disaccharide analogs.
Figure 1. Target Araf a(1 ? 5) Araf disaccharides.

874
A. K. Pathak et al. / Bioorg. Med. Chem. 17 (2009) 872–881
Pr-
i
HO
OH
AcO
OAc
OAc
OH
O
AcO
HO
ref. 11
O
O
OH
i
-Pr
a
O
O
b
O
Si
O
OAc
a
O
O
OC8H17
O
OC8H17
i
11
O
OH
OAc
OAc
9
OH
10
Si
O
OC8H17
OC8H17
6
i
-Pr
21
Pr-
AcO
OAc
OH
O
OBz
b
STol
OAc
Pr-
i
O
i
-Pr
20
O
AcO
OAc
HO
OH
HO
Si
O
HO
d
O
O
c
O
O
OC8H17
d
c
OPiv
OH
OC8H17
O
O
Si
O
i
OC8H17
O
O
OBz
OAc
OH
OH
i
-Pr
Pr-
14
OC8H17
OC8H17
13
22
7
12
Scheme 3. Reagents and conditions: (a) 14, NIS, Sn(OTf)2, CH2Cl2, 0 °C, 30 min, 85%;
(b) 7 N NH3/MeOH, rt, overnight, 85%; (c) 19, NIS, Sn(OTf)2, CH2Cl2, 0 °C, 30 min,
87%; (d) 7 N NH3/MeOH, rt, overnight, 87%; (e) NaOMe, MeOH, rt, overnight, 75%.
Scheme 1. Reagents and conditions: (a) TIPSCl2, pyridine, 0 °C, 4 h, 87%; (b) i—CS2,
NaH, MeI, DMF, 0 °C–rt, 30 min, ii—Bu3SnH, AIBN, rt, 3 h, overall yield 86%; (c)
Et4N+Fꢀ, THF, rt, 3 h, 89%; (d) i—TrCl, DMAP, pyridine, 45 °C, overnight, ii—BzCl,
pyridine, 50 °C, overnight; iii—5% TFA/CHCl3, ꢀ20 °C, 4 h, overall yield 65%.
cation. Disaccharide 21 was fully characterized by the 1H NMR
spectrum and anomeric proton signals were evident as a doublet
at d 5.28 ppm (H-1, J1,2ax = 4.7 Hz) and as a singlet at d 5.13 ppm
(H-10), suggesting 1,2-trans glycosylation. The assigned structure
was further supported by the 13C NMR spectrum in which the sig-
nals from the anomeric carbons C-1 and C-10 were observed at d
104.02 ppm and d 105.68 ppm, respectively. Disaccharide 21 was
deprotected using 7 N NH3/MeOH to produce the 2-deoxygenated
disaccharide 6. The 1H NMR spectrum of 6 in CD3OD at 600 MHz
showed the anomeric proton H-1 as a doublet of doublets at d
5.11 ppm (J1,2eq = 1.9 Hz, J1,2ax = 5.5 Hz) and the H-1’ proton at d
4.89 ppm as a doublet (J1’,2’ = 1.2 Hz). The 13C NMR spectrum of 6
Pr-
i
Pr-
i
OPiv
OPiv
O
O
OH
HO
b
a
i-Pr
i
-Pr
O
O
O
Si
O
Si
O
OC8H17
OC8H17
OC8H17
Si
O
i
Si
O
i
OH
i
-Pr
i
-Pr
Pr-
Pr-
16
15
11
c
OPiv
OPiv
TrO
OPiv
TrO
HO
O
O
d
O
e
OC8H17
OC8H17
OC8H17
showed anomeric carbons C-1’ and C-1 at
105.24 ppm respectively.
d 109.68 and
OH
19
18
17
The synthesis of 3-deoxygenated disaccharide 22 was accom-
plished by the reaction of glycosyl acceptor 19 and glycosyl donor
Scheme 2. Reagents and conditions: (a) (CH3)3CCOCl, pyridine, rt, overnight, 97%;
(b) Et4N+Fꢀ, THF, rt, 3 h, 91%; (c) TrCl, DMAP, Pyridine, 45 °C, 3 h, 85%; (d) i—CS2,
NaH, MeI, DMF, 0 °C, rt, 30 min, ii—Bu3SnH, AIBN, rt, 3 h, overall yield 83%; (e) 5%
TFA/CHCl3, ꢀ20 °C, 4 h, 83%.
20 similar to the synthesis of 21. The
a-glycosylation was sup-
ported by the 1H NMR spectrum of 22 that showed the anomeric
H-10 signal at 5.09 ppm, but the H-1 signal was unresolved from
the H-30 proton signal. The 13C NMR spectrum of 22 showed ano-
meric carbons C-1 and C-10 signals at 106.04 and 105.65 ppm
respectively. Global de-protection of disaccharide 22 with 25%
NaOMe in MeOH solution resulted in disaccharide 7. The 1H NMR
spectrum of 7 at 600 MHz in CD3OD showed the anomeric protons
H-1 and H-10 at d 4.87 ppm and d 4.92 ppm as a singlet and doublet
(J1’,2’ = 1.2 Hz), respectively. The 13C NMR spectrum of 7 showed
anomeric carbons at d 110.09 and d 109.56 ppm assigned to C-1
and C-10, respectively.
showed the anomeric proton at d 5.27 ppm as a doublet of doublets
(J1,2eq = 0.9 Hz, J1,2ax = 5.2 Hz). The H-2 axial and equatorial protons
in 14 were observed as ddd at d 2.43 ppm and d 2.21 ppm, respec-
tively. These assignments were made on the basis of coupling con-
stant correlation and nOe experiments. The decoupled and DEPT
13C NMR spectra of 14 were also supportive of the structure, and
showed the anomeric carbon and C-2 at
39.31 ppm, respectively.
d 103.64 and d
The synthesis of 3-deoxy acceptor glycoside 19 began from gly-
coside 11 (Scheme 2). The 2-hydroxyl group in 11 was protected by
a pivaloyl group to give 15 that, on desilylation using Et4N+Fꢀ in
THF, produced 16 in excellent yield. The 5-OH group of compound
16 was then selectively tritylated to produce glycoside 17. Com-
pound 17 was deoxygenated via a free radical pathway in two
steps as described for 12 to give the 3-deoxy glycoside 18 in 83%
yield after purification. To access the desired 3-deoxy glycosyl
acceptor 19, compound 18 was detritylated at ꢀ20 °C using TFA
in CHCl3. The structure of 3-deoxy glycoside 19 was supported
by the 1H NMR spectrum that showed the anomeric proton at d
4.99 ppm as a singlet and signals from axial and equatorial protons
at C-3 were observed as ddd at d 1.72 and d 2.47 ppm, respectively.
The 13C NMR spectra of 19 showed the anomeric carbon and C-3 at
d 105.97 and d 31.51 ppm, respectively.
The synthesis of 2-fluoro disaccharide 8 was achieved starting
from known glycoside 2-deoxy-2-fluoro-1,3,5-tri-O-benzoyl-
a-D-
arabinofuranose 2328 as presented in Scheme 4.
The 2-fluoro Araf 23 was treated with n-octanol in CH3CN in
presence of Lewis acid SnCl4 similar to a reported glycosylation
method29 and resulted in a mixture of
a
- and b-octyl glycosides.
After chromatographic purification on SiO2, the desired -octyl
-configuration
a
Araf glycoside 24 was obtained in 54% yield. The
a
in 24 was supported by the 1H NMR spectrum that showed the
anomeric proton as a doublet at d 5.30 ppm (J1,F = 10.3 Hz) and
the H-2 proton at d 5.10 as a doublet of doublets (J2,3 = 0.7 Hz,
J2,F = 49.66 Hz). The 13C NMR decoupled spectrum of 24 showed
C-1 and C-2 as doublets at
d 105.16 (J1,F = 34.8 Hz) and d
98.25 ppm (J2,F = 181.9 Hz), respectively. The synthesis of glycoside
25 was achieved in an overall yield of 81% by a three step reaction
sequence in one reaction vessel. In the first step, glycoside 24 was
debenzoylated by NH3/MeOH. After completion of the reaction, it
was concentrated under vacuum to syrup that was utilized without
purification. In the second step, the 5-hydroxyl group was selec-
Both 2-deoxy and 3-deoxy Araf glycosyl acceptors 14 and 19
were subjected to glycosylation using the known thioglycoside do-
nor 2027 as represented in Scheme 3. Glycosyl acceptor 14 on reac-
tion with thioglycoside 20 in the presence of activator NIS and
Lewis acid Sn(OTf)2 gave disaccharide 21 in 85% yield after purifi-

A. K. Pathak et al. / Bioorg. Med. Chem. 17 (2009) 872–881
875
F
F
F
BzO
TrO
BzO
O
O
O
a
b
d
OC8H17
OC8H17
OBz
OBz
OBz
OBz
25
23
24
c
AcO
OAc
HO
OH
O
O
F
HO
e
O
F
F
O
O
O
O
OAc
OH
OC8H17
OBz
OC8H17
OC8H17
8
OBz
OH
27
26
Scheme 4. Reagents and conditions: (a) C8H17OH, SnCl4, CH3CN, rt, 30 min, 54%; (b)
i—7 N NH3/MeOH, rt, overnight, ii—TrCl, DMAP, pyridine, 45 °C, 48 h, iii—BzCl,
pyridine, rt, overnight, overall yield 81%; (c) 5% TFA/CHCl3, ꢀ20 °C, 4 h, 81%; (d) 20,
NIS, Sn(OTf)2, CH2Cl2, 0 °C, 30 min, 88%; (e) 7 N NH3/MeOH, rt, overnight, 88%.
tively tritylated using TrCl in pyridine at 50 °C. In the third step, the
tritylated glycoside was benzoylated by adding BzCl in the same
reaction vessel to give glycoside 25. Purified 25 was treated with
5% TFA in CHCl3 at ꢀ20 °C to remove the trityl group to give accep-
tor glycoside 26 in 81% yield. The structure of glycoside 26 was
characterized by 1H NMR, 13C NMR and HR-ESIMS spectra.
Coupling of the glycosyl acceptor 26 and thioglycosyl donor 20
was carried out in the presence of activator NIS and the Lewis acid
Sn(OTf)2 at 0 °C to yield disaccharide 27 in 88% yield. The 1,2-trans
glycosylation was supported by the 1H NMR spectrum that dis-
played anomeric signals as a doublet at d 5.25 ppm (J1,F = 10.4 Hz,
H-1) and a signlet at d 5.17 ppm (H-10). The 13C NMR spectrum
27 showed anomeric carbons at
d 105.51 as a singlet and
d 105.01 ppm as a doublet (2J1,F = 34.8 Hz). Finally, removal of all
acyl protecting groups in 27 by NH3/MeOH produced the final
2-deoxy-2-fluoro glycoside 8 in 88% yield. The 1H NMR spectrum
in CD3OD of disaccharide 8 showed anomeric signals at d 5.06
(d, J1,F = 12.2 Hz) and
d 4.94 (d, J1’,2’ = 1.2 Hz) whereas the
13C NMR spectrum showed anomeric carbons at d 109.68 (C-1’)
2
and d 106.5 (d, JF,C-1 = 127.6 Hz).
All new compounds were characterized using 1H NMR, 13C NMR
and HR-ESIMS. The NMR shifts of mono- and di-saccharides were
compared with literature results.10,12,15,30 nOe, decoupling and
D2O exchange experiments were performed to confirm NMR
assignments as needed.
Figure 2. TLC autoradiograms of reaction products produced through the inclusion
of 6, 7 and 8, mycobacterial membranes and DP[14C]A. The TLC/autoradiography
performed using CHCl3–MeOH–NH4OH–H2O (65:25:0.4:3.6) and products revealed
through exposure to Kodak X-Omat film at ꢀ70 °C for 3 days.
3. Biological activity
3.1. In vitro activity
weak competitive inhibitors of their native acceptor 1a [Ara-
f(1 ? 5)Araf-O-C8H17] in the AraTs assay resulting in an IC50 values
of 2.45 mM, 2.15 mM, 3.58 mM, respectively.
The arabinofuranosyl disaccharides were screened against
M. tuberculosis (MTB H37Ra) and M. avium (NJ 211) in vitro as
reported.31 Disaccharides 6, 7 and 8 were active in the concentra-
tion range >12.8 6 128 lg/mL for both strains.
4. Conclusion
3.2. Cell free assay
In conclusion, we have efficiently prepared three new a(1 ? 5)–
Based on the previous use of specific Araf-based neoglycolipid
acceptors,32 compounds 6–8 were assessed as acceptors and com-
petitive inhibitors. Assays were performed in the presence of mem-
branes, resulting in [14C]Araf incorporation from the established
enzymatic Araf donor DP-[14C]Araf by compounds 6, 7 and 8. TLC
autoradiography (Fig. 2) demonstrated the enzymatic conversion
of 6, 7 and 8 to their corresponding homologated products. The
enzymatic kinetic analysis for compounds 6, 7 and 8 (Fig. 3) gave
Km values of 0.15 mM, 0.11 mM and 0.56 mM, respectively. Further
competition-based, experiments established that 6, 7 and 8 were
linked Araf disaccharides 6, 7 and 8 possessing deoxygenation at
the 2- or 3-position of the reducing sugar as biochemical probes
to study substrate specificity of AraTs in MTB. Deoxygenation re-
moves a potential metabolic site, but, additionally, the added
hydrophobicity may play an important role for inhibiting AraTs
as has been noted in reports by us and others.11–14 The activity
of these disaccharides as acceptors and competitive inhibitors
was evaluated, and only moderate activities were seen against
the AraTs in a cell free assay system as well as in the in vitro anti-
bacterial assay against MTB H37Ra and MAC strain NJ211. Clearly,

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A. K. Pathak et al. / Bioorg. Med. Chem. 17 (2009) 872–881
Currently, bis(cyclohexylmethyl)amino substituted Araf disaccha-
ride 3 is the only reported disaccharide that has shown both en-
zyme inhibition and prevention of mycobacterial growth.12 The
results from this investigation also clearly indicate that the reduc-
ing end sugar can be altered to other similar pentose ring systems
to effectively bind in the catalytic region of AraTs enzymes.
5. Experimental
5.1. Synthesis
All reactions were performed under a dry argon atmosphere and
reaction temperatures were measured externally. All chemicals and
anhydrous solvents from Aldrich were used as is in the reactions.
Whenever necessary, compounds and starting materials were dried
by azeotropic removal of water with toluene under reduced pres-
sure. Reactions were monitored by thin-layer chromatography
(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 chromatography
were reagent grade from Fisher Scientific. Flash chromatography
was carried out on Fischer silica gel 60 (230–400 Mesh). The 1H
and 13C NMRspectra were recorded on NicoletNT 300NB instrument
at 300 MHz and 75 MHz, respectively. Coupling constants (J) values
arereportedin Hzandchemicalshifts arein ppm(d) relativetoresid-
ual solvent peak or internal standard (TMS). The peak assignments in
NMR were done on the basis of coupling constant values and when-
ever needed nOe experiments were performed. The HR-ESIMS were
recorded on a BioTof-2 time-of-flight mass spectrometer.
5.2. Octyl 3,5-O-1,1,3,3-tetra-iso-propyldisilyl-a-D-
arabinofuranoside (11)25
Octyl
a-D
-arabinofuranoside 1011 (3.00 g, 11.5 mmol) was dis-
solved in dry pyridine (100 mL) under argon atmosphere and
cooled to 0 °C. TIPSCl2 (4.0 mL, 12.6 mmol) was added and the
reaction mixture was stirred at 0 °C for 4 h. The solvent was evap-
orated to dryness and the remaining oil was dissolved in 150 mL
CHCl3. The CHCl3 solution was washed with deionized water
(2 ꢁ 50 mL) and dried over Na2SO4. It was concentrated to a syrup
that on purification by column chromatography over silica gel
using CHCl3–MeOH (95:5) gave compound 11 (4.44 g, 77% yield).
HR-ESIMS: m/z Found 527.3193 [M+Na]+, calcd 527.3200 for
C25H52O6Si2Na. 1H NMR (CDCl3): d 4.87 (1H, dd, J1,2 = 0.7 Hz, J1,2-
OH = 2.5 Hz, H-1), 4.18 (1H, dd, J3,4 = 3.0 Hz, J2,3 = 6.3 Hz H-3), 4.14
(1H, ddd, J1,2 = 0.7 Hz, J2,2-OH = 2.5 Hz, J2,3 = 6.3 Hz, H-2), 3.99 (1H,
dd, J4,5a = 3.1 Hz, J5a,5b = 12.5 Hz, H-5a), 3.93 (1H, dd, J4,5b = 3.7 Hz,
J5a,5b = 12.5 Hz, H-5b), 3.88 (1H, ddd, J3,4 = 3.0 Hz, J4,5a = 3.1 Hz,
J4,5b = 3.7 Hz, H-4), 3.70 (1H, m, OCH2), 3.41 (1H, m, OCH2), 2.00
(1H, d, J1,2-OH = J2,2-OH = 2.5 Hz, 2-OH), 1.58 (2H, m, CH2), 1.29
(10H, m, 5ꢁ CH2), 1.05 (28H, m, 4ꢁ CH, 8ꢁ CH3), 0.88 (3H, m,
CH3). 13C NMR (CDCl3): d 106.75 (C-1), 82.64 (C-4), 80.71 (C-2),
76.94 (C-3), 68.36 (OCH2), 61.55 (C-5), 31.82, 29.63, 29.36, 29.24,
26.07, 22.65 (CH2), 17.46, 17.33, 17.26, 17.19, 17.14, 17.09, 17.05,
16.99, 14.09 (CH3), 13.50, 13.15, 12.81, 12.56 (CH).
Figure 3. Kinetic analysis of acceptors 6, 7 and 8. The inset illustrates the double
reciprocal plot for substrates for the mycobacterial AraTs.
the deoxygenated disaccharides can act as modest acceptors, and
can affect bacterial growth. In a previous study, compounds 1b
and 1c (possessing methyl and benzyl protecting groups at the
reducing end of an Araf disaccharide), the reported IC50 values
were 3.70 and 1.12 mM, respectively.11 The IC50 values found for
three new deoxy disaccharides 6, 7 and 8 were comparable to val-
ues obtained for compounds 1b and 1c. Deoxygenation, that is,
alteration in hydrophilicity, at the reducing end in the disaccha-
rides 6, 7 and 8 did not significantly alter AraTs substrate or com-
petitive inhibitory activity when compared to other disaccharides
bearing substitution at the reducing and non-reducing ends.11–14
5.3. Octyl 2-deoxy-3,5-di-O-1,1,3,3-tetra-iso-propyldisilyl-a-D-
arabinofuranoside (12)
A solution of compound 11 (1.60 g, 3.17 mmol) was dissolved in
dry DMF (25 mL) under argon atmosphere and cooled to 0 °C. CS2
(1.1 mL, 18.38 mmol) was added and the reaction mixture was stir-
red at 0 °C for 15 min followed by addition of NaH (380 mg,
9.51 mmol). The reaction mixture was further stirred for 30 min
and MeI (2.0 mL, 31.70 mmol) was added dropwise. The solution

A. K. Pathak et al. / Bioorg. Med. Chem. 17 (2009) 872–881
877
was stirred for 20 min at 0 °C and allowed to warm to room tem-
perature over 30 min. Deionized water (50 mL) was added to the
reaction mixture followed by extraction with EtOAc (3ꢁ 100 mL).
The combined EtOAc extract was washed with deionized water
(50 mL), brine solution (50 mL), dried over Na2SO4 and concen-
trated to yellow oil. The oil was dried under vacuum at room tem-
perature for 1 h and was used further without purification. The
crude thiocarbonyl intermediate product was dissolved in dry tol-
uene (30 mL) and Bu3SnH (3.2 mL, 12.01 mmol) followed by AIBN
(113 mg, 0.69 mmol) were added. The reaction mixture was re-
fluxed for 3 h, cooled to room temperature and concentrated under
vacuum. The crude product was purified over silica gel column
using cyclohexane–EtOAc (9:1) as the eluent to produce compound
12 (1.33 g, yield 86%) with slight impurities of tributyltin salts and
was used as such for next step. ESIMS: m/z Found 511.3249
[M+Na]+, calcd 511.3251 for C25H52O5Si2Na. 1H NMR (CDCl3): d
5.04 (1H, dd, J1,2eq = 3.5 Hz, J1,2ax = 5.7 Hz, H-1), 4.29 (1H, m,
J3,4 = 6.8 Hz, J2eq,3 = 7.0 Hz, J2ax,3 = 8.5 Hz, H-3), 4.02 (1H, m, H-5a),
3.85 (1H, m, H-4, H-5b), 3.70 (1H, m, OCH2), 3.39 (1H, m, OCH2),
2.45 (1H, ddd, J1,2ax = 5.7 Hz, J2ax,3 = 8.5 Hz, J2ax,2eq = 13.3 Hz, H-
2ax), 1.94 (1H, ddd, J1,2eq = 3.5 Hz, J2eq,3 = 7.0 Hz, J2ax,2eq = 13.3 Hz,
H-2eq), 1.55 (2H, m, CH2), 1.27 (10H, m, 5ꢁ CH2), 1.05 (28H, m,
4ꢁ CH, 8ꢁ CH3), 0.88 (3H, m, CH3).
J5a,5b = 11.8 Hz, H-5b), 3.72 (1H, m, OCH2), 3.43 (1H, m, OCH2), 2.43
(1H, ddd, J1,2ax = 5.2 Hz, J2ax,3 = 5.9 Hz, J2ax,2eq = 13.4 Hz, H-2ax), 2.21
(1H, ddd, J1,2eq = 0.9 Hz, J2eq,3 = 2.2 Hz, J2ax,2eq = 13.4 Hz, H-2eq), 1.59
2H, (m, CH2), 1.30 (10H, m, 5ꢁ CH2), 0.85 (3H, m, CH3). 13C NMR
(CDCl3): d 166.69 (C@O), 133.15 (CH, Ar), 129.86 (C, Ar), 129.70,
128.32 (each CH, Ar), 103.64 (C-1), 83.55 (C-4), 74.86 (C-3), 67.46
(OCH2), 62.74 (C-5), 39.31 (C-2), 31.79, 29.34, 29.39, 29.25, 26.24,
22.62 (each CH2), 14.04 (CH3).
5.6. Octyl 2-O-pivaloyl-3,5-O-1,1,3,3-tetra-iso-propyldisilyl-a-D-
arabinofuranoside (15)
Compound 11 (2 g, 3.96 mmol) was dissolved in dry pyridine
(10 mL) and pivaloyl chloride (0.6 mL, 4.75 mmol) and DMAP
(48 mg, 0.40 mmol) were added. The reaction mixture was stirred
overnight at room temperature and TLC showed completion of the
reaction. It was concentrated to a syrup, and purification by column
chromatography over silica gel using cyclohexane–EtOAc (95:5)
gave compound 15 (2.26 g) in 97% yield. HR-ESIMS: m/z Found
611.3760 [M+Na]+, calcd 611.3775 for C30H60O7Si2Na. 1H NMR
(CDCl3): d 5.10 (1H, dd, J1,2 = 1.1 Hz, J2,3 = 4.4 Hz, H-2), 4.78 (1H, d,
J1,2 = 1.1 Hz, H-1), 4.32 (1H, dd, J2,3 = 4.4 Hz, J3,4 = 7.0 Hz, H-3), 3.98
(3H, m, H-4, H2-5), 3.64 (1H, m, OCH2), 3.41 (1H, m, OCH2), 1.58
(2H, m, CH2), 1.27 (10H, m, 5ꢁ CH2), 1.20 (9H, s, 3ꢁ CH3), 1.05
(28H, m, 4ꢁ CH, 8ꢁ CH3), 0.87 (3H, m, CH3). 13C NMR (CDCl3): d
177.42 (C@O), 105.48 (C-1), 84.16 (C-2), 81.43 (C-4), 76.55 (C-3),
67.79 (OCH2), 62.20 (C-5), 38.61 (C), 31.83, 29.50, 29.34, 29.24
(CH2), 27.03 (CH3), 26.04, 22.65 (CH2), 17.42, 17.32, 17.29, 17.13,
17.05, 16.98, 16.94, 14.06 (CH3), 13.58, 13.22, 12.78, 12.53 (CH2).
5.4. Octyl 2-deoxy-a-D-arabinofuranoside (13)
Slightly impure compound 12 (1.20 g, 2.66 mmol) was dis-
solved in a mixture of dry THF (20 mL) and CH3CN (20 mL) under
argon atmosphere, and Et4N+Fꢀ (834 mg, 5.59 mmol) was added.
The reaction mixture was stirred at room temperature for 3 h
and concentrated to syrup. Purification over silica gel column using
CHCl3–MeOH (9:1) gave pure compound 13 (538 mg, 89%). HR-
ESIMS: m/z Found 269.1727 [M+Na]+, calcd 269.1729 for
C13H26O4Na. 1H NMR (DMSO-d6): d 4.98 (1H, dd, J1,2eq = 2.6 Hz,
J1,2ax = 5.8 Hz, H-1), 4.81 (1H, d, J3,3-OH = 5.3 Hz, 3-OH), 4.62 (1H, t,
J5a,5-OH = J5b,5-OH = 5.7 Hz, 5-OH), 3.90 (1H, dddd, J2eq,3 = 4.9 Hz,
J3,4 = 5.2 Hz, J3,3-OH = 5.3 Hz, J2ax,3 = 8.4 Hz, H-3), 3.67 (1H, ddd,
J4,5a = 3.5 Hz, J3,4 = 5.2 Hz, J4,5b = 5.7 Hz, H-4), 3.57 (1H, m, OCH2),
3.49 (1H, ddd, J4,5a = 3.5 Hz, J5a,5-OH = 11.4 Hz, H-5a), 3.35 (1H, dd,
J4,5b = J5,5-OH = 5.7 Hz, J5a,5b = 11.4 Hz, H-5b), 3.27 (1H, m, OCH2),
2.27 (1H, ddd, J1,2ax = 5.8 Hz, J2ax,3 = 8.4 Hz, J2ax,2eq = 13.4 Hz, H-
2ax), 1.62 (1H, dddd, J1,2eq = 2.6 Hz, J2eq,3 = 4.9 Hz, J = 7.6 Hz, J2ax,2-
eq = 13.4 Hz, H-2eq), 1.47 (2H, m, CH2), 1.27 (10H, m, 5ꢁ CH2),
0.87 (3H, m, CH3).
5.7. Octyl 2-O-pivaloyl-a-D-arabinofuranoside (16)
Compound 15 (2.20 g, 3.74 mmol) was treated with Et4N+Fꢀ
(1.00 g, 6.73 mmol) as described in the synthesis of compound
13. Purification over silica gel column using CHCl3–MeOH (95:5)
gave pure compound 16 (1.18 g, 91%). HR-ESIMS: m/z Found
369.2249 [M+Na]+, calcd 369.2253 for C18H34O6Na. 1H NMR
(CDCl3): d 5.06 (1H, s, H-1), 4.81 (1H, dd, J1,2 = 0.9 Hz, J2,3 = 2.5 Hz,
H-2), 4.13 (1H, ddd, J4,5b = 3.3 Hz, J4,5a = 4.6 Hz, J3,4 = 5.8 Hz, H-4),
3.96 (1H, ddd, J2,3 = 2.5 Hz, J3,4 = J3,3-OH = 5.8 Hz, H-3), 3.89 (1H,
ddd, J4,5a = 4.6 Hz, J5a,5-OH = 7.9 Hz, J5a,5b = 11.9 Hz, H-5a), 3.73 (2H,
m, H-5b, OCH2), 3.44 (1H, m, OCH2), 3.16 (1H, d, J3,3-OH = 5.8 Hz,
3-OH), 1.82 (1H, dd, J5b,5-OH = 5.2 Hz, J5a,5-OH = 7.9 Hz, 5-OH), 1.59
(2H, m, CH2), 1.27 (10H, m, 5ꢁ CH2), 1.22 (9H, s, 3ꢁ CH3), 0.88
(3H, m, CH3). 13C NMR (CDCl3): d 178.91 (C@O), 105.19 (C-1),
85.32 (C-2), 84.15 (C-4), 76.44 (C-3), 67.90 (OCH2), 62.02 (C-5),
38.71 (C), 31.79, 29.45, 29.31, 29.21 (CH2), 27.04 (CH3), 26.06,
22.63 (CH2), 14.07 (CH3).
5.5. Octyl 3-O-benzoyl-2-deoxy-a-D-arabinofuranoside (14)
To a dry pyridine (10 mL) solution of compound 13 (497 mg,
2.01 mmol) were added TrCl (672 mg, 2.40 mmol) and DMAP
(25 mg, 0.20 mmol) at room temperature. The reaction mixture
was heated at 45 °C overnight and TLC confirmed the completion
of the reaction. It was cooled to room temperature and BzCl
(0.36 mL, 3.07 mmol) was added dropwise. The reaction mixture
was heated at 50 °C overnight, cooled to room temperature and con-
centrated under vacuum to give a syrup. The syrup was dissolved in
CHCl3 (30 mL)andcooledtoꢀ20 °C, and5%TFAinCHCl3 (11 mL)was
added dropwise. The reaction mixture was stirred for 4 h at the same
temperature, co-evaporated with ethanol (2ꢁ 20 mL), and concen-
trated to syrup. Column chromatography using cyclohexane–EtOAc
(1:1) ontheoil gaveglycoside14(396 mg, overall yieldin three steps
65%) as a colorless syrup. HR-ESIMS: m/z Found 373.1996 [M+Na]+,
calcd 373.1985 for C20H30O5Na. 1H NMR (CDCl3): d 8.06 (2H, m,
Ar), 7.57 (1H, m, Ar), 5.33 (1H, ddd, J2eq,3 = 2.2 Hz, J3,4 = 3.8 Hz,
J2ax,3 = 5.9 Hz, H-3), 5.27 (1H, dd, J1,2eq = 0.9 Hz, J1,2ax = 5.2 Hz, H-1),
4.25 (1H, dd, J3.4 = J4,5a = 3.8 Hz, J4,5b = 4.0 Hz, H-4), 3.88 (1H, dd,
J4,5a = 3.8 Hz, J5a,5b = 11.8 Hz, H-5a), 3.83 (1H, dd, J4,5b = 4.0 Hz,
5.8. Octyl 2-O-pivaloyl-5-O-trityl-a-D-arabinofuranoside (17)
To a dry pyridine (20 mL) solution of compound 16 (1.18 g,
3.41 mmol) were added trityl chloride (1.16 g, 4.16 mmol) and
DMAP (42 mg, 0.34 mmol), and the reaction mixture was stirred
at 50 °C overnight. Co-evaporation with toluene (2ꢁ 50 mL) re-
sulted in an oil that was dissolved in CHCl3 (100 mL), washed with
water (2ꢁ 20 mL), dried over Na2SO4 and concentrated. Column
chromatography (cyclohexane–EtOAc 95:5) gave compound 17 as
an oil (1.70 g, 85%). HR-ESIMS: m/z Found 611.3339 [M+Na]+, calcd
611.3349 for C37H48O6Na. 1H NMR (CDCl3): d 7.46 (6H, m, Ar), 7.37
(9H, m, Ar), 5.05 (1H, s, H-1), 4.81 (1H, dd, J1,2 = 0.7 Hz, J2,3 = 2.2 Hz,
H-2), 4.28 (1H, dd, J3,4 = 4.7 Hz, J4,5b = 5.3 Hz, J4,5a = 5.8 Hz, H-4),
3.96 (1H, ddd, J2,3 = 2.2 Hz, J3,4 = 4.7 Hz, J3,3-OH = 7.1 Hz, H-3), 3.77
(1H, m, OCH2), 3.29 (1H, dd, J4,5a = 5.8 Hz, J5a,5b = 9.7 Hz, H-5a),
3.19 (1H, dd, J4,5b = 5.3 Hz, J5a,5b = 9.7 Hz, H-5b), 3.04 (1H, d, J3,3-
OH = 7.1 Hz, 3-OH), 1.60 (2H, m, CH2), 1.31 (10H, m, 5ꢁ CH2), 1.08

878
A. K. Pathak et al. / Bioorg. Med. Chem. 17 (2009) 872–881
(9H, s, 3ꢁ CH3), 0.88 (3H, m, CH3). 13C NMR (CDCl3): d 178.43
(C@O), 143.81 (C), 128.66, 127.79, 126.98 (CH), 105.10 (C-1),
86.58 (C), 83.95 (C-2), 83.86 (C-4), 77.22 (C-3), 67.80 (OCH2),
63.82 (C-5), 38.53 (C), 31.78, 29.51, 29.31, 29.20 (CH2), 26.94
(CH3), 26.08, 22.61 (CH2), 14.06 (CH3).
(200 mg) in dry CH2Cl2 (15 mL) were cooled at 0 °C under argon
atmosphere. The mixture was stirred for 15 min, and NIS
(327 mg, 1.45 mmol) followed by Sn(OTf)2 (51 mg, 0.12 mmol)
was added to initiate coupling. It was stirred for 30 min at rt,
and the reaction was quenched by addition of Et3N (1 mL). The
reaction mixture was diluted with CH2Cl2 (20 mL) and filtered
through a Celite pad. The filtrate was washed with 10% Na2S2O3
(20 mL), followed by washing with saturated aqueous NaHCO3
(20 mL). The organic layer was dried over Na2SO4, the solvent
was removed under vacuum, and the residue was purified by col-
umn chromatography (cyclohexane–EtOAc, 3:1) to give pure disac-
charide 21 as a colorless oil (627 mg, 85%). HR-ESIMS: m/z Found
631.2741 [M+Na]+, calcd 631.2725 for C31H44O12Na. 1H NMR
(CDCl3): d 8.05 (2H, m, Ar), 7.57 (1H, m, Ar), 7.43 (2H, m, Ar),
5.43 (1H, ddd, J3,4 = 3.0 Hz, J2eq,3 = 4.6 Hz, J2ax,3 = 7.7 Hz, H-3), 5.28
(1H, d, J1,2ax = 4.7 Hz, H-1), 5.13 (1H, s, H-1’), 5.11 (1H, d,
5.9. Octyl 3-deoxy-2-O-pivaloyl-5-O-trityl-a-D-arabinofuranoside
(18)
A solution of compound 17 (1.70 g, 2.89 mmol) was dissolved in
dry DMF (25 mL) under argon atmosphere and cooled to 0 °C. CS2
(1.01 mL, 16.75 mmol) was added and reaction mixture was stirred
at 0 °C for 15 min followed by addition of NaH (347 mg,
8.67 mmol). Reaction mixture was further stirred for 30 min, MeI
(1.8 mL, 28.9 mmol) was added dropwise. The reaction mixture
was stirred for 20 min at 0 °C and allowed to warm to room tem-
perature in 30 min. Deionized water (50 mL) was added to reaction
mixture and extracted with EtOAc (3ꢁ 100 mL). The combined
EtOAc extract was washed with deionized water (50 mL), aq. satd.
NaCl solution (50 mL), dried over Na2SO4 and concentrated to a
yellow oil. It was dried under vacuum at room temperature for
1 h and was used further without purification. The crude interme-
diate product was dissolved in dry toluene (30 mL) and Bu3SnH
(2.0 mL, 7.35 mmol) followed by AIBN (69 mg, 0.42 mmol) were
added. The reaction mixture was refluxed for 3 h, cooled to room
temperature and concentrated under vacuum. The crude product
was purified over silica gel column using cyclohexane–EtOAc
(98:2) as the eluent to produce compound 18 (1.37 g, yield 83%).
HR-ESIMS: m/z Found 595.3405 [M+Na]+, calcd 595.3394 for
C37H48O5Na. 1H NMR (CDCl3): d 7.45 (6H, m, Ar), 7.26 (9H, m,
Ar), 4.98 (1H, dd, J2,3ax = 1.3 Hz, J2,3eq = 6.2 Hz, H-2), 4.96 (1H, s, H-
1), 4.51 (1H, dddd, J3ax,4 = 4.3 Hz, J4,5b = 5.8 Hz, J4,5a = 6.4 Hz,
J3eq,4 = 8.5 Hz, H-4), 3.68 (1H, m, OCH2), 3.41 (1H, m, OCH2), 3.31
J2’,3’ = 1.3 Hz, H-2’), 4.97 (1H, dd, J2’,3’ = 1.3 Hz, J3’,4’ = 4.6 Hz, H-3’),
4.43 (1H, dd, J4’5a’ = 3.0 Hz, J5a’,5b’ = 11.3 Hz, H-5a’), 4.36 (1H, dd,
J3,4 = J4,5a = 3.0 Hz, J4,5b = 5.7 Hz, H-4), 4.27 (1H, ddd, J4’5a’ = 3.0 Hz,
J3’,4’ = 4.6 Hz, J4’5b’ = 5.7 Hz, H-4’), 4.21 (1H, dd, J4’5b’ = 5.7 Hz,
J5a’5b’ = 11.3 Hz, H-5b’), 4.00 (1H, dd, J4.5a = 3.0 Hz, J5a5b = 11.1 Hz,
H-5a), 3.71 (2H, m, H-5b, OCH2), 3.42 (1H, m, OCH2), 2.46 (1H,
ddd, J1,2ax = 4.7 Hz, J2ax,3 = 7.7 Hz, J2ax,2eq = 13.1 Hz, H-2ax), 2.10
(1H, m, H-2eq), 2.09, 2.10, 2.11 (each 3H, s, 3ꢁ CH3), 1.58 (2H, m,
CH2), 1.31 (10H, m, 5ꢁ CH2), 0.86 (3H, m, CH3). 13C NMR (CDCl3):
d 170.52, 170.07, 169.42, 166.36 (4ꢁ C=O), 133.04 (CH, Ar),
130.00 (C, Ar), 129.63, 128.28 (each CH2, Ar), 105.68 (C-1’),
104.02 (C-1), 82.50 (C-4), 80.90 (C-4’), 80.85 (C-2’), 77.06 (C-3’),
74.83 (C-3), 67.42 (OCH2), 66.67 (C-5), 63.26 (C-5’), 39.25 (C-2),
31.77, 29.76, 29.37, 29.23, 26.23, 22.58 (6ꢁ CH2), 20.71, 20.68,
20.64, 14.02 (4ꢁ CH3).
5.12. Octyl 2,3,5-tri-O-acetyl-a-D-arabinofuranosyl-(1 ? 5)-3-
(1H, dd, J4,5a = 6.4 Hz, J5a,5b = 9.2 Hz, H-5a), 3.00 (1H, dd, J4,5b
5.8 Hz, J5a,5b = 9.2 Hz, H-5b), 2.48 (1H, ddd, J2,3eq = 6.2 Hz, J3eq,4
=
=
deoxy-2-O-pivaloyl- -arbinofuranoside (22)
a-D
8.5 Hz, J3ax,3eq = 14.2 Hz, H-3eq), 1.70 (1H, ddd, J2,3ax = 1.3 Hz,
J3ax,4 = 4.3 Hz, J3ax,3eq = 14.2 Hz, H-3ax), 1.54 (2H, m, CH2), 1.30
(10H, m, 5ꢁ CH2), 1.02 (9H, s, 3ꢁ CH3), 0.88 (3H, m, CH3).
Donor glycoside 20 (503 mg, 1.32 mmol), acceptor glycoside 19
(290 mg, 0.90 mmol) were coupled as described for the synthesis
of disaccharide 21. The crude disaccharide was purified by column
chromatography (cyclohexane–EtOAc, 3:1) to give pure disaccha-
ride 22 (449 mg, 87%) as a colorless oil. HR-ESIMS: m/z Found
611.3038 [M+Na]+, calcd 611.3001 for C29H48O12Na. 1H NMR
(CDCl3): d 5.13 (1H, d, J = 1.8 Hz, H-20), 5.09 (1H, s, H-10), 5.03
(1H, dd, J2,3ax = 1.7 Hz, J2,3eq = 6.4 Hz, H-2), 4.98 (2H, m, H-1, H-30),
5.10. Octyl 3-deoxy-2-O-pivaloyl-a-D-arabinofuranoside (19)
Compound 18 (1.00 g, 1.75 mmol) was dissolved in CHCl3
(30 mL) and cooled to ꢀ20 °C, and 5% TFA in CHCl3 (11 mL) was
added dropwise. The reaction mixture was stirred for 4 h, co-evapo-
rated with ethanol (2ꢁ 20 mL), and concentrated to syrup. Column
chromatography (cyclohexane–EtOAc, 3:1) yielded compound 19
(479 mg, yield 83%) as a colorless syrup. HR-ESIMS: m/z Found
353.2296 [M+Na]+, calcd 353.2304 for C18H34O5Na. 1H NMR (CDCl3):
d 5.05 (1H, dd, J2,3ax = 1.3 Hz, J2,3eq = 6.4 Hz, H-2), 4.99 (1H, s, H-1),
4.31 (1H, m, H-4), 3.77 (1H, ddd, J4,5a = 3.1 Hz, J5a,OH = 5.7 Hz,
J5a,5b = 11.8 Hz, H-5a), 3.66 (1H, m, OCH2), 3.60 (1H, ddd,
J4,5b = 5.5 Hz, J5b,OH = 6.7 Hz, J5a,5b = 11.8 Hz, H-5b), 3.41 (1H, m,
OCH2), 2.47 (1H, ddd, J2,3eq = 6.4 Hz, J3eq,4 = 8.8 Hz, J3ax,3eq = 14.4 Hz,
H-3eq), 1.87 (1H, dd, J5a,OH = 5.7 Hz, J5b,OH = 6.7 Hz, 5-OH), 1.72 (1H,
ddd, J2,3ax = 1.3 Hz, J3ax,5a = 5.7 Hz, J3ax,5b = 6.7 Hz, H-3ax), 1.58 (2H,
m, CH2), 1.28 (10H, m, 5ꢁ CH2), 1.19 (9H, s, 3ꢁ CH3), 0.88 (3H, m,
CH3). 13C NMR (CDCl3): d 177.60 (C@O), 105.97 (C-1), 78.34 (C-4),
77.44 (C-2), 67.39, (OCH2), 64.76 (C-5), 38.57 (C), 31.80 (CH2),
31.51 (C-3), 29.51, 29.33, 29.21 (CH2), 27.03 (CH3), 26.07, 22.62
(CH2), 14.07 (CH3).
4.42 (1H, dd, J4 5a = 2.1 Hz, J5a ,5b = 10.6 Hz, H-5a0), 4.36 (1H, m,
H-4), 4.24 (2H, m, H-40, H-5b0), 3.82 (1H, dd, J4,5a = 6.2 Hz,
J5a,5b = 10.3 Hz, H-5a), 3.65 (1H, m, OCH2), 3.51 (1H, dd,
J4,5b = 5.5 Hz, J5a,5b = 10.3 Hz, H-5b), 3.40 (1H, m, OCH2), 2.47 (1H,
ddd, J2,3eq = 6.4 Hz, J3eq,4 = 8.2 Hz, J3ax,3eq = 14.2 Hz, H-3eq), 2.10
(6H, s, 2ꢁ CH3), 2.09 (3H, s, CH3), 1.71 (1H, ddd, J2,3ax = 1.7 Hz,
J = 5.0 Hz, J3ax,3eq = 14.2 Hz, H-3ax), 1.55 (2H, m, CH2), 1.28 (10H,
m, 5ꢁ CH2), 1.19 (9H, s, 3ꢁ CH3), 0.88 (3H, m, CH3). 13C NMR
(CDCl3): d 177.69, 170.56, 170.12, 169.54 (C@O), 106.04 (C-1),
105.65 (C-10), 81.19 (C-20), 80.22 (C-40), 77.32 (C-2), 77.10 (C-30),
76.12 (C-4), 69.66 (OCH2), 67.41 (C-5), 63.18 (C-50), 38.53 (C),
32.51 (C-3), 31.79, 29.50, 29.33, 29.21 (CH2), 27.02 (CH3), 26.05
(CH2), 22.61 (CH2), 20.76, 20.72 (CH3), 14.06 (CH3).
0
0
0
0
5.13. Octyl 2-deoxy-2-fluoro-3,5-O-dibenzoyl-a-D-
arabinofuranoside (24)
Compound 2328 (900 mg, 1.94 mmol) was dissolved in dry
CH3CN (200 mL) was added SnCl4 (0.27 mL, 2.33 mmol) at room
temperature and the mixture was stirred for 15 min. To this solu-
tion, n-octanol (0.37 mL, 2.33 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 ice-water bath and a saturated
5.11. Octyl 2,3,5-tri-O-acetyl-
a-D-arabinofuranosyl-(1 ? 5)-2-
deoxy-3-O-benzoyl- -arbinofuranoside (21)
a-D
Donor glycoside 20 (695 mg, 1.82 mmol), acceptor glycoside 14
(425 mg, 1.21 mmol) and activated, powdered 4 Å molecular sieves

A. K. Pathak et al. / Bioorg. Med. Chem. 17 (2009) 872–881
879
2
aqueous NaHCO3 solution was added dropwise to precipitate tin
salts. After complete precipitation, the mixture was filtered
through Celite and washed with chloroform (2ꢁ 10 mL), concen-
trated to a syrup, and re-dissolved in CHCl3 (100 mL). The solution
was next washed with water (2ꢁ 25 mL) and brine (2ꢁ 25 mL),
dried over Na2SO4 and concentrated under vacuum to give a crude
oil. Column chromatography (cyclohexane–EtOAc, 7:1) gave the
129.89 (CH), 129.81 (C), 128.52 (CH), 104.84 (d, J1,F = 34.8 Hz, C-
1
3
1), 98.43 (d, J2,F = 181.9 Hz, C-2), 83.29 (d, J4,F = 1.8 Hz, C-4),
77.30 (d, J3,F = 29.0 Hz, C-3), 67.44 (OCH2), 62.32 (C-5), 31.81,
29.49, 29.37, 29.26 (CH2), 26.14 (CH2), 22.65 (CH2), 14.07 (CH3).
2
5.16. Octyl 2,3,5-tri-O-acetyl- -arabinofuranosyl-(1 ? 5)-2-
deoxy-2-fluoro-3-O-benzoyl- -D-arbinofuranoside (27)
a
-D
a
desired
a-isomer 24 as a colorless oil (403 mg, 54% yield). HR-
ESIMS: m/z Found 495.2153 [M+Na]+, calcd 495.2154 for
C27H33FO6Na. 1H NMR (CDCl3): d 8.05 (4H, m, Ar), 7.57 (2H, m,
Ar), 7.43 (4H, m, Ar), 5.48 (1H, ddd, J2,3 = 0.7 Hz, J3,4 = 4.7 Hz,
J3,F = 22.4 Hz, H-3), 5.30 (1H, d, J1,2 = 0 Hz, J1,F = 10.3 Hz, H-1), 5.10
(1H, dd, J1,2 = 0.0 Hz, J2,3 = 0.7 Hz, J2,F = 49.66 Hz, H-2), 4.74 (1H,
dd, J4,5a = 3.5 Hz, J5a,5b = 11.9 Hz, H5a), 4.62 (1H, dd, J4,5b = 4.6 Hz,
J5a,5b = 11.9 Hz, H5b), 4.50 (1H, dd, J4,5a = 3.5 Hz, J4,5b = 4.6 Hz, H-4),
3.76 (1H, m, OCH2), 3.49 (1H, m, OCH2), 1.59 (2H, m, CH2), 1.30
(10H, m, 5ꢁ CH2), 0.86 (3H, m, CH3). 13C NMR (CDCl3): d 166.37,
165.77 (2ꢁ C=O), 133.68, 133.18, 129.97, 129.90, 128.59, 128.44
(Ar), 105.16 (J1,F = 34.8 Hz, C-1), 98.25 (J2,F = 181.9 Hz, C-2), 81.00
(J4,F = 1.8 Hz, C-4), 77.63 (J3,F = 30.5 Hz, C-3), 67.61 (OCH2), 63.83
(C-5), 31.90, 29.58, 29.45, 29.35, 26.23, 22.73 (CH2), 14.16 (CH3).
Glycosyl donor 20 (47 mg, 0.12 mmol), glycosyl acceptor 26
(30 mg, 0.08 mmol) were coupled as described for the synthesis of
disaccharide 21. The crude disaccharide was purified by column
chromatography (cyclohexane–EtOAc, 3:1) to give pure disaccharide
27 (45 mg, 88%) as a colorless oil. HR-ESIMS: m/z Found 649.2620
[M+Na]+, calcd 649.2631 for C31H43FO12Na. 1H NMR (CDCl3): d 8.03
(2H, m, Ar), 7.59 (1H, m, Ar), 7.45 (2H, m, Ar), 5.51 (1H, ddd,
J2,3 = 0.9 Hz, J3,4 = 5.1 Hz, J3,F = 22.3 Hz, H-3), 5.25 (1H, d,
J1,F = 10.6 Hz, H-1), 5.18 (1H, d, J2’,3’ = 1.3 Hz, H-2’), 5.17 (1H, s, H-1’),
5.04 (1H, dd, J2,3 = 0.9 Hz, J2,F = 43.8 Hz, H-2), 4.95 (1H, m, H-3’),
4.44 (1H, dd, J4’,5’a = 3.4 Hz, J5’a,5’b = 11.6 Hz, H-5’a), 4.33 (2H, m, H-4,
H-4’), 4.21 (1H, dd, J4’,5’b = 5.6 Hz, J5’a,5’b = 11.6 Hz, H-5’b), 4.03 (1H,
dd, J4,5a = 3.8 Hz, J5a,5b = 11.3 Hz, H-5a), 3.83 (1H, dd, J4,5b = 3.1 Hz,
J5a,5b = 11.3 Hz, H-5b), 3.74 (1H, m, OCH2), 3.47 (1H, m, OCH2), 2.09
(6H, s, 2ꢁ OCH3), 2.08 (3H, s, OCH3), 1.58 (2H, m, CH2), 1.28 (10H,
m, 5ꢁ CH2), 0.86 (3H, m, CH3). 13C NMR (CDCl3): d 170.62, 170.43,
169.38 (COCH3), 165.58 (COC6H5), 133.53, 129.82 (CH), 129.17 (C),
5.14. Octyl 2-deoxy-2-fluoro-3-O-benzoyl-5-O-trityl-a-D-
arabinofuranoside (25)
2
To a solution of saccharide 24 (260 mg, 0.55 mmol) in dry meth-
anol (5 mL) was added 7 N NH3/MeOH (10 mL) and the reaction mix-
ture was stirred overnight at room temperature. TLC showed no
starting material and it was concentration under vacuum to an oil.
The crude product was dissolved in dry pyridine (4 mL) and trityl
chloride (108 mg, 0.39 mmol) and DMAP (4 mg, 0.03 mmol) were
added. The reaction mixture was stirred at 50 °C overnight, cooled
to room temperature and BzCl (0.05 mL, 0.44 mmol) was added.
The reaction mixture was heated overnight at 50 °C. Co-evaporation
with toluene (2ꢁ 25 mL) resulted in an oil that was dissolved in
CHCl3 (100 mL), washed with water (2ꢁ 20 mL), dried over Na2SO4
and concentrated. Column chromatography (cyclohexane–EtOAc,
95:5) gave compound 25 as oil (272 mg, overall yield 81%). HR-
ESIMS: m/z Found 633.2997 [M+Na]+, calcd 633.2987 for C39H43FO5-
Na. 1H NMR (CDCl3): d 8.03 (2H, m, Ar), 7.51 (9H, m, Ar), 7.20 (9H, m,
Ar), 5.41 (1H, ddd, J2,3 = 1.0 Hz, J3,4 = 5.1 Hz, J3,F = 22.6 Hz, H-3), 5.25
(1H, d, J1,F = 10.6 Hz, H-1), 4.99 (1H, dd, J2,3 = 1.0 Hz, J2,F = 49.7 Hz,
H-2), 4.32 (1H, dd, J4,5a = 2.5 Hz, J = 9.5 Hz, H-5a), 3.75 (1H, ddd,
J4,5b = 6.6 Hz, J = 9.5 Hz, H-5b), 3.49 (1H, dd, J4,5a = 2.5 Hz,
J4,5b = 6.6 Hz, H-4), 3.41 (2H, m, OCH2), 1.59 (2H, m, CH2), 1.30
(10H, m, CH2), 0.86 (3H, m, CH3).
128.49 (CH), 105.51 (C-1’), 105.01 (d, J1,F = 34.8 Hz, C-1), 98.70 (d,
2J2,F = 182.5 Hz, C-2), 81.38 (d, 3J4,F = 1.2 Hz, C-4), 81.04 (C-4’), 80.74
2
(C-2’), 77.00 (C-3’), 76.61 (d, J3,F = 18.9 Hz, C-3), 67.47 (OCH2),
65.49 (C-5), 63.38 (C-5’), 31.80, 29.46, 29.36, 29.26, 26.13, 22.63
(CH2), 20.78, 20.74, 20.62 (COCH3), 14.06 (CH3).
5.17. Octyl
a-D-arabinofuranosyl-(1 ? 5)-2-deoxy-a-D-
arbinofuranoside (6)
To a solution of disaccharide 21 (200 mg, 0.33 mmol) in dry
methanol (5 mL) was added 7 N NH3/MeOH (5 mL) and the reaction
mixture was stirred overnight at room temperature. Concentration
in vacuum, and column chromatography (CHCl3–MeOH, 9:1) gave
the de-blocked disaccharide containing a minor impurity. For the fi-
nal purification, an aqueous solution (5 mL) of the disaccharide was
passed through a small column packed with Bio-BeadsTM SM-4 (20–
50 mesh) and lyophilized to afford 6 as a hygroscopic solid
(105 mg, 85%). HR-ESIMS: m/z Found 401.2156 [M+Na]+ calcd
401.2164 for C18H34O8Na. 1H NMR (CD3OD): d 5.11 (1H, dd,
J1,2eq = 1.9 Hz, J1,2ax = 5.5 Hz, H-1), 4.89 (1H, d, J1’,2’ = 1.2 Hz, H-1’),
4.15 (1H, ddd, J2eq,3 = 5.6 Hz, J2ax,3 = 7.9 Hz, J3,4 = 8.6 Hz, H-3), 4.00
(1H, ddd, J4,5a = J4,5b = 4.6 Hz, J3,4 = 8.6 Hz, H-4), 3.95 (1H, dd,
5.15. Octyl 2-deoxy-2-fluoro-3-O-benzoyl-
a-
D-
J1’,2’ = 1.2 Hz, J2’,3’ = 3.3 Hz, H-2’), 3.94 (1H, ddd, J4’,5’a = 3.3 Hz,
arabinofuranoside (26)
J4’,5’b = 5.6 Hz, J3’,4’ = 6.3 Hz, H-4’), 3.81 (1H, dd, J2’,3’ = 3.3 Hz,
J3’,4’ = 6.3 Hz, H-3’), 3.78 (1H, dd, J4,5a = 4.6 Hz, J5a,5b = 11.8 Hz, H-
5a), 3.73 (1H, dd, J4’,5’a = 3.3 Hz, J5’a,5’b = 11.8 Hz, H-5a’), 3.68 (1H, m,
OCH2), 3.62 (1H, dd, J4’,5’b = 5.6 Hz, J5’a,5’b = 11.8 Hz, H-5b’), 3.54
(1H, dd, J4,5b = 4.6 Hz, J5a,5b = 11.8 Hz, H-5b), 3.38 (1H, m, OCH2),
2.35 (1H, ddd, J1,2ax = 5.5 Hz, J2ax,3 = 7.9 Hz, J2ax,2eq = 13.6 Hz, H-2ax),
Compound 25 (80 mg, 0.13 mmol) was dissolved in CHCl3
(3 mL) and cooled to ꢀ20 °C, and 5% TFA in CHCl3 (1.3 mL) was
added dropwise. The reaction mixture was stirred for 1 hr and cold
aqueous satd. NaHCO3 solution (10 mL) was added. It was ex-
tracted with CHCl3 (2ꢁ 15 mL), washed with deionized water
(2ꢁ 10 mL) and dried over Na2SO4. After concentration and column
chromatography (cyclohexane–EtOAc, 5:1) compound 26 (38 mg,
yield 81%) was obtained. HR-ESIMS: m/z Found 391.1891
[M+Na]+, calcd 391.1891 for C20H29FO5Na. 1H NMR (CDCl3): d
8.04 (2H, m, Ar), 7.60 (1H, m, Ar), 7.46 (2H, m, Ar), 5.38 (1H, ddd,
J1,3 = 0.9 Hz, J3,4 = 4.8 Hz, J3,F = 23.0 Hz, H-3), 5.25 (1H, d,
J1,2 = 0 Hz, J1,F = 10.4 Hz, H-1), 5.10 (1H, dd, J1,4 = 1.1 Hz,
J2,F = 49.7 Hz, H-2), 4.24 (1H, dd, J = 4.3 Hz, 8.4 Hz, H-4), 3.93 (2H,
m, H2-5), 3.74 (1H, m, OCH2), 3.48 (1H, m, OCH2), 2.15 (1H, dd,
J = 5.3, 7.9 Hz, 5-OH), 1.60 (2H, m, CH2), 1.29 (10H, m, 5ꢁ CH2),
0.86 (3H, m, CH3). 13C NMR (CDCl3): d 166.01 (C=O), 133.63,
1.82 (1H, dddd, J1,2eq = 1.9 Hz, J = 3.7 Hz, J2eq,3 = 5.6 Hz, J2ax,2eq
=
13.6 Hz, H-2eq), 1.55 (2H, m, CH2), 1.32 (10H, m, 5ꢁ CH2), 0.89 (3H,
m, CH3). 13C NMR (CD3OD): d 109.68 (C-1’), 105.24 (C-1), 85.79 (C-
4’), 84.89 (C-4), 83.31 (C-2’), 78.84 (C-3’), 72.82 (C-3), 68.88
(OCH2), 68.31 (C-5), 63.08 (C-5’), 42.10 (C-2), 33.00, 30.75, 30.49,
30.41, 27.31, 23.70 (6ꢁ CH2), 14.42 (CH3).
5.18. Octyl
a-D-arabinofuranosyl-(1 ? 5)-3-deoxy-a-D-
arbinofuranoside (7)
To a solution of disaccharide 22 (160 mg, 0.35 mmol) in dry
methanol (6 mL) was added 25% w/v NaOMe–MeOH (0.15 mL),

880
A. K. Pathak et al. / Bioorg. Med. Chem. 17 (2009) 872–881
and the reaction mixture was stirred overnight at room tempera-
ture. Concentration under vacuum and column chromatography
(CHCl3–MeOH, 9:1) gave the de-blocked disaccharide. For the final
purification, an aqueous solution (5 mL) of the disaccharide was
passed through a small column packed with Bio-BeadsTM SM-4
(20-50 mesh), and the eluted fractions were lyophilized to afford
7 as a hygroscopic solid (77 mg, 75%). HR-ESIMS: m/z Found
401.2108 [M+Na]+, calcd 401.2146 for C18H34O8Na. 1H NMR
(CD3OD): d 4.92 (1H, d, J1’,2’ = 1.2 Hz, H-1’), 4.87 (1H, s, H-1), 4.29
(1H, m, H-4), 4.08 (1H, dd, J2,3ax = 1.8 Hz, J2,3eq = 3.7 Hz, H-2), 3.96
(1H, m, H-4’), 3.97 (1H, dd, J1’,2’ = 1.2 Hz, J2’,3’ = 3.3 Hz, H-2’), 3.82
(1H, dd, J2’,3’ = 3.3 Hz, J3’,4’ = 5.8 Hz, H-3’), 3.80 (1H, dd,
J4,5a = 5.3 Hz, J5a,5b = 10.5 Hz, H-5a), 3.73 (1H, dd, J4’,5’a = 3.4 Hz,
J5’a,5’b = 11.8 Hz, H-5’a), 3.67 (1H, m, OCH2), 3.62 (1H, dd,
J4’,5’b = 5.3 Hz, J5’a,5’b = 11.8 Hz, H-5’b), 3.55 (1H, dd, J4,5b = 6.5 Hz,
J5a,5b = 10.5 Hz, H-5b), 3.37 (1H, m, OCH2), 2.34 (1H, ddd,
J2,3eq = 3.7 Hz, J3eq,4 = 8.7 Hz, J3ax,3eq = 13.5 Hz, H-3eq), 1.67 (1H,
ddd, J2,3ax = 1.8 Hz, J3ax,4 = 4.7 Hz, J = 13.5 Hz, H-3ax), 1.52 (2H, m,
CH2), 1.30 (10H, m, 5ꢁ CH2), 0.89 (3H, m, CH3). 13C NMR (CD3OD):
d 110.09 (C-1), 109.56 (C-1’), 86.06 (C-4’), 83.08 (C-2’), 78.72 (C-3’),
78.29 (C-4), 76.11 (C-2), 70.66 (C-5), 68.21 (OCH2), 63.07 (C-5),
35.61 (C-3), 33.01, 30.70, 30.41, 27.28, 23.71 (CH2), 14.43 (CH3).
move any residual solvent. The dried constituents of the assay
were then resuspended in 8 L of a 1% aqueous solution of Igepal.
The remaining constituents of the AraTs assay containing 50 mM
l
MOPS (adjusted to pH 8.0 with KOH), 5 mM b-mercaptoethanol,
10 mM MgCl2, 1 mM ATP, membranes (250
nal reaction volume of 80 L. The reaction mixtures were then
incubated at 37 °C for 1 h. A CHCl3–CH3OH (1:1, 533 L) solution
lg) were added to a fi-
l
l
was then added to the incubation tubes and the entire contents
centrifuged 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-equilibrated [C2H5OH–H2O (1:1)]
1 mL Whatmann 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-buta-
nol saturated water, the pooled extracts were back-washed twice
with water saturated with n-butanol (3 mL). The n-butanol-satu-
rated water fraction was dried and re-suspended in 200 lL of n-
butanol. The total cpm of radiolabeled material extractable into
the n-butanol phase was measured by scintillation counting using
10% of the labelled material and 10 mL of EcoScintA (National
Diagnostics, Atlanta). The incorporation of [14C]Araf was deter-
mined by subtracting counts present in control assays (incubation
of the reaction components in the absence of the compounds). An-
other 10% of the labelled material was subjected to thin-layer chro-
matography (TLC) in CHCl3–CH3OH–NH4OH–H2O (65:25:0.5:3.6)
on aluminium backed Silica Gel 60 F254 plates (E. Merck, Darms-
tadt, Germany). Autoradiograms were obtained by exposing TLCs
to X-ray film (Kodak X-Omat) for 3 days. IC50 values for 6, 7 and
8 were evaluated by competition-based experiments performed
by mixing either 6, 7 or 8 at various concentrations 0.5, 0.75, 1.0,
5.19. Octyl
a-D-arabinofuranosyl-(1 ? 5)-2-deoxy-2-fluoro-a-D-
arbinofuranoside (8)
To a solution of disaccharide 27 (40 mg, 0.06 mmol) in dry
methanol (3 mL) was added 7 N NH3/MeOH (5 mL), and the
reaction mixture was stirred overnight at room temperature.
Concentration under vacuum and column chromatography
(CHCl3–MeOH 9:1) gave disaccharide 8 as an oil (22 mg, 88%).
HR-ESIMS: m/z Found 419.2051 [M+Na]+, calcd 419.2055 for
C18H33FO8Na. 1H NMR (300 MHz, CD3OD): 5.06 (1H, d,
3J1,F = 12.2 Hz, H-1), 4.94 (1H, d, J1’,2’ = 1.2 Hz, H-1’), 4.75 (1H, dd,
2.0, 4.0 and 6.0 mM with Araf
a(1 ? 5)Araf-O-C8H17 at 0.4 mM
2
concentration followed by thin-layer chromatography/autoradiog-
raphy (TLC not shown) as described earlier, and the extent of prod-
uct formation was determined. The Rf on TLC of product formed by
native acceptor 1a was found to be 0.33 and readily distinguishable
from other product bands; the Rf values of products formed by 6, 7
and 8 were 0.40, 0.42 and 0.45, respectively.
J2,3 = 2.0 Hz, J2,F = 52.2 Hz, H-2), 4.14 (1H, ddd, J2,3 = 2.5 Hz, J3,4
=
=
3
6.7 Hz, J3,F = 26.3 Hz, H-3), 4.03 (1H, ddd, J3,4 = 6.7 Hz, J4,5b
3.4 Hz, J4,5a = 5.9 Hz, H-4), 4.00 (1H, dd, J1’,2’ = 3.3 Hz, J2’,3’ = 3.3 Hz,
H-2’), 3.97 (1H, ddd, J4’,5a’ = 3.4 Hz, J4’,5b’ = 4.9 Hz, J3’,4’ = 6.1 Hz, H-
4’), 3.86 (1H, dd, J4’5’a = 4.9 Hz, J5’a5’b = 11.3 Hz, H-5’a), 3.83 (1H,
dd, J2’,3’ = 3.3 Hz, J3’,4’ = 6.1 Hz, H-3’), 3.75 (1H, dd, J4,5a = 3.4 Hz,
J5a,5b = 11.9 Hz, H-5a), 3.70 (1H, m, OCH2), 3.67 (1H, dd,
J4,5b = 3.4 Hz, J5a,5b = 11.3 Hz, H-5b), 3.64 (1H, dd, J4,5b = 5.9 Hz,
J5a,5b = 11.9 Hz, H-5b), 3.43 (1H, m, OCH2), 1.58 (2H, m, CH2), 1.34
(10H, m, 5ꢁ CH2), 0.90 (3H, m, CH3). 13C NMR (CD3OD): d 109.68
(C-1’), 106.5 (d, 2JF,C-1 = 127.6 Hz, C-1), 103.37 (d, 1JF,C-
Acknowledgments
This work was supported by NIH/NIAID Grant R01AI45317.
We acknowledge Dr. Kamal Tiwari for providing precursor com-
pound 23. GSB acknowledges support in the form of a Personal
Research Chair from Mr. James Bardrick, a Royal Society Wolfson
Research Merit Award, as a former Lister Institute-Jenner Re-
search Fellow, the Medical Research Council (G9901077 and
G0500590) and The Welcome Trust (081569/2/06/2). The M. avi-
um strain (NJ211) was kindly provided by Dr. Leonid Heifets, Na-
tional Jewish Center for Immunology and Respiratory Diseases,
Denver, CO, USA.
3
2 = 181.3 Hz, C-2), 85.80 (C-4’), 83.25 (d, JF,C-4 = 4.9 Hz, C-4),
2
83.24 (C-2’), 78.84 (C-3’), 77.21 (d, JF,C-3 = 26.3 Hz, C-3), 68.69
(OCH2), 67.48 (C-5), 63.06 (C-5’), 33.00, 30.56, 30.46, 30.40,
27.21, 23.70 (6ꢁ CH2), 14.43 (CH3).
6. Biological
6.1. In vitro assay
References and notes
In vitro inhibition assays31 of the Araf disaccharides 6, 7 and 8
were performed on Mycobacterium tuberculosis (MTB H37Ra, ATCC
25177) and Mycobacterium avium (NJ 211).
1. (a) World Health Organization. Available form: http://www.who.int/
mediacentre/factsheets/fs104/en/index.html.; (b) Palmer, M. V. Curr. Top.
Microbiol. Immunol. 2007, 315, 195; (c) Onyebujoh, P. C.; Ribeiro, I.; Whalen,
C. C. J. Infect. Dis. 2007, 196, S35; (d) Churchyard, G. J.; Scano, F.; Grant, A. D.;
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