Disaccharide analogs as probes for glycosyltransferases in Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmc.2007.04.012

Glycosyltransferases (GTs) play a crucial role in mycobacterial cell wall biosynthesis and are necessary for the survival of mycobacteria. Hence, these enzymes are potential new drug targets for the treatment of tuberculosis (TB), especially multiple drug

Full text of DOI:10.1016/j.bmc.2007.04.012

Bioorganic & Medicinal Chemistry 15 (2007) 5629–5650
Disaccharide analogs as probes for glycosyltransferases
in Mycobacterium tuberculosis
Ashish K. Pathak,a,ꢀ Vibha Pathak,a,ꢀ Lainne Seitz,a Sudagar S. Gurcha,b
Gurdyal S. Besra,b James M. Riordana and Robert C. Reynoldsa,*
aDrug Discovery Division, Southern Research Institute, PO Box 55305, Birmingham, AL 35255, USA
bSchool of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Received 19 January 2007; revised 2 April 2007; accepted 4 April 2007
Available online 10 April 2007
Abstract—Glycosyltransferases (GTs) play a crucial role in mycobacterial cell wall biosynthesis and are necessary for the survival of
mycobacteria. Hence, these enzymes are potential new drug targets for the treatment of tuberculosis (TB), especially multiple drug-
resistant TB (MDR-TB). Herein, we report the efficient syntheses of Araf(a 1 ! 5)Araf, Galf(b 1 ! 5)Galf, and Galf(b 1 ! 6)Galf
disaccharides possessing a 5-N,N-dimethylaminonaphthalene-1-sulfonamidoethyl (dansyl) unit that were prepared as fluorescent
disaccharide acceptors for arabinosyl- and galactosyl-transferases, respectively. Such analogs may offer advantages relative to radi-
olabeled acceptors or donors for studying the enzymes and for assay development and compound screening. Additionally, analogs
possessing a 5-azidonaphthalene-1-sulfonamidoethyl unit were prepared as photoaffinity probes for their potential utility in studying
active site labeling of the GTs (arabinosyl and galactosyl) in Mycobacterium tuberculosis (MTB). Beyond their preparation, initial
biological testing and kinetic analysis of these disaccharides as acceptors toward glycosyltransferases are also presented.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
for HIV and attendant opportunistic diseases due to
the large number of different agents and an increased
likelihood of drug interactions.4 MTB is an obligate
intracellular pathogen that persists within macrophages
in the human host, and these cells are involved in dis-
semination of infection.5 Intracellular bacilli are more
resistant to treatment due to limited access of drugs to
bacteria within macrophages, necessitating chronic
treatment with high therapeutic doses of multiple antibi-
otics lasting six months or longer for effective control
and treatment of the disease.6 For these reasons, recent
research in tuberculosis has focused on the identification
of new drug targets in TB, with a particular emphasis on
druggable targets and the development of new, safer
agents for the treatment of this disease.
Tuberculosis continues to be a major public health issue
throughout the world.1 Disease incidence remains high,
and there are particular concerns relative to the appear-
ance and increase of multi-drug-resistant forms of tuber-
culosis (MDR-TB).2 The emergence of drug-resistance
strains of mycobacteria is oftentimes a result of patient
non-compliance to treatment protocols, and can be
attributed in part to the lengthy treatment times and side
effects of current therapies. With advanced HIV disease,
immunocompromised patients have a higher risk of
developing active tuberculosis—either from new expo-
sure to TB or reactivation of quiescent mycobacteria.
Without the aid of an active immune system, treatment
can be more difficult and the disease may be more resis-
tant to therapy.3 TB treatment can be exacerbated in
AIDS patients undergoing chronic, combined therapies
Among the front line drugs for treatment of TB, two
drugs isoniazid (INH) and ethambutol (EMB) target
the assembly of the mycobacterial cell wall that is essen-
tial for the survival of pathogen.7 The structure of the
cell wall has now been thoroughly elucidated in terms
of its essential complex polysaccharides, the specific
chemical linkages therein, and the macromolecular
structure of the mycolylarabinogalactan complex.8 The
cell wall core of members of the Mycobacterium genus
consists of an extensively cross-linked peptidoglycan to
Keywords: Arabinofuranose; Galactofuranose; Fluorescent probe;
Photoaffinity probe; Glycosyltransferases; Arabinosyltransferases;
Galactosyltransferases; Tuberculosis.
*
Corresponding author. Tel.: +1 205 581 2454; fax: +1 205 581
2877; e-mail: reynolds@sri.org
ꢀ
Present address: Department of Chemistry, Western Illinois Univer-
sity, Macomb, IL 61455, USA.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.04.012

5630
A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
which is attached the linear D-galactan composed of
alternating 5- and 6-linked b-D-Galf units. Chemical
analysis of the degradation fragments arising from the
reducing end of the arabinogalactan (AG) obtained
from MTB, Mycobacterium bovis BCG, and Mycobacte-
rium leprae demonstrated the existence of the terminal
sequence (b1!5)-D-Galf-(b1 ! 6)-D-Galf-(b1 ! 5)-D-
Galf-(b1 ! 4)-L-Rhap-(a1 ! 3)-D-GlcpNAc. This unit
is crucial to the cell wall infrastructure and anchors
the exterior, waxy mycolate units, known targets of
the first line agent INH, to the interior peptidoglycan.
The mycobacterial cell wall oligosaccharide is composed
of three major sugars, mannopyranose (Manp), galac-
tofuranose (Galf), and arabinofuranose (Araf) in a vari-
ety of defined glycosidic linkages. The two major
oligosaccharide portions are AG and lipoarabinoman-
nan (LAM). AG is composed of alternate (b1 ! 5)
and (b1 ! 6) linked Galf units, linear (a1 ! 5) linked
Araf, and branched arabinan hexasaccharide at the ter-
minus that consists of (a1 ! 5), (a1 ! 3), (b1 ! 2)
linked Araf units (Fig. 1). LAM is Manp capped arab-
inan. The AG complex is critical for the survival of
Mycobacterium tuberculosis, and is a critical part of
the cell wall barrier that protects the bacillus within
the macrophage and functions as an effective barrier to
antibiotics that are commonly used in the treatment of
typical Gram-positive bacteria.
of arabinosyltransferases (AraTs) that promote
the polymerization of decaprenolphosphoarabinose
(DPA) as the donor (represented in Fig. 2). The
galactan portion is prepared through the actions of
a bifunctional galactosyltransferase (GalT) enzyme
preparing both the (b1 ! 5) and (b1 ! 6) linkages
along with another recently characterized enzyme
(Rv3782), and both enzymes utilize the donor uridin-
ediphosphate-galactofuranose
(UDP-Galf)
(repre-
sented in Fig. 3).8,9 UDP-Galf is produced by the
conversion of UDP-galactopyranose (UDP-Galp) by
the enzyme UDP-Galp mutase. Mycobacterial viabil-
ity requires an intact arabinan and galactan, and
thus compounds that inhibit these glycosyltransferas-
es are both useful biochemical tools as well as
potential lead compounds for new anti-tuberculosis
agents.
We have pursued disaccharide analogs designed as
probes for mycobacterial cell wall biochemistry and,
specifically, as potential glycosyltransferase substrates
and inhibitors.10 As an example, photoaffinity probes
can play an important role in specific labeling and
identification of target biomolecules, and they have
been useful in the identification of receptors and bind-
ing sites for various ligands and in locating functional
sites of macromolecules.11 Cory et al. utilized the
sulfonamide derivatives for N-terminal amino acid
labeling of proteins.12 This technique uses photoacti-
vatable, heterobifunctional reagents, which are gener-
The assembly of the arabinan portions of cell wall
polysaccharides in mycobacteria involves a family
OH
OH
(β 1 2)
O
OH
O
HO
HO
O
HO
O
HO
O
O
HO
O
OH
O
n~2
Arabinan
OH
O
O
HO
O
O
Rha-GlcNAc
Linker
O
OH
O
OH
OH
O
HO
OH
O
OH
HO
H
OH
(β 1 5)
OH
O
n~7
O
OH
O
OH
(α 1 3)
OH
O
HO
O
O
(α 1 5)
HO
O
O
HO
HO
O
OH
O
OH
O
H
O
OH
O
O
O
(β 1 6)
O
OH
O
HO
OH
HO
O
HO
O
OH
n~2
H
OH
O
O
O
OH
O
OH
HO
HO
O
OH
OH
HO
O
H
O
O
Galactan
HO
OH
H
OH
Arabinan
OH
O
O
O
HO
O
H
OH
O
HO
O
Arabinan
HO
H
OH
O
O
HO
O
H
OH
O
HO
O
HO
H
OH
HO
O
HO
O
H
OH
HO
HO
n~11
H
OH
Figure 1. Structure representation of M. tuberculosis arabinogalactan (AG) structure.

A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
5631
O
O
P
O
OH
HO
O
O
OH
HO
8
O
OH
HO
OH
O
DPA
AraT
OH
O
Arabinan
O
OH
OR
OR
OH
OH
Figure 2. Prototypical arabinosyltransferase-catalyzed reaction.
O
OH
O
Rha-GlcNAc
Linker
Rha-GlcNAc
Linker
O
O
HO
HO
O
UDP-Gal
f
OH
H
HO
HO
OH
(β 1 5)
O
Galactosyl
Transferases
HO
HO
H
OH
H
OH
UDP-Galf,
Galactosyl
Transferases
O
HO
O
Rha-GlcNAc
Linker
O
HO
Galactan
HO
H
OH
O
(β 1 5)
HO
HO
O
O
H
OH
HO
HO
(β 1 6)
H
OH
Figure 3. Prototypical galactosyltransferase-catalyzed reaction.
ally azide derivatives that, upon exposure to ultravio-
let light, photo decompose into a reactive nitrene
intermediate that subsequently forms a cross-link
between the ligand and its binding protein under
physiological conditions. These adducts can then be
analyzed using gel electrophoresis. This technique is
potentially useful as a marker because of its high sen-
sitivity and ease of handling.13
2. Results and discussion
2.1. Strategy
We have previously described syntheses of Araf and
Galf disaccharides based on linkages present in the cell
wall of MTB as acceptors/inhibitors of specific myco-
bacterial glycosyltransferases (GTs).10 In earlier reports,
we briefly communicated the synthesis of fluorescent
Araf disaccharides[14] and related photoaffinity probes,15
and a Manp photoaffinity probe16 as well as acceptor
activity for their respective GTs. The present account,
while using a similar synthetic strategy, reports detailed
methods with modifications for improving the prepara-
tion of final targets in both the photoaffinity and fluores-
cent disaccharide series.
In the present communication, we would like to pro-
vide a detailed account of the synthesis of fluores-
cent disaccharide probes (1–3) bearing
a highly
fluorescent 5-N,N-dimethylaminonaphthalene-1-sulfon-
amido14 (dansyl) function (Chart 1). These substrates
were designed for the development of non-radiola-
beled second generation competition based acceptor
assays that can potentially eliminate the use of
radiolabeled donors (DPA and UDP-Galp). Further-
more, this paper describes the preparation of related
disaccharides (4–6) bearing 1-azido-5-naphthalene sul-
fonamido13 functionality as potential pro-fluorescent
photoaffinity probes to study the nature of AraTs
and GalT (Chart 1). Synthetic details and prelimin-
ary biological data relating to acceptor activity of
these disaccharide probes are discussed.
Several different strategies were adopted for Araf
disaccharide preparation to introduce an ethylamino
functionality for attachment of naphthylsulfonamido
group at the anomeric center (Fig. 4). Starting from
the readily synthesized Araf thiocresylglycoside 7, a
nitroethyl group was introduced at the anomeric cen-
ter by reaction with commercially available 1-nitroeth-
anol that afforded nitroethyl glycoside 8 in excellent

5632
A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
OH
HO
O
O
S
OH
O
O
OH
O
N
O
R
H
OH
1. R = NMe2
4. R = N3
O
O
HO
HO
O
O
O
O
HO
O
O
HN
S
HN
S
HO
OH
HO
O
O
O
H
HO
H
OH
OH
O
HO
HO
HO
HO
H
OH
3. R = NMe2
6. R = N3
R
2. R = NMe2
5. R = N3
H
R
Chart 1. Fluorescent (1–3) and Photoaffinity (4–6) probes for glycosyltransferases in MTB.
and photoaffinity disaccharides Araf(a1 ! 5)Araf,
Galf(b1 ! 5)Galf, and Galf(b1 ! 6)Galf probes (1–6)
were readily synthesized as described (Scheme 1–5).
OAc
OAc
AcO
AcO
O
O
O
STol
NO2
OAc
OAc
2.2. Synthesis of fluorescent probe for AraT’s (Scheme 1)
7. Tol =
p
-methylphenyl
8
2.2.1. Synthesis of building blocks. Compound 10,
1,2,3,5-tetra-O-acetyl-D-arabinofuranoside, was obtained
from D-arabinose by reported methods.17 Reaction of 10
with chloroethanol in the presence of SnCl4 gave 11 as
the pure a-isomer, anomeric proton as singlet at d
5.48, after silica gel column chromatography.18 The
chloroethyl compound 11 was then heated with NaN3
in dry DMF for 6 h followed by deacetylation with
7 N NH3/MeOH to give 12 in an overall yield of 70%
(two steps) after purification. The 5-position was selec-
tively blocked using 1.0 equiv of benzoyl chloride in
dry pyridine at ꢀ78 ꢁC followed by warming to room
temperature and leaving overnight. Standard workup
and chromatography on Silica gel G gave compound
13 in moderate (58%) yield. Both the 2- and 3-positions
were blocked with a TBDMS group to give glycoside 14
followed by treatment with 7 N NH3/MeOH to obtain
the acceptor 15 in high yield. The trichloroacetimidate
donor 16 was synthesized as reported.19
OAc
O
AcO
OTBDMS
O
O
O
OAc
O
N
OTBDMS
O
9
Figure 4.
yield. Unfortunately, deacetylation of 8 using several
different reagents proved ineffective, and only
poor yield (48%) of deacetylated glycoside was
a
achieved using K2CO3 at ꢀ20 ꢁC. Alternatively, glyco-
side
7 was reacted with commercially available
N-(2-hydroxyethyl)phthalimide to afford the desired
ethylphthalimido a-glycoside. This product on deacet-
ylation followed by blocking of the hydroxyl groups
with tert-butyldimethylsilyl (TBDMS) groups and
selective deprotection of the 5-OTBDMS group
yielded the acceptor saccharide containing 5-OH
group. This acceptor sugar was coupled with thiogly-
coside donor 7 resulting in the desired a 1 ! 5-linked
disaccharide 9. The dephthaloylation of disaccharide 9
with hydrazine hydrate under several conditions failed
to give the desired disaccharide possessing a free
ethylamino disaccharide as single product, yielding
an inseparable mixture of the blocked amino byprod-
ucts as a result of acetyl group migration.
2.2.2. Glycosylation and reduction. The trichloroacetimi-
date donor 16 and the acceptor azidoethyl-2,3-O-di-tert-
butyldimethylsilyl-a-D-arabinofuranoside (15) were
reacted for 2 h in the presence of the Lewis acid pro-
moter BF3ÆEt2O (1.0 equiv, dissolved in 2 mL of dry
CH2Cl2). Additions were done at 0 ꢁC, and the reaction
was carried out under an inert atmosphere in dry
˚
CH2Cl2 at room temperature over powdered 4 A molec-
ular sieves. Column chromatography on Silica gel G
afforded the pure disaccharide 17 in 66% yield. The azi-
do group was reduced by heating with Ph3P in a ben-
zene/H2O mixture followed by rapid purification via
flash chromatography to afford compound 18.
Finally, a chloroethyl group was introduced at the gly-
cosidic linkage on the reducing end followed by dis-
placement of chloride with azide to synthesize the
desired a(1 ! 5) linked disaccharide. This approach
was also utilized to prepare the 1-O-ethylazido glyco-
sides in the Galf disaccharide probes. The fluorescent
2.2.3. Incorporation of the fluorescent group. The relative
instability of 18 necessitated immediate reaction with
dansyl chloride in the presence of N-methylimidazole
to produce the fluorescently labeled disaccharide 19 in

A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
OR1
5633
OR2
AcO
OAc
BzO
R3O
HO
OTBDMS
O
O
O
O
c
a
e
OAc
O
O
N3
R1
O
N3
OAc
10
OR1
OR2
OTBDMS
13. R1 = H
14. R1 = TBDMS
15
11. R1 = Cl, R2 = R3 =Ac
12. R1 = N3, R2 = R3 = H
b
d
BzO
OBz
O
f
OC(NH)CCl3
OBz
16
R2O
OR2
BzO
OBz
O
O
h
j
O
OR1
O
OTBDMS
O
O
1
OR2
OBz
O
O
O
N
S
O
R
H
OR1
OTBDMS
N
17. R = N3
18. R = NH2
19. R1 = TBDMS, R2 = Bz
20. R1 = TBDMS, R2 = H
i
g
Scheme 1. Reagents and conditions: (a) Cl(CH2)2OH, SnCl4, CH3CN, rt, 85%; (b) NaN3, DMF, 85 ꢁC; 7 N NH3/MeOH, rt, 70% in two steps; (c)
BzCl, Py, ꢀ78 ꢁC to rt, 58%; (d) TBDMSCl, DMF, imidazole, 85 ꢁC, 81%; (e) 7 N NH3/MeOH, rt, 93%; (f) BF3ÆEt2O, CH2Cl2, ꢀ20 ꢁC, 66%; (g)
Ph3P, benzene, H2O, 50 ꢁC, 87%; (h) dansyl chloride, N-methylimidazole, CH2Cl2, 0 ꢁC, 95%; (i) 7 N NH3–MeOH, rt, 93%; (j) Et4N+Fꢀ, THF, rt,
97%.
AcO
OAc
OTBDMS
OTBDMS
TBDMSO
HO
O
OH
HO
O
O
c
b
O
a
O
OTBDMS
O
O
N3
O
N3
O
N3
OAc
OTBDMS
OTBDMS
15
OH
O
R
12
21
OTBDMS
22. R = N3
23. R = NH2
d
e
RO
OR
O
O
OTBDMS
O
OR
O
S
O
4
O
N
H
OTBDMS
g
N3
24. R = Ac
25. R = H
f
Scheme 2. Reagents and conditions: (a) TBDMSCl, DMF, imidazole, 60 ꢁC, 90%; (b) TFA/water (1:1), 0 ꢁC, 68%; (c) 7, NIS, Sn(OTf)2, CH2Cl2,
ꢀ20 ꢁC, 91%; (d) HCO2NH4, MeOH, 5% Pd/C, rt, 87%; (e) 5-azidonaphthalenesulfonyl chloride, N-methylimidazole, CH2Cl2, 0 ꢁC, 81%; (f) 7 N
NH3–MeOH, rt, 87%; (g) Et4N+Fꢀ, THF, rt, 80%.
high yield. Lastly, compound 20 obtained from 19 was
deprotected to give the final fluorescent target 1.
achieved in one step with a yield of 68% after column
purification.
2.3. Synthesis of photoaffinity probe for AraTs (Scheme
2)
2.3.2. Glycosylation and reduction. The 1-thiocresyl-
2,3,5-tri-O-acetyl-a-D-arabinofuranoside donor 720
and the acceptor azidoethyl-2,3-O-di-tert-butyldimeth-
ylsilyl-a-D-arabinofuranoside 15 were reacted for 2 h
in the presence of the Lewis acid promoter Sn(OTf)2
and activator N-iodosuccinimide. Addition of the re-
agents, and the subsequent reaction, was carried out
at ꢀ20 ꢁC under an argon atmosphere in dry CH2Cl2
2.3.1. Synthesis of building blocks. The synthesis of the
target probe 4 is represented in Scheme 2. A slightly dif-
ferent approach was adapted that was shorter and more
efficient than the synthesis of 22 via selective protection
of the 5-position with a benzoyl group as shown in
Scheme 1. Compound 19 was first reacted with 3.5 equiv
TBDMSCl in the presence of imidazole at 60 ꢁC for two
days affording compound 21 in 90% yield, followed by
selective deprotection of only the 5-OTBDMS group
using TFA/water (1:1) system at 0 ꢁC. The preparation
of compound 15 from compound 21 was successfully
˚
over powdered 4 A molecular sieves. Standard work-
up followed by column chromatographic purification
on Silica gel G afforded the pure disaccharide 22 in
91% yield. The trans glycosidation (a-linked disaccha-
ride) was confirmed by the appearance of a singlet
1
anomeric proton in HNMR spectrum.15 The azido

5634
A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
O
MPMO
O
HO
MPMO
N3
H
OMPM
33
g
STol
O
AcO
R2O
O
O
RO
MPMO
O
O
O
O
f
a
AcO
AcO
R2O
R2O
R1
O
HO
HO
N3
N3
O
H
H
OAc
OR2
H
H
d
OR
OMPM
32
26
27. R1 = Cl, R2 = Ac
28. R1 = N3, R2 = Ac
29. R1 = N3, R2 = H
30. R = H
31. R = MPM
e
b
c
h
O
MPMO
O
MPMO
O
O
MPMO
HO
R1O
TrO
N3
N3
H
OMPM
36
H
j
OMPM
34. R1 = H
35. R1 = MPM
i
Scheme 3. Reagents and conditions: (a) HOCH2CH2Cl, NIS, Sn(OTf)2, CH2Cl2, ꢀ20 ꢁC to rt, 86%; (b) NaN3, DMF, 70 ꢁC, quant.; (c) 7 N NH3–
MeOH, rt, quant.; (d) 2,2-dimethoxypropane, camphor sulfonic acid, acetone, rt, 82%; (e) p-methoxybenzyl chloride (MPMCl), NaH, DMF, 0 ꢁC to
rt, 98%; (f) 60% aq AcOH, 60 ꢁC, 77%; (g) Bu2SnO, MPMCl, toluene, reflux to rt, 90%; (h) TrCl, DMAP, Py, rt–50 ꢁC, 80%; (i) MPMCl, NaH, THF,
0 ꢁC–rt, 86%; (j) 5% TFA in CH3Cl, 0 ꢁC, 70%.
O
R1O
O
O
O
HN
S
R1O
R2O
c
2
O
H
OR1
O
R2O
R2O
f
H
NMe2
OR2
O
MPMO
O
39. R1 = MPM, R2 = Ac
40. R1 = MPM, R2 = H
e
O
R
MPMO
AcO
H
a
OMPM
O
33
O
R1O
AcO
AcO
O
O
O
HN
S
H
OAc
R1O
R2O
O
d
H
5
37. R = N3
38. R = NH2
OR1
b
O
R2O
R2O
f
H
N3
OR2
41. R1 = MPM, R2 = Ac
42. R1 = MPM, R2 = H
e
Scheme 4. Reagents and conditions: (a) 26, NIS, triflic acid, CH2Cl2, ꢀ20 ꢁC, 66%; (b) Ph3P, benzene, H2O, 50 ꢁC, 85%; (c) dansyl chloride, N-
methylimidazole, CH2Cl2, 0 ꢁC, 77%; (d) azidonaphthalenesulfonyl chloride, N-methylimidazole, CH2Cl2, 0 ꢁC, 68%; (e) 7 N NH3–MeOH, rt, 40:
quant., 42: 92%; (f) SnCl4, CH2Cl2, thiophenol, ꢀ78 to ꢀ20 ꢁC, 2: 33%, 5: 78%.
group was reduced by reaction with ammonium for-
mate (H2CO2NH4) in dry methanol using 5% Pd/C
as catalyst for 2 h at room temperature followed by
rapid purification via flash chromatography to afford
compound 23 in 87% yield. Again, the relative insta-
bility (probably a result of the acetyl blocking groups)
of 23 necessitated immediate reaction of the free
amine.
2.3.3. Incorporation of fluorescent group. Disaccharide 23
was reacted immediately with 5-azidonaphthalenesulfo-
nyl chloride in the presence of N-methylimidazole at
0 ꢁC over 4 h. The disaccharide 24 was obtained in good
yield (81%). Lastly, compound 24 was deprotected via
25 to give the final target compound (5-azidonaphtha-
lene-1-sulfonamidoethyl)-5-O-(a-D-arabinofuranosyl)-a-
D-arabinofuranoside 4.15

A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
5635
O
R1O
O
O
R1O
O
HN
S
c
R2O
3
O
O
H
OR1
R2O
R2O
f
H
OR2
NMe2
O
45. R1 = MPM, R2 = Ac
46. R1 = H, R2 = Ac
MPMO
e
O
MPMO
O
R
AcO
O
H
OMPM
a
36
AcO
AcO
O
R1O
H
OAc
O
O
R1O
O
HN
S
d
43. R = N3
44. R = NH2
b
6
R2O
O
O
H
OR1
R2O
R2O
f
H
OR2
N3
47. R1 = MPM, R2 = Ac
48. R1 = H, R2 = Ac
e
Scheme 5. Reagents and conditions: (a) 26, NIS, trflic acid, CH2Cl2, ꢀ20 ꢁC, 43%; (b) Ph3P, benzene, H2O, 50 ꢁC, 75%; (c) dansyl chloride, N-
methylimidazole, CH2Cl2, 0 ꢁC, 60%; (d) azidonaphthalenesulfonyl chloride, N-methylimidazole, CH2Cl2, 0 ꢁC, 68%; (e) SnCl4, CH2Cl2, thiophenol,
ꢀ78 to ꢀ20 ꢁC, 46: 83%, 48: 81%; (f) 7 N NH3–MeOH, rt, 3: quant., 6: 80%.
2.4. Synthesis of probes for GT
oxybenzylated at the 6-position. Glycoside 33, possess-
ing a free 5-OH for coupling, was produced in 90%
yield after silica gel flash column chromatography.
2.4.1. Synthesis of building blocks. Thiocresyl 2,3,5,6-tet-
ra-O-acetyl-b-D-galactofuranoside 26, the building
block for disaccharides Galf(b 1 ! 5)Galf (2) and a
Galf(b 1 ! 5)Galf (3), was readily obtained from
D-galactose by reported method.10d,21 In a reaction
sequence starting from thioglycoside 26, both accep-
tors 33 and 36 were prepared possessing either a free
5-OH or 6-OH for coupling with thioglycoside donor
26 as represented in Scheme 3. Compound 26 was
reacted with 1-chloroethanol in presence of NIS and
Lewis acid promoter Sn(OTf)2 in dichloromethane at
ꢀ20 ꢁC to produce 27 in 86% yield and a-glycosida-
Glycoside 32 was selectively blocked using trityl chloride
in pyridine at 50 ꢁC to give the 6-tritylated derivative 34
in 72% yield. Glycoside 34 was then p-methoxybenzylat-
ed to give 35 in 86% yield. Finally, detritylation with
10% CF3COOH in chloroform and further purification
using column chromatography gave glycoside 36 pos-
sessing free 6-OH group in 70% yield.
2.4.2. Glycosylation and deprotection. Once the synthesis
of acceptor glycosides 33 and 36 was achieved, disaccha-
rides 37 and 43 were prepared using thioglycoside 26 as
the donor, NIS as the promoter, and triflic acid as the
Lewis acid as represented in Schemes 4 and 5,
respectively.10d
1
tion was confirmed by H NMR spectra. Compound
27 on further reaction with NaN3 in DMF at 70 ꢁC
gave the glycoside 28 possessing the ethylazido func-
tionality. Compound 28 was deacetylated using 7 N
NH3/MeOH to give
a quantitative yield of the
deblocked glycoside 29. The 5,6-hydroxyl groups in
29 were first blocked with an isopropylidene group
using 2,20-dimethoxypropane in the presence of a
catalytic amount of (1S)-(+)-10-camphorsulfonic acid
to yield 30. Treatment of glycoside 30 with p-meth-
oxybenzyl chloride and NaH afforded compound 31
in 98% yield. The ether protecting group was used
due to the potential for ester group (benzoyl)
migration between hydroxyl groups,10d and the
p-methoxybenzyl protecting group was used specifi-
cally allowing ready deprotection of the disaccharides
without affecting the azido group.
2.4.2.1. Synthesis of Galf(b 1 ! 5)Galf disaccharide.
The synthesis of b1 ! 5-linked Galf disaccharide was
carried out by coupling the acceptor glycoside 33 with
thiocresyl glycoside donor 26 in dry dichloromethane
at ꢀ20 ꢁC in presence of promoter NIS and triflic acid
to produce disaccharide 37 in 66% yield after column
chromatography. b-Glycosidation was confirmed by
the appearance of a singlet anomeric proton.14 The azi-
do group was reduced to the amine to produce disaccha-
ride 38 by reduction with H2CO2NH4 in the presence of
5% Pd/C in dry MeOH. Rapid purification gave pure
disaccharide 38 in 92% yield as a colorless oil that was
stored in a freezer (ꢀ20 ꢁC) for further use to prepare
the b1 ! 5-linked Galf probes.
Saccharide 32 was regio-selectively benzylated via stan-
nylated intermediates after reaction with dibutyltin
oxide in boiling toluene and azeotropic removal of
water.10d Evaporation gave the crude 5,6-O-stannylene
acetal intermediate which was then selectively p-meth-
2.4.2.2. Synthesis of Galf(b 1 ! 6)Galf disaccharide.
The synthesis of the b1 ! 6-linked Galf disaccharide
was carried out by coupling glycoside 36 and the thio-

5636
A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
cresyl glycoside donor 26 in dry dichloromethane at
ꢀ20 ꢁC in the presence of promoter NIS and trifluorom-
ethanesulfonic acid to produce disaccharide 43 in 65%
yield after column chromatography. Again, the azido
group in disaccharide 43 was reduced to an amino group
using H2CO2NH4 over 5% Pd/C in dry MeOH and puri-
fication gave disaccharide 44 in 98% yield. It was stored
in the freezer (ꢀ20 ꢁC) for later use.
cases the coupling constant was found to be 0.0 Hz),
suggesting trans-glycosylation and formation of a-
linked disaccharides. Similar trends were seen in 13C
NMR spectra of disaccharides with C-1 present at high-
er ppm value (low field region) as compared to C-10 in
the range 110–105 ppm. The b-stereochemistry, trans-
glycosylation, of all of the D-Galf glycosides was evident
by their appearance as a singlet or doublet with 3J1,2 va-
lue 62.0 Hz in the range of 4.98–5.08 ppm.24 It is nota-
ble that H-1 appeared as ddd at 4.00 ppm with
3J1,2 = 2.4 Hz in compound 29 (D2O as solvent) with
additional long-range coupling with H-3 and H-4 pro-
tons (J = values less 61.0 Hz), and this observation
may be attributable to conformational change in a polar
2.5. Synthesis of fluorescent and photoaffinity probes for
GT
2.5.1. Galf(b1 ! 5)Galf fluorescent disaccharide (2) and
photoaffinity probe (5). Disaccharide 2 was prepared by
reaction of 38 with dansyl chloride at 0 ꢁC in the pres-
ence of N-methylimidazole and dry CH2Cl2 to give
disaccharide 39 in 77% yield after chromatography as
shown in Scheme 4. Disaccharide 39 was deacetylated
using 7N NH3 in methanol to produce 40 in quantitative
yield after purification. Treatment of 40 with ceric
ammonium nitrate to remove the p-methoxybenzyl
group in dichloromethane under standard conditions22
was not effective. Addition of tin (IV) chloride and thi-
ophenol to a cold dichloromethane solution of disaccha-
ride 40 at ꢀ78 ꢁC and further reaction at ꢀ20 ꢁC
overnight provided the deblocked disaccharide 2 in
moderate 33% yield after purification. In a similar fash-
ion, photoaffinity probe 5 was prepared by reaction of
38 with 5-azidonaphthalene sulfonyl chloride to produce
disaccharide 41. Deacetylation followed by debenzyla-
tion under conditions used to prepare 2 gave the target
5 in 78% yield.
1
solvent. H NMR spectra of Galf disaccharides showed
a singlet or doublet due to anomeric H-1 proton in the
range of 4.61–5.05 ppm and anomeric H-10 in the range
of
4.90–5.42 ppm
with
coupling
constants
3J1,2 6 2.0 Hz. As with the Araf disaccharides, the ano-
meric proton of the reducing sugar in Galf disaccharides
is seen at higher field as compared to a non-reducing su-
gar. In 13C NMR spectra of Galf glycosides the C-1 sig-
nal appeared between 108.6 and 105.4 ppm. However, in
Galf disaccharides the C-1 proton is seen between 105.8
and 109.9 ppm and C-10 at higher field in a range be-
tween 105.2 and 109.5 ppm. Detailed NMR data (1H
and 13C NMR) on all disaccharides (sugar portion)
are presented in Tables 1–4 and the values confirmed
the proposed configuration at the anomeric centers
and the structures of synthesized disaccharides.
2.7. Glycosyltransferase activity of disaccharides
2.5.2. Galf(b1 ! 6)Galf fluorescent disaccharide (3) and
photoaffinity probe (6). Synthesis of fluorescently labeled
b1 ! 6-linked Galf disaccharide 3 and photoaffinity
probe 6 was carried out in a similar fashion to the
b1 ! 5-linked Galf disaccharides as represented in
Scheme 5. Disaccharide 44 was reacted with dansyl
chloride or 5-azidonaphthalene sulfonyl chloride and
N-methylimidazole to produce 45 and 47, respectively.
These two disaccharides (45 and 47) were first debenzy-
lated with SnCl4 and thiophenol at sub zero tempera-
tures to produce disaccharides 46 and 48 in 83% and
81% respective yields followed by deacetylation to give
3 (quantitative) and 6 (80%).
The effectiveness of these compounds as photoaffinity
and fluorescent probes to study the mycobacterial glyco-
syltransferases (AraT’s and GalT) requires that they act
as enzyme substrates. As such, a cell-free assay system
using the membrane fraction from Mycobacterium
smegmatis was used to evaluate their acceptor
activity.10c,d,24
2.7.1. Arabinosyltransferase activity. Based on the previ-
ous use of specific arabinose based neoglycolipid accep-
tors,10 probe disaccharides 1 and 4 were assayed for
acceptor activity. As expected, both compounds showed
acceptor activity in assays performed in the presence of
mycobacterial membrane fractions containing GT activ-
ity and resulted in [14C]Araf incorporation from DP-
[14C]A as the arabinose donor. TLC/autoradiography
for the reaction products from 1 is shown in Figure 5.
Data for probe 4 were described earlier.15 Both probes
demonstrated the enzymatic conversion of the disaccha-
rides to their corresponding trisaccharide products. Dif-
ferent concentrations of 1 (2 mM maximum) and of 4
(8 mM maximum) were used for arabinosyltransferase
activity. Calculation of kinetic constants revealed that
probe 1 possessed a Km value 0.68 mM, whereas probe
4 possessed a Km value 3.07 mM. Based on these data,
both probes are clearly substrates for the arabi-
nosyltransferases. Compound 1 may be useful in the
development of competition-based acceptor assay for
the discovery of inhibitors of a crucial mycobacterial en-
zyme. Disaccharide 4, on the other hand, may be useful
2.6. Structural elucidation
All mono- and disaccharides were characterized using
NMR, MS, and CHN analysis. Whenever necessary,
NOE, decoupling, and DEPT experiments were per-
formed in order to confirm NMR assignments and ste-
reochemistry at the anomeric center of all sugars.
Coupling constant correlations were used to assign the
individual protons. In Araf glycosides the anomeric pro-
3
ton is seen as singlet or doublet with the J1,2 value
62.0 Hz, suggesting the formation of the a-stereocenter
with the D-arabino configuration.23 In Araf disaccha-
rides the H-1 anomeric protons of the reducing sugar
(4.58–4.83 ppm) are generally seen at lower ppm as com-
pared to non-reducing sugar H-10 (4.87–5.38 ppm) with
3
3
0
0
coupling constant J1,2 and J1 ;2 6 2:0 Hz (in most

A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
Table 1. 1H NMR chemical shifts for Araf disaccharides
5637
Compound
Shifts d (ppm), J (Hz)
Unit
H-1 (J1,2
)
H-2 (J2,3
)
H-3 (J3,4
)
H-4 (J4,5a
)
H-5a (J5a,5b
)
H-5b (J4,5b)
17
18
19
20
22
23
24
25
1
Araf
Araf0
Araf
Araf0
Araf
Araf0
Araf
Araf0
Araf
Araf0
Araf
Araf0
Araf
Araf0
Araf
Araf0
Araf
Araf0
Araf
Araf0
4.83 (d) (1.4)
5.38 (s) (0)
4.06 (dd) (3.7)
5.59 (d) (1.1)
4.04 (dd) (3.4)
5.58 (s) (0)
3.99 (dd) (6.5)
5.56 (d) (4.8)
3.99 (dd) (5.8)
5.57 (d) (7.0)
3.91 (m) (nd)
5.56 (d) (4.0)
3.77 (m) (nd)
3.99 (m) (2.3)
3.97 (dd) (6.4)
4.97 (dd) (4.8)
3.99 (m) (nd)
4.97 (dd) (4.7)
3.91 (m) (nd)
4.99 (dd) (4.9)
3.93 (dd) (6.3)
3.86 (ddd) (6.6)
3.77 (m) (nd)
3.99 (m) (4.5)
3.84 (m) (5.8)
3.84 (m) (5.8)
4.08 (m) (5.2)
4.62 (ddd) (3.2)
4.07 (m) (5.4)
4.62 (ddd) (3.1)
3.98 (m) (4.6)
4.62 (ddd) (3.1)
3.99 (m) (nd)
4.17 (dd) (2.4)
4.01 (ddd) (4.6)
4.29 (ddd) (3.0)
3.99 (m) (4.6)
4.28 (ddd) (2.9)
3.91 (m) (4.5)
4.29 (ddd) (3.1)
3.71 (m) (nd)
3.94 (ddd) (3.2)
3.99 (m) (nd)
4.17 (dd) (2.3)
3.96 (ddd) (3.3)
3.84 (m) (4.8)
3.89 (dd) (11.2)
4.84 (dd) (11.8)
3.89 (dd) (11.0)
4.84 (dd) (11.8)
3.84 (dd) (10.9)
4.83 (dd) (11.6)
3.77 (m) (10.8)
3.86 (dd) (11.8)
3.81 (dd) (11.2)
4.42 (dd) (11.3)
3.82 (dd) (10.6)
4.42 (dd) (11.2)
3.78 (dd) (10.8)
4.43 (dd) (11.4)
3.71 (m) (nd)
3.75 (dd) (3.7)
4.62 (dd) (4.7)
3.72 (dd) (4.3)
4.69 (dd) (4.7)
3.61 (dd) (3.5)
4.68 (dd) (4.8)
3.58 (dd) (3.2)
3.77 (m) (2.3)
3.63 (dd) (3.8)
4.22 (dd) (5.6)
3.60 (dd) (4.2)
4.22 (dd) (5.8)
3.51 (m) (nd)
4.23(5.6)
4.83 (d) (1.5)
5.37 (s) (0)
4.69 (d) (1.8)
5.32 (s) (0)
4.04 (dd) (3.3)
5.57 (s) (0)
4.68 (d) (1.5)
5.07 (s) (0)
3.92 (dd) (3.4)
4.07 (d) (0.8)
4.05 (dd) (3.6)
5.15 (d) (1.3)
3.99 (m) (nd)
5.14 (d) (1.5)
3.91 (m) (nd)
5.14 (d) (1.5)
3.80 (dd) (4.1)
3.99 (dd) (4.0)
3.92 (dd) (3.4)
4.05 (d) (0.8)
3.79 (dd) (2.8)
3.96 (dd) (3.5)
4.83 (d) (1.5)
5.11 (s) (0)
4.82 (d) (1.4)
5.09 (s) (0)
4.66 (d) (1.9)
5.07 (s) (0)
4.58 (d) (1.8)
4.87 (d) (1.8)
4.68 (d) (1.5)
5.07 (s) (0)
3.62 (m) (nd)
3.63 (dd) (4.9)
3.58 (dd) (3.2)
3.77 (m) (2.3)
3.63 (dd) (5.5)
3.52 (dd) (3.8)
3.74 (dd) (11.9)
3.77 (m) (10.7)
3.86 (dd) (11.8)
3.73 (dd) (11.9)
3.76 (dd) (10.9)
4
4.63 (d) (1.2)
4.89 (d) (1.3)
Table 2. 13C NMR chemical shifts for Araf disaccharides
likely enzymatic conversion of all Galf disaccharides to
their corresponding trisaccharide products and incorpo-
ration of [14C]Gal to the 50-OH of 2 and 5 and the 60-OH
of 3 and 6. Galf disaccharides 3, 4, and 6 gave rise to a
second, slower migrating band (Figs. 6 and 8) which,
based on relative migration profiles, would be antici-
pated to be a tetrasaccharide product resulting from fur-
ther elongation of the trisaccharide products at the 60-
OH or 50-OH consistent with the alternating linkage
pattern of AG. Calculation of kinetic constants (Figs.
7–9) revealed that 2 and 3 possessed Km values 7.33
and 2.38 mM, respectively, whereas 5 and 6 possessed
Km values 2.77 and 1.98 mM, respectively. The acceptor
data clearly suggested that disaccharide probes 2 and 3
can be used for the development of competition-based
assays for inhibitor discovery, and that disaccharides 5
and 6 may be useful for probing the bi-functional
galactosyltransferase.
Compound
Unit
Shifts d (ppm)
C-1
C-2
C-3
C-4
C-5
17
18
19
20
22
24
25
1
Araf
Araf0
Araf
Araf0
Araf
Araf0
Araf
Araf0
Araf
Araf0
Araf
Araf0
Araf
Araf0
Araf
Araf0
Araf
Araf0
108.5
105.9
108.3
105.9
108.3
106.1
108.5
107.5
108.5
105.9
108.4
106.0
109.6
109.8
107.7
107.6
109.6
109.6
82.1
82.1
87.4
82.1
82.6
82.1
83.5
79.2
84.1
81.0
82.6
81.2
85.3
83.4
82.5
81.0
83.1
83.1
79.1
78.1
79.2
78.0
78.6
77.9
79.1
78.0
78.8
77.1
78.6
77.1
79.9
78.4
76.9
76.8
79.0
78.7
84.1
81.2
83.8
81.2
83.5
81.1
82.5
86.9
82.0
80.5
83.4
80.5
83.2
85.3
81.2
84.2
85.9
84.3
66.5
63.7
66.8
63.7
66.6
63.7
65.8
61.9
66.4
63.3
66.5
63.3
67.4
62.7
61.5
66.7
67.5
63.1
4
2.8. Conclusion
In summary, we reported a detailed account of the syn-
theses of Araf (a1 ! 5) Araf, Galf (b1 ! 5) Galf, and
Galf (b1 ! 6) Galf disaccharides as potential fluorescent
and photoaffinity probes (1–6). All probe disaccharides
were found to be very stable at room temperature but
were stored in the refrigerator at 4 ꢁC in dark bottles
when not in use. These disaccharides have shown accep-
tor activity in cell free assay systems for the target glyco-
syltransferases. Extensive developmental work utilizing
these compounds is in progress and will be reported else-
where. Competition-based assays are being developed to
substitute a fluorescence-based assay for the reported as-
says based on radiolabeled decaprenolphosphoarabi-
nose (14C-DPA) for AraTs and UDP-[14C]Galf (used
for in situ preparation of UDP-[14C]Galp) for GalT.
Recently, we have reported the use of Galf (b1 ! 5)
Galf and Galf (b1 ! 6) Galf disaccharides as potential
as a photoaffinity probe for labeling active site residues
of the arabinosyltransferase.
2.7.2. Galactosyltransferase activity. All four Galf
probes 2, 3, 5, and 6 were screened for acceptor activ-
ity.10d Assays were performed in the presence of mem-
branes and the cell wall enzymatic fraction P60 and
resulted in excellent [14C]Galf incorporation from
UDP-[14C]Galp, following endogenous conversion to
UDP-[14C]Galf and transferase activity for all Galf
probes. Different concentrations of probes 2 and 3 in
the range of 0–4 mM were used for galactosyltransferase
activity. In the case of probes 5 and 6, a concentration
range of 0–6 mM was utilized. TLC/autoradiography
(Figs. 6 and 8) demonstrated acceptor activity and the

5638
A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
Table 3. 1H NMR chemical shifts for Galf disaccharides
Compound
Shifts d (ppm), J (Hz)
H-4 (J4,5
4.00 (dd) (3.5) 4.07 (m) (3.6)
4.35 (dd) (4.2) 5.32 (ddd) (3.6) 4.24 (dd) (12.0) 4.13 (dd) (7.8)
4.06 (m) (nd) 4.06 (m) (3.6) 3.62 (dd) (10.4) 3.59 (dd) (3.6)
4.36 (dd) (4.2) 5.32 (ddd) (3.6) 4.24 (dd) (12.0) 4.14 (dd) (7.8)
3.97 (m) (nd) 3.97 (m) (7.2) 3.55 (dd) (10.4) 3.50 (dd) (3.6)
4.35 (dd) (3.6) 5.31 (ddd) (3.6) 4.24 (dd) (12.0) 4.12 (dd) (7.8)
4.01 (dd) (6.0) 3.96 (ddd) (4.2) 3.44 (dd) (nd) 3.44 (dd) (6.0)
Unit H-1 (J1,2
)
H-2 (J2,3
)
H-3 (J3,4
)
)
H-5 (J5,6a
)
H-6a (J6a,6b
)
H-6b (J5,6b)
37
38
39
40
41
42
43
44
45
46
47
48
2
Galf 5.05 (s) (0)
Galf0 5.24 (s) (0)
Galf 5.04 (s) (1.2)a 3.98a(dd) (3.0) 3.99 (m) (n.d.)
Galf0 5.42 (s) (0)
Galf 4.87 (s) (1.2)a 3.83a(dd) (2.4) 3.97 (m) (n.d.)
Galf0 5.39 (s) (0)
Galf 4.88 (s) (0)
Galf0 5.26 (s) (0)
4.01 (s) (0)
5.17 (d) (1.8)
4.07 (m) (5.9)
4.96 (dd) (5.4)
3.62 (dd) (10.2) 3.59 (dd) (3.6)
5.17 (d) (1.8)
4.95 (dd) (4.8)
5.15 (d) (1.8)
3.81 (d) (1.8)
4.01 (s) (0)
4.95 (dd) (5.4)
4.06 (dd) (2.4)
3.79 (m) (5.4)
3.98 (m) (nd)
4.95 (dd) (5.6)
3.98 (dd) (5.4) 3.79 (m) (6.6)
3.98 (m) (nd) 3.98 (m) (6.1)
3.62 (dd) (11.4) 3.56 (dd) (nd)
3.54 (dd) (10.7) 3.49 (dd) (4.5)
Galf 4.87 (d) (1.2) 3.79 (m) (nd)
Galf0 5.37 (s) (0)
Galf 4.81 (s) (0)
Galf0 5.05 (s) (0)
5.14 (d) (1.6)
4.34 (m) (3.6) 5.31 (ddd) (3.6) 4.25 (dd) (12.2) 4.12 (dd) (7.5)
4.17 (dd) (3.2) 3.97 (ddd) (4.9) 3.86 (dd) (12.2) 3.78 (dd) (5.8)
4.45 (dd) (3.4) 5.39 (ddd) (4.4) 4.39 (dd) (11.9) 4.22 (dd) (6.9)
4.06 (dd) (3.6) 3.71 (ddd) (4.8) 3.82 (dd) (10.2) 3.61 (dd) (7.2)
4.23 (dd) (3.6) 5.38 (ddd) (4.2) 4.32 (dd) (12.0) 4.17 (dd) (7.8)
3.93c (dd) (1.5) 4.09 (dd) (3.8)
5.34 (d) (1.6) 4.06 (dd) (5.1)
Galf 5.04 (s) (1.8) 4.00b (dd) (3.6) 3.96 (dd) (7.2)
Galf0 5.06 (d) (1.2) 5.07(dd) (2.4)
Galf 5.04 (s) (0) 3.95 (m) (nd)
Galf0 5.04 (s) (0.5) 5.07c(dd) (1.8)
4.95 (dd) (5.4)
3.95 (m) (6.3)
4.98 (dd) (5.6)
3.91 (dd) (6.6)
4.98 (dd) (5.4)
4.10 (d) (2.1)
5.04 (m) (5.8)
3.90 (dd) (6.6)
4.97 (dd) (5.8)
4.14 (u) (nd)
4.04 (dd) (3.0) 3.61 (m) (nd)
3.76 (m) (10.4) 3.45 (dd) (5.0)
4.24 (dd) (4.0) 5.38 (ddd) (4.0) 4.32 (dd) (11.9) 4.17 (dd) (7.4)
4.01 (dd) (3.0) 3.67 (ddd) (4.8) 3.79 (dd) (10.2) 3.56 (dd) (7.2)
4.22 (dd) (3.6) 5.36 (ddd) (4.2) 4.30 (dd) (12.0) 4.17 (dd) (7.2)
Galf 4.86 (s) (0)
Galf0 5.01 (s) (0)
Galf 4.80 (s) (0)
Galf0 5.06 (s) (0)
Galf 4.83 (s) (0)
Galf0 5.01 (s) (0)
Galf 4.83 (s) (0)
Galf0 5.06 (s) (0)
3.81 (d) (3.0)
5.07 (d) (1.8)
3.88 (s) (0)
4.01 (t) (2.1)
4.31 (dd) (3.6) 5.36 (ddd) (4.2) 4.37 (dd) (12.0) 4.20 (dd) (7.8)
3.98 (dd) (3.1) 3.66 (ddd) (3.1) 3.77 (m) (nd) 3.56 (m) (6.6)
4.30 (dd) (3.7) 5.37 (ddd) (3.7) 4.27 (dd) (11.8) 4.16 (dd) (7.4)
4.07 (u) (nd) 4.03 (u) (4.0) 3.80 (dd) (10.5) 3.58 (dd) (8.5)
4.03 (ddd) (3.8) 3.81 (dd) (10.5) 3.58 (dd) (8.6)
5.04 (m) (nd)
3.77 (m) (3.0)
5.07 (d) (2.1)
3.92 (u) (nd)
5.04 (d) (2.4)
5.06 (dd) (5.9)
4.04 (dd) (6.1)
3.99 (m) (nd)
4.31 (dd) (3.7) 5.37 (ddd) (4.2) 4.37 (dd) (11.9) 4.20 (dd) (7.1)
3.91 (dd) (3.7) 3.86 (ddd) (6.6) 3.67 (dd) (11.8) 3.64 (dd) (5.2)
Galf 4.66 (d) (1.5) 3.82 (dd) (3.3)
Galf0 5.16 (d) (0.6) 3.99 (m) (nd)
Galf 4.67 (d) (1.5) 3.81 (dd) (3.1)
Galf0 4.91 (d) (1.6) 3.98 (dd) (3.3)
Galf 4.61 (d) (1.6) 3.77 (dd) (3.3)
Galf0 5.15 (d) (1.5) 3.99 (m) (nd)
Galf 4.63 (d) (1.4) 3.76 (dd) (3.1)
Galf0 4.90 (d) (1.4) 3.98 (dd) (3.3)
4.10 (u) (3.1)
3.72 (ddd) (6.5) 3.64 (dd) (11.2) 3.61 (dd) (6.6)
3.82 (m) (4.6) 3.74 (dd) (10.2) 3.48 (dd) (7.0)
4.02d (ddd) (5.8) 3.99 (dd) (3.2) 3.72 (ddd) (5.7) 3.64 (dd) (11.2) 3.61 (dd) (6.9)
3
3.96d (ddd) (5.4) 3.82 (m) (nd)
5
4.02 (dd) (5.9)
3.99 (m) (nd)
3.95 (dd) (5.7)
4.02 (dd) (5.9)
3.86 (dd) (nd) 3.84 (m) (6.4)
3.66 (dd) (11.8) 3.62 (m) (n.d.)
4.09 (m) (5.9) 3.71 (ddd) (3.1) 3.62 (m) (11.1) 3.60 (dd) (6.6)
3.77 (dd) (3.8) 3.81 (ddd) (5.0) 3.72 (dd) (10.4) 3.46 (dd) (7.2)
6
3.99 (dd) (3.2) 3.72 (dd) (5.6)
3.63 (dd) (11.2) 3.61 (dd) (6.9)
nd, not determined due to complexity of signal; u, unresolved signal.
a Showed J1,2 = 1.2 Hz.
b Showed J1,2 = 1.8 Hz.
c Showed J1,2 = 0.5 Hz.
d Showed J1,3 = 0.5 Hz.
photoaffinity probes to identify the galactosyltransferase
(glfT) in MTB.ꢁ Very recently the isolation and purifica-
tion of glfT in milligram quantities has been reported by
expression of the Rv3808c gene in Escherichia coli C41
(DE3) and purification of protein from the culture.26
These probes may also be useful for labeling and identi-
fication of active site residues.
under reduced pressure. The reactions were monitored
by thin-layer chromatography (TLC) on precoated 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 of reagent grade. Flash col-
umn chromatography was carried out on silica gel 60
(230–400 Mesh). 1H and 13C NMR spectra were re-
corded at 300 and 75 MHz, respectively. Specific
1HNMR spectra were recorded at 600 MHz as required.
The coupling constants (J) are reported in Hz and chem-
ical shifts are in ppm (d) relative to a residual solvent
peak or an internal standard. FAB mass spectra were re-
corded either by adding NBA (3-nitrobenzyl alcohol) or
LiCl and in some cases ESI-MS spectra were recorded
on a BioTof-2 time-of-flight mass spectrometer. The
presence of water, when presented in the analytical re-
sults, was confirmed by 1H NMR as per journal
requirements.
3. Experimental
3.1. Synthesis
3.1.1. General procedures. The reactions were performed
under a dry argon atmosphere and reaction tempera-
tures were measured externally. Anhydrous solvents
were directly purchased and used as such in reactions.
Whenever necessary, compounds and starting materials
were dried by azeotropic removal of water with toluene
ꢁ
Alderwick, L. J.; Dover, L. G.; Veerapen, N.; Gurcha, S. S.; Kremer,
L.; Roper, D. L.; Pathak, A. K.; Reynolds, R. C.; Besra, G. S.
(communicated).
3.1.1.1. Chloroethyl 2,3,5-tri-O-acetyl-a-D-arabinofu-
ranoside (11). To a dry CH3CN (800 mL) solution of

A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
Table 4. 13C NMR chemical shifts for Galf disaccharides
Compound Unit Shifts d (ppm)
C-3 C-4
106.2 87.9 83.2 80.5 73.9 70.5
5639
which was stirred for another 1 h at room temperature.
Celite (5.0 g) was added, the suspension was cooled in an
ice-water bath, and a saturated aqueous NaHCO3 solu-
tion was added dropwise to precipitate tin salts. After
complete precipitation, the mixture was filtered through
Celite and extracted with chloroform (2· 500 mL). The
chloroform layer was again extracted with a cold-satu-
rated solution of NaHCO3 (2· 100 mL) followed by
water (100 mL) and brine (100 mL). The organic layer
was dried over Na2SO4 and concentrated to an oil. Col-
umn chromatography (CHCl3 100%) afforded pure a-
isomer 11 (22.1 g, 85%) as an oil. FABMS: m/z 525
[M+H]+. Anal. Calcd for C13H19ClO8: C, 46.14; H,
C-1
C-2
C-5
C-6
37
38
39
40
41
42
43
44
45
46
47
2
Galf
Galf0 105.1 80.9 76.5 80.4 69.4 63.0
Galf 106.0 87.8 83.2 80.5 74.0 70.5
Galf0 105.1 80.9 76.5 80.4 69.3 63.0
Galf 105.8 87.4 82.9 81.1 74.1 70.3
Galf0 105.2 81.0 76.4 80.3 69.3 63.0
Galf 106.6 86.2 83.5 81.9 73.4 69.3
Galf0 105.3 87.4 78.7 78.6 70.9 63.8
Galf 105.9 87.4 82.9 81.3 74.3 70.2
Galf0 105.2 81.1 76.5 80.3 69.4 63.1
Galf 107.8 81.2 77.6 84.7 77.1 62.7
Galf0 105.6 80.1 76.2 81.8 69.2 61.6
Galf 106.5 88.1 82.1 80.5 75.1 67.8
Galf0 105.7 81.1 76.5 80.1 69.3 62.7
Galf 105.9 87.4 81.9 81.1 75.5 67.7
Galf0 105.7 81.2 76.5 80.0 69.3 62.7
Galf 108.3 86.1 79.2 78.5 71.0 69.3
Galf0 106.3 81.8 76.0 79.9 69.1 62.6
Galf 106.0 87.4 81.9 81.2 75.5 67.6
Galf0 105.7 81.1 76.5 80.1 69.3 62.8
Galf 108.2 85.7 79.4 78.1 69.7 69.1
Galf0 106.2 79.9 76.1 81.7 69.2 62.7
Galf 109.4 83.1 78.5 84.3 77.2 64.3
Galf0 109.2 82.6 78.8 85.0 72.3 62.8
Galf 109.9 82.9 78.9 85.0 72.6 70.6
Galf0 109.5 82.8 78.8 85.6 71.1 64.5
Galf 109.3 82.6 78.5 84.2 77.1 64.2
Galf0 109.1 83.1 78.7 84.9 72.2 62.7
Galf 109.9 82.9 78.9 85.5 71.0 70.5
Galf0 109.4 82.8 78.8 85.0 72.5 64.4
1
5.65. Found C, 46.09; H, 5.69. H NMR (DMSO-d6):
d 5.61 (d, 1H, J = 4.4 Hz, H-3), 5.48 (s, 1H, H-1), 5.41
(d, 1H, J = 1.2 Hz, H-2), 4.76 (dd, 1H, J = 5.0,
13.8 Hz, H-5a), 4.64 (dd, 1H, J = 4.7, 13.8 Hz, H-5b),
4.65 (m, 1H, H-4), 3.95 (m, 1H, OCH2), 3.87 (m, 3H,
OCH2, CH2Cl).
3.1.1.2. Azidoethyl a-D-arabinofuranoside (12). To a
dry DMF (500 mL) solution of compound 11
(31.0 g, 91.52 mmol) was added NaN3 (8.9 g,
137.28 mmol). The reaction mixture was heated at
85 ꢁC for 6 h, left at rt for 2 days at which time
300 mL of acetone/ether (2:1) was added. It was fil-
tered through Celite, and the solid was washed with
acetone/ether mixture (20 mL). The combined filtrate
was concentrated to an oil. 7 N NH3/MeOH
(200 mL) was added under inert atmosphere and the
mixture was stirred overnight at rt. The reaction mix-
ture was concentrated to an oil and purified by col-
umn chromatography using CHCl3/MeOH (95:5) as
the eluting solvent to yield compound 12 (14.4 g,
70%). FABMS: m/z 220 [M+H]+. Anal. Calcd for
C7H13N3O5Æ1.0 H2O: C, 35.44; H, 6.37; N, 17.71.
Found C, 35.39; H, 6.57; N, 17.89. 1H NMR
(DMSO-d6): d 5.36 (d, 1H, J = 5.4 Hz, 2-OH), 5.12
3
5
6
2,3,5,6-tetra-O-acetyl-a-D-arabinofuranoside 10 (26.0 g,
81.69 mmol) was added SnCl4 dropwise and the mixture
was stirred for 30 min at room temperature. 1-Chloro-
ethanol (7.5 mL) was added dropwise to the reaction
Figure 5. Kinetic analysis of acceptor 1 [SRI#20309—the inset shows the double reciprocal plot for 1 as a substrate for the arabinosyltransferase.
The TLC autoradiogram shows the fluorescent trisaccharide reaction product mixture resulting from a(1 ! 5) and b(1 ! 2) arabinofuranosyl-
transferase activities as reported earlier25 through the inclusion of 1 at the highest concentration (2 mM), mycobacterial membranes and [14C]-DPA.
TLC/autoradiography was performed using chloroform/methanol/NH4OH/H2O (65:25:0.5:3.6) and products revealed through exposure to Kodak
X-Omat film at ꢀ70 ꢁC for 48 h.

5640
A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
Figure 6. An autoradiogram of reaction products produced through
the inclusion of disaccharides 2 (SRI 20413, upper panel) and 3 (SRI
20414, lower panel), mycobacterial membranes and UDP[14C]Galf.
Lane 1, no acceptor; lane 2, 0.25 mM; lane 3, 0.5 mM; lane 4,
0.75 mM; lane 5, 1.0 mM; lane 6, 1.5 mM; lane 7, 2.0 mM; and lane 8,
4.0 mM. TLC/autoradiography was 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.
(d, 1H, J = 5.4 Hz, 3-OH), 4.77 (d, 1H, J1,2 = 1.9 Hz,
H-1), 4.72 (t, 1H, J = 5.6 Hz, 5-OH), 3.83 (dd, 1H,
J1,2 = 1.9 Hz, J2,3 = 4.2 Hz, H-2), 3.78 (m, 2H, H-4,
OCH2), 3.68 (dd, 1H, J2,3 = 4.2, J3,4 = 7.1 Hz, H-3),
3.60 (dd, 1H, J4,5a = 3.0 Hz, J5a,5b = 11.9 Hz, H-5a),
3.57 (m, 1H, OCH2), 3.46 (dd, 1H, J4,5b = 5.7 Hz,
J5a,5b = 11.9 Hz, H-5b), 3.44 (m, 2H, CH2N3).
Figure 7. Kinetic analysis of acceptor disaccharides 2 (SRI 20413)
and (SRI 20414). The inset illustrates the double reciprocal
4
plot for disaccharides as
galactosyltransferase.
a substrate for the mycobacterial
3.1.1.4. Azidoethyl 2,3-O-di-tert-butyldimethylsilyl-5-
O-benzoyl-a-D-arabinofuranoside (14). Compound 13
(12.4 g, 38.39 mmol) was dissolved in dry DMF
(500 mL) and to it were added TBDMSCl (21.5 g,
141 mmol) and imidazole (19.2 g, 282 mmol). The reac-
tion mixture was heated at 60 ꢁC for one day and con-
centrated. The residue was dissolved in CHCl3 (40 mL)
and washed with water (10 mL). Organic layer was dried
over Na2SO4 and concentrated to syrup. Column chro-
matography (cyclohexane/EtOAc, 9:1) afforded com-
pound 21 (17.1 g, 81%) as an oil. FABMS (NBA): m/z
558 [M+H]+. Anal. Calcd for C26H45N3O6Si2: C,
56.59; H, 8.23; N, 7.62. Found C, 56.58; H, 8.19; N,
7.70. 1H NMR (CDCl3): d 8.07, 7.57, 7.44 (each m,
Ph), 4.89 (d, 1H, J1,2 = 1.1 Hz, H-1), 4.57 (dd, 1H,
J4,5a = 3.3 Hz, J5a,5b = 11.9 Hz, H-5a), 4.36 (dd, 1H,
J4,5b = 5.7 Hz, J5a,5b = 11.9 Hz, H-5b), 4.23 (ddd, 1H,
J4,5a = 3.3 Hz, J4,5b = 5.7 Hz, J3,4 = 6.2 Hz, H-4), 4.10
(dd, 1H, J1,2 = 1.3 Hz, J2,3 = 3.3 Hz, H-2), 4.04 (dd,
1H, J2,3 = 3.3 Hz, J3,4 = 6.2 Hz, H-3), 3.91 (m, 1H,
OCH2), 3.59 (m, 1H, OCH2), 3.46 (m, 1H, CH2N3),
3.35 (m, 1H, CH2N3).
3.1.1.3. Azidoethyl 5-O-benzoyl-a-D-arabinofuranoside
(13). The compound 12 (14.4 g, 65.69 mmol) was azeotr-
oped with pyridine and re-dissolved in 500 mL of dry
pyridine. It was cooled to ꢀ78 ꢁC and BzCl (7.6 mL,
65.69 mmol) was added dropwise. After complete addi-
tion, the reaction mixture was stirred overnight at rt. It
was poured in ice-water mixture and extracted with
CHCl3 (200 mL). The organic layer was washed with
water (2· 50 mL), dried over Na2SO4, and concentrated
to oil. Column chromatography using CHCl3/MeOH
(98:2) as the eluting solvent gave compound 13 (12.4 g,
58%). FABMS (LiCl): m/z 330 [M+Li]+. Anal. Calcd
for C14H17N3O6Æ1.0 H2O: C, 49.25; H, 5.61; N, 12.31.
1
Found C, 49.29; H, 5.58; N, 12.29. H NMR (DMSO-
d6): d 7.98, 7.67, 7.54 (each m, Ph), 5.54 (d, 1H,
J = 5.1 Hz, 2-OH), 5.43 (d, 1H, J = 5.3 Hz, 3-OH),
4.85 (d, 1H, J1,2 = 2.0 Hz, H-1), 4.51 (dd, 1H,
J4,5a = 2.9 Hz, J5a,5b = 11.9 Hz, H-5a), 4.31 (dd, 1H,
J4,5b = 5.9 Hz, J5a,5b = 11.9 Hz, H-5b), 4.07 (ddd, 1H,
J3,4 = 2.9 Hz, J4,5a = 2.9 Hz, J4,5b = 5.9 Hz, H-4), 3.87
(ddd, 1H, J1,2 = 2.0 Hz, J2,3 = 4.6 Hz, H-2), 3.78 (m,
2H, H-3, OCH2), 3.59 (m, 1H, OCH2), 3.40 (m, 2H,
CH2N3).
3.1.1.5. Azidoethyl 2,3-O-di-tert-butyldimethylsilyl-a-
D-arabinofuranoside (15). Method A: Compound 14

A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
5641
Figure 8. An autoradiogram of reaction products produced through
the inclusion of disaccharides 5 (SRI 20668) and 6 (SRI 20669),
mycobacterial membranes, and UDP[14C]Galf. Lane 1, no acceptor;
lane 2, 0.5 mM; lane 3, 0.75 mM; lane 4, 1.0 mM; lane 5, 2.0 mM; lane
6, 4.0 mM; and lane 7, 6.0 mM. TLC/autoradiography was 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.
Figure 9. Kinetic analysis of acceptor disaccharides 5 (SRI 20668)
and 6 (SRI 20669). The inset illustrates the double reciprocal
plot for disaccharides as
galactosyltransferase.
a substrate for the mycobacterial
(17.0 g, 30.52 mmol) was dissolved in 7 N NH3/MeOH
(200 mL) under inert atmosphere and stirred overnight
at rt. The reaction mixture was concentrated to an oil
and subjected to column chromatography using cyclo-
hexane/EtOAc (9:1) as the eluting solvent to give com-
pound 15 (12.5 g, 93%) as a colorless oil.
(1.2 g, 60%) was obtained as an oil. Spectral and analyt-
ical data have been reported.14
3.1.1.7. Aminoethyl 5-O-(2,3,5-tri-O-benzoyl-a-D-ara-
binofuranosyl)-2,3-O-di-tert-butyldimethylsilyl-a-D-arabi-
nofuranoside (18). Compound 17 (1.0 g, 1.16 mmol) was
dissolved in 50 mL of benzene, Ph3P (608 mg,
2.32 mmol) was added, and the reaction mixture was
heated to 50 ꢁC. After 1 h, water (2.8 mL) was added
and heating was continued for 5 h. The reaction mixture
was concentrated and dissolved in CHCl3, washed with
water (2· 10 mL) and brine (10 mL). The organic layer
was dried over Na2SO4, concentrated, and chromato-
graphed (CHCl3/MeOH, 95:5) to give 18 (845 mg,
87%) as a colorless oil. Spectral and analytical data have
been reported.13
Method B: To a THF (8.86 mL) solution of compound
21 (1.8 g, 3.20 mmol) was added TFA/H2O (1:1,
17.5 mL) at 0 ꢁC and the solution was stirred for 4 h.
A saturated aqueous solution of NaHCO3 (20 mL)
was added and the mixture was diluted with CHCl3
(50 mL). The organic layer was washed with 15 mL of
deionized water and concentrated. Purification by col-
umn chromatography (cyclohexane/EtOAc, 3:1) affor-
ded 15 (980 mg, 68%) as colorless oil. Spectral and
analytical data reported earlier.13
3.1.1.6. Azidoethyl 5-O-(2,3,5-tri-O-benzoyl-a-D-ara-
binofuranosyl)-2,3-O-di-tert-butyldimethylsilyl-a-D-arabi-
nofuranoside (17). A dry CH2Cl2 (20 mL) solution of 15
(1.00 g, 2.24 mmol) and 16 (1.63 g, 2.69 mmol) was
cooled to ꢀ20 ꢁC and stirred for 15 min. To it BF3ÆEt2O
(0.34 mL, 2.69 mmol) was added dropwise and the reac-
tion mixture was stirred for another 2 h at ꢀ20 ꢁC. The
reaction mixture was poured into a cold, saturated solu-
tion of NaHCO3 (20 mL). The organic layer was washed
with water (2· 25 mL), brine (20 mL) and dried over
Na2SO4. After concentration and column chromatogra-
phy (cyclohexane/EtOAc, 5:1), pure disaccharide 17
3.1.1.8. 5-N,N-Dimethylaminonaphthalene-1-sulfonam-
idoethyl 5-O-(2,3,5-tri-O-benzoyl-a-D-arabinofuranosyl)-
2,3-O-di-tert-butyldimethylsilyl-a-D-arabinofuranoside (19).
To a dry CH2Cl2 (25 mL) solution of 18 (805 mg,
0.93 mmol) was added N-methylimidazole and the mix-
ture was cooled to 0 ꢁC. To it 5-N,N-dimethylamino-
naphthalene-sulfonyl chloride (378 mg, 1.40 mmol) was
added and the reaction mixture was stirred at 0 ꢁC for
3 h. The mixture was washed with water and brine,
and the organic layer was dried over Na2SO4. Concen-

5642
A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
tration followed by column chromatography (cyclohex-
ane/EtOAc, 5:1) gave 19 (970 mg, 95%) as a light
yellow oil. Spectral and analytical data have been
reported.13
sugar signals, d 170.6, 170.1, 169.4 (COCH3), 66.4
(OCH2), 50.8 (CH2N3), 25.8, 25.7 (C), 20.75, 20.72,
20.7 (COCH3), 17.83, 17.79 (CH3), ꢀ4.3, ꢀ4.7, ꢀ4.8,
ꢀ4.9 (CH3).
3.1.1.9. 5-N,N-Dimethylaminonaphthalene-1-sulfonam-
idoethyl 5-O-(a-D-arabinofuranosyl)-2,3-O-di-tert-butyl-
dimethylsilyl-a-D-arabinofuranoside (20). Compound 19
(900 mg, 0.82 mmol) was debenzoylated with 7 N
NH3/MeOH (30 mL) as described for the preparation
of 15. Purification by column chromatography
(CHCl3/MeOH, 96:4) afforded 20 (567 mg, 88%) as a
light yellow oil. FABMS (LiCl): m/z 793.5 [M+Li]+.
Anal. Calcd for C36H62N2O11SSi2: C, 54.94; H, 7.95;
3.1.1.12. Aminoethyl 5-O-(2,3,5-tri-O-acetyl-a-D-ara-
binofuranosyl)-2,3-O-di-tert-butyldimethylsilyl-a-D-arabi-
nofuranoside (23). Disaccharide 22 (500 mg, 0.71 mmol)
was dissolved in dry MeOH (10 mL) and 5% Pd/C was
added (500 mg) under argon atmosphere. H2CO2NH4
(179 mg, 2.83 mmol) was added and the reaction mix-
ture was stirred at rt for 2 h. TLC showed complete con-
version, and the reaction mixture was filtered through a
short Celite pad and concentrated to syrup. The concen-
trate was dissolved in CHCl3 (20 mL), washed with
deionized water (10 mL), and dried over Na2SO4. Evap-
oration of the organic layer afforded the crude disaccha-
ride 23 (420 mg) that was used as such to prepare 24. 1H
NMR (CDCl3): major non-sugar signals in crude sam-
ple, d 3.71 (m, 1H, OCH2), 3.43 (m, 1H, OCH2), 2.86
(m, 2H, CH2NH), 2.10 (s, 6H, CH3), 2.09 (s, 3H,
CH3), 0.90 (s, 12H, CH3), 0.88 (s, 12H, CH3), 0.10,
0.09, 0.08 (CH3’s).
1
N, 3.56. Found: C, 54.98; H, 7.92; N, 3.50. H NMR
(CDCl3): non-sugar signals, d 8.53 (d, 1H, J = 8.6 Hz,
Ph), 8.29 (d, 1H, J = 8.7 Hz, Ph), 8.22 (dd, 1H,
J = 1.2, 7.4 Hz, Ph), 7.53 (m, Ph), 7.18 (d, 1H,
J = 7.6 Hz, Ph), 5.30 (m, 1H, NH, D2O exchangeable),
3.50 (m, 2H, OCH2), 3.25 (d, 1H, J = 11.4 Hz, 30-OH),
3.08 (m, 2H, CH2NH), 2.89 (s, 2H, 2· NCH3), 0.87,
0.86 (s, 9H each 6· CH3), 0.08 (s, 3H, CH3), 0.06 (s,
6H, CH3), 0.05 (s, 3H, CH3). 13C NMR (CDCl3): non-
sugar signals, d 151.9, 134.7, 130.4, 129.9, 129.6, 129.5,
128.3, 123.1, 118.9, 115.2 (Ph), 66.8 (OCH2), 45.4
(NCH3), 43.3 (CH2NH), 25.7, 25.7 (CH3), 17.83, 17.82
(C), ꢀ4.3, ꢀ4.61, ꢀ4.67, ꢀ4.8 (CH3).
3.1.1.13. 5-Azidonaphthalene-1-sulfonamidoethyl 5-O-
(2,3,5-tri-O-acetyl-a-D-arabinofuranosyl)-2,3-O-di-tert-
butyldimethylsilyl-a-D-arabinofuranoside (24). Crude 24
(420 mg, 0.62 mmol) was coupled with 5-azi-
donaphthalenesulfonyl chloride (248 mg, 0.93 mmol) in
the presence of N-methylimidazole (102 mg, 1.24 mmol)
using dry CH2Cl2 (50 mL) as solvent as described for
the preparation of 19. Purification by column chromatog-
raphy (cyclohexane/EtOAc, 3:1) afforded 24 (453 mg,
81%) as a light yellow oil (light sensitive). Spectral and
analytical data have been reported.14
3.1.1.10. Azidoethyl 2,3,5-tri-O-tert-butyldimethylsi-
lyl-a-D-arabinofuranoside (21). Compound 12 (780 mg,
3.56 mmol) was silylated by reaction with TBDMSCl
(1.84 mg,
12.5 mmol)
and
imidazole
(1.5 mg,
21.36 mmol) in dry DMF (40 mL) as described for the
preparation of 14. Column chromatography (cyclohex-
ane/EtOAc, 5:1) afforded 21 (1.80 g, 90%) as an oil.
Spectral and analytical data have been reported.14
3.1.1.14. 5-Azidonaphthalene-1-sulfonamidoethyl 5-O-
(a-D-arabinofuranosyl)-2,3-O-di-tert-butyldimethylsilyl-a-
D-arabinofuranoside (25). Compound 24 (430 mg,
0.47 mmol) was deacetylated by 7 N NH3/MeOH
(6 mL) as described for the preparation of 20. Column
chromatography (CHCl3/MeOH, 95:5) afforded pure
25 (322 mg, 87%) as a light sensitive, yellow oil. Spectral
and analytical data have been reported.14
3.1.1.11. Azidoethyl 5-O-(2,3,5-tri-O-acetyl-a-D-arabi-
nofuranosyl)-2,3-O-di-tert-butyldimethylsilyl-a-D-arabino-
furanoside (22). Alcohol 15 (980 g, 2.05 mmol),
thioglycoside donor 1-deoxy-1-thiocresyl-2,3,5-tri-O-
acetyl-a-D-arabinofuranoside19 7 (942 mg, 2.46 mmol),
˚
and activated, powdered 4 A molecular sieves (100 mg)
in dry CH2Cl2 (30 mL) were cooled at 0 ꢁC under argon
atmosphere. The mixture was stirred for 15 min, and
NIS (553 mg, 2.46 mmol) followed by Sn(OTf)2 (86 mg,
0.21 mmol) were added to initiate coupling. The reaction
mixture was stirred for 30 min at rt, and the reaction was
quenched by addition of Et3N (5 mL), diluted with
CH2Cl2 (60 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 column chromatography (cyclohexane/EtOAc 3:1) to
give pure disaccharide 22 (1.33 g, 91%) as an oil. ESI-
MS: m/z 728.3233 [M+Na]+, Calcd 728.3216 [M+Na]+
3.1.1.15. Chloroethyl 2,3,5,6-tetra-O-acetyl-b-D-galac-
tofuranoside (27). b-D-Galactofuranose pentaacetate
(26)21 (20.0 g, 51.28 mmol) was treated with SnCl4
(6.0 mL, 51.28 mmol) and 1-chloroethanol (4.12 mL,
61.52 mmol) in dry CH3CN (150 mL) as described for
the preparation of 11. Purification by column chroma-
tography (cyclohexane/EtOAc, 2:1) yielded 27 (13.44 g,
64%) as an oil. FABMS (LiCl): m/z 417 [M+Li]+. Anal.
Calcd for C16H23ClO10: C, 46.78; H, 5.64. Found: C,
46.69; H, 5.60. 1H NMR (300 MHz, CDCl3) d: 5.39
(ddd, 1H, J4,5 = 3.7 Hz, J5,6a = 4.7 Hz, J5,6b = 7.0 Hz,
H-5), 5.09 (dd, 1H, J1,2 = 0.7 Hz, J2,3 = 2.0 Hz, H-2),
5.08 (s, 1H, H-1), 5.01 (ddd, 1H, J1,3 = 0.7 Hz,
J2,3 = 2.0 Hz, J3,4 = 6.0 Hz, H-3), 4.34 (dd, 1H,
J5,6a = 4.7 Hz, J6a,6b = 11.8 Hz, H-6a), 4.33 (ddd, 1H,
J1,4 = 0.3 Hz, J4,5 = 3.5 Hz, J3,4 = 6.0 Hz, H-4), 4.21
(dd, 1H, J5,6b = 7.0 Hz, J6a,6b = 11.8 Hz, H-6b), 3.90
(m, 1H, OCH2), 3.78 (m, 1H, OCH2), 3.66 (m, 2H,
1
for C30H55N3O12Si2. H NMR (CDCl3): non-sugar sig-
nals, d 3.88 (m, 1H, OCH2), 3.57 (m, 1H, OCH2), 3.44
(m, 1H, CH2N3), 3.34 (m, 1H, CH2N3), 2.10 (s, 6H, 2·
COCH3), 2.09 (s, 3H, CH3), 0.99 (s, 9H, 3· CH3), 0.88
(s, 9H, 3· CH3), 0.102 (9H, s, 3· CH3), 0.099 (s, 6H,
2· CH3), 0.081 (s, 3H, CH3). 13C NMR (CDCl3): non-

A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
5643
CH2Cl), 2.14, 2.12, 2.10, 2.07 (each s, each 3H, 4·
COCH3). 13C NMR (75 MHz, CDCl3):
170.5,
170.00, 169.95, 169.6 (COCH3), 105.5 (C-1), 81.3 (C-
2), 80.1 (C-4), 76.3 (C-3), 70.0 (C-5), 67.7 (OCH2),
62.4 (C-6), 42.6 (CH2Cl), 20.8, 20.72, 20.68, 20.63 (4·
COCH3).
After 1 h of stirring, the pH was adjusted to 7 by adding
Et3N. Concentration under vacuum gave the product
which was dissolved in CHCl3 (250 mL), washed with
saturated NaHCO3 (50 mL) and water (50 mL), dried
over Na2SO4, and concentrated to an oil. Purification
by column chromatography (CHCl3/MeOH, 99:1)
yielded 30 (2.46 g, 82%) as an oil. FABMS (NBA): m/z
290 [M+H]+. Anal. Calcd for C11H19N3O6Æ0.5 H2O: C,
44.29; H, 6.76; N, 14.09. Found: C, 44.29; H, 6.79; N,
d
3.1.1.16. Azidoethyl 2,3,5,6-tetra-O-acetyl-b-D-galac-
tofuranoside (28). To a dry DMF (70 mL) solution of
compound 27 (4.80 g, 11.66 mmol) was added NaN3
(1.89 g, 29.2 mmol), and the reaction mixture was stirred
at 70 ꢁC overnight. The mixture was concentrated to
near dryness and 100 mL of acetone/ether (2:1) was
added and the mixture was stirred for 20 min at rt. It
was filtered and concentrated to dryness. The resulting
oil was re-dissolved in CHCl3 (200 mL), washed with
water (2· 40 mL), dried over Na2SO4, and concentrated.
Column chromatography (cyclohexane/EtOAc, 2:1)
gave compound 28 as pale yellow oil (4.88 g, quant.).
FABMS (LiCl): m/z 424 [M+Li]+. Anal. Calcd for
C16H23N3O10: C, 46.04; H, 5.56; N, 10.07. Found: C,
1
14.01. H NMR (300 MHz, CDCl3): d 5.08 (s, 1H, H-
1), 4.37 (ddd, 1H, J4,5 = 1.4 Hz, J5,6a = 6.5 Hz,
J5,6b = 7.2 Hz, H-5), 4.14 (dd, 1H, J3,4 = 1.8 Hz,
J4,5 = 1.4 Hz, H-4), 4.12 (dd, 1H, J5,6a = 6.5 Hz,
J6a,6b = 8.3 Hz, H-6a), 4.05 (m, 3H, H-2, H-3, 3-OH),
4.02 (dd, 1H, J5,6b = 7.2 Hz, J6a,6b = 8.3 Hz, H-6b),
3.93 (m, 1H, OCH2), 3.65 (m, 1H, OCH2), 1.46 (m,
2H, CH2N3), 2.88 (d, 1H, J = 11.6 Hz, 2-OH). 13C
NMR (75 MHz, CDCl3): d 110.0 (C), 108.5 (C-1), 85.6
(C-4), 78.6 (C-3), 78.4 (C-2), 75.5 (C-5), 65.9 (C-6),
65.5 (OCH2), 50.6 (CH2N3), 25.5, 25.9 (2· CH3).
1
46.06; H, 5.62; N, 9.99. H NMR (300 MHz, CDCl3):
3.1.1.19. Azidoethyl 5,6-isopropyledine-2,3-di-O-p-
methoxybenzyl-b-D-galactofuranoside (31). To a dry
DMF solution (50 mL) of compound 30 (1.50 g,
5.19 mmol) was added NaH (60% suspension in mineral
oil ꢀ498 mg, 20.76 mmol) followed by p-methoxybenzyl
chloride (2.81 mL, 20.76 mmol) dropwise with cooling
at 0 ꢁC. The reaction mixture was stirred at rt overnight.
The reaction mixture was cooled to 0 ꢁC and MeOH
(23 mL) was added to the reaction. The mixture was
evaporated to near dryness and re-dissolved in CHCl3
(200 mL). It was washed with water (50 mL), dried over
Na2SO4, and purified by column chromatography
(cyclohexane/EtOAc, 5:1) to give 31 (2.69 g, 98%) as
an oil. FABMS (LiCl): m/z 536 [M+Li]+. Anal. Calcd
for C27H35N3O8: C, 61.24; H, 6.66; N, 7.93. Found: C,
d
5.41 (ddd, 1H, J4,5 = 3.7 Hz, J5,6a = 4.7 Hz,
J5,6b = 7.0 Hz, H-5), 5.08 (d, 1H, J2,3 = 1.9 Hz, H-2),
5.07 (s, 1H, H-1), 5.02 (ddd, 1H, J1,3 = 0.4 Hz, J2,3
=
1.9 Hz, J3,4 = 5.7 Hz, H-3), 4.34 (dd, 1H, J5,6a = 4.7 Hz,
J6a,6b = 11.8 Hz, H-6a), 4.32 (ddd, 1H, J4,5 = 3.7 Hz,
J3,4 = 5.7 Hz, H-4), 4.23 (dd, 1H, J5,6b = 7.0 Hz,
J6a,6b = 11.8 Hz, H-6b), 3.89 (ddd, 1H, J = 3.6, 6.9,
10.6 Hz, OCH2), 3.66 (ddd, 1H, J = 3.4, 6.1, 10.6 Hz,
OCH2), 3.45 (ddd, 1H, J = 3.4, 6.9, 13.3 Hz, CH2N3),
3.36 (ddd, 1H, J = 3.6, 6.1, 13.3 Hz, CH2N3), 2.14, 2.12,
2.09, 2.07 (each s, each 3H, 4· COCH3). 13C NMR
(75 MHz, CDCl3): d 170.3, 169.9, 169.8, 169.4 (COCH3),
105.4 (C-1), 81.1 (C-2), 80.2 (C-4), 76.2 (C-3), 69.1 (C-5),
66.5 (OCH2), 62.3 (C-6), 50.3 (CH2N3), 20.6, 20.54, 20.48,
20.43 (4· COCH3).
1
61.11; H, 6.49; N, 7.77. H NMR (300 MHz, CDCl3):
d 7.29, 7.18 (m, each 2H, Ph), 6.89 (m, 4H, Ph), 5.07
(s, 1H, H-1), 4.44 (m, 4H, 2· CH2Ph), 4.14 (ddd, 1H,
J4,5 = 6.8 Hz, J5,6a = 6.9 Hz, J5,6b = 7.3 Hz, H-5), 4.04
(dd, 1H, J1,2 = 1.0 Hz, J2,3 = 2.9 Hz, H-2), 4.00 (dd,
1H, J3,4 = 7.0 Hz, J4,5 = 6.8 Hz, H-4), 3.94 (m, 1H,
OCH2), 3.83 (dd, 1H, J5,6a = 6.9 Hz, J6a,6b = 6.4 Hz,
H-6a), 3.81, 3.80 (s, each 3H, 2· OCH3), 3.77 (dd, 1H,
J5,6b = 7.3 Hz, J6a,6b = 6.4 Hz, H-6a), 3.72 (dd, 1H,
J2,3 = 2.9 Hz, J3,4 = 3.4 Hz, H-3), 3.61 (m, 1H, OCH2),
3.47 (m, 1H, CH2N3), 3.32 (m, 1H, CH2N3), 1.40, 1.35
(s, each 3H, 2· CH3). 13C NMR (75 MHz, CDCl3) d:
159.4, 159.3 (2· C), 129.7 (CH), 129.3 (C), 113.8,
113.7 (2· CH), 109.6 (C), 106.4 (C-1), 87.5 (C-2), 83.5
(C-3), 82.0 (C-4), 76.5 (C-6), 71.8, 71.6 (2· CH2Ph),
66.3 (OCH2), 65.3 (C-6), 55.2, 55.2 (2· OCH3), 50.5
(CH2N3), 26.4, 25.4 (2· CH3).
3.1.1.17. Azidoethyl b-D-galactofuranoside (29). Com-
pound 28 (4.48 g, 10.7 mmol) was debenzoylated with
7 N NH3/MeOH (10 mL) as described for the prepara-
tion of 20. Purification by column chromatography
(CHCl3/MeOH, 5:1) afforded 29 (2.67 g, 98%) as an
oil. FABMS (NBA): m/z 250 [M+H]+. Anal. Calcd for
C8H15N3O6Æ1.5 H2O: C, 34.78; H, 6.57; N, 15.21.
Found: C, 34.77; H, 6.59; N, 15.20. 1H NMR
(300 MHz, D2O): d 4.00 (ddd, 1H, J1,2 = 2.4 Hz,
J1,3 = 0.6 Hz, J1,4 = 0.7 Hz, H-1), 3.98 (m, 2H, H-2,
H-3), 3.90 (ddd, 1H, J1,4 = 0.7 Hz, J3,4 = 6.0 Hz,
J4,5 = 4.0 Hz, H-4), 3.80 (m, 1H, OCH2), 3.73 (ddd,
1H, J4,5 = 4.0 Hz, J5,6a = 4.5 Hz, J5,6b = 7.4 Hz, H-5),
3.64 (m, 1H, OCH2), 3.60 (dd, 1H, J5,6a = 4.5 Hz,
J6a,6b = 11.7 Hz, H-6a), 3.55 (dd, 1H, J5,6b = 7.4 Hz,
J6a,6b = 11.7 Hz, H-6b), 3.42 (m, 2H, CH2N3). 13C
NMR (75 MHz, D2O): d 107.6 (C-1), 83.0 (C-4), 81.3
(C-2), 76.9 (C-3), 70.9 (C-5), 66.9 (OCH2), 63.0 (C-6),
50.6 (CH2N3).
3.1.1.20. Azidoethyl 2,3-di-O-p-methoxybenzyl-b-D-
galactofuranoside (32). A solution of compound 31
(2.50 g, 4.72 mmol) in 60% aqueous acetic acid
(75 mL) was stirred for 4 h at 60 ꢁC. The solution was
concentrated in vacuum, coevaporated with diluted
CHCl3 (20 mL), and neutralized with saturated NaH-
CO3 solution. The organic phase was washed with water
(2· 50 mL), dried over Na2SO4, and purified by column
chromatography (CHCl3/MeOH, 95:5) to give 32
3.1.1.18. Azidoethyl 5,6-isopropyledine-b-D-galacto-
furanoside (30). To a solution of compound 29 (2.60 g,
10.4 mmol) in dry acetone (20 mL) were added 2,20-
dimethoxypropane (2.57 mL, 20.9 mmol) and (1S)-(+)-
10-camphorsulfonic acid (242 mg, 1.04 mmol) at rt.

5644
A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
(1.78 g, 77%) as an oil. FABMS: m/z 507 [M+NH4]+.
Anal. Calcd for C24H31N3O8Æ0.5 H2O: C, 57.82; H,
6.47; N, 8.43. Found: C, 57.85; H, 6.47; N, 8.39. H
NMR (300 MHz, CDCl3): d 7.27, 7.20 (each m, each
2H, Ph), 6.88 (m, 4H, Ph), 5.04 (s, 1H, H-1), 4.46 (m,
chromatography (cyclohexane/EtOAc, 2:1) gave pure
34 (1.54 g, 80%) as an oil. FABMS (LiCl): m/z 737
[M+Li]+. Anal. Calcd for C43H45N3O8: C, 70.57; H,
1
1
6.20; N, 5.74. Found: C, 70.67; H, 6.23; N, 5.79. H
NMR (300 MHz, CDCl3): d 7.42 (m, 6H, Ph), 7.25 (m,
11H, Ph), 7.16 (m, 2H, Ph), 6.85 (m, 4H, Ph), 5.01 (s,
1H, H-1), 4.42 (m, 4H, CH2Ph), 4.17 (m, 1H, H-4),
4.00 (d, 1H, J2,3 = 2.4 Hz, H-2), 3.98 (dd, 1H,
J2,3 = 2.4 Hz, J3,4 = 5.2 Hz, H-3), 3.84-3.76 (m, 2H, H-
5, OCH2), 3.80, 3.79 (s, each 3H, 2· OCH3), 3.54 (m,
1H, OCH2), 3.40 (m, 1H, CH2N3), 3.26 (m, 2H, H-6a,
CH2N3), 3.17 (dd, 1H, J5,6b = 5.9 Hz, J6a,6b = 9.4 Hz,
H-6b), 2.32 (d, 1H, J = 7.1 Hz, 5-OH). 13C NMR
(75 MHz, CDCl3) d: 159.4, 159.2 (2· C), 143.8, 129.7
(2· C), 129.6, 129.4 (2· CH), 129.2 (C), 128.6, 127.7,
126.9 (3· CH), 113.8, 113.7 (2· CH), 106.5 (C-1), 87.2
(C-2), 86.6 (C), 83.0 (C-3), 81.7 (C-4), 71.9, 71.6 (2·
CH2Ph), 70.0 (C-5), 66.2 (OCH2), 64.8 (C-6), 55.2
(OCH3), 50.5 (CH2N3).
4H, 2· CH2Ph), 4.08 (dd, 1H, J3,4 = 6.8 Hz, J4,5
=
3.9 Hz, H-4), 4.03 (dd, 1H, J1,2 = 1.1 Hz, J2,3 = 2.9 Hz,
H-2), 4.00 (dd, 1H, J2,3 = 2.9 Hz, J3,4 = 6.8 Hz, H-3),
3.85 (m, 1H, OCH2), 3.81, 3.79 (s, each 3H, 2·
OCH3), 3.73 (m, 1H, H-5), 3.65 (m, 2H, H2-6), 3.59
(m, 1H, OCH2), 3.46 (m, 1H, CH2N3), 3.33 (m, 1H,
CH2N3), 2.51 (d, 1H, J = 7.0 Hz, 5-OH), 2.23 (dd, 1H,
J = 5.1, 7.3 Hz, 6-OH). 13C NMR (75 MHz, CDCl3) d:
159.4, 159.32 (2· C), 129.7, 129.5 (3· CH), 129.4,
129.1 (2· C), 113.8, 113.8 (2· CH), 106.6 (C-1), 87.1
(C-2), 83.0 (C-3), 82.2 (C-4), 71.9, 71.8 (2· CH2Ph),
71.4 (C-5), 66.3 (OCH2), 64.3 (C-6), 55.2, 55.2 (2·
OCH3), 50.5 (CH2N3).
3.1.1.21. Azidoethyl 2,3,6-tri-O-p-methoxybenzyl-b-D-
galactofuranoside (33). A toluene (150 mL) solution of
compound 32 (749 mg, 1.53 mmol) and dibutyltin oxide
(380 mg, 1.53 mmol) was refluxed overnight with azeo-
tropic removal of water. The reaction mixture was
cooled and evaporated to dryness. The residue was dis-
solved in dry DMF (10 mL) and anhydrous CsF
(465 mg, 3.06 mmol) and p-methoxybenzyl chloride
(0.45 mL, 3.06 mmol) were added. The resulting mixture
was stirred overnight at rt under argon, concentrated
under vacuum, re-dissolved in chloroform (150 mL),
and washed with 10% aqueous KF solution and water
(10 mL). The combined organic phase was dried, con-
centrated, and chromatographed (cyclohexane/EtOAc,
3:1) to afford pure 33 (840 mg, 90%) as an oil. FABMS
(LiCl): m/z 616 [M+Li]+. Anal. Calcd for
C32H39N3O9Æ0.5 H2O: C, 62.12; H, 6.52; N, 6.79. Found:
C, 62.15; H, 6.53; N, 6.75. 1H NMR (300 MHz, CDCl3):
d 7.23 (m, 6H, Ph), 6.87 (m, 6H, Ph), 5.04 (s, 1H, H-1),
4.44 (m, 6H, 3· CH2Ph), 4.08 (ddd, 1H, J1,4 = 0.8 Hz,
J3,4 = 6.9 Hz, J4,5 = 3.5 Hz, H-4), 4.01 (d, 1H,
J2,3 = 2.6 Hz, H-2), 4.00 (dd, 1H, J2,3 = 2.6 Hz,
J3,4 = 6.9 Hz, H-3), 3.86 (m, 1H, H-5), 3.84-3.76 (m,
1H, OCH2), 3.80, 3.795, 3.790 (each s, each 3H, 3·
OCH3), 3.57 (m, 1H, OCH2), 3.48 (d, 2H, J = 6.0 Hz,
H2-6), 3.43 (m, 1H, CH2N3), 3.31 (m, 1H, CH2N3),
2.40 (d, 1H, J = 5.9 Hz, 5-OH). 13C NMR (75 MHz,
CDCl3) d: 159.3, 159.2, 159. 1 (3· C), 129.9, 129.6 (2·
C), 129.6, 129.4, 129.2 (3· CH), 128.4 (C), 113.7,
113.67, 113.64 (3· CH), 106.4 (C-1), 87.2 (C-2), 82.8
(C-3), 81.5 (C-4), 72.9 (C-6), 71.7, 71.6, 71.1 (3·
CH2Ph), 69.9 (C-5), 66.2 (OCH2), 55.15, 55.13, 55.12
(3· OCH3), 50.4 (CH2N3).
3.1.1.23. Azidoethyl 2,3,5-tri-O-p-methoxybenzyl-6-O-
trityl-b-D-galactofuranoside (35). To a dry THF solution
(50 mL) of compound 34 (1.54 g, 2.11 mmol) was added
NaH (60% suspension in mineral oil ꢀ152 mg,
6.32 mmol) followed by p-methoxybenzyl chloride
(0.85 mL, 6.32 mmol) dropwise with cooling at 0 ꢁC.
The reaction mixture was stirred at rt overnight. MeOH
(20 mL) was added to the reaction, the mixture was
evaporated to near dryness and re-dissolved in CHCl3
(250 mL). It was washed with water (2· 50 mL), dried
over Na2SO4, and purified by column chromatography
(cyclohexane/EtOAc, 6:1) to give 35 (1.53 g, 86%) as
an oil. FABMS (LiCl): m/z 857 [M+Li]+. Anal. Calcd
for C51H53N3O9Æ0.15 H2O: C, 72.28; H, 6.18; N, 4.86.
Found: C, 72.25; H, 6.22; N, 4.89. 1H NMR
(300 MHz, CDCl3): d 7.41 (m, 6H, Ph), 7.21 (m, 13H,
Ph), 7.06 (m, 2H, Ph), 6.82 (m, 6H, Ph), 4.98 (d, 1H,
J1,2 = 1.4 Hz, H-1), 4.45 (m, 6H, 3· CH2Ph), 4.10 (dd,
1H, J3,4 = 7.6 Hz, J4,5 = 3.1 Hz, H-4), 3.96 (dd, 1H,
J1,2 = 1.4 Hz, J2,3 = 3.8 Hz, H-2), 3.89 (dd, 1H,
J2,3 = 3.8 Hz, J3,4 = 7.6 Hz, H-3), 3.80, 3.76, 3.75 (s,
each 3H, OCH3), 3.72 (m, 1H, OCH2), 3.63 (ddd, 1H,
J4,5 = 3.1 Hz, J5,6a = J5,6b = 5.9 Hz, H-5), 3.50 (m, 1H,
OCH2), 3.35 (m, 2H, H-6a, CH2N3), 3.22 (m, 2H, H-
6b, CH2N3). 13C NMR (75 MHz, CDCl3) d: 159.3,
159.19, 159.17 (3· C), 143.9, 130.3, 129.93 (3· C),
129.89 (CH), 129.7 (C), 129.6, 129.5, 128.7, 127.7,
126.9, 113.7, 113.7, 113.6 (CH), 106.1 (C-1), 88.3 (C-
2), 86.9 (C), 82.3 (C-3), 80.6 (C-4), 75.7 (C-5), 72.9,
71.7, 71.7 (3· CH2Ph), 66.1 (OCH2), 63.6 (C-6), 55.23,
55.20, 55.1 (3· OCH3), 50.5 (CH2N3).
3.1.1.24. Azidoethyl 2,3,5-tri-O-p-methoxybenzyl-b-D-
galactofuranoside (36). Compound 35 (1.40 g,
1.64 mmol) in CHCl3 (25 mL) was chilled to 0 ꢁC. 5%
TFA/CHCl3 (11 mL) was added dropwise over a period
of 1.5 h at 0 ꢁC. The reaction mixture was then stirred
for another 5 h at 0 ꢁC, neutralized with satd. NaHCO3,
and extracted with CHCl3 (200 mL). The organic layer
was washed with water (2· 50 mL), dried over Na2SO4,
and concentrated. Purification by column chromatogra-
phy (cyclohexane/EtOAc, 3:1) afforded 36 (700 mg,
70%) as an oil. FABMS (LiCl): m/z 616 [M+Li]+. Anal.
3.1.1.22. Azidoethyl 2,3-di-O-p-methoxybenzyl-6-O-
trityl-b-D-galactofuranoside (34). Compound 32 (1.29 g,
2.64 mmol) was dissolved in dry pyridine (45 mL) and
trityl chloride (1.47 g, 5.28 mmol) was added followed
by DMAP (32 mg, 0.26 mmol). The reaction mixture
was stirred overnight at 50 ꢁC. The solution was concen-
trated to dryness and re-dissolved in CHCl3 (200 mL).
The CHCl3 layer was washed with water (2· 50 mL),
brine (2· 50 mL) and dried over Na2SO4. Concentration
of the combined organic layers followed by column

A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
5645
Calcd for C32H39N3O9: C, 63.04; H, 6.45; N, 6.89.
Found: C, 63.00; H, 6.42; N, 6.85. 1H NMR
(300 MHz, CDCl3): d 7.23 (m, 6H, Ph), 6.86 (m, 6H,
Ph), 5.06 (s, 1H, H-1), 4.44 (m, 6H, 3· CH2Ph), 4.16
(dd, 1H, J3,4 = 7.4 Hz, J4,5 = 4.8 Hz, H-4), 4.02 (dd,
1H, J1,2 = 1.1 Hz, J2,3 = 3.3 Hz, H-2), 3.95 (dd, 1H,
J2,3 = 3.3 Hz, J3,4 = 7.4 Hz, H-3), 3.87 (m, 1H, OCH2),
3.81, 3.78, 3.77 (each s, each 3H, 3· OCH3), 3.64 (m,
4H, H-5, H2-6, OCH2), 3.47 (m, 1H, CH2N3), 3.33 (m,
1H, CH2N3), 2.19 (dd, 1H, J = 4.8, 7.8 Hz, 6-OH). 13C
NMR (75 MHz, CDCl3) d: 159.3, 159.2, 159.1 (C),
130.2 (C), 129.6, 129.5 (2· CH), 129.3, 129.2 (2· C),
113.8, 113.7 (2· CH), 106.2 (C-1), 87.7 (C-2), 82.6 (C-
3), 81.6 (C-4), 77.7 (C-5), 72.3, 71.8, 71.6 (3· CH2Ph),
66.2 (OCH2), 62.0 (C-6), 55.2, 55.1 (3· OCH3), 50.5
(CH2N3).
(CHCl3/MeOH, 9:1) yielded 38 (2.69 g, 92%) as an oil.
FABMS (NBA): m/z 914 [M+H]+. Anal. Calcd for
C46H59NO18Æ0.5 H2O: C, 59.86; H, 6.55; N, 1.52. Found
C, 59.90; H, 6.49; N, 1.48. 1H NMR (600 MHz, CDCl3):
non-sugar signals, d 7.21 (m, 12H, Ph), 6.86 (m, 6H, Ph),
4.48, 4.40 (m, each 6H, 3· CH2Ph), 3.81 (s, 3H, OCH3),
3.79 (s, 6H, 2· OCH3), 3.69 (m, 1H, OCH2), 3.46 (m,
1H, OCH2), 2.87 (m, 2H, CH2NH2), 2.08, 2.06, 1.98,
1.97 (each s, each 3H, 4· COCH3). 13C NMR
(75 MHz, CDCl3): non-sugar signals d 170.4, 169.9,
169.9, 169.2 (4· COCH3), 159.3, 159.2, 159.0 (4· C),
130.0, 129.7, 129.3 (3· C), 129.6, 129.3, 129.0 (3· CH),
113.7 (2· CH), 113.6 (CH), 72.9 (CH2Ph), 71.7 (2·
CH2Ph), 69.2 (OCH2), 63.0 (C-60), 55.2 (OCH3), 55.1
(2· OCH3), 41.9 (CH2NH2), 20.7, 20.6, 20.5, 20.4 (4·
COCH3).
3.1.1.25. Azidoethyl 5-O-(2,3,5,6-tetra-O-acetyl-b-D-
galactofuranosyl)-2,3,6-tri-O-p-methoxybenzyl-b-D-galac-
tofuranoside (37). Compound 33 (2.92 g, 4.78 mmol) was
3.1.1.27. 5-N,N-Dimethylaminonaphthalene-1-sulfo-
namidoethyl 5-O-(2,3,5,6-tetra-O-acetyl-b-D-galactofur-
anosyl)-2,3,6-tri-O-p-methoxybenzyl-b-D-galactofurano-
side (39). Compound 38 (350 mg, 0.38 mmol) was mixed
with N-methylimidazole (63 mg, 0.76 mmol) and dansyl
chloride (155 mg, 0.57 mmol) in dry CH2Cl2 (40 mL) at
0 ꢁC as described for the preparation of 18. Purification
by column chromatography (cyclohexane/EtOAc, 1:1)
afforded the 39 (340 mg, 77%) as an oil. FABMS
(NBA): m/z 1148 [M+H]+. Anal. Calcd for
C58H70N2O20SÆ0.5 H2O: C, 60.25; H, 6.19; N, 2.42.
Found C, 60.19; H, 6.18; N, 2.41. 1H NMR
(600 MHz, CDCl3): non-sugar signals d 8.53 (d, 1H, J
8.4 Hz, Ph), 8.27 (d, 1H, J = 8.4 Hz, Ph), 8.23 (dd, 1H,
J 0.6, 7.8 Hz, Ph), 7.50 (dd, 1H, J = 7.8, 8.4 Hz, Ph),
7.48 (dd, 1H, J = 7.2, 8.4 Hz, Ph), 7.19 (m, 6H, Ph),
7.12 (d, 1H, J = 7.2 Hz, Ph), 6.87 (d, 4H, J = 8.4 Hz,
Ph), 6.83 (d, 2H, J = 8.4 Hz, Ph), 5.33 (m, 1H, NH),
4.39 (m, 6H, 3· CH2Ph), 3.799, 3.795, 3.791 (each s,
each 3H, 3· OCH3), 3.59 (m, 1H, OCH2), 3.40 (m,
1H, OCH2), 3.07 (m, 2H, CH2NH), 2.86 (s, 6H,
N(CH3)2), 2.08, 2.06, 1.98, 1.94 (each s, each 3H, 4·
COCH3). 13C NMR (75 MHz, CDCl3): non-sugar sig-
nals d 170.5, 169.9, 169.2 (4· COCH3), 159.4, 159.3,
159.0 (C), 151.9 (C) 134.8 (C), 130.3 (CH), 130.1
129.8, 129.7 (C), 129.6, 129.5, 129.4 (CH), 129.2 (C),
129.1, 128.3, 123.1, 118.7, 115.1, 113.8, 113.6 (CH),
72.9 (CH2Ph), 71.7 (2· CH2Ph), 65.7 (OCH2), 55.2,
55.2, 55.1 (3· OCH3), 45.3 (N(CH3)2), 43.0 (CH2NH),
20.8, 20.7, 20.6, 20.5 (4· COCH3).
˚
dissolved in dry CH2Cl2 (50 mL) and powdered 4 A
molecular sieves (ꢁ500 mg) were added under argon.
The mixture was then cooled to ꢀ20 ꢁC. The glycosyla-
tion donor 26 (3.02 g, 7.18 mmol) in 15 mL dry CH2Cl2
was added dropwise under argon. The mixture was stir-
red for 15 min, and NIS (2.16 g, 9.56 mmol) followed by
triflic acid (4.0 lL, 0.48 mmol) were added. The reaction
mixture was allowed to stir for 30 min at ꢀ20 ꢁC,
quenched by addition of Et3N (5 mL), and filtered
through a Celite pad. The filtrate was washed with
10% Na2S2O3 (30 mL), followed by washing with satu-
rated aqueous NaHCO3 (20 mL). The organic layer
was dried over Na2SO4, the solvent was removed under
vacuum, and the residue was purified by column chro-
matography (cyclohexane/EtOAc, 2:1) to give 37
(3.72 g, 83%) as an oil. FABMS (LiCl): m/z 946.4
[M+Li]+. Anal. Calcd for C46H57N3O18Æ0.5 H2O: C,
58.22; H, 6.16; N, 4.23. Found C, 58.19; H, 6.19; N,
1
4.27. H NMR (600 MHz, CDCl3): non-sugar signals,
d 7.22 (m, 6H, Ph), 6.86 (m, 6H, Ph), 4.50, 4.46, 4.42,
4.41, 4.39 (each d, 6H, J = 11.4 Hz, 3· CH2Ph), 3.83
(m, 1H, OCH2), 3.80, 3.794, 3.793 (each s, each 3H,
3· OCH3), 3.58 (m, 1H, OCH2), 3.44 (m, 1H,
CH2N3), 3.32 (m, 1H, CH2N3). 13C NMR (75 MHz,
CDCl3): non-sugar signals, d 170.4, 170.03, 170.02,
169.3 (4· COCH3), 159.4, 159.2, 159.1 (3· C), 129.8,
129.3, 129.1, 113.8, 113.7, 113.6 (12· CH), 72.9,
71.9, 71.7 (3· CH2Ph), 66.3 (OCH2), 55.2, 55.2 (3·
OCH3), 50.5 (CH2N3), 20.8, 20.7, 20.6, 20.5 (4·
COCH3).
3.1.1.28. 5-N,N-Dimethylaminonaphthalene-1-sulfonam-
idoethyl 5-O-(b-D-galactofuranosyl)-2,3,6-tri-O-p-meth-
oxybenzyl-b-D-galactofuranoside (40). Disaccharide 39
(300 mg, 0.26 mmol) in dry methanol (5 mL) was treated
with 7 N NH3/MeOH (10 mL) overnight at rt. Concen-
tration under vacuum followed by column chromatogra-
phy (CHCl3/MeOH, 9:1) yielded 40 (285 mg, 98%) as an
oil. FABMS (NBA): m/z 980 [M+H]+. Anal. Calcd for
C50H62N2O16SÆ1.0 H2O: C, 60.23; H, 6.47; N, 2.81.
Found C, 60.19; H, 6.43; N, 2.84. 1H NMR
(600 MHz, CDCl3): non-sugar signals d 8.51 (d, 1H, J
8.4 Hz, Ph), 8.25 (d, 1H, J = 9.0 Hz, Ph), 8.19 (dd, 1H,
J 1.2, 8.4 Hz, Ph), 7.48 (m, 1H, Ph), 7.46 (m, 1H, Ph),
7.18 (m, 4H, Ph), 7.13 (dd, 2H, J = 2.4, 10.8 Hz, Ph),
7.11 (d, 1H, J = 7.2 Hz, Ph), 6.85 (m, 6H, Ph), 4.35
3.1.1.26. Aminoethyl 5-O-(2,3,5,6-tetra-O-acetyl-b-D-
galactofuranosyl)-2,3,6-tri-O-p-methoxybenzyl-b-D-galac-
tofuranoside (38). Disaccharide 37 (3.00 g, 3.19 mmol)
was dissolved in dry MeOH (50 mL) and 5% Pd/C was
added (1.30 g) under argon atmosphere. H2CO2NH4
(807 mg, 12.8 mmol) was added and the reaction mixture
was stirred at rt for 4 h. TLC showed complete conver-
sion, and the mixture was filtered through a short Celite
pad and concentrated to a syrup. It was dissolved in
CHCl3 (100 mL), washed with deionized water
(20 mL), and dried over Na2SO4. Concentration affor-
ded the crude disaccharide and chromatography

5646
A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
(m, 6H, 3· CH2Ph), 3.79, 3.78, 3.76 (s, each 3H, 3·
OCH3), 3.56 (m, 2H, H-60b, OCH2), 3.36 (m, 1H,
OCH2), 3.03 (m, 2H, CH2NH), 2.85 (s, 6H, N(CH3)2).
13C NMR (75 MHz, CDCl3): non-sugar signals d
159.5, 159.3, 159.2 (3· C), 151.8 (C), 134.7 (C), 130.3,
129.8 (CH), 129.7 (C), 129.6 (CH), 129.5 (C), 129.3
(CH), 129.2, 128.9 (C), 128.3, 123.1, 118.8, 115.1,
113.9, 113.8, 113.7 (10· CH), 73.0, 71.7, 71.6 (3·
CH2Ph), 65.3 (OCH2), 55.3, 55.22, 55.20 (3· OCH3),
45.3 (N(CH3)2), 42.8 (CH2NH).
124.3, 121.0, 114.8 (CH), 66.0 (OCH2), 42.9 (CH2NH),
20.8, 20.7, 20.5 (OCH3).
3.1.1.31. Azidoethyl 6-O-(2,3,5,6-tetra-O-acetyl-b-D-
galactofuranosyl)-2,3,5-tri-O-p-methoxybenzyl-b-D-galac-
tofuranoside (43). Glycosylation was carried out by the
reaction of acceptor 36 (2.20 g, 3.61 mmol) and donor
26 (2.30 g, 5.41 mmol) in the presence of NIS (1.60 g,
7.22 mmol) and triflic acid (30 lL, 0.36 mmol) in dry
CH2Cl2 (60 mL) ꢀ20 ꢁC as described for 37. Purification
by column chromatography (cyclohexane/EtOAc, 2:1)
yielded disaccharide 43 (2.35 g, 69%) as an oil. FABMS
(LiCl): m/z 946 [M+Li]+. Anal. Calcd for C46H57N3O18:
C, 58.78; H, 6.11; N, 4.47. Found C, 58.76; H, 6.08; N,
4.43. 1H NMR (600 MHz, CDCl3): non-sugar signals, d
7.27 (dd, 2H, J = 0.3, 9.0 Hz, Ph), 7.22 (dd, 2H, J
0.3 Hz, 9.0 Hz, Ph), 7.13 (dd, 2H, J = 0.3, 9.0 Hz, Ph),
6.89 (dd, 2H, J = 0.3, 8.4 Hz, Ph), 6.82 (dd, 2H,
J = 0.3, 9.0 Hz, Ph), 6.81 (dd, 2H, J = 0.3, 9.0 Hz, Ph),
4.60, 4.53, 4.44, 4.42, 4.38, 4.21 (each d, each 1H,
J = 11.4 Hz, 3· CH2Ph), 3.85 (m, 1H, OCH2), 3.81,
3.764, 3.757 (s, each 3H, 3· OCH3), 3.58 (m, 1H,
OCH2), 3.44 (m, 1H, CH2N3), 3.34 (m, 1H, CH2N3),
2.12, 2.10, 2.05, 2.03 (each s, each 3H, 4· COCH3).
13C NMR (75 MHz, CDCl3): non-sugar signals, d
170.5, 169.9, 169.5 (4· COCH3), 159.3, 159.22, 159.19
(3· C), 130.2 (C), 129.8 (CH), 129.7 (C), 129.6 (CH),
129.5 (C), 113.7, 113.67, 113.61 (16· CH), 73.0, 71.7,
71.6 (3· CH2Ph), 66.5 (OCH2), 55.2, 55.20, 55.17 (3·
OCH3), 50.5 (CH2N3), 20.8, 20.7, 20.6, 20.6 (4·
COCH3).
3.1.1.29. 5-Azidonaphthalene-1-sulfonamidoethyl 5-O-
(2,3,5,6-tetra-O-acetyl-b-D-galactofuranosyl)-2,3,6-tri-O-
p-methoxybenzyl-b-D-galactofuranoside (41). Compound
38 (1.70 g, 1.86 mmol) was mixed with N-methylimidaz-
ole (0.3 mL, 3.72 mmol) and 1-azido-5-naphtha-
lenesulfonyl chloride (747 mg, 2.79 mmol) in dry
CH2Cl2 (50 mL) at 0 ꢁC as described for the preparation
of 18. Purification by column chromatography (cyclo-
hexane/EtOAc, 1:1) afforded the 41 (1.22 g, 68%) as an
oil. ESI-MS: m/z 1167.37 [M+Na]+. Anal. Calcd for
C56H64N4O20SÆ0.5 H2O: C, 58.28; H, 5.68; N, 4.85.
Found C, 58.27; H, 5.59; N, 4.80. 1H NMR
(300 MHz, CDCl3): non-sugar signals, d 8.38 (m, 2H,
Ph), 8.27 (dd, 1H, J = 1.2, 8.4 Hz, Ph), 7.55 (dd, 1H,
J = 3.7, 7.6 Hz, Ph), 7.52 (dd, 1H, J = 3.4, 7.4 Hz, Ph),
7.26 (dd, 1H, J = 0.7, 15.5 Hz, Ph), 7.19 (m, 6H, Ph),
6.85 (m, 6H, Ph), 5.39 (t, 1H, J = 5.2 Hz, NH), 4.38
(m, 3· CH2Ph), 3.79 (m, 3· OCH3), 3.59 (m, 1H,
OCH2), 3.40 (m, 1H, OCH2), 3.07 (m, 2H, CH2NH),
2.08, 2.06, 1.98, 1.94 (s, each 3H, 4· COCH3). 13C
NMR (75 MHz, CDCl3): non-sugar signals d 170.6,
170.0, 169.4 (4· COCH3), 159.5, 159.4, 159.1 (C),
137.6 (C) 134.9 (C), 130.3 (CH), 130.1 129.6 (C),
129.6, 129.4 (CH), 129.2 (C), 129.2, 128.5, 128.2 (CH),
127.2 (C), 124.3, 121.0, 114.8, 113.9, 113.7 (CH), 73.0,
71.8, 71.7 (3· CH2Ph), 65.7 (OCH2), 55.3, 55.2 (3·
OCH3), 43.2 (CH2NH), 20.8, 20.7, 20.6, 20.5 (4·
COCH3).
3.1.1.32. Aminoethyl 6-O-(2,3,5,6-tetra-O-acetyl-b-D-
galactofuranosyl)-2,3,5-tri-O-p-methoxybenzyl-b-D-galac-
tofuranoside (44). Disaccharide 43 (2.35 g, 2.50 mmol)
was dissolved in dry MeOH (50 mL) and 5% Pd/C was
added (2.0 g) under argon atmosphere. H2CO2NH4
(630 mg, 10.0 mmol) was added and the reaction mix-
ture was stirred at rt for 4 h. TLC showed complete con-
version, and the mixture was filtered through a short
Celite pad and concentrated to syrup. It was dissolved
in CHCl3 (100 mL), washed with deionized water
(20 mL), and dried over Na2SO4. Concentration affor-
ded the crude disaccharide. Column chromatography
(CHCl3/MeOH, 9:1) gave 44 (2.20 g, 98%) as an oil.
FABMS (NBA): m/z 914 [M+H]+. Anal. Calcd for
C46H59NO18: C, 60.45; H, 6.51; N, 1.53. Found C,
3.1.1.30. 5-Azidonaphthalene-1-sulfonamidoethyl 5-O-
(2,3,5,6-tetra-O-acetyl-b-D-galactofuranosyl)-b-D-galacto-
furanoside (42). Disaccharide 41 (1.17 g, 1.02 mmol) was
dissolved in 110 mL of CH2Cl2/H2O (10:1) and DDQ
(1.16 g, 5.1 mmol) was added at rt. The reaction mixture
was stirred for 4 h, dried over Na2SO4, and concentrated
to syrup. Purification over silica gel using CHCl3/MeOH
(95:5) afforded pure disaccharide 42 (734 mg, 92%). ESI-
MS: m/z 785.20 [M+H]+. Anal. Calcd for C32H40N4O17-
SÆ1.5 H2O: C, 47.35; H, 5.34; N, 6.90. Found C, 47.29;
1
60.48; H, 6.47; N, 1.54. H NMR (600 MHz, CDCl3):
non-sugar signals d 7.26, 7.23, 7.13 (m, each 2H, Ph),
6.88 (m, 2H, Ph), 6.82 (m, 4H, Ph), 4.61 (d, 1H,
J = 11.5 Hz, CH2Ph), 4.51 (d, 1H, J = 11.4 Hz, CH2Ph),
4.43, 4.42 (each d, 1H, J = 11.5 Hz, CH2Ph), 4.38 (d,
1H, J = 11.4 Hz, CH2Ph), 4.22 (d, 1H, J = 11.5 Hz,
CH2Ph), 3.81, 3.77, 3.76 (s, each 3H, 3· OCH3), 3.81-
3.70 (m, 3H, OCH2), 2.87 (t, 2H, J = 5.3 Hz, CH2NH2),
2.12, 2.10, 2.05, 2.03 (s, each 3H, 4· COCH3).
1
H, 5.33; N, 6.87. H NMR (300 MHz, CDCl3): non-su-
gar signals d 8.41 (ddd, 1H, J = 0.7, 0.8, 8.8 Hz, Ph),
8.39 (ddd, 1H, J = 0.8, 1.2, 8.6 Hz, Ph), 8.27 (dd, 1H,
J = 1.2, 7.4 Hz, Ph), 7.64 (dd, 1H, J = 7.6, 8.8 Hz, Ph),
7.54 (dd, 1H, J = 7.4, 8.6 Hz, Ph), 7.35 (dd, 1H,
J = 0.7, 7.6 Hz, Ph), 5.91 (t, 1H, J = 5.9 Hz, NH), 3.69
(m, 1H, OCH2), 3.44 (m, 1H, OCH2), 3.28 (d, 1H,
J = 6.5 Hz, 3-OH), 3.18 (d, 1H, J = 9.3 Hz, 2-OH), 3.09
(dd, 2H, J = 5.1, 10.4 Hz, CH2NH), 2.87 (br s, 1H, 6-
OH), 2.14, 2.13 (each s, each 3H, OCH3), 2.08 (s, 6H,
2· OCH3). 13C NMR (75 MHz, CDCl3): non-sugar sig-
nals, d 171.2, 170.4, 170.2 (4· COCH3), 137.6, 134.7 (C),
130.2 (CH), 129.1 (C), 128.5, 128.1 (CH), 127.1 (C),
3.1.1.33. 5-N,N-Dimethylaminonaphthalene-1-sulfonam-
idoethyl 6-O-(2,3,5,6-tetra-O-acetyl-b-D-galactofurano-
syl)-2,3,5-tri-O-p-methoxybenzyl-b-D-galactofuranoside
(45). Coupling of 44 (240 mg, 0.26 mmol) with dansyl
chloride (106 mg, 0.40 mmol) was carried out in the
presence of N-methylimidazole (43 mg, 0.52 mmol) in

A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
5647
dry CH2Cl2 (30 mL) at 0 ꢁC as described for 39. Purifi-
cation by column chromatography (cyclohexane/
EtOAc, 1:1) afforded 45 (180 mg, 60%) as an oil. FAB-
MS (NBA): m/z 1148 [M+H]+. Anal. Calcd for
C58H70N2O20S: C, 60.72; H, 6.15; N, 2.44. Found C,
oil. ESI-MS: m/z 807.40 [M+Na]+. Anal. Calcd for
C56H64N4O20S: C, 58.73; H, 5.63; N, 4.89. Found C,
1
58.76; H, 5.69; N, 4.87. H NMR (300 MHz, CDCl3):
non-sugar signals, d 8.40 (d, 1H, J = 8.7 Hz, Ph), 8.37
(d, 1H, J = 8.3 Hz, Ph), 8.27 (dd, 1H, J = 1.0, 7.2 Hz,
Ph), 7.53 (m, 2H, Ph), 7.27 (m, 1H, Ph), 7.17 (m, 6H,
Ph), 6.84 (m, 6H, Ph), 5.48 (t, 1H, J = 5.8 Hz, NH),
4.60, 4.42, 4.37, 4.31, 4.26, 4.22 (each d, each 1H,
J = 11.4 Hz, CH2Ph), 3.82, 3.78, 3.76 (each s, each 3H,
3· OCH3), 3.56 (m, 1H, OCH2), 3.33 (m, 1H, OCH2),
3.08 (m, 2H, CH2NH), 2.12, 2.09, 2.02, 2.01 (each s,
each 3H, 4· COCH3). 13C NMR (75 MHz, CDCl3):
1
60.69; H, 6.13; N, 2.39. H NMR (600 MHz, CDCl3):
non-sugar signals, d 8.52 (d, 1H, J = 8.4 Hz, Ph), 8.27
(d, 1H, J = 8.4 Hz, Ph), 8.22 (dd, 1H, J = 1.2, 7.2 Hz,
Ph), 7.31 (m, 2H, Ph), 7.21 (m, 5H, Ph), 7.14 (d, 1H,
J = 8.4 Hz, Ph), 7.13 (d, 1H, J = 7.2 Hz, Ph), 6.88 (m,
3H, Ph), 6.84 (m, 2H, Ph), 6.81 (m, 1H, Ph), 5.40 (t,
1H, J = 6.0 Hz, NH), 4.60, 4.43, 4.39, 4.38, 4.34, 4.26
(each d, each 1H, J = 11.4 Hz, CH2Ph), 3.81 (s, 3H,
OCH3), 3.77, 3.75 (each s, each 3H, 2· OCH3), 3.61
(m, 1H, OCH2), 3.35 (m, 1H, OCH2), 3.04 (m, 2H,
CH2NH), 2.86 (s, 6H, N(CH3)2), 2.11, 2.09, 2.01, 2.01
(each s, each 3H, 4· COCH3). 13C NMR (75 MHz,
CDCl3): non-sugar signals, d 170.5, 170.0, 169.9, 169.5
(4· COCH3), 159.3, 159.2, 159.1 (C), 148.1, 134.8 (C),
130.3 (CH), 130.2 (C), 129.8 (CH), 129.7 (C), 129.6,
129.5, 128.2, 123.2, 118.9, 115.2, 113.8, 113.7, 113.6
(CH), 72.9, 71.6, 71.5 (3· CH2Ph), 65.7 (OCH2), 55.2,
55.2, 55.1 (3· OCH3), 45.4 (NCH3), 42.9 (CH2NH),
20.8, 20.7, 20.6, 20.5 (4· COCH3).
non-sugar signals,
d 170.6, 170.1, 170.00, 169.5
(COCH3), 159.3, 159.3, 159.2 (C), 137.5, 134.8 (C),
130.3 (CH), 130.2 (C), 129.8 (CH), 129.6 (C), 129.5,
129.4 (CH), 129.3, 129.1 (C), 128.4, 128.1 (CH), 127.1
(C), 124.3, 121.0, 114.8, 113.8, 113.7, 113.6 (CH), 72.9,
71.7, 71.5 (3· CH2Ph), 65.6 (OCH2), 55.2, 55.2, 55.1
(3· OCH3), 43.0 (CH2NH), 20.8, 20.7, 20.6, 20.5 (4·
COCH3).
3.1.1.36. 5-Azidonaphthalene-1-sulfonamidoethyl 6-O-
(2,3,5,6-tetra-O-acetyl-b-D-galactofuranosyl)-b-D-galacto-
furanoside (48). Disaccharide 47 (1.30 g, 1.14 mmol) was
dissolved in 110 mL of CH2Cl2/H2O (10:1) and DDQ
(1.30 g, 5.7 mmol) was added at room temperature.
The reaction mixture was stirred for 4 h, dried over
Na2SO4, and concentrated to syrup. Column chromatog-
raphy (CHCl3/MeOH, 95:5) afforded 48 (720 mg, 81%)
as oil. FABMS (NBA): m/z 639.14 [M+Na]+. Anal.
Calcd for C32H40N4O17SÆ1.0 H2O: C, 48.42; H, 5.21; N,
7.06. Found: C, 48.46; H, 5.27; N, 7.01. 1H NMR
(600 MHz, CDCl3): non-sugar signals d 8.41 (d, 1H,
J = 8.6 Hz, Ph), 8.40 (d, 1H, J = 8.3 Hz, Ph), 8.28 (dd,
1H, J = 1.0, 7.3 Hz, Ph), 7.65 (dd, 1H, J = 7.6, 8.6 Hz,
Ph), 7.55 (dd, 1H, J = 7.3, 8.3 Hz, Ph), 7.37 (d, 1H,
J = 7.6 Hz, Ph), 5.87 (t, 1H, J = 6.0 Hz, NH), 4.23 (br
s, 1H, OH), 3.68 (m, 1H, OCH2), 3.61 (br s, 1H, OH),
3.41 (m, 1H, OCH2), 3.21 (d, 1H, J = 8.3 Hz, OH),
3.09 (dd, 2H, J = 5.3, 10.4 Hz, CH2NH), 2.14, 2.12,
2.07, 2.06 (each s, each 3H, 4· COCH3). 13C NMR
(75 MHz, CDCl3): non-sugar signals, d 170.8, 170.4,
170.1, 17.1 (COCH3), 137.6, 134.7 (C), 130.2 (CH),
129.1 (C), 128.5, 128.2 (CH), 127.1 (C), 124.3, 122.0,
114.9 (CH), 65.8 (OCH2), 42.8 (CH2NH), 20.8, 20.77,
20.72, 20.6 (4· COCH3).
3.1.1.34. 5-N,N-Dimethylaminonaphthalene-1-sulfo-
namidoethyl 6-O-(2,3,5,6-tetra-O-acetyl-b-D-galactofur-
anosyl)-b-D-galactofuranoside (46). A solution of 45
(150 mg, 0.13 mmol) and thiophenol (0.05 mL,
0.46 mmol) in dry CH2Cl2 (10 mL) was cooled to
ꢀ78 ꢁC under argon. SnCl4 (0.05 mL, 0.40 mmol) was
added and the mixture was stirred for 1 h at ꢀ78 ꢁC
and further overnight at ꢀ20 ꢁC. A saturated NaHCO3
solution (5 mL) was added and the reaction mixture was
further diluted with CHCl3 (30 mL). The organic layer
was washed with water (2· 10 mL), dried over Na2SO4,
and concentrated to an oil. Chromatography
(CHCl3/MeOH, 95:5) yielded 46 (85 mg, 83%) as an
oil. FABMS (NBA): m/z 787 [M+H]+. Anal. Calcd for
C34H46N2O17SÆ1.0 H2O: C, 50.74; H, 6.01; N, 3.48.
Found: C, 50.77; H, 6.00; N, 3.44. 1H NMR
(300 MHz, CDCl3, D2O exchanged): non-sugar signals,
d 8.54 (d, 1H, J = 8.6 Hz, Ph), 8.26 (d, 1H, J = 8.6 Hz,
Ph), 8.54 (dd, 1H, J = 1.2, 7.4 Hz, Ph), 7.54 (m, 2H,
Ph), 7.19 (d, 1H, J = 7.4 Hz, Ph), 5.65 (br s, 1H, NH),
3.68 (m, 1H, OCH2), 3.40 (m, 1H, OCH2), 3.08 (dd,
2H, J = 4.6, 5.4 Hz, CH2NH), 2.89 (s, 6H, N(CH3)2),
2.14, 2.12, 2.07, 2.06 (each s, each 3H, 4· COCH3).
13C NMR (75 MHz, CDCl3): non-sugar signals, d
170.7, 170.3, 170.1, 170.0 (4· COCH3), 151.9, 134.7
(C), 130.5 (CH), 129.9, 129.5 (C), 129.4, 128.4, 123.2,
118.8, 115.2 (CH), 65.8 (OCH2), 43.00 (CH2NH), 31.8,
29.4 (N(CH3)2), 20.8, 20.76, 20.72, 20.6 (4· COCH3).
3.1.1.37. 5-N,N-Dimethylaminonaphthalene-1-sulfo-
namidoethyl 5-O-(a-D-arabinofuranosyl)-a-D-arabinofu-
ranoside (1). Compound 20 (540 mg, 0.69 mmol) was
dissolved in dry THF (20 mL) and tetraethylammonium
fluoride (3.9 mg, 2.07 mmol) was added. The reaction
mixture was stirred overnight at rt and concentrated to
oil. Column chromatography (CHCl3/MeOH, 5:1) gave
compound 1 as an oil that was dissolved in deionized
water (5 mL), frozen, and lyophilized to yield a light yel-
low, hygroscopic solid (372 mg, 97%). Spectral and ana-
lytical data have been reported.13
3.1.1.35. 5-Azidonaphthalene-1-sulfonamidoethyl 6-O-
(2,3,5,6-tetra-O-acetyl-b-D-galactofuranosyl)-2,3,5-tri-O-
p-methoxybenzyl-b-D-galactofuranoside (47). Compound
44 (2.2 g, 2.41 mmol) was reacted with N-methylimidaz-
ole (0.38 mL, 4.82 mmol) and 1-azido-5-naphtha-
lenesulfonyl chloride (967 mg, 0.40 mmol) in dry
CH2Cl2 (30 mL) at 0 ꢁC for 4 h as described for 41. Puri-
fication by column chromatography (cyclohexane/
EtOAc, 1:1) gave disaccharide 47 (1.64 g, 68%) as an
3.1.1.38. 5-N,N-Dimethylaminonaphthalene-1-sulfo-
namidoethyl 5-O-(b-D-galactofuranosyl)-b-D-galactofur-
anoside (2). Compound 40 (125 mg, 0.13 mmol) was
reacted with thiophenol (0.05 mL, 0.46 mmol) and

5648
A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
SnCl4 (0.05 mL, 0.40 mmol) in dry CH2Cl2 (10 mL) as
described for 46. Column chromatography (CHCl3/
MeOH 3:1) gave 2 as a light sensitive oil. An aqueous
solution (5 mL) of the oil was passed through a small
column packed with Bio-BeadsTM SM-4 (20–50 mesh),
and the solution was frozen and lyophilized to afford 2
as a hygroscopic solid (27 mg, 33%). FABMS (NBA):
m/z 619 [M+H]+. Anal. Calcd for C26H38N2O13SÆ1.0
H2O: C, 49.05; H, 6.33; N, 4.40. Found C, 49.00; H,
6.36; N, 4.38. 1H NMR (600 MHz, CD3OD): non-sugar
signals, d 8.56 (dt, 1H, J = 1.2, 8.4 Hz, Ph), 8.34 (dt, 1H,
J = 0.6, 9.0 Hz, Ph), 8.20 (dd, 1H, J = 1.2, 7.2 Hz, Ph),
7.59 (dd, 1H, J = 8.4, 9.0 Hz, Ph), 7.58 (dd, 1H,
J = 8.4, 9.0 Hz, Ph), 7.28 (dd, 1H, J = 0.6, 7.2 Hz, Ph),
3.55 (m, 1H, OCH2), 3.31 (m, 1H, OCH2), 3.05 (dd,
2H, J = 5.4, 7.2 Hz, CH2NH), 2.88 (s, 6H, N(CH3)2).
13C NMR (75 MHz, CD3OD): non-sugar signals, d
153.2, 137.1 (2· C), 131.2 (CH), 131.2, 130.9 (2· C),
130.0, 129.1, 124.3, 120.6, 116.5 (5· CH), 67.6
(OCH2), 45.8 (N(CH3)2), 43.9 (CH2NH).
The aqueous solution that remained after brief evapora-
tion was frozen and lyophilized to yield a light sensitive
solid (375 mg, 78%). ESI-MS: m/z 639.17 [M+Na]+.
Anal. Calcd for C24H32N4O13SÆ1.0 H2O: C, 45.42; H,
5.40; N, 8.53. Found C, 45.49; H, 5.39; N, 8.61. 1H
NMR (600 MHz, CD3OD): non-sugar signals d 8.49
(dt, 1H, J = 0.8, 0.9, 8.7 Hz, Ph), 8.40 (dt, 1H, J = 0.9,
1.2, 8.5 Hz, Ph), 8.26 (dd, 1H, J = 1.2, 7.3 Hz, Ph),
7.71 (dd, 1H, J = 7.6, 8.7 Hz, Ph), 7.60 (dd, 1H,
J = 7.4, 8.5 Hz, Ph), 7.48 (dd, 1H, J = 0.8, 7.6 Hz, Ph),
3.51 (m, 1H, OCH2), 3.28 (m, 1H, OCH2), 3.07 (t, 2H,
J = 5.6 Hz, CH2NH). 13C NMR (75 MHz, CD3OD):
138.8, 137.1 (C), 130.9 (CH), 130.4 (C), 129.1 (CH),
128.4 (C), 125.5, 122.6, 116.1 (CH), 67.5 (OCH2), 43.9
(CH2NH).
3.1.1.42. 5-Azidonaphthalene-1-sulfonamidoethyl 6-O-
(b-D-galactofuranosyl)-b-D-galactofuranoside (6). Disac-
charide 48 (700 mg, 0.89 mmol) was deacetylated in
7 N NH3/MeOH (20 mL) as described for the prepara-
tion of 40. An aqueous solution (5 mL) of crude
disaccharide was passed through a small column of
Bio-BeadsTM SM-4 (25 g, 20–50 mesh) and eluted with
MeOH/H2O (0–100%). The aqueous solution that
remained after brief evaporation was frozen and lyoph-
ilized to a solid (439 mg, 80%). ESI-MS: m/z 619
[M+H]+. Anal. Calcd for C24H32N4O13SÆ1.0 H2O: C,
45.42; H, 5.40; N, 8.53. Found C, 45.49; H, 5.41; N,
3.1.1.39. 5-N,N-Dimethylaminonaphthalene-1-sulfo-
namidoethyl 6-O-(b-D-galactofuranosyl)-b-D-galactofur-
anoside (3). Disaccharide 46 (80 mg, 0.10 mmol) was
deacetylated by 7 N NH3/MeOH (5 mL) as described
for 40. Column chromatography (CHCl3/MeOH, 3:1)
followed by a short column of Bio-BeadsTM SM-4
(20–50 mesh), freezing, and lyophilization afforded 3
as yellow fluffy solid (44 mg, 70%). FABMS (NBA):
m/z 619 [M+H]+. Anal. Calcd for C26H38N2O13SÆ1.0
H2O: C, 49.05; H, 6.33; N, 4.40. Found C, 48.99; H,
6.32; N, 4.37. 1H NMR (600 MHz, CD3OD): non-sugar
signals, d 8.56 (1H, dt, J = 1.2, 8.4 Hz, Ph), 8.34 (1H, dt,
J = 1.2, 8.4 Hz, Ph), 8.20 (1H, dd, J = 1.2, 7.2 Hz, Ph),
7.59 (1H, dd, J = 7.2, 8.4 Hz, Ph), 7.58 (1H, dd,
J = 7.2, 8.4 Hz, Ph), 7.28 (1H, dd, J = 1.2, 8.4 Hz, Ph),
3.59 (1H, m, OCH2), 3.31 (1H, m, OCH2), 3.06 (2H, t,
J = 5.4 Hz, CH2NH) 2.88 (6H, s, N(CH3)2). 13C NMR
(75 MHz, CD3OD): non-sugar signals, d 153.2, 137.1
(2· C), 131.2 (CH), 131.2, 130.9 (2· C), 130.0, 129.1,
124.3, 120.6, 116.5 (5· CH), 67.5 (OCH2), 45.8
(N(CH3)2), 43.9 (CH2NH).
1
8.57. H NMR (600 MHz, CD3OD): non-sugar signals,
d 8.50 (d, 1H, J = 8.5 Hz, Ph), 8.41 (d, 1H, J = 8.5 Hz,
Ph), 8.27 (dd, 1H, J = 1.2, 7.4 Hz, Ph), 7.72 (dd, 1H,
J = 7.6, 8.7 Hz, Ph), 7.62 (dd, 1H, J = 7.4, 8.5 Hz, Ph),
7.50 (d, 1H, J = 7.6 Hz, Ph), 3.56 (m, 1H, OCH2), 3.29
(m, 1H, OCH2), 3.08 (t, 2H, J = 5.5 Hz, CH2NH). 13C
NMR (75 MHz, CD3OD): non-sugar signals, d 138.8,
137.2 (C), 130.9 (CH), 130.5 (C), 129.1 (CH), 128.4
(C), 125.5, 122.6, 116.1 (CH), 67.4 (OCH2), 43.9
(CH2NH).
3.2. Biological activity
3.2.1. Arabinosyltransferase assay. Compounds 1 at a
range of concentrations from 0.25 to 2.0 mM and 2 from
0.5 to 8.0 mM stored as 100 mM ethanol stocks and
DP[14C]A (20,000 cpm, 9 mM, 10 lL [stored in chloro-
form/methanol, 2:1]) were dried under a stream of argon
in a microcentrifuge tube (1.5 mL) and placed in a vac-
uum desiccator for 15 min to remove any residual sol-
vent. The dried constituents of the assay were then re-
suspended in 8 ll of a 1% aqueous solution of Igepal.
The remaining constituents of the arabinosyltransferase
assay containing 50 mM MOPS (adjusted to pH 8.0
with KOH), 5 mM b-mercaptoethanol, 10 mM MgCl2,
1 mM ATP, and M. smegmatis membranes (250 lg)
were added to a final reaction volume of 80 lL. The
reaction mixtures were then incubated at 37 ꢁC for 1 h.
A CHCl3/CH3OH (1:1, 533 lL) solution was then added
to the incubation tubes and the entire contents centri-
fuged 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-equil-
ibrated (C2H5OH/H2O [1:1]) 1 mL Whatman strong an-
ion exchange (SAX) cartridge which was washed with
3.1.1.40. 5-Azidonaphthalene-1-sulfonamidoethyl 5-O-
(a-D-arabinofuranosyl)-a-D-arabinofuranoside (4). Com-
pound 25 (300 mg, 0.38 mmol) was dissolved in dry
THF (20 mL) and Et4N+Fꢀ (171 mg, 1.15 mmol) was
added. The reaction mixture was stirred overnight and
concentrated to oil. Column chromatography (CHCl3/
MeOH, 5:1) gave compound 4 as an oil that was dis-
solved in deionized water (5 mL), frozen, and lyophi-
lized to get a light sensitive, hygroscopic solid (128 mg,
60%). Spectral and analytical data have been reported.14
3.1.1.41. 5-Azidonaphthalene-1-sulfonamidoethyl 5-O-
(b-D-galactofuranosyl)-b-D-galactofuranoside (5). A solu-
tion of compound 42 (610 mg, 0.78 mmol) in dry MeOH
(15 mL) was cooled to 0 ꢁC under argon and treated
with 7 N NH3/MeOH. The reaction mixture was stirred
overnight at room temperature and concentrated to dry-
ness. An aqueous solution (5 mL) was passed through a
small column of Bio-BeadsTM SM-4 (25 g, 20–50 mesh)
and product 5 was eluted with MeOH/H2O (0/100%).

A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
5649
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 H20 (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 lL of n-butanol. The total cpm of radiolabeled mate-
rial extractable into the n-butanol phase was measured by
scintillation counting using 10% of the labeled material
and 10 mL of EcoScintA (National Diagnostics, Atlan-
ta). The incorporation of [14C]Araf was determined by
subtracting counts present in control assays (incubation
of the reaction components in the absence of the com-
pounds). Another 10% of the labeled material was sub-
jected to thin-layer chromatography (TLC) in CHCl3/
CH3OH/NH4OH/H2O (65:25:0.5:3.6) on aluminum
backed Silica Gel 60 F254 plates (E. Merck, Darmstadt,
Germany). Autoradiograms were obtained by exposing
TLCs to X-ray film (Kodak X-Omat) for 3 days.
the form of a Personal Chair, the Lister Institute as a for-
mer Jenner Research Fellow, the Medical Research
Council (UK), and the Wellcome Trust.
References and notes
1. (a) Rattan, A.; Kalia, A.; Ahmad, N. Emerging Infect. Dis.
1998, 4, 195–209; (b) Butler, D. Nature 2000, 406, 670–
672; (c) Raviglione, M. C.; Snider, D. E.; Kochi, A. JAMA
1995, 273, 220–226; (d) NIAID, Web Site, http://
www.niaid.nih.gov/factsheets/tb.htm; http://www.niaid.nih.
gov/factsheets/tbrsch.htm and http://www.who.int/gtb/
publications/factsheet/index.htm.
2. (a) Van Scoy, R. E.; Wilkowske, C. J. Mayo Clin. Proc.
1999, 74, 1038–1048; (b) Ravilione, M. C. Scot. Med. J.
2000, 45, 52–55; (c) Long, R. CMAJ 2000, 163, 425–428.
3. (a) Walsh, C. Nature 2000, 406, 775–781; (b) Smith, M. B.;
Boyaromatics, M. C.; Veasey, S.; Woods, G. L. Aromat-
icch. Pathol. Lab. Med. 2000, 124, 1267–1274; (c) Pozniak,
A. Int. J. Tuberc. Lung Dis. 2000, 4, 993–994.
4. De Jong, B. C.; Israelski, D. M.; Corbett, E. L.; Small, P.
M. Annu. Rev. Med. 2004, 55, 283–301.
5. Sibille, Y.; Reynolds, H. Am. Rev. Respir. Dis. 1990, 141,
47–50.
3.2.2. Galactosyltransferase assay. All four disaccharides
at a range of concentrations from 0.5 to 6.0 mM were
incubated with 0.5 lCi of UDP-[14C]Galp (Amersham,
257 mCi/mmol) in a buffer containing 50 mM MOPS,
pH 7.9, 5 mM 2-mercaptoethanol, 10 mM MgCl2,
62.5 lM ATP, and 50 lL of a membrane preparation
from M. smegmatis (0.5 mg of protein) in a total volume
of 160 lL for 1 h at 37 ꢁC. A chloroform/methanol (1:1,
1.07 mL) solution was then added to the incubation
tubes and the entire contents centrifuged at
14,000 rpm. The supernatant was recovered, dried under
a stream of argon, re-suspended in ethanol:water (1:1,
1 mL), and loaded onto a 1 mL Whatman strong anion
exchange (SAX) cartridge. The cartridge was then
washed with ethanol (3 mL). The eluants were com-
bined, dried, and the resulting products partitioned be-
tween the two phases arising from n-butanol (3 mL)
and water (3 mL). The organic phase was recovered
and back washed with water saturated with n-butanol
twice (3 mL each). The n-butanol fraction was
dried, re-suspended in 200 lL of n-butanol-saturated
with water. Fifty microliters of this solution was sub-
jected to scintillation counting, and another 50 lL was
applied to an analytical thin-layer chromatogram devel-
oped in chloroform/methanol/ammonium hydroxide/water
(65:25:0.5:3.6) and subjected to autoradiography. The
incorporation of [14C]Gal was determined by subtract-
ing counts present in control assays (incubation of the
reaction components in the absence of the acceptors).
6. (a) Mauch, H. Zentralblzhyg Umweltmed. 1993, 194, 152–
161; (b) Bass, J. B.; Farer, I. S.; Hopewell, P. C.; Obrien,
R.; Jacobs, R. F.; Ruben, F.; Snider, D. E.; Thornton, G.
Am. J. Respir. Crit. Care Med. 1994, 149, 1359–1371.
7. (a) Blanchard, J. S. Annu. Rev. Biochem. 1996, 65, 215–
239; (b) Lowary, T. L. Mycobacterial Cell Wall Compo-
nents. In Glycoscience: Chemistry and Chemical Biology;
Fraser-Reid, B., Tatsuta, K., Thiem, J., Eds.; Springer:
Berlin, 2001; pp 2005–2080.
8. (a) Abou-Zeid, C.; Voiland, A.; Michel, G.; Cocito, C.
Eur. J. Biochem. 1982, 128, 363–370; (b) Daffe, M.;
Brennan, P. J.; McNeil, M. J. Biol. Chem. 1990, 265,
6734–6743; (c) Brennen, P. J.; Nikaido, H. Annu. Rev.
Biochem. 1995, 64, 29–63; (d) Besra, G. S.; Khoo, K.-H.;
McNeil, M. R.; Dell, A.; Morris, H. R.; Brennan, P.
Biochemistry 1995, 34, 4257–4266; (e) Lee, R. E.; Brennan,
P. J.; Besra, G. S. Curr. Top. Microbiol. Immunol. 1996,
215, 1–27; (f) Mikusova, K.; Yagi, T.; Stern, R.; McNeil,
M. R.; Besra, G. S.; Crick, D. C.; Brennan, P. J. J. Biol.
Chem. 2000, 275, 33890.
´ ˇ
´
´
´
9. Mikusova, K.; Belanova, M.; Kordulakova, J.; Honda,
K.; McNeil, M. R.; Mahapatra, S.; Crick, D. C.; Brennan,
P. J. J. Bacteriology 2006, 188, 6592–6598.
10. (a) Pathak, A. K.; Besra, G. S.; Crick, D.; Maddry, J. A.;
Morehouse, C. B.; Suling, W. J.; Reynolds, R. C. Bioorg.
Med. Chem. 1999, 7, 2407–2413; (b) Reynolds, R. C.;
Bansal, N.; Rose, J.; Friedrich, J.; Suiling, W. J.; Maddry,
J. A. Carbohydr. Res. 1999, 317, 164–179; (c) Pathak, A.
K.; Pathak, V.; Maddry, J. A.; Suling, W. J.; Gurcha, S.
S.; Besra, G. S.; Reynolds, R. C. Bioorg. Med. Chem. 2001,
9, 3145–3151; (d) Pathak, A. K.; Pathak, V.; Seitz, L.;
Maddry, J. A.; Gurcha, S. S.; Besra, G. S.; Suling, W. J.;
Reynolds, R. C. Bioorg. Med. Chem. 2001, 9, 3129–3143;
(e) Pathak, A. K.; Pathak, V.; Suling, W. J.; Gurcha, S. S.;
Morehouse, C. B.; Besra, G. S.; Maddry, J. A.; Reynolds,
R. C. Bioorg. Med. Chem. 2002, 10, 923–928; (f) Pathak,
A. K.; Pathak, V.; Kulshrestha, M.; Kinnaird, D.; Suling,
W. J.; Gurcha, S. S.; Besra, G. S.; Reynolds, R. C.
Tetrahedron 2003, 59, 10239–10248; (g) Kremer, L.;
Dover, L. G.; Gurcha, S. S.; Pathak, A. K.; Reynolds,
R. C.; Besra, G. S. Carbohydr. Bioeng. 2002, 178–185.
11. (a) Bayley, H.; Knowles, J. R. Methods Enzymol. 1977, 46,
69–114; (b) Hazum, E. Methods Enzymol. 1983, 103, 58–
Acknowledgments
This work was supported by NIH/NIAID Grant
R01AI45317. The authors are thankful to Mr. M. D.
Richardson for FABMS, ESI-MS and Ms. Joan Bearden
for CHN analysis. The 600 MHz NMR work was
supported by NCI Grant CA-13148 (the University of
Alabama at Birmingham shared NMR Core Facility).
GSB acknowledges support from Mr. James Bardrick in

5650
A. K. Pathak et al. / Bioorg. Med. Chem. 15 (2007) 5629–5650
71; (c) Eberle, A. N.; deGraan, P. N. E. Methods Enzymol.
1985, 109, 129–156.
12. Cory, P. P.; Becker, R. R.; Rosenbluth, R.; Isenberg, I. J.
Am. Chem. Soc. 1968, 1643–1647.
18. Pathak, A. K.; El-Kattan, Y. A.; Bansal, N.; Maddry, J. A.;
Reynolds, R. C. Tetrahedron Lett. 1998, 39, 1497–1500.
19. Pathak, A. K.; Pathak, V.; Khare, N. K.; Maddry, J. A.;
Reynolds, R. C. Carbohydr. Lett. 2001, 4, 117–122.
20. D’Souza, F. W.; Cheshev, P. E.; Ayers, J. D.; Lowary, T.
L. J. Org. Chem. 1998, 63, 9037–9044.
21. Chittenden, G. J. F. Carbohydr. Res. 1972, 25, 35–41.
22. Greene, T. W.; Wuts, P. G. M. In Protective Groups in
Organic Synthesis, 3rd ed.; John Wiley, 1999; pp 86–91.
23. Mizutani, K.; Kasai, R.; Nakamura, M.; Tanaka, O.;
Matsuura, H. Carbohydr. Res. 1989, 185, 27–38.
24. Cyr, N.; Perlin, A. S. Can. J. Chem. 1979, 57, 2504–2511.
25. Lee, R. E.; Brennan, P. J.; Besra, G. S. Glycobiology 1997,
7, 1121–1128.
13. (a) Muramoto, K.; Kamiya, H. Agric. Biol. Chem. 1984,
48, 2695–2699; (b) Muramoto, K.; Kamiya, H. Agric. Biol.
Chem. 1988, 52, 547–554.
14. Pathak, A. K.; Pathak, V.; Bansal, N.; Maddry, J. A.;
Reynolds, R. C. Tetrahedron Lett. 2001, 42, 979–982.
15. Pathak, A. K.; Pathak, V.; Gurcha, S. S.; Besra, G. S.;
Reynolds, R. C. Bioorg.Med.Chem.Lett.2002,12, 2749–2752.
16. Pathak, A. K.; Pathak, V.; Riordan, J. M.; Gurcha, S. S.;
Besra, G. S.; Reynolds, R. C. Carbohydr. Res. 2004, 339,
683–691.
17. (a) Guthrie, R. D.; Smith, S. C. Chem. Ind. (London) 1968,
547–548; (b) Kam, B. L.; Barascut, J.-L.; Imbach, J.-L.
Carbohydr. Res. 1979, 69, 135–142.
26. Rose, N. L.; Completo, G. C.; Lin, S.-J.; McNeil, M.;
Palcic, M. M.; Lowary, T. L. J. Am. Chem. Soc. 2006, 128,
6721–6729.