Studies on (β,1→5) and (β,1→6) linked octyl Galf disaccharides as substrates for mycobacterial galactosyltransferase activity

Article abstract of DOI:10.1016/S0968-0896(01)00179-1

The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (MTB) and the continuing pandemic of tuberculosis emphasizes the urgent need for the development of new anti-tubercular agents with novel drug targets. The recent structural elucidation of the mycobacterial cell wall highlights a large variety of structurally unique components that may be a basis for new drug development. This publication describes the synthesis, characterization, and screening of several octyl Galf(β,1→5)Galf and octyl Galf(β,1→6)Galf derivatives. A cell-free assay system has been utilized for galactosyltransferase activity using UDP[¹⁴C]Galf as the glycosyl donor, and in vitro inhibitory activity has been determined in a colorimetric broth microdilution assay system against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC). Certain derivatives showed moderate activities against MTB and MAC. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing end showed better activity as compared to totally deprotected disaccharides that more closely resemble the natural substrates in cell wall biosynthesis.

Full text of DOI:10.1016/S0968-0896(01)00179-1

Bioorganic & Medicinal Chemistry 9 (2001) 3129–3143
Studies on (ꢀ,1!5) and (ꢀ,1!6) Linked Octyl Galf
Disaccharides as Substrates for Mycobacterial
Galactosyltransferase Activity
Ashish K. Pathak,a Vibha Pathak,a Lainne Seitz,a Joseph A. Maddry,a
Sudagar S. Gurcha,b Gurdyal S. Besra,b William J. Sulingc and Robert C. Reynoldsa,*
aDepartment of Organic Chemistry, Southern Research Institute, PO Box 55305, Birmingham, AL 35255-5305, USA
bDepartment of Microbiology & Immunology, University of Newcastle upon Tyne, Newcastle upon Tyne, NE24HH, UK
cDepartment of Biochemistry, Southern Research Institute, PO Box 55305, Birmingham, AL 35255-5305, USA
Received 2 April 2001; accepted 10 May 2001
Abstract—The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (MTB) and the continuing pan-
demic of tuberculosis emphasizes the urgent need for the development of new anti-tubercular agents with novel drug targets. The
recent structural elucidation of the mycobacterial cell wall highlights a large variety of structurally unique components that may be
a basis for new drug development. This publication describes the synthesis, characterization, and screening of several octyl
Galf (b,1!5)Galf and octyl Galf (b,1!6)Galf derivatives. A cell-free assay system has been utilized for galactosyltransferase
activity using UDP[14C]Galf as the glycosyl donor, and in vitro inhibitory activity has been determined in a colorimetric broth
microdilution assay system against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC). Certain
derivatives showed moderate activities against MTB and MAC. The biological evaluation of these disaccharides suggests that more
hydrophobic analogues with a blocked reducing end showed better activity as compared to totally deprotected disaccharides that
more closely resemble the natural substrates in cell wall biosynthesis. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
Traditionally, the cell wall of mycobacteria has been
one of the major targets for treatment of tuberculosis as
is evidenced by the presently accepted mechanism of
action of the clinically effective agents isoniazid (INH),
ethambutol (EMB), and ethionamide.5 The value of
drugs that target the cell wall is also reinforced by the
fact that the waxy nature of the mycobacterial cell wall
acts as a protective barrier against both host cellular
immune response and typical antibacterial agents.6 In
recent years, the structure of the cell wall has been
thoroughly studied in terms of its constituent complex
polysaccharides and the specific chemical linkages
therein.5,6b,7 The macromolecular structure of the
mycolylarabinogalactan complex has been described.8
Clues regarding the initiation of mycolylar-
abinogalactan biosynthesis have arisen from earlier
work that showed the arabinogalactan hetero-
polysaccharide chains are attached through phospho-
diester linkages to C-6 of a proportion of muramic acid
residues of mycobacterial cell walls.9 The cell wall core
of members of the Mycobacterium genus consists of
extensively cross linked peptidoglycan to which is
attached the linear d-galactan composed of alternating
In spite of the ready availability of highly active agents
for the treatment of tuberculosis, this disease remains
one of the primary killers worldwide.1 Furthermore, the
resurgence of tuberculosis in developed nations as well
as the appearance of multiple drug-resistant forms of
the disease throughout the world has raised the concern
that this disease may once again resurface as the white
plague. These factors have led to an increased aware-
ness of the disease, and public health officials are now
calling for increased funding for the development of
new drugs and effective vaccines for the treatment of
tuberculosis.2 In particular, there is a critical awareness
that new classes of drugs with mechanisms of action
that are unique from presently used anti-tubercular
agents will be needed to treat drug resistant forms of the
disease.3 In fact, there are few treatment options for
patients infected with the highly intractable forms of
MDR-TB.4
*Corresponding author. Tel.: +1-205-581-2454; fax: +1-205-581-2877;
e-mail: reynolds@sri.org (R.C. Reynolds).
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00179-1

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A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3129–3143
Figure 1. Sugar linkages in cell wall polysaccharide of Mycobacterium tuberculosis.
Figure 2. Structures of b(1!6) and b(1!5) linked galactofuranosyl disaccharides.
5- and 6-linked a-d-Galf units.8b,10a More recently,
chemical analysis of degradation fragments arising from
the reducing end of the arabinogalactan obtained from
Mycobacterium tuberculosis, Mycobacterium bovis BCG
and Mycobacterium leprae demonstrated the existence
of the terminal sequence !5)-d-Galf-(b1!6)-d-Galf-
(b1!5)-d-Galf-(b1!4)-l-Rhap-(a1!3)-d-GlcNAc (Fig.
1).10 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 pepti-
doglycan. Since the component sugars l-rhamanose, d-
arabinofuranose and d-galactofuranose are not found
in mammalian cells, the biosynthesis and utilization of
these particular structures offer a new set of drug targets
that will be potentially highly active and selective.
glycosyltransferases, we report the synthesis, character-
ization, and biological evaluation of several galactofur-
anose disaccharides having b1!5 and b1!6 glycosidic
linkages similar to those present in the natural galactan.
Results and Discussion
Synthesis
The b1!6 and b1!5 linked galactofuranosyl dis-
accharide derivatives (Fig. 2) were synthesized using
several glycosylation techniques with different donors
(Fig. 3) and acceptors. Crystalline d-(+)-galactofur-
anose pentaacetate (1) was prepared from d-(+)-galac-
tose by a sulfuric acid catalyzed reaction in methanol
and subsequent acetylation using acetic anhydride in
pyridine.13 The Lewis acid-mediated glycosylation14 of 1
using SnCl4 with n-octanol yielded octyl 2,3,5,6-tetra-O-
acetyl-b-d-galctofuranoside (2) in 72% yield that on
deacetylation with sodium methoxide in methanol gave
the octyl b-d-galactofuranoside (3) in 95% yield. From
compound 3 various acceptors were prepared. Initially,
Several workers have extensively studied the biosynth-
esis of the galactofuranose chain in the cell wall.5,11 The
in vitro studies have shown that uridine-50-dipho-
sphogalactofuranose (UDP-d-Galf) is the substrate for
the galactosyltransferases involved in mycobacterial
galactan biosynthesis.11b The galactosyl residues in the
pyranose form are produced through the conversion of
UDP-Glcp to UDP-Galp by the enzyme UDP-glucose
4-epimerase. UDP-Galf is obtained from UDP-Galp by
the unprecedented reaction catalyzed by the enzyme
UDP-Galp mutase.11fÀh The structure of the substrate
was confirmed by NMR techniques11g and X-ray crys-
tallography.11i Again, the absence of d-Galf residues in
humans suggests that inhibitors of the enzymes involved
either in the formation (mutase) or in the transfer onto
the growing galactan chains (galactosyltransferases)
may be ideal drug targets.
In a continuation of earlier work,12 on the synthesis of
disaccharides as substrates/inhibitors of mycobacterial
Figure 3. Structure of galactofuranosyl acceptors.

A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3129–3143
3131
we started with the synthesis of octyl 2,3,5-tri-O-ben-
zoyl-b-d-galactofuranoside (6a) and octyl-2,3,6-tri-O-
benzoyl-b-d-galactofuranoside (6b) as acceptors for the
synthesis of b1!6 and b1!5 linked galactofuranose
disaccharide derivatives by a route similar to that
reported earlier15 (Scheme 1). Compound 3 was selec-
tively blocked via tritylation with trityl chloride in pyri-
dine at 50 ꢀC to give the 6-tritylated derivative 4 in 72%
yield, and 4 was then benzoylated to give 5a in 87%
yield. Treatment with 80% acetic acid gave the mixture
of 6a and 6b that were separated using column chro-
matography to give the pure aglycons in 58 and 39%
yields, respectively. Each isomer was characterized using
1H and 13C NMR spectroscopy. The byproduct 6b is
formed through migration of the benzoyl group on the
5-position to the 6-hydroxy group during detritylation.
The glycosidation reactions with acceptors 6a and 6b
were carried out with 2,3,5,6-tetra-O-acetyl-b-d-galac-
tofuranosyl chloride16 (11) as the donor and HgBr2 as
the promoter in dichloromethane over 4 A molecular
sieves,15a and gave an excellent yield (99%) for the
b1!5 linked disaccharide (21). An inseparable anome-
ric mixture [(55:45) of b1!6 (15) and b1!5 (21) as
d-galactofuranoside17 (12) as the glycosylation donor
with acceptors 6a and 6b in the presence of NIS and
TESOTf/TMSOTf, but no major products were
observed even at different reaction temperatures. The
use of either NIS/TfOH or NIS/Sn(OTf)2 as acid
promoters, however, resulted in an ꢁ79% yield of
disaccharide 15. An attempt to prepare 21 under the
same conditions gave an inseparable mixture of
disaccharides 15 and 21 in 82% yield in a ratio of 55:45,
respectively, and, again, benzoyl migration in acidic
medium is probably the cause.
From earlier anti-mycobacterial studies of dis-
accharides,12c it was observed that disaccharides having
a fully blocked reducing end gave better activity than
the related totally deblocked disaccharides. Hence, we
synthesized (Schemes 1 and 2) the protected acceptors
6c, 6d, 10a, and 10b using benzyl or methyl groups in
order to probe steric tolerance in the transferase active
site. Compounds 6c and 6d were synthesized as descri-
bed for compound 6a. Compound 4 was benzylated/
methylated using NaH and BnBr/MeI to obtain 5b and
5c that on acid hydrolysis (80% acetic acid) gave the
aglycon 6c/6d in 78 and 99% yields, respectively. Com-
pounds 10a and 10b were prepared starting from 3. The
5,6-hydroxyl groups were first blocked with an iso-
propylidene group using 2,20-dimethoxypropane and
(1S)-(+)-10-camphor sulphonic acid to yield 7, and on
treatment with benzyl bromide/methyl iodide in pre-
sence of NaH afforded compounds 8a and 8b in 80
and 81% yields, respectively. Acid hydrolysis, using
1
determined by H NMR] was obtained in the coupling
reaction of 6a and 11 to get the b1!6 disaccharide.
Hence, a Lewis acid promoted coupling using SnCl4 and
2,3,5,6-tetra-O-acetyl-b-d-galactofuranose tetraacetate
(1) as the donor and 6a and 6b as acceptors was
attempted, but only moderate yields of disaccharides 15
and 21 were obtained.14 In search for other glycosyl
donors, we tried the n-pentenyl 2,3,5,6-tetra-O-acetyl-b-
Scheme 1. Synthesis of various galactofuranosyl acceptors for b(1!6) disaccharides. Reagents & Conditions: (i) SnCl4, CH3(CH2)7OH, CH3CN,
0ꢀC-rt, 30 min, 72%; (ii) NaOMe, MeOH, rt, 7 h, 95%; (iii) TrCl, Py, 50ꢀC, 48 h, 72%; (iv) BzCl, Py, rt, overnight, 87%; (v) BnBr, NaH, DMF,
0ꢀC-rt, 3 h, 99%; (vi) Mel, NaH, THF, rt, overnight, 90%; (vii) CH3COOH, H2O, 60ꢀC, 30 min, 6a: 58%, 6b: 39%, 6c: 78%, 6d: 99%.
Scheme 2. Synthesis of various galactofuranosyl acceptors for b(1!5) disaccharides. Reagents & Conditions: (i) 2,20-dimethoxypropane, (1S)-(+)-
10-camphorsulphonic acid, acetone, rt, 30 min, 98%; (ii) BnBr, NaH, DMF, 0ꢀC-rt, overnight, 80%; (iii) Mel, NaH, THF, rt, overnight, 81%;
(iv) TFA containing 1% H2O, CHCl3, rt, 30 min, 9a: 92%, 9b: 99%; (v) Bu2SnO, Toluene, reflux, overnight, BnBr, CsF, DMF, rt, 8 h, 82%;
(vi) Bu2SnO, Toluene, reflux, overnight, Mel, CsF, DMF, rt, overnight, 59%.

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A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3129–3143
Table 1. Inhibitor activities of disaccarides against Mycobacterium tuberculosis and Mycobactrium avium
Compound no.
MICa (mg/mL)
MTB H37Ra
MAC NJ 168
MAC NJ 211
MAC NJ 3404
17
18
19
20
22
23
24
25
26
>128
>128
NDb
>128
>128
ND
>12.8
ND
>12.8
>128
>128
>128
>128
>128
>128
>128
>12.8
>128
>12.8
>128
>12.8ꢃ128
>12.8ꢃ128
>128
>128
>128
>128
>12.8
>128
>128
>12.8
>12.8ꢃ128
>12.8ꢃ128
>12.8
>12.8
>128
>128
aMIC, minimum inhibitory concentration.
bND, not determined.
trifluoroacetic acid (TFA) containing 1% H2O in
chloroform at room temperature, gave 9a and 9b in 92
and 99% respectively. The saccharides 9a and 9b were
regio-selectively benzylated/methylated via stannylated
intermediates18 formed with dibutyltin oxide in boiling
toluene and azeotropic removal of water. Evaporation
gave the crude 5,6-O-stannylene acetals which were
then selectively benzylated/methylated at the 6-positions
to give 10a or 10b as major products in 82 and 59%
yields, as well as minor products 6c or 6d in 8 and
10%, respectively, after silica gel flash column chro-
matography. Each set of isomers was characterized
using 1H NMR and 13C NMR spectroscopy. In the
carbon spectrum of compound 6c, C-5 and C-6 reso-
nated at d 78.23 and 62.30, respectively, whereas in
compounds 10a and 10b C-5 and C-6 resonated at d
73.37 and 70.06, respectively. Further, APT and 2D-
NMR experiments also supported the assignments of
both isomers.
preparations.20 We prepared 1-thiocresyl-2,3,5,6-tetra-
O-acetyl-b-d-galactofuranoside (14) as glycosyl
a
donor, but the use of TMSOTf or TESOTf did not give
any disaccharide product in coupling attempts several
acceptors. The use of triflic acid (TfOH) or Sn(OTf)2,
however, gave excellent yields in short reaction times.
We have exclusively used Sn(OTf)2 rather than TfOH
due to ease of use and handling for the preparation of
the disaccharides 16, 17, 22, and 23. The structures
1
of these four disaccharides were verified by H NMR,
13C NMR and FABMS spectroscopy. H-1 and H-10
resonated in the range of d 5.17 and 5.19, respectively,
with coupling constants of ꢁ0.7–1.5 Hz whereas the
C-1 and C-10 were observed in the range of d 105.33
and 105.90, respectively, suggesting the b-glycosidic
linkage. Deacytlation of the blocked products with
7N NH3 in MeOH afforded b1!6 and b1!5 linked
disaccharides 18, 19, 24, and 25 having a protected
reducing end. Further, catalytic hydrogenation of
disaccharides 18 and 24 over Pd/C in MeOH gave the
totally deblocked b1!6 and b1!5 linked disaccharides
20 and 26, respectively. The deacylation of dis-
accharides 15, and impure 21, with 7N NH3 in MeOH
also gave pure, fully deblocked b1!6 and b1!5 linked
disaccharides 20 and 26, respectively, after column
chromatography. NMR, FABMS and, whenever
necessary, 2D NMR experiments were performed to
characterize all compounds.
During this work, we investigated other glycosyl donors
of galactofuranose for disaccharide synthesis. The tri-
chloroacetimidate derivatives have been widely used as
donors for glycosylation reactions in pyranose sugars
and recently many furanoses trichloroimidates were
reported due to gaining interest in penta- and hexafur-
anosides.19 We prepared the 2,3,5,6-tetra-O-acetyl-b-d-
galactofuranosyl trichloroacetaimidate (13) in two steps
from compound 1 via intermediate 2,3,5,6-tetra-O-ace-
tyl-b-d-galactofuranoside. The galactofuranose tri-
chloroacetimidate has recently been prepared and used
as glycosyl donor in disaccharide synthesis.19d,e The
b1!6 and b1!5 linked disaccharides 16 (89%) and 22
In-vitro assay
In-vitro antibacterial assays21 of disaccharides were
performed with MTB H37Ra and three clinical isolates
of MAC. The results are summarized in Table 1. Mod-
erate activity was seen with 18 and 24 against MTB and
one strain of MAC. Both compounds are totally
blocked at the reducing end.
ꢂ
(87%) were obtained in high yields using BF3 Et2O as
the promoter in dichloromethane. The only dis-
advantage to this procedure was a low yield of 2,3,5,6-
tetra-O-acetyl-b-d-galactofuranoside with HBr/AcOH/
H2O due to ring opening which resulted in some pyr-
anose formation. It was then reacted with tri-
chloroacetonitrile in the presence of K2CO3 to yield 13.
We also investigated 1,2-trans-thiofuranosyl derivatives
of b-d-galactofuranoside as glycosylation donors. Sev-
eral different sugar donors based on thioethyl or thio-
phenyl pyranoses and galactofuranoses have been
reported and used successfully for different disaccharide
Development of
syltransferase assay
a
simple mycobacterial galacto-
Based on previous use of specific neoglycolipid accep-
tors compounds 20 and 26 were synthesized corre-
sponding to the two major structural motifs found
within the galactan of arabinogalactan. Assays per-

A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3129–3143
3133
formed in the presence of membranes and the cell wall
enzymatic fraction P60 resulted in excellent [14C]Galf
incorporation from UDP-[14C]Galp, following endo-
genous conversion to UDP-[14C]Galf and transferase
activity for both 26 and 20 (see Fig. 4A and B). A
concentration of 4 mM for both acceptors resulted in
optimum galactosyltransferase activity. TLC/auto-
radiography (Fig. 5A) demonstrated the enzymatic
conversion of both the disaccharide 26 and 20 acceptors
to their corresponding trisaccharide products, [14C]Gal
to the 50-OH of 20 and 60-OH of 26. Compound 20 gave
rise to a second, slower migrating band (Fig. 5A, lane 3)
which based on relative migration profiles would be
anticipated to be a tetrasaccharide product resulting from
further elongation of the above trisaccharide precursor at
the 60-OH consistent with the alternating linkage pat-
tern of arabinogalactan. The complete chemical char-
acterization of the enzymatically synthesized, products
along with the identity of the galactosyltransferase gene
product are reported in a separate communication.22
Compounds 18 and 24 were not recognized as sub-
strates for the galactosyltransferase enzyme, presumably
due to the bulky benzyl ether protecting groups on the
reducing sugar. In contrast, 25 and 19, which possessed
methyl ether protecting groups on the reducing sugar,
were recognized as substrates without significant loss of
acceptor recognition (Figs 2C, D and 4B). Interestingly,
compound 19 produced a single trisaccharide product
Figure 4. Kinetic analysis of acceptors 26 (panel A), 20 (panel B), 25 (panel C) and 19 (panel D). The insets illustrate the double reciprocal plots for
each compound. The inhibitory properties of acceptors 25 (panel E) and 19 (panel F) are shown in a dose–response with compounds 26 and 20 at a
fixed concentration of 0.5 mM, respectively.

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A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3129–3143
Figure 5. An autoradiogram of reaction products produced through the inclusion of 26 and 20 (panel A), and 25 and 19 (panel B), mycobacterial
membranes and UDP-[14C]Gal. TLC/autoradiography was performed using chloroform/methanol/ammonium hydroxide/water (65:25:0.5:3.6) and
products revealed through exposure to Kodak X-Omat film at À70 ꢀC for 48 h.
([14C]Gal to the 50-OH of 19, whereas the fully de-
blocked 20 produced a mixture of tri- and tetra-
saccharide products as described above (Fig. 2A and B).
Experimental
Chemical synthesis
Calculation of kinetic constants (Fig. 4A–D) revealed
that 26 and 20 possessed Km values 3.77 and 2.60 mM,
respectively. In contrast, 25 and 19 possessed Km values
5.95 and 31.73 mM, respectively. The unexpected high
value Km value for 19, in addition to its singular pro-
duct was surprising and may offer some insight into the
catalytic mechanism of the bi-functional galactosyl-
transferase.22 Further competition based, experiments
established that 25 and 19 were effective inhibitors of
their native acceptors (26 and 20) in the galactosyl-
transferase assay (Fig. 4E and F) resulting in IC50
values of 3.32 and 3.65 mM for 25 and 19, respectively.
The disaccharides 18 and 24 possessed no inhibitory
activity in the above assays.
General procedure. All manipulations were conducted
under a dry argon atmosphere. Reaction temperatures
were measured externally. Anhydrous solvents from
Aldrich were used without further drying. Whenever
necessary, compounds and starting reagents were dried
by azeotropic removal of water with toluene under
reduced pressure. Reactions were monitored by thin-
layer chromatography (TLC) on pre-coated E. Merck
silica gel (60F254) plates (0.25 mm) and visualized using
UV light (254 nm) and/or heating after spray with
(NH4)2SO4 solution (150 g ammonium sulphate, 30 mL
H2SO4, 750 mL H2O). All solvents used for work up
and chromatography were reagent grade from Fisher
Scientific. Flash chromatography was carried out on
Fischer silica gel 60 (230–400 mesh). Melting points
were determined with a Mel-Temp II capillary melting
points apparatus and are uncorrected. 1H and 13C
NMR spectra were recorded on Nicolet NT 300NB
It is intriguing that both 18 and 24 show activity in the
in vitro, whole cell assay, but are not active, either as
acceptors or inhibitors, in the cell free glycosyltransferase
assay. Although, we have not measured the ability of
these molecules to bind and lyse cells as nonspecific
surfactants, it is possible that both compounds are hav-
ing activity against the whole bacteria through this
mechanism. Clearly, the benzyl groups are too large to
allow enzyme binding and inhibition, and they may lead
to adverse nonspecific activity as dipolar, uncharged
surfactants. The methyl substituted disaccharides, 19
and 25, on the other hand, appear to compete with the
more natural acceptors, 20 and 26, and show modest
IC50 values in agreement with our earlier conclusions
from octyl b(1!4) Galf-Rhap disaccharides;12c more
hydrophobic disaccharides that are blocked with less
sterically demanding blocking groups at the reduc-
ing end show acceptor activity as well as binding and
inhibition of the mycobacterial galactosyltransferases.
These results are in concordance with our efforts to
eventually make later generation inhibitors of the
mycobacterial glycosyltransferases that are less ‘sugar-
like’ and more ‘drug-like.’
1
instrument at 300 and 75 MHz, respectively. Certain H
NMR spectra were recorded on a Bruker Advance 600
System at 600 MHz. The coupling constants (J) are
reported in Hz, and chemical shifts are reported in ppm
(d) relative to residual solvent peak or internal standard.
Microanalyses were performed on a Perkin–Elmer 2400
CHN analyzer. FABMS were recorded on a Varian/
MAT 311A double-focusing mass spectrometer either
by adding NBA/LiCl.
Octyl 2,3,5,6-tetra-O-acetyl-ꢀ-D-galactofuranoside (2).
1
To a solution of b-d-galactofuranose pentaacetate13
(20.0 g, 51.28 mmol) in dry CH3CN (100 mL) was added
SnCl4 (6.0 mL, 51.28 mmol) at 0 ꢀC and the mixture was
stirred for 30 min. To the cold solution, n-octanol
(8.88 mL, 56.40 mmol) was added dropwise over a per-
iod of 30 min. It was again stirred for 1 h at room tem-
perature. Celite 10.0 g was added, the suspension was
cooled in an ice-water bath, and a saturated aqueous
NaHCO3 solution was added dropwise to precipitate tin

A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3129–3143
3135
salts. After complete precipitation, the mixture was fil-
tered through Celite and washed with chloroform
(2Â10 mL). Concentration gave syrup that was redis-
olved in CHCl3 (400 mL). The solution was washed with
water (2Â50 mL), brine (2Â50 mL), dried over Na2SO4
and concentrated in vacuo to give a crude oil. Flash
chromatography (cyclohexane/EtOAc, 2:1) gave 2 as a
colorless oil (16.97 g, 72%). Rf=0.51 (cyclohexane/
EtOAc, 1:1). FAB-MS (LiCl) m/e 467 [M+Li]+. 1H
NMR (300 MHz, CDCl3) d 5.39 (1H, ddd, J4,5=3.5 Hz,
J5,6a=4.5 Hz, J5,6b=7.3 Hz, H-5), 5.04 (1H, dd,
J1,2=0.7 Hz, J2,3=2.0 Hz, H-2), 5.01 (1H, s, H-1), 4.99
(1H, ddd, J1,3=0.3 Hz, J2,3=2.0 Hz, J3,4=5.8 Hz, H-3),
4.34 (1H, dd, J5,6a=4.5 Hz, J6a,6b=11.9 Hz, H-6a), 4.24
(1H, dd, J4,5=3.5 Hz, J3,4=5.8 Hz, H-4), 4.22 (1H, dd,
J5,6b=7.3 Hz, J6a,6b=11.9 Hz, H-6), 3.65 (1H, m,
OCH2), 3.42 (1H, m, OCH2), 2.14 (3H, s, OAc), 2.11
(3H, s, OAc), 2.09 (3H, s, OAc), 2.06 (3H, s, OAc), 1.56
(2H, m, CH2), 1.28 (10H, m, 5ÂCH2), 0.88 (3H, m,
CH3). 13C NMR (75 MHz, CDCl3): d 170.52, 170.07,
170.04, 169.69 (C¼O), 105.48 (C-1), 81.47 (C-2), 79.79
(C-4), 76.64 (C-3), 69.33 (C-5), 67.87 (OCH2), 62.72 (C-
6), 31.84, 29.40, 29.34, 29.27, 26.06, 26.01, 22.68
(6ÂCH2), 20.86, 20.81, 20.73 (3ÂOAc), 14.11 (CH3).
solved in 25 mL of dry pyridine, and benzoyl chloride
(1.1 mL, 9.36 mmol) was added. The reaction mixture
was stirred overnight at room temperature, poured into
an ice–water mixture (25 mL), and extracted with
CHCl3 (2Â25 mL). The combined CHCl3 layers were
washed with brine, dried over Na2SO4 and con-
centrated. Column chromatography (cyclohexane/
EtOAc, 10:1) gave pure 5a (1.36 g, 87%) as a colorless
syrup. Rf=0.80 (cyclohexane/EtOAc 1:1). FAB-MS
(LiCl) m/e 853 [M+Li]+. 1H NMR (300 MHz, CDCl3):
d 8.18–7.16 (30H, m, aromatic), 5.77 (1H, ddd,
J4,5=4.0 Hz, J5,6a=6.3 Hz, J5,6b=5.1 Hz, H-5), 5.45
(1H, d, J3,4=5.3 Hz, H-3), 5.37 (1H, d, J1,2=1.1 Hz, H-
2), 5.20 (1H, s, H-1), 4.72 (1H, dd, J4,5=4.0 Hz,
J3,4=5.3 Hz, H-4), 3.71 (1H, m, OCH2), 3.56 (1H, dd,
J5,6a=6.3 Hz, J6a,6b=9.9 Hz, H-6a), 3.43 (1H, dd,
J5,6b=5.1 Hz, J6a,6b=9.9 Hz, H-6b), 3.52–3.47 (1H, m,
OCH2), 1.60–1.55 (2H, m, CH2), 1.28–1.20 (10H, m,
5ÂCH2), 0.90–0.84 (3H, m, CH3).
Octyl 2,3,5-tri-O-benzyl-6-O-trityl-ꢀ-D-galactofurano-
side (5b). Compound 4 (2.36 g, 4.42 mmol) was dis-
solved in dry DMF (30 mL) and NaH (60% dispersion
in mineral oil) was added (382 mg, 15.91 mmol), and the
mixture was stirred at room temperature for 15 min.
The reaction was cooled to 0oC and benzyl bromide
(1.89 mL, 15.91 mmol) was added dropwise, and the
solution stirred for 3 h at room temperature. Next,
MeOH (10 mL) was added, and the reaction was con-
centrated to dryness. The resulting oil was redissolved in
CHCl3 (200 mL), washed with water (2Â50 mL) fol-
lowed by brine (50 mL), and dried over Na2SO4. Con-
centration of the combined organic layers, and flash
chromatography of the product (cyclohexane/EtOAc
20:1) gave compound 5b as a colorless oil (3.53 g, 99%).
Rf=0.73 (cyclohexane/EtOAc 2:1). FAB-MS (LiCl) m/e
811 [M+Li]+. 1H NMR (300 MHz, CDCl3): d 7.31
(40H, m, aromatic), 4.97 (1H, s, H-1), 4.54 (6H, m,
3ÂCH2-aromatic), 4.11 (1H, dd, J=3.3, 6.8 Hz, H-4),
3.92 (2H, m, H-2, H-3), 3.66 (1H, m, H-5), 3.58 (1H, m,
OCH2), 3.40 (1H, dd, J5,6a=6.6 Hz, J6a,6b=9.9 Hz, H-
6a) 3.31 (2H, m, H-6b, OCH2), 1.56 (2H, m, CH2), 1.25
(10H, m, 5ÂCH2), 0.87 (3H, m, CH3).
Octyl ꢀ-D-galactofuranoside (3). To a solution of com-
pound 2 (13.5 g, 24.87 mmol) in dry methanol (50 mL)
was added NaOMe/MeOH (25% w/v, 9.0 mL) dropwise
while cooling in an ice-bath. The reaction mixture was
stirred at room temperature for 1 h. Concentration in
vacuo gave a syrup. Flash chromatography (CHCl3/
MeOH 10:1) gave 3 as a solid (8.57 g, 95%). Rf=0.31
(CHCl3/MeOH, 7:1), mp 88–90 ꢀC. FAB-MS (NBA) m/
e 292 [M+H]+. 1H NMR (300 MHz, DMSO-d6): d 5.26
(1H, d, J=5.7 Hz, 2-OH, D2O exchanged), 5.04 (1H, d,
J=5.5 Hz, 3-OH), 4.67 (1H, d, J=2.4 Hz, H-1), 4.51
(1H, 2H, m, 5-OH, 6-OH), 3.81 (1H, dd, J2,3=4.8 Hz,
J3,4=7.4 Hz, H-3), 3.74 (1H, dd, J1,2=2.4 Hz,
J2,3=4.8 Hz, H-2), 3.70 (1H, dd, J4,5=2.9 Hz,
J3,4=7.4 Hz, H-4), 3.56 (1H, m, OCH2), 3.49 (1H, m,
H-5), 3.31 (3H, m, H2-6, OCH2), 1.49 (2H, m, CH2),
1.25 (10H, m, 5ÂCH2), 0.86 (3H, m, CH3).
Octyl 6-O-trityl-ꢀ-D-galactofuranoside (4). To a dry
pyridine (60 mL) solution of compound 3 (5.2 g,
17.8 mmol) was added trityl chloride (7.44 g,
26.7 mmol), and the reaction mixture was stirred at
50 ꢀC overnight. Co-evaporation with toluene
(2Â50 mL) removed all traces of pyridine. The resulting
oil was redissolved in CHCl3 (150 mL), washed with
water (2Â20 mL), dried over Na2SO4 and concentrated.
Column chromatography (CHCl3/MeOH 95:5) gave
compound 4 as a pale yellow oil (6.84 g, 72%). Rf=0.65
(CHCl3/MeOH 95:5). FAB-MS (LiCl) m/e 541
[M+Li]+. 1H NMR (300 MHz, CDCl3): d 7.43 (6H, m,
aromatic), 7.30 (9H, m, aromatic), 5.05 (1H, s, H-1),
4.00 (4H, m, H-2, H-3, H-4, H-5), 3.65 (1H, m, OCH2),
3.37 (3H, m, H2-6, OCH2), 2.98 (1H, d, J=11.9 Hz, 3-
OH), 2.78 (1H, br s, 2-OH), 2.17 (1H, s, 5-OH) 1.57 (2H,
m, CH2), 1.24 (10H, m, 5ÂCH2), 0.86 (3H, m, CH3).
Octyl 2,3,5-tri-O-methyl-6-O-trityl-ꢀ-D-galactofurano-
side (5c). Compound 4 (1.16 g, 2.17 mmol) was dis-
solved in dry THF (20 mL) and NaH (60% dispersion in
mineral oil, 157 mg, 6.52 mmol) was added and the
mixture was stirred at room temperature for 15 min.
The reaction was cooled to 0 ꢀC and methyl iodide
(0.41 mL, 6.52 mmol) was added dropwise to above
solution and stirred overnight at room temperature.
Methanol (10 mL) was added, and the solution was
concentrated to dryness. The resulting oil was redis-
solved in CHCl3 (150 mL), washed with water
(2Â25 mL), brine (25 mL) and dried over Na2SO4.
Concentration of the combined organic layers followed
by flash chromatography (cyclohexane/EtOAc 5:1) gave
compound 5c as a colorless oil (1.12 g, 90%). Rf=0.55
(cyclohexane/EtOAc 2:1). FAB-MS (LiCl) m/e 583
[M+Li]+. 1H NMR (300 MHz, CDCl3): d 7.46 (6H, m,
aromatic), 7.28 (12H, m, aromatic), 4.93 (1H, s, H-1),
4.01 (1H, dd, J4,5=3.9 Hz, J3,4=7.0 Hz, H-4), 3.65 (1H,
Octyl 2,3,5-tri-O-benzoyl-6-O-trityl-ꢀ-D-galactofurano-
side (5a). Compound 4 (1.00 g, 1.87 mmol) was dis-

3136
A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3129–3143
dd, J1,2=1.3 Hz, J2,3=3.1 Hz, H-2), 3.59 (2H, m, H-3,
OCH2), 3.51 (3H, s, OCH3), 3.47 (1H, m, H-5), 3.35
(2H, m, H-6a, OCH2), 3.36, 3.31 (each 3H, s, 2ÂOCH3),
3.21 (1H, dd, J5,6b=5.1 Hz, J6a,6b=9.7 Hz, H-6b), 1.53
(2H, m, CH2), 1.23 (10H, m, 5ÂCH2), 0.87 (3H, m, CH3).
EtOAc 2:1). FAB-MS (LiCl) m/e 341 [M+Li]+. 1H
NMR (300 MHz, CDCl3): d 4.99 (1H, s, H-1), 4.08 (1H,
dd, J4,5=5.5 Hz, J3,4=6.6 Hz, H-4), 3.79 (2H, m, H2-6),
3.69 (3H, m, H-2, H-3, OCH2), 3.53, 3.43 (each 3H, s,
2ÂOCH3), 3.42 (2H, m, H-5, OCH2), 2.35 (1H, dd,
J=4.6, 8.6 Hz, 6-OH), 1.57 (2H, m, CH2), 1.27 (10H, m,
5ÂCH2), 0.88 (3H, m, CH3).
Octyl 2,3,5-tri-O-benzoyl-ꢀ-D-galactofuranoside (6a)
and Octyl 2,3,6-tri-O-benzoyl-ꢀ-D-galactofuranoside
(6b). Compound 5a (1.36 g, 1.61 mmol) was dissolved
in CHCl3, cooled to 0 ꢀC, and trifluoroacetic acid (10%
in CHCl3) was added dropwise. The reaction mixture
was stirred for 1 h, co-evaporated with ethanol
(2Â20 mL), and concentrated to give a syrup. Flash
chromatography (cyclohexane/EtOAc 10:1) yielded 6a
(566 mg, 58%) and 6b (376 mg, 39%) as separate, pure
and colorless syrups. Rf=0.48 (6a) and 0.53 (6b)
(cyclohexane/EtOAc 2:1). FAB-MS (LiCl) m/e 611
[M+Li]+. Compound 6a. 1H NMR (300MHz, CDCl3): d
8.12–7.94 (6H, m, aromatic), 7.64–7.26 (9H, m, aromatic),
5.64 (1H, ddd, J5,6a=J5,6b=4.8 Hz, J4,5=4.2 Hz, H-5),
5.59 (1H, d, J3,4=4.8 Hz, H-3), 5.47 (1H, d,
J1,2=0.9 Hz, H-2), 5.31 (1H, s, H-1), 4.64 (1H, dd,
J4,5=4.2 Hz, J3,4=4.8 Hz, H-4), 4.08 (2H, d,
J5,6a=J5,6b=4.8 Hz, H-6a, H-6b), 3.77 (1H, m, OCH2),
3.56 (1H, m, OCH2), 1.64 (2H, m, CH2), 1.27 (10H, m,
Octyl 5,6-isopropyledine-ꢀ-D-galactofuranoside (7). To a
solution of compound 3 (1.50 g, 5.14 mmol) in dry ace-
tone (30 mL) was added 2,20-dimethoxypropane
(0.95 mL, 7.70 mmol) and (1S)-(+)-10-camphorsulfonic
acid (120 mg, 0.51 mmol) at room temperature. After
30 min of stirring, the pH was adjusted to pH 7 by
adding Et3N. Concentration in vacuo gave the crude oil
which was dissolved in CHCl3 (100 mL), washed with
saturated NaHCO3 (20 mL) and water (20 mL), dried
over Na2SO4, and concentrated to an oil. This oil was
further purified by flash chromatography (cyclohexane/
EtOAc, 5:1) to give 7 as a colorless oil (1.60 g, 94%).
Rf=0.81 (CHCl3/MeOH, 8:1). FABMS (NBA) m/e 333
1
[M+H]+. H NMR (300 MHz, CDCl3): d 5.02 (1H, s,
H-1), 4.36 (1H, dd, J=1.3, 1.5, 7.8 Hz, H-5), 4.03 (6H,
m, H-2, H-3, H-4, H2-6, 3-OH), 3.73 (1H, m, OCH2),
3.44 (1H, m, OCH2), 2.06 (1H, d, J=11.9 Hz, 2-OH),
1.57 (2H, m, CH2), 1.42, 1.39 (2Â3H, s, 2ÂCH3), 1.28
(10H, m, 5ÂCH2), 0.88 (3H, m, CH3), 13C NMR
(75 MHz, CDCl3): d 110.13 (C), 108.38 (C-1), 85.53 (C-
2), 78.6 (C-3), 78.04 (C-4), 75.71 (C-5), 67.80 (O-CH2),
65.71 (C-6), 31.76, 29.46, 29.25, 29.16, 26.06 (5ÂCH2),
25.58, 225.52 (2ÂCH3), 22.59 (CH2), 14.03 (CH3).
1
5ÂCH2), 0.88 (3H, m, CH3). Compound 6b. H NMR
(300 MHz, CDCl3) d 8.08–8.02, 7.65–7.39 (15H, m,
aromatic), 5.63 (1H, d, J3,4=4.8 Hz, H-3), 5.51 (1H, d,
J1,2=1.1 Hz, H-2), 5.26 (1H, s, H-1), 4.60 (1H, dd,
J5,6a=7.9 Hz, J6a,6b=12.3 Hz, H-6a), 4.51–4.46 (2H, m,
H-5, H-6b), 4.37 (1H, dd, J4,5=2.4 Hz, J3,4=4.8 Hz, H-
4), 3.72 (1H, m, O-CH2), 3.50 (1H, m, OCH2), 1.63 (2H,
m, CH2), 1.21 (10H, m, 5ÂCH2), 0.85 (3H, m, CH3).
Octyl 5,6- isopropyledine-2,3-di-O-benzyl-ꢀ-D-galacto-
furanoside (8a). To a dry DMF solution (20 mL) of
compound 7 (1.50 g, 4.52 mmol) was added NaH (60%
suspension in mineral oil—325 mg, 13.55 mmol) fol-
lowed by benzyl bromide (1.61 mL, 13.55 mmol) drop-
wise with cooling at 0 ꢀC. The reaction mixture was
stirred at room temperature overnight. MeOH (5 mL)
was added to the reaction, the mixture was evaporated
to near dryness, and redissolved in CHCl3 (100 mL). It
was washed with water (20 mL) and brine (10 mL), dried
over Na2SO4, and purified by flash column chromato-
graphy (cyclohexane/EtOAc 10:1) to give 8a as a color-
less oil (1.83 g, 79%). Rf=0.55 (cyclohexane/EtOAc
2:1). FABMS (LiCl) m/e 519 [M+Li]+. 1H NMR
(300 MHz, CDCl3): d 7.37–7.28 (10H, aromatic), 5.05
(1H, s, H-1), 4.52 (4H, m, 2ÂCH2), 4.18 (1H, ddd,
J4,5=6.6 Hz, J5,6a=J5,6b=6.8 Hz, H-5), 4.01 (1H, dd,
J4,5=6.6 Hz, J3,4=6.8 Hz, H-4), 4.02 (1H, d, J1,2=1.3 Hz,
H-2), 3.85 (2H, dd, J5,6a=J5,6b=6.8 Hz, H-6a, H-6b), 3.76
(2H, m, H-3, OCH2), 3.41 (1H, m, OCH2), 1.57 (2H, m,
CH2), 1.41, 1.35 (each 3H, s, 2ÂCH3), 1.27 (10H, m,
5ÂCH2), 0.87 (3H, m, CH3). 13C NMR (75 MHz,
CDCl3) d: 137.52, 137.46 (C, aromatic), 128.45, 128.36,
127.95, 127.82, 127.74 (CH, aromatic), 109.60 (C),
105.98 (C-1), 88.01 (C-2), 83.99 (C-3), 81.52 (C-4), 76.49
(C-5), 72.07, 72.04 (2ÂCH2-aromatic), 67.72 (O–CH2),
65.37 (C-6), 31.82, 29.52, 29.35, 29.25, 26.11, 22.65
(6ÂCH2), 26.48 (CH3), 25.41 (CH3), 14.07 (CH3).
Octyl 2,3,5-tri-O-benzyl-ꢀ-D-galactofuranoside (6c).
Compound 5b in glacial acetic acid (25 mL) was heated
to 60 ꢀC for 30 min. Water (5 mL) was added dropwise
while heating, and the reaction mixture was then cooled
to room temperature and stirred for another 30 min
followed by co-evaporation with ethanol (2Â100 mL).
Flash chromatography of the resulting oil (cyclohexane/
EtOAc 5:1) gave 6c as a colorless oil (1.84 g, 74%).
Rf=0.48 (cyclohexane/EtOAc 2:1). FAB-MS (LiCl) m/e
1
659 [M+Li]+. H NMR (300 MHz, CDCl3): d 7.34–
7.25 (15H, m, aromatic), 5.05 (1H, s, H-1), 4.69–4.39
(6H, m, 3ÂCH2-aromatic), 4.19 (1H, m, H-5), 4.08 (1H,
dd, J4,5=3.1 Hz, J3,4=5.0 Hz, H-4), 4.00 (1H, d,
J1,2=0.9 Hz, H-2), 3.68 (4H, m, H-3, H2-6, OCH2), 3.39
(1H, m, OCH2), 2.27 (1H, dd, J=4.5, 8.5 Hz, 6-OH),
1.57 (2H, m, CH2), 1.27 (10H, br s, 5ÂCH2), 0.88 (3H,
m, CH3). 13C NMR (75 MHz, CDCl3) d: 138.28, 137.52,
137.40 (C, aromatic), 128.45, 128.37, 128.04, 128.01,
127.99, 127.92, 127.83, 127.72 (CH, aromatic), 105.95
1
(C-1, JCH=170.9 Hz), 88.11 (C-2), 83.21 (C-3), 81.77
(C-4), 78.23 (C-5), 72.71, 72.10, 72.06 (3ÂCH2-aro-
matic), 67.75 (O-CH2), 62.30 (C-6), 31.83, 29.51, 29.36,
29.27, 26.16, 22.65 (6ÂCH2), 14.08 (CH3).
Octyl 2,3,5-tri-O-methyl-ꢀ-D-galactofuranoside (6d).
Prepared from compound 5c (1.00 g, 1.74 mmol) using
the procedure listed for 6a. Flash chromatography of
the product (cyclohexane/EtOAc 2:1) yielded 6d as a
colorless oil (580 mg, 99%). Rf=0.23 (cyclohexane/
Octyl 5,6-isopropyledine-2,3-di-O-methyl-ꢀ-D-galacto-
furanoside (8b). Compound 8b was prepared by the

A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3129–3143
3137
procedure of 8a using 7 (1.75 g, 5.27 mmol) and methyl
iodide (1.00 mL, 15.81 mmol). Workup and flash col-
umn chromatography (cyclohexane/EtOAc 5:1) gave
compound 8b as a colorless oil (1.79 g, 95%). Rf=0.59
(cyclohexane/EtOAc 1:1). FAB-MS (LiCl) m/e 367
2.75 mmol) and dibutyltin oxide (821 mg, 3.3 mmol) was
refluxed for 6 h with azeotropic removal of water. The
reaction mixture was cooled and evaporated to dryness.
The residue was dissolved in dry DMF (10 mL) and to it
was anhydrous CsF (835 mg, 5.5 mmol) and benzyl
bromide (0.4 mL, 3.3 mmol). The resulting mixture was
stirred overnight under argon, then concentrated in
vacuo, redissolved in diethylether (150 mL), and washed
with 10% aqueous KF solution and water (10 mL). The
combined organic phase was dried, concentrated and
subjected to flash chromatography (cyclohexane/EtOAc
10:1) to give compound 10a as a colorless oil (1.31 g,
85%). Rf=0.51 (cyclohexane/EtOAc 2:1). FAB-MS
(LiCl) m/e 569 [M+Li]+. 1H NMR (300 MHz, CDCl3):
d 7.35–7.28 (15H, m, aromatic), 5.03 (1H, s, H-1), 4.52
(6H, m, 3ÂCH2–aromatic), 4.10 (1H, dd, J4,5=3.2 Hz,
J3,4=6.6 Hz, H-4), 4.05 (1H, dd, J2,3=2.6 Hz,
J3,4=6.6 Hz, H-3), 4.00 (1H, dd, J1,2=1.1 Hz,
J2,3=2.6 Hz, H-2), 3.92 (1H, m, H-5), 3.66 (1H, m,
OCH2), 3.54 (2H, m, H2-6), 3.38 (1H, m, O-CH2), 2.40
(1H, d, J5,OH=6.4 Hz, 5-OH), 3.41 (1H, m, OCH2), 1.56
(2H, m, CH2), 1.27 (10H, m, 5ÂCH2), 0.88 (3H, m,
CH3). 13C NMR (75 MHz, CDCl3): d 138.06, 137.81,
137.41 (C, aromatic), 128.43, 128.36, 127.94, 127.89,
127.80, 127.74, 127.66 (CH, aromatic), 106.16 (C-1),
87.73 (C-2), 83.25 (C-3), 81.15 (C-4), 73.37 (C-6), 72.20,
71.91, 71.61 (3ÂCH2-aromatic), 70.06 (C-5), 67.69 (O-
CH2), 31.83, 29.51, 29.37, 29.27, 26.14, 22.65 (6ÂCH2),
14.09 (CH3).
1
[M+Li]+. H NMR (300 MHz, CDCl3): d 5.00 (1H, s,
H-1), 4.24 (1H, ddd, J4,5=6.2 Hz, J5,6b=6.6 Hz,
J5,6a=6.8 Hz, H-5), 4.01 (1H, dd, J5,6a=6.8 Hz,
J6a,6b=8.4 Hz, H-6a), 3.96 (1H, dd, J3,4=6.3 Hz,
J4,5=6.2 Hz, H-4), 3.88 (1H, dd, J5,6b=6.6 Hz,
J6a,6b=8.4 Hz, H-6b), 3.71 (2H, m, H-2, OCH2), 3.46
(2H, m, H-3, OCH2), 3.40 (6H, s, 2ÂOCH3), 1.60–1.54
(2H, m, CH2), 1.45, 1.38 (each 3H, s, 2ÂCH3), 1.27
(10H, m, 5ÂCH2), 0.90–0.85 (3H, m, CH3). 13C NMR
(75 MHz, CDCl3): d 109.72 (C), 105.80 (C-1), 89.91 (C-
2), 86.09 (C-3), 81.77 (C-4), 76.27 (C-5), 67.80 (O-CH2),
65.48 (C-6), 57.92, 57.44 (2ÂOCH3), 31.87, 29.61, 29.39,
29.27 (4ÂCH2), 26.51 (CH3), 26.16 (CH2), 25.39 (CH3),
22.66 (CH2), 14.03 (CH3).
Octyl 2,3-di-O-benzyl-ꢀ-D-galactofuranoside (9a). To a
solution of compound 8a (1.75 g, 3.42 mmol) in CHCl3
(25 mL) was added TFA containing 1% H2O (5 mL)
and the reaction mixture was stirred for 30 min. Next,
ethanol (50 mL) was added. The solution was con-
centrated in vacuo, coevaporated with toluene
(2Â10 mL) to remove traces of TFA, and flash chroma-
tographed (cyclohexane/EtOAc 2:1) to give 9a as a col-
orless oil (1.46 g, 91%). Rf=0.25 (cyclohexane/EtOAc
2:1). FAB-MS (LiCl) m/e 479 [M+Li]+. 1H NMR
(300 MHz, CDCl3): d 7.37–7.27 (10H, m, aromatic),
5.03 (1H, d, J1,2=1.1 Hz, H-1), 4.55 (4H, m, 2ÂOCH2-
aromatic), 4.06 (2H, m, H-3, H-4), 4.01 (1H, s, H-2),
3.67 (4H, m, H-5, H2-6, OCH2), 3.38 (1H, m, OCH2),
1.56 (2H, m, CH2), 1.28 (13H, m, 5ÂCH2), 0.89 (3H, m,
CH3). 13C NMR (75 MHz, CDCl3): d 137.55, 137.21 (C,
aromatic), 128.49, 128.01, 127.86 (CH, aromatic),
106.42 (C-1), 87.58 (C-2), 83.41 (C-3), 82.24 (C-4),
72.35, 72.05 (2ÂCH2-aromatic), 71.10 (C-5), 67.87 (O-
CH2), 64.71 (C-6), 31.82, 29.50, 29.35, 29.27, 26.12,
22.65 (6ÂCH2), 14.09 (CH3).
Octyl 2,3,6-tri-O-methyl-ꢀ-D-galactofuranoside (10b).
From compound 9b (570 mg, 1.78 mmol) by the same
procedure to prepare 10a. Flash chromatography
(cyclohexane/EtOAc 2:1) gave compound 10b as color-
less oil (351 mg, 59%). Rf=0.65 (CHCl3/MeOH 95:5).
FAB-MS (LiCl) m/e 341[M+Li]+. 1H NMR (300 MHz,
CDCl3) d 5.00 (1H, s, H-1), 3.98 (1H, dd, J4,5=3.5 Hz,
J3,4=6.0 Hz, H-4), 3.90 (1H, dddd, J4,5=3.5 Hz,
J5,6a=5.3 Hz, J5,OH=5.9 Hz, J5,6b=6.7 Hz, H-5), 3.74
(1H, ddd, J1,3=0.6 Hz, J2,3=2.4 Hz, J3,4=6.0 Hz, H-3),
3.71 (1H, dd, J1,2=0.9 Hz, J2,3=2.4 Hz, H-2), 3.66 (1H,
m, OCH2), 3.50 (1H, dd, J5,6a=5.3 Hz, J6a,6b=9.8 Hz,
H-6a), 3.46 (1H, dd, J5,6b=6.7 Hz, J6a,6b=9.8 Hz, H-
6b), 3.43–3.91 (1H, m, OCH2), 3.42, 3.41, 3.40 (each
3H, s, 3ÂOAc), 3.16 (1H, d, J5,OH=5.9 Hz, 5-OH),
1.60–1.56 (2H, m, CH2), 1.27 (10H, br s, 5ÂCH2), 0.90–
0.86 (3H, m, CH3). 13C NMR (75 MHz, CDCl3): d
105.74 (C-1), 89.33 (C-4), 85.42 (C-2), 81.40 (C-3), 74.03
(C-6), 70.12 (C-5), 67.70 (O-CH2), 59.15, 58.08, 57.41
(3ÂOCH3), 31.83, 29.45, 29.34, 29.25, 26.07, 22.66
(6ÂCH2), 14.11 (CH3).
Octyl 2,3-di-O-methyl-ꢀ-D-galactofuranoside (9b). Pre-
pared from compound 8c (1.75 g, 5.27 mmol) by the
same procedure as reported for compound 9a. Flash
chromatography (cyclohexane/EtOAc 1:1) gave com-
pound 9b as a colorless oil (1.46 g, 91%). Rf=0.25
(cyclohexane/EtOAc 1:1). FABMS (LiCl) m/e 327
[M+Li]+. 1H NMR (300 MHz, CDCl3): d 5.01 (1H, m,
H-1), 4.00 (1H, dd, J5,6a=4.6 Hz, J6a,6b=6.4 Hz, H-6a),
3.82 (1H, ddd, J4,5=3.3 Hz, J5,6a=4.6 Hz, J5,6a=8.1 Hz,
H-5), 3.76–3.73 (3H, m, H-3, H-4, H-6b), 3.71 (1H, dd,
J1,2=0.8 Hz, J2,3=2.5 Hz, H-2), 3.68–3.63 (1H, m,
OCH2), 3.46–3.39 (1H, m, OCH2), 3.43, 3.42 (each 3H,
s, 2ÂOCH3), 1.57 (2H, m, CH2), 1.28 (10H, m, 5ÂCH2),
0.88 (3H, m, CH3). 13C NMR (75 MHz, CDCl3): d
106.00 (C-1), 89.17 (C-2), 85.56 (C-3), 82.57 (C-4), 71.16
(C-5), 67.87 (O-CH2), 64.68 (C-6), 58.12, 57.47
(2ÂOCH3), 31.81, 29.43, 29.32, 29.24, 26.05, 22.65
(6ÂCH2), 14.08 (CH3).
1-Trichloroacetimidoyl-2,3,5,6-tetra-O-acetyl-ꢀ-D-galac-
tofuranoside (13). d-(+)-Galactofuranose pentaacetate
(1) (2.00 g, 5.1 mmol) was dissolved in 50 mL of glacial
acetic acid, followed by dropwise addition of 33% HBr
in acetic acid (10 mL) over a 15 min period with cooling
(ice/water bath). After complete addition, the reaction
mixture was stirred at room temperature for 1 h. Deio-
nized water (5 mL) was added, the reaction was stirred
overnight, and was then poured into an ice–water mix-
ture. Next, this mixture was extracted with chloroform,
and the combined chloroform extract was washed with
Octyl 2,3,6-tri-O-benzyl-ꢀ-D-galactofuranoside (10a). A
toluene (150 mL) solution of compound 9a (1.39 g,

3138
A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3129–3143
cold saturated aq NaHCO3 followed by cold water. The
organic layer was dried and concentrated to syrup. The
desired product was purified by Silica gel G (70–230
mesh) column chromatography using cyclohexane/ethyl
acetate (2:1) as the eluent to give 1.53 g (73%) of 2,3,5,6-
tetra-O-acetyl-b-d-galactofuranoside as a mobile syrup.
129.65, 129.04 (4Â aromatic), 90.54 (C-1), 81.01 (C-4),
79.42 (C-2), 76.38 (C-3), 68.93 (C-5), 62.37 (C-6), 21.01,
20.66, 20.62, 20.54 (CH3, 4ÂOAc).
Octyl 6-O-(2,3,5,6-tetra-O-acetyl-ꢀ-D-galactofuranosyl)-
2,3,5-tri-O-benzoyl-ꢀ-D-galactofuranoside (15). Com-
pound 6a (300 mg, 0.50 mmol) and activated, powdered
4 A molecular sieves (100 mg) in dry CH2Cl2 (10 mL)
were cooled at 0 ꢀC. The glycosylation donor 12
(250 mg, 0.6 mmol) in 5 mL dry CH2Cl2 was added
dropwise under an Ar atmosphere. The mixture was
stirred for 15 min, and NIS (135 mg, 0.6 mmol) followed
by Sn(OTf)2 (25 mg, 0.06 mmol) were added to initiate
coupling. The reaction mixture was allowed to stir for
30 min at rt, and the reaction was quenched by addition
of Et3N (1 mL), diluted with CH2Cl2 (10 mL) and fil-
tered through a Celite pad. The filtrate was washed with
10% Na2S2O3 (10 mL), followed by washing with satu-
rated aqueous NaHCO3 (10 mL). The organic layer was
dried over Na2SO4, the solvent was removed in vacuo,
and the residue was purified by column chromato-
graphy (cyclohexane/EtOAc 2:1) to give a mixture of
disaccharides 15 and 21 (55:45) as a colorless oil
(373 mg, 79%). Rf=0.60 (cyclohexane/EtOAc 1:2).
FABMS (LiCl) m/e 941 [M+Li]+. 1H NMR (300 MHz,
CDCl3, selected peaks for 15): d 5.83 (1H, m, ddd,
J4,5=3.6 Hz, J5,6a=5.5 Hz, J5,6b=6.8 Hz, H-5), 5.55
(1H, d, J3,4=4.9 Hz, H-3), 5.43 (1H, d, J1,2=1.0 Hz, H-
1
FABMS (LiCl): 355 (M+Li)+. H NMR (mixture of a
and b isomers, 1:4): d 5.52 (1H, dd, J=1.8, 4.8 Hz, H-
1a), 5.38 (4H, m, H-1b, H-2a, H-5a, H-5b), 5.13 (1H, t,
J=5.1 Hz, H-3a), 5.05 (2H, m, H-2b, H-3b), 4.43 (1H, t,
J=4.6 Hz, H-4b), 4.35 (2H, m, H-6aa,b), 4.19 (2H, m, H-
4a, H-6bb), 4.07 (1H, dd, J=4.6, 6.4 Hz, H-6ba), 3.48
(1H, dd, J=2.0, 6.8 Hz, 1-OHa), 3.09 (1H, d, J=3.5 Hz,
1-OHb), 2.16, 2.15, 2.14, 2.13, 2.12, 2.10, 2.09, 2.06
(each 3H, s, 8ÂOAc).
The 2,3,5,6-tetra-O-acetyl-b-d-galactofuranoside (1.20 g,
3.45 mmol) was dissolved in dry CH2Cl2 (30 mL) fol-
lowed by the addition of anhydrous K2CO3 (700 mg)
and trichloroacetonitrile (0.86 mL, 8.63 mmol) at room
temperature. The reaction mixture was stirred for 6 h at
room temperature, filtered through a Celite pad, and
concentrated to a syrup. It was purified by Silica gel G
(70–230 mesh) column chromatography with cyclohex-
ane/ethyl acetate (2:1) as the eluent to give the desired
pure compound 13 as a syrup (1.70 g, 80%). Rf=0.48
(cyclohexane/EtOAc 1:1). FAB-MS (LiCl): 498
1
(M+Li)+. H NMR: d 8.64 (1H, s, NH), 6.35 (1H, s,
0 0 0 0
2), 5.36 (1H, ddd, J4 ,5 =3.6 Hz, J5 ,6 a=6.8 Hz,
H-1), 5.42 (1H, ddd, J4,5=4.4 Hz, J5,6a=4.6 Hz,
J5,6b=7.2 Hz, H-5), 5.35 (1H, dd, J1,2=1.3 Hz,
J2,3=2.0 Hz, H-2), 5.11–5.09 (1H, ddd, J1,3=0.5 Hz,
J2,3=2.0 Hz, J3,4=4.2 Hz, H-3), 4.46 (1H, dd,
J3,4=4.2 Hz, J4,5=4.4 Hz, H-4), 4.37 (1H, dd,
J5,6a=4.6 Hz, J6a,6b=11.9 Hz, H-6a), 4.21 (1H, dd,
J5,6b=7.2 Hz, J6a,6b=11.9 Hz, H-6b), 2.16, 2.14, 2.10,
2.05 (each 3H, s, 4ÂOAc).
J5 ,6 b=5.5 Hz, H-50), 5.27 (1H, s, H-1), 5.09 (1H, s, H-
0
0
0
10), 4.99 (1H, dd, J1 ,2 =1.2 Hz, J2 ,3 =1.5 Hz,0 H-2 ),
0
0
0
0
0
0
0
0
4.94 (1H, dd, J2 ,3 =1.5 Hz, J3 ,4 =5.6 Hz, H-3 ), 4.59
(1H, dd, J3,4=4.9 Hz, J4,5=3.6 Hz, H-4), 4.18 (1H, dd,
J5 ,6 a=6.8 Hz, J6 a,6 b=11.9 Hz, H-60a), 4.05 (1H, dd,
J5,6a=5.5 Hz, J6a,6b=10.5 Hz, H-6a), 3.88 (1H, dd,
J5,6b=6.8 Hz, J6a,6b=10.5 Hz, H-6b), 2.10, 2.02, 1.99,
1.95 (4ÂOCH3).
0
0
0
0
1-Deoxy-1-thiocresyl-2,3,5,6-tetra-O-acetyl-ꢀ-D-galacto-
furanoside (14). d-(+)-Galactofuranose pentaacetate (1)
(4.75 g, 12.17 mmol) was dissolved in dry CH2Cl2
(60 mL) and cooled to 0 ꢀC, and p-thiocresol (1.77 gm,
14.6 mmol) was then added. After the mixture was stir-
Octyl 6-O-(2,3,5,6-tetra-O-acetyl-ꢀ-D-galactofuranosyl)-
2,3,5 - tri - O - benzyl - ꢀ - D - galactofuranoside (16). The
synthesis of 16, starting from donor 14 (1.44 g,
3.42 mmol) and acceptor 6c (1.60 g, 2.85 mmol), was
carried out as described for compound 15 (reaction
time=15 min). Column chromatography (cyclohexane/
EtOAc 5:1) gave the expected product as colorless oil
(2.26 g, 89%). Rf=0.49 (cyclohexane/EtOAc 1:1).
FABMS (LiCl) m/e 899 [M+Li]+. Anal. calcd for
C49H64O15: C, 65.90; H, 7.22. Found C, 65.36; H, 7.11.
1H NMR (300 MHz, CDCl3): d 7.36–7.20 (15H, m,
ꢂ
red for 20 min under argon, BF3 Et2O (3.85 mL,
30.43 mmol) was added dropwise. The reaction was
neutralized after 30 min with Et3N (4.4 mL,
30.43 mmol), diluted with CH2Cl2, and then washed
with water (50 mL) and brine (50 mL). The solution was
dried over Na2SO4 and evaporated. The crude product
was purified by column chromatography (cyclohexane/
EtOAc 3:1) to give 14 (5.13 g, 93%) as a clear syrup.
Rf=0.48 (cyclohexane/EtOAc 1:1). FAB-MS (LiCl):
461.2 (M+Li)+. Anal. calcd for C21H26O9S: C, 55.49;
H, 5.77. Found C, 55.20; H, 5.76. 1H NMR: d 7.40–7.36
(2H, d, aromatic), 7.14–7.11 (2H, d, aromatic), 5.43
(1H, d, J1,2=2.5 Hz, H-1), 5.39 (1H, ddd, J4,5=3.8 Hz,
J5,6a=4.6 Hz, J5,6b=7.0 Hz, H-5), 5.21 (1H, dd,
J1,2=2.5 Hz, J2,3=2.7 Hz, H-2), 5.07 (1H, ddd,
J1,3=0.6 Hz, J2,3=2.7 Hz, J3,4=6.1 Hz, H-3), 4.47 (1H,
ddd, J1,4=0.5 Hz, J4,5=3.8 Hz, J3,4=6.1 Hz, H-4), 4.32
(1H, dd, J5,6a=4.6 Hz, J6a,6b=11.9 Hz, H-6a), 4.18 (1H,
dd, J5,6b=7.0 Hz, J6a,6b=11.9 Hz, H-6b), 2.34 (3H, s,
CH3), 2.13, 2.11, 2.10, 2.05 (4ÂOAc). 13C NMR: d
170.32, 169.84, 169.76, 169.45 (4ÂOAc), 138.13, 132.82,
0
0
0
0
aromatic), 5.37 (1H, ddd, J4 ,5 =3.9 Hz, J5 ,6 a=4.2 Hz,
J5 ,6 b=7.3 Hz, H-50), 5.08 (1H, dd0, J1 ,2 =0.7 Hz,
0
0
0
0
0
J1,2=1.6 Hz, H-1), 4.99 (1H, ddd, J1 ,3 =0.4 Hz,
0
0
J2 ,3 =2.0 Hz, H-2 ), 5.06 (1H, s, H-1 ), 5.04 (1H, d,
0
0
0
0
0
0
0
J2 ,3 =2.0 Hz, J3 ,4 =5.7 Hz, H-3 ), 4.70, 4.58, 4.52 (each
1H, d, J=11.8 Hz, 3ÂCH2–aromatic) 4.48 (2H, d,
J=11.8 Hz, 3ÂCH2-aromatic), 4.31 (1H, d, J=11.8 Hz,
0
0
3ÂCH2-aromatic), 4.28 (1H, dd, J5 ,6 a=4.2 Hz,
J6 a,6 b=11.9 Hz, H-60a), 4.23 (1H, dd, J4 ,5 =3.9 Hz,
0
0
0
0
J3 ,4 =5.7 Hz, H-40), 4.19 (1H, dd, J5 ,6 b=7.3 Hz,
0
0
0
0
J6 a,6 b=11.9 Hz, H-60b), 4.07 (1H, dd, J4,5=3.0 Hz,
J3,4=6.7 Hz, H-4), 4.00 (1H, dd, J2,3=3.3 Hz,
J3,4=6.7 Hz, H-3), 3.97 (1H, dd, J1,2=1.6 Hz,
J2,3=3.3 Hz, H-2), 3.85 (1H, dd, J5,6a=4.1 Hz,
0
0

A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3129–3143
3139
J6a,6b=9.9 Hz, H-6a), 3.76 (1H, ddd, J4,5=3.0 Hz,
J5,6a=4.1 Hz, J5,6b=7.3 Hz, H-5), 3.70–3.62 (1H, m, O-
CH2), 3.66 (1H, dd, J5,6b=7.3 Hz, J6a,6b=9.9 Hz, H-6b),
3.38 (1H, m, OCH2), 2.12, 2.08, 2.05, 2.03 (each 3H, s,
4ÂOAc), 1.59–1.54 (2H, m, CH2), 1.27 (10H, br s,
5ÂCH2), 0.90–0.85 (3H, m, CH3). 13C NMR (75 MHz,
CDCl3): d 170.36, 169.92, 169.88, 169.42 (4ÂC¼O),
138.22, 137.78, 137.59 (4ÂC–aromatic), 128.32, 128.24,
128.19, 128.17, 127.87, 127.81, 127.72, 127.66, 127.57
(CH–aromatic), 105.97 (C-1), 105.73 (C-10), 88.30 (C-2),
82.59 (C-3), 81.07 (C-20), 80.38 (C-4), 80.15 (C-40), 76.48
(C-30), 76.18 (C-5), 73.37, 71.94, 71.79 (3ÂCH2-aro-
matic), 69.33 (C-50), 68.07 (C-6), 67.72 (OCH2), 62.61
(C-60), 31.76, 29.41, 29.33, 29.21, 26.10, 22.59 (6ÂCH2),
20.77, 20.66, 20.60, 20.56 (4ÂOAc), 14.04 (CH3).
127.99, 127.91, 127.83, 127.80 (CH, aromatic), 108.25
(C-10, JCH=176.9 Hz), 105.98 (C-1), 88.06 (C-2), 87.46
1
(C-40), 82.89 (C-3), 80.79 (C-4), 78.70 (C-20), 78.46 (C-
30), 75.95 (C-5), 72.79, 72.05, 71.97 (3ÂCH2–aromatic),
70.67 (C-50), 67.85 (OCH2), 67.00 (C-6), 63.94 (C-60),
31.82, 29.43, 29.36, 29.27, 26.14, 22.65 (6ÂCH2), 14.10
(CH3).
Octyl 6-O-(ꢀ-D-galactofuranosyl)-2,3,5-tri-O-methyl-ꢀ-
D-galactofuranoside (19). Prepared from compound 17
(236 mg, 0.36 mmol) by the procedure described for 18.
Flash chromatography (CHCl3/MeOH 9:1) gave 19 as a
colorless oil (155 mg, 88%). Rf=0.45 (CHCl3/MeOH,
5:1). FAB-MS (LiCl) m/e 503 [M+Li]+. Anal. calcd for
C23H44O11ꢂ 0.75H2O: C, 54.18; H, 8.70. Found C, 54.14;
1
H, 8.54. H NMR (600 MHz, CDCl3): d 5.02 (1H, s, H-
Octyl 6-O-(2,3,5,6-tetra-O-acetyl-ꢀ-D-galactofuranosyl)-
2,3,5-tri-O-methyl-ꢀ-D -galacto-furanoside (17). The
synthesis 17, starting from donor 14 (396 mg, 0.94 mmol)
and acceptor 6d (260 mg, 0.78 mmol), by the procedure
described for compound 15 (reaction time=30 min).
Column chromatography (cyclohexane/EtOAc 3:1)
gave disaccharide 17 as colorless oil (369 mg, 90%).
Rf=0.40 (cyclohexane/EtOAc 1:1). FAB-MS (LiCl) m/e
671 [M+Li]+. Anal. calcd for C31H52O15: C, 56.01; H,
7.88. Found C, 56.05; H, 7.76. 1H NMR (300 MHz,
10), 4.97 (1H, s, H-1), 4.42 (1H, br s, OH), 4.14 (1H, t,
0
dd, J3,4=7.0 Hz, J4,5=4.3 Hz, H-4), 4.01 (1H, d,
0
0
0
0
0
J3 ,4 =J4 ,5 =4.7 Hz, H-4 ), 4.05 (1H, m, H-3 ), 4.02 (1H,
0
0
0
0
0
J2 ,3 =2.4 Hz, H-2 ), 3.91 (1H, ddd, J4 ,5 =4.7 Hz,
0
J5 ,6 a=6.3 Hz, J5 ,6 b=4.0 Hz, H-50), 3.90 (1H, dd,
J5,6a=4.1 Hz, J6a,6b=10.9 Hz, H-6a), 3.86 (1H, br s,
0
0
0
0
0
0
0
OH), 3.79 (1H, dd, J5 ,6 a=6.3 Hz, J6 a,6 b=11.2 Hz, H-
60a), 3.73 (1H, dd, J5 ,6 b=4.0 Hz, J6 a,6 b=11.2 Hz, H-
60b), 3.69 (1H, dd, J1,2=1.0 Hz, J2,3=2.9 Hz, H-2), 3.67
(1H, m, OCH2), 3.65 (1H, dd, J2,3=2.9 Hz,
J3,4=7.0 Hz, H-3), 3.58 (1H, dd, J5,6b=5.1 Hz,
J6a,6b=10.9 Hz, H-6b), 3.50 (3H, s, OMe), 3.50 (1H,
ddd, J4,5=4.3 Hz, J5,6a=4.1 Hz, J5,6b=5.1 Hz, H-5),
3.42 (3H, s, OMe), 3.41 (1H, m, OCH2), 3.40 (3H, s,
OMe), 1.95 (1H, br s, OH), 1.57 (2H, m, CH2), 1.30 (10,
m, 5ÂOCH2), 0.88 (3H, m, CH3). 13C NMR (75 MHz,
CDCl3): 108.17 (C-10), 105.40 (C-1), 89.47 (C-2), 85.46
(C-40), 85.35 (C-3), 80.71 (C-4), 79.96 (C-30), 79.19 (C-
5), 78.06 (C-20), 71.05 (C-50), 67.66 (OCH2), 66.70 (C-6),
63.99 (C-60), 58.87, 57.93, 57.41 (3ÂOCH3), 31.76,
29.30, 29.18, 26.02, 22.59 (6ÂCH2), 14.05 (CH3).
0
0
0
0
0
0
0
0
CDCl3): d 5.38 (1H, ddd, J4 ,5 =3.9 Hz, J5 ,6 a=4.2 Hz,
J5 ,6 a=7.3 Hz, H-50), 5.08 (2H, br s, H-10, H-20), 5.01
0
0
0
(1H, dd, J1 ,3 =0.7 Hz, J2 ,3 =2.2 Hz, J3 ,4 =5.7 Hz, H-
0
0
0
0
0
30), 4.99 (1H, br s, H-1), 4.33 (1H, dd, J5 ,6 a=4.2 Hz,
0
0
J6 a,6 b=11.9 Hz, H-60a), 4.27 (1H, dd, J4 ,5 =3.9 Hz,
0
0
0
0
J3 ,4 =5.7 Hz, H-40), 4.21 (1H, dd, J5 ,6 b=7.3 Hz,
0
0
0
0
J6 a,6 b=11.9 Hz, H-60b), 3.99 (1H, m, H-3), 3.82 (1H,
dd, J5,6a=4.2 Hz, J6a,6b=10.5 Hz, H-6a), 3.71–3.64 (3H,
m, H-2, H-4, OCH2), 3.63 (1H, dd, J5,6b=7.2 Hz,
J6a,6b=10.5 Hz, H-6b), 3.54 (1H, m, H-5), 3.53 (3H, s,
5-OCH3), 3.41 (1H, m, OCH2), 3.42, 3.40 (each 3H, s 2-
OCH3, 3-OCH3), 2.13, 2.11, 2.09, 2.06 (each 3H, s,
4ÂOAc), 1.56 (2H, m, CH2), 1.27 (10H, m, 5ÂCH2),
0.90–0.85 (3H, m, CH3).
0
0
Octyl 6-O-(ꢀ-D-galactofuranosyl)-ꢀ-D-galactofuranoside
(20)
Method A. To a solution of disaccharide 15 (150 mg,
0.16 mmol) in dry methanol (10 mL) was added 7 N
NH3/MeOH (5 mL) dropwise, and the reaction mixture
was stirred at room temperature for 6 h. Concentration
in vacuo, and flash chromatography (CHCl3/MeOH
5:1) gave 20 as a colorless oil (67 mg, 92%). Rf=0.35
(CHCl3/MeOH 5:1). FAB-MS (LiCl) m/e 461
[M+Li]+. Anal. calcd for C20H38O11ꢂ 1/2 H2O: C,
Octyl 6-O-(ꢀ-D-galactofuranosyl)-2,3,5-tri-O-benzyl-ꢀ-
D-galactofuranoside (18). To a solution of disaccharide
16 (1.70 g, 1.91 mmol) in dry methanol (25 mL) was
added 7 N NH3/MeOH (5 mL) dropwise, and the reac-
tion mixture was stirred at room temperature overnight.
Concentration in vacuo to a syrup, followed by flash
chromatography (CHCl3/MeOH 10:1), gave 18 as a low
melting solid (1.11 g, 81%). Rf=0.51 (CHCl3/MeOH,
7:1). Mp 42–44 ꢀC. FAB-MS (LiCl) m/e 731 [M+Li]+.
Anal. calcd for C23H42O11.0.5H2O: C, 67.08; H, 7.83.
51.82; H, 8.26. Found C, 51.20; H, 8.58. 1H NMR
0
0
0
(600 MHz, D2O): d 4.93 (1H, d, J1 ,2 =1.3 Hz, H-1 ),
4.85 (1H, d, J1,2=1.6 Hz, H-1), 4.02 (1H, dd,
J1,2=1.6 Hz, J2,3=3.0 Hz, H-2), 3.97 (2H, m, H-3, H-
1
Found C, 66.94; H, 7.85. H NMR (300 MHz, CDCl3):
d 7.36–7.20 (m, aromatic), 5.03 (1H, s, H-1), 4.95 (1H, s,
0
30), 3.94 (1H, dd, J1 ,2 =1.3 Hz, J2 ,3 =5.4 Hz,0 H-2 ),
0
0
0
0
H-10), 4.48 (6H, m, 3ÂCH2–aromatic), 4.11 (1H, dd,
3.90 (1H, dd, J3 ,4 =6.0 Hz, J4 ,5 =4.2 Hz, H-4 ), 3.88
(1H, ddd, J4,5=4.2 Hz, J5,6a=3.6 Hz, J5,6b=7.8 Hz, H-
5), 3.82 (1H, dd, J3,4=6.0 Hz, J4,5=4.2 Hz, H-4), 3.74
(2H, m, H-50, H-6a), 3.62 (1H, m, OCH2), 3.61 (1H, dd,
0
0
0
0
0
J3,4=7.1, J4,5=3.4 Hz, H-4), 4.06 (1H, dd, J3 ,4 =2.4,
0
0
0
0
0
0
J4 ,5 =2.2 Hz, H-4 ), 3.97 (3H, m, H-2 , H-3, H-3 ), 3.94
(1H, d, J1,2=0.9 Hz, H-2), 3.88 (1H, dd, J5,6a=3.7,
J6a,6b=10.3 Hz, H-6a), 3.81 (1H, m, H-50), 3.62 (6H, m,
H-5, H-6b, H-60a, H-60b, OCH2, OH), 3.37 (2H, m,
OCH2, OH), 3.24, 2.23 (each 1H, br s, OH), 1.57 (2H,
m, CH2), 1.27 (10H, m, 5ÂCH2), 0.88 (3H, m, CH3).
13C NMR (75 MHz, CDCl3): d 137.88, 137.62, 137.44
(C, aromatic), 128.44, 128.38, 128.35, 128.08, 128.06,
J5 ,6 a=4.8 Hz, J6 a,6 b=11.8 Hz, H-60a), 3.56 (1H, dd,
0
0
0
0
J5 ,6 a=7.3 Hz, J6 a,6 b=11.8 Hz, H-60b), 3.53 (1H, dd,
J5,6b=7.8 Hz, J6a,6b=10.8 Hz, H-6b), 3.36 (1H, m,
OCH2), 1.50 (2H, m, CH2), 1.21 (10H, m, 5ÂCH2), 0.79
(3H, m, CH3). 13C NMR (75 MHz, D2O): 108.25 (C-10),
107.51 (C-1), 83.31, 83.07 (C-4, C-40), 81.43, 81.24 (C-2,
0
0
0
0

3140
A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3129–3143
C-20), 77.08 (C-3, C-30), 71.09 (C-50), 69.87 (C-6), 69.74
(C-5), 68.55 (OCH2), 63.10 (C-60), 31.88, 29.38, 29.28,
25.98, 22.67 (6ÂCH2), 13.99 (CH3).
3H, s, 4ÂOAc), 1.56 (2H, m, CH2), 1.27 (10H, br s,
5ÂCH2), 0.89–0.85 (3H, m, CH3). 13C NMR (75 MHz,
CDCl3): d 170.44, 170.05, 170.04 (C¼O), 138.11, 137.88,
137.45 (C, aromatic), 128.40, 128.38, 128.31, 128.14,
128.08, 127.93, 127.72, 127.64, 127.53, 127.49 (CH,
aromatic), 105.90(C-10), 105.33(C-1), 88.43 (C-40), 83.65
(C-20), 81.05 (C-4), 80.59 (C-30), 80.09 (C-2), 77.20 (C-
3), 74.06 (C-50), 73.39, 72.15, 72.09 (3ÂCH2-aromatic),
71.17 (C-60), 69.51 (C-5), 67.79 (O-CH2), 63.00 (C-6),
31.85, 29.55, 29.40, 29.28, 26.19, 22.66 (6ÂCH2), 20.81,
20.72, 20.63, 20.53 (CH3, OAc), 14.11 (CH3).
Method B. To a methanol solution (15 mL) of dis-
accharide 18 (750 mg, 1.00 mmol) was added Pd/C
(10%, 300 mg), and the mixture was stirred at room
temperature under hydrogen (130 mL, 24 h). The reac-
tion was filtered through a Celite pad and concentrated
to give viscous, colorless oil. Flash chromatography
(CHCl3/MeOH 5:1) gave 20 as low melting hygroscopic
solid (406 mg, 86%).
Octyl 5-O-(2,3,5,6-tetra-O-acetyl-ꢀ-D-galactofuranosyl)-
2,3,6-tri-O-methyl-ꢀ-D -galacto-furanoside (23). Dis-
accharide 23 was synthesized starting with donor 14
(295 mg, 0.70 mmol) and acceptor 10b (243 mg,
0.58 mmol) by the procedure described for compound
15 (reaction time=30 min). Column chromatography
(cyclohexane/EtOAc 4:1) gave 23 as a colorless oil
(478 mg, 99%). Rf=0.40 (cyclohexane/EtOAc 1:1).
FAB-MS (LiCl) m/e 671 [M+Li]+. Anal. calcd for
Octyl 5-O-(2,3,5,6-tetra-O-acetyl-ꢀ-D-galactofuranosyl)-
2,3,6-tri-O-benzoyl-ꢀ-D-galactofuranoside (21). Com-
pound 21 was prepared starting with 12 (319 mg,
0.76 mmol) and acceptor 6b (380 mg, 0.63 mmol) by the
method described for compound 15 (reaction time
=30 min). Chromatography (cyclohexane/EtOAc 2:1)
gave the 21 as a colorless oil (695 mg, 92%). Rf=0.60
(cyclohexane/EtOAc 1:2). FAB-MS (LiCl) m/e 941
[M+Li]+. Anal. calcd for C49H58O18ꢂ 1/2 H2O: C,
62.34; H, 6.30. Found C, 62.27; H, 6.28. 1H NMR
(300 MHz, CDCl3): 8.07 (6H, m, aromatic), 7.51 (9H,
1
4
C31H52O15. H2O: C, 55.63; H, 7.91. Found C, 55.80;
1
H, 7.69. H NMR (300 MHz, CDCl3): d 5.42 (1H, ddd,
J4 ,5 =3.6 Hz, J5 ,6 a=4.1 Hz, J5 ,6 b=7.6 Hz, H-50), 5.42
0
0
0
0
0
0
(1H, s, H-10), 5.17 (1H, d, J2 ,3 =1.5 Hz,0H-2 ), 4.98 (1H,
0
0
0
0 0 0 0
dd, J2 ,3 =1.5 Hz, J3 ,4 =5.3 Hz, H-3 ), 4.97 (1H, d,
m, aromatic), 5.70 (1H, d, J3,4=5.1 Hz, H-3), 5.50 (1H,
0
0
d, J1 ,2 =1.0 Hz, H-1 ), 5.50 (1H, s, H-2), 5.32 (1H, ddd,
0
0
J1,2=1.3 Hz, H-1), 4.39 (1H, dd, J4 ,5 =3.6 Hz,
0
J4 ,5 =3.8 Hz, J5 ,6 a=4.4 Hz, J5 ,6 b=7.4 Hz, H-50), 5.24
0
0
0
0
0
0
0
0
0
0
0
J3 ,4 =5.3 Hz, H-4 ), 4.37 (1H, dd, J5 ,6 a=4.1 Hz,
(1H, s, H-1), 5.23 (1H, dd, J1 ,2 =1.0 Hz, J2 ,3 =2.4 Hz,
J6 a,6 b=11.9 Hz, H-60a), 4.20 (1H, dd, J5 ,6 b=7.6 Hz,
0
0
0
0
0
0
0
0
H-20), 4.99 (1H, dd, J2 ,3 =2.4 Hz, J3 ,4 =5.9 Hz, H-3 ),
4.68 (1H, dd, J5,6a=4.9 Hz, J6a,6b=11.5 Hz, H-6a), 4.63
(1H, dd, J5,6b=5.9 Hz, J6a,6b=11.5 Hz, H-6a), 4.58 (1H,
ddd, J4,5=3.4 Hz, J5,6a=4.9 Hz, J5,6b=5.9 Hz, H-5),
4.46 (1H, dd, J3,4=5.1 Hz, J4,5=3.4 H0z, H-4), 4.42 (1H,
J6 a,6 b=11.9 Hz, H-60b), 4.01 (1H, ddd, J4,5=3.5 Hz,
J5,6a=7.0 Hz, J5,6b=4.4 Hz, H-5), 3.97 (1H, dd,
J4,5=3.5 Hz, J3,4=6.9 Hz, H-4), 3.74 (1H, dd, J2,3=3.1
Hz, J3,4=6.9 Hz, H-3), 3.68 (1H, dd, J1,2=1.3 Hz,
J2,3=3.1 Hz, H-2), 3.66 (1H, m, OCH2), 3.61 (1H, dd,
J5,6a=7.0 Hz, J6a,6b=10.4 Hz, H-6a), 3.56 (1H, dd,
J5,6b=4.4 Hz, J6a,6b=10.4 Hz, H-6b), 3.41 (1H, m,
OCH2), 3.40, 3.38, 3.35 (each 3H, s, 3ÂOCH3), 2.13,
2.11, 2.09, 2.05 (each 3H, s, 3xOAc), 1.57 (2H, m, CH2),
1.28 (10H, m, 5ÂCH2), 0.88 (3H, m, CH3). 13C NMR
(75 MHz, CDCl3): d 170.44, 170.02, 169.96, 169.29
(4ÂC¼O), 105.35 (C-1), 104.95 (C-10), 89.96 (C-2),
84.90 (C-3), 80.97, 80.52, 80.10 (C-4, C-20, C-40), 76.62
(C-30), 73.62 (C-5), 73.26 (C-6), 69.41 (C-50), 67.61
(OCH2), 62.84 (C-60), 59.06, 57.63, 57.39 (3ÂOCH3),
31.74, 29.41, 29.26, 29.16, 26.04, 22.26 (6ÂCH2), 20.74,
20.70, 20.58, 20.49 (4ÂOAc), 14.01 (CH3).
0
0
0
0
0
0
0
0
0
0
0
dd, J3 ,4 =5.9 Hz, J4 ,5 =3.8 Hz, H-4 ), 4.30 (1H, dd,
J5 ,6 a=4.4 Hz, J6 a,6 b=11.9 Hz, H-60a), 4.14 (1H, dd,
0
0
0
0
J5 ,6 b=7.4 Hz, J6 a,6 b=11.9 Hz, H-60b), 3.73 (1H, m,
OCH2), 3.51 (1H, m, OCH2), 2.04, 2.00, 1.96, 1.78 (each
3H, s, 4ÂOCH3), 1.62 (2H, m, CH2), 1.25 (10H, m,
5ÂCH2), 0.86 (3H, m, CH3).
0
0
0
0
Octyl 5-O-(2,3,5,6-tetra-O-acetyl-ꢀ-D-galactofuranosyl)-
2,3,6 - tri - O - benzyl - ꢀ - D - galactofuranoside (22). Dis-
accharide 22 was synthesized starting with donor 14
(540 mg, 1.28 mmol) and acceptor 10a (600 mg,
1.07 mmol) by the procedure described for compound
15 (reaction time=15 min). Column chromatography
(cyclohexane/EtOAc 5:1) gave 22 as a colorless oil
(933 mg, 98%). Rf=0.55 (cyclohexane/EtOAc 1:1).
FAB-MS (LiCl) m/e 899 [M+Li]+. Anal. calcd for
C49H64O15: C, 65.98; H, 7.17. Found C, 65.56; H, 7.43.
1H NMR (300 MHz, CDCl3): d 7.30 (15H, m, aro-
Octyl-5-O-(ꢀ-D-galactofuranosyl)-2,3,6-tri-O-benzyl-ꢀ-D
- galactofuranoside (24). To a solution of disaccharide
22 (1.00 g, 0.11 mmol) in dry methanol (15 mL) was
added 7 N NH3/MeOH (3 mL) dropwise and the reac-
tion mixture was stirred at room temperature for 3 h.
Concentration in vacuo and flash chromatography
(CHCl3/MeOH 10:1) gave 24 as a colorless oil (810 mg,
99%). Rf=0.53 (CHCl3/MeOH, 7:1). FAB-MS (LiCl)
m/e 731 [M+Li]+. Anal. calcd for C41H56O11ꢂ H2O: C,
66.29; H, 7.87. Found C, 66.59; H, 7.89. 1H NMR
(600 MHz, CDCl3): d 7.37–7.21 (15H, m, aromatic),
5.34 (1H, s, H-10), 4.98 (1H, s, H-1), 4.58–4.35 (6H, m,
3ÂCH2–aromatic), 4.10 (1H, br s, H-3), 4.04 (3H, m, H-
5, H-30, H-40), 4.01 (1H, br s, H-2), 3.97 (1H, d, 1.8 Hz,
H-20), 3.83 (2H, m, H-4, H-50), 3.67 (1H, dd,
matic), 5.47 (1H, s, H-10), 5.32 (1H, ddd, J4 ,5 =4.2 Hz,
0
0
J5 ,6 a=3.7 Hz, J5 ,6 b=7.5 Hz, H-50), 5.19 (1H, d,
0
0
0
0
0
0
J1,2=1.6 Hz, H-1), 5.03 (1H, d, J2 ,3 =1.5 Hz, H-2 ),
0
0
0
0
0
0
4.96 (1H, dd, J2 ,3 =1.5 Hz, J3 ,4 =5.4 Hz, H-3 ), 4.59–
4.47 (6H, m, 3ÂCH2–aromatic), 3.52 (1H, dd,
J
3a,4a=5.4 Hz, H-40), 4.31 (1H, dd,
0
J4 ,5 =4.2,
0
J5 ,6 a=3.7 Hz, J6 a,6 b=11.9 Hz, H-60a), 4.13 (1H, dd,
0
0
0
0
J5 ,6 b=7.5 Hz, J6 a,6 b=11.9 Hz, H-60b), 4.12–4.02 (4H,
m, H-3, H-4, H-5), 3.99 (1H, dd, J1,2=1.6 Hz,
J2,3=3.1 Hz, H-2), 3.72–3.61 (3H, m, H2-6, OCH2),
3.42–3.34 (1H, m, OCH2), 2.07, 2.04, 1.96, 1,94 (each
0
0
0
0
J5 ,6 a=6.1 Hz, J6 a,6 b=11.3 Hz, H-60a), 3.57 (4H, m, H-
0
0
0
0

A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3129–3143
3141
6a, H-6b, H-60b, OCH2), 3.33 (1H, m, OCH2), 1.54 (2H,
m, CH2), 1.29 (10H, m, CH2), 0.87 (3H, m, CH3). 13C
NMR (75 MHz, CDCl3): d 137.74, 137.47, 137.05 (C,
aromatic), 128.52, 128.41, 128.37, 128.20, 128.12,
127.81, 127.72, 127.65 (CH, aromatic), 106.83 (C-10),
105.60 (C-1), 87.90 (C-2), 87.36 (C-40), 84.56 (C-3),
80.76 (C-4), 78.97, 78.79 (C-20, C-30), 73.66 (C-5), 73.44,
72.14, 71.89 (3ÂCH2-aromatic), 70.92 (C-50), 70.00 (C-
6), 67.66 (OCH2), 64.12 (C-60), 31.82, 29.42, 29.36,
29.25, 26.10, 22.66 (6ÂCH2), 14.10 (CH3).
62.07 (C-60), 31.78, 29.34, 29.22, 29.16, 25.88, 22.60
(6ÂCH2), 13.91 (CH3).
Method B. To a methanol solution (15 mL) of dis-
accharide 24 (725 mg, 1.00 mmol) was added Pd/C
(10%, 300 mg), and the mixture was stirred at room
temperature under hydrogen (105 mL, 10 h). Filtration
through a Celite pad and concentration in vacuo gave a
viscous, colorless oil. Flash chromatography (CHCl3/
MeOH 5:1) gave 26 as low melting, hygroscopic solid
(410 mg, 90%).
Octyl 5-O-(ꢀ-D-galactofuranosyl)-2,3,6-tri-O-methyl-ꢀ-
D-galactofuranoside (25). This product was prepared
from compound 23 (220 mg, 0.33 mmol) by the proce-
dure described for compound 24. Flash chromato-
graphy (CHCl3/MeOH 95:5) gave 25 as a colorless oil
(152 mg, 92%). Rf=0.51 (CHCl3/MeOH, 9:1). FABMS
(LiCl) m/e 503 [M+Li]+. C23H44O11.1.33 H2O: C,
53.16; H, 8.99. Found C, 53.09; H, 8.73. 1H NMR
Biological studies
Method for in-vitro activity. Assays were performed
using a colorimetric microdilution broth assay by the
previously reported method.21 Each compound was
assayed in duplicate at either 2 log10 or 4 log10 dilutions
consisting of 1.28 and 12.8 or 0.128, 1.28, 12.8 and
128 mg/mL respectively. The minimum inhibitory con-
centration (MIC) was recorded as the lowest drug con-
centration that inhibited the growth completely.
(300MHz, CDCl3): d 5.32 (1H, s, H-10), 4.98 (1H, s, H-1),
0
4.15 (1H, m, H-40), 4.07 (1H, d, J1 ,2 =1.0 Hz, H-2 ),
0
0
4.05 (1H, m, H-5), 4.03 (1H, m, H-30), 3.97 (1H, m, H-
4), 3.91 (1H, m, H-50), 3.76 (1H, dd, J5 ,6 a=6.2 Hz,
J6 a,6 b=11.4 Hz, H-60a), 3.75 (1H, dd, J5 ,6 b=4.5 Hz,
0
0
0
0
0
0
J6 a,6 b=11.4 Hz, H-60b), 3.69 (1H, d, J1,2=1.0 Hz, H-2),
3.61 (4H, m, H-3, H2-6, OCH2), 3.40, 3.39, 3.37 (each
3H, s, OCH3), 3.39 (1H, m, OCH2), 1.56 (2H, m, CH2),
1.27 (1H, m, 5ÂCH2), 0.88 (3H, m, CH3). 13C NMR
(75 MHz, CDCl3): d 107.17 (C-10), 104.96 (C-1), 89.16
(C-2), 87.44 (C-40), 86.49 (C-3), 81.03 (C-4), 78.85 (C-
20), 78.71 (C-30), 73.58 (C-5), 72.71 (C-6), 71.16 (C-50),
67.64 (OCH2), 64.02 (C-60), 59.19, 57.84, 57.55 (3
ÂOCH3), 31.77, 29.36, 29.29, 29.18, 26.01, 22.60
(6ÂCH2), 14.05 (CH3).
Bacterial strains and growth conditions. M. smegmatis
mc2155 was a generous gift from W. R. Jacobs, Albert
Einstein College of Medicine, Bronx, New York.23
Liquid cultures of M. smegmatis were grown at 37 ꢀC in
Luria Bertoni (LB) broth medium (Difco) supplemented
with 0.05% Tween 80, biomass harvested, washed with
phosphate buffered saline (PBS) and stored at À20 ꢀC
until further use.
0
0
Galactosyl transferases
Preparation of membrane and cell wall enzyme frac-
tions. M. smegmatis cells (10 g wet weight) were washed
and re-suspended in 30 mL of buffer A, containing
50 mM MOPS (adjusted to pH 8.0 with KOH), 5 mM b-
mercaptoethanol and 10 mM MgCl2 at 4 ꢀC and sub-
jected to probe sonication (Soniprep 150, MSE Sanyo
Gallenkamp, Crawley, Sussex, UK; 1 cm probe) for a
total time of 10 min in 60 s pulses and 90 s cooling
intervals between pulses. The sonicate was centrifuged at
27,000g for 60 min at 4 ꢀC. The resulting mycobacterial
cell wall pellets were re-suspended in buffer A. Percoll
(Pharmacia, Sweden) was added to yield a 60% sus-
pension and centrifuged at 27,000g for 1 h at 4 ꢀC. The
upper, particulate diffuse cell wall enzymatically active
(P60) band was collected and washed three times with
buffer A and re-suspended in buffer A at a final protein
concentration of 10 mg/mL. Membrane fractions were
obtained by centrifugation of the 27,000g supernatant
at 100,000g for 1 h at 4 ꢀC. The supernatant was care-
fully removed and the membranes gently re-suspended
in buffer A at a protein concentration of 20 mg/mL.
Protein concentrations were determined using the BCA
Protein Assay Reagent kit (Pierce Europe, Oud-Beijer-
land, The Netherlands).
Octyl 5-O-(ꢀ-D-galactofuranosyl)-ꢀ-D-galactofuranoside
(26)
Method A. To a solution of disaccharide 21 (200 mg,
0.21 mmol) in dry methanol (15 mL) was added 7 N
NH3/MeOH (5 mL) and the reaction mixture was stir-
red at room temperature for 6 h. Concentration in
vacuo and flash chromatography (CHCl3/MeOH 5:1)
gave 24 as a colorless oil (96 mg, 99%). Rf=0.45
(CHCl3/MeOH, 2:1). FAB-MS (LiCl) m/e 461
[M+Li]+. Anal. calcd for C20H38O11.H2O: C, 50.84; H,
8.10. Found C, 50.89; H, 8.70. 1H NMR (600 MHz,
0
0
0
CDCl3): d 5.14 (1H, d, J1 ,2 =1.8 Hz, H-1 ), 4.81 (1H, d,
J1,2=1.8 Hz, H-1), 4.07 (1H, dd, J1,2=1.8 Hz,
J2,3=3.6 Hz, H-2), 4.02 (1H, dd, J3,4=6.6 Hz,
0
J4,5=4.2 Hz, H-4), 3.97 (1H, dd, J3 ,4 =6.6 Hz,
0
J4 ,5 =4.2 Hz, H-40), 3.96 (1H, dd, J2 ,3 =4.2 Hz,
0
0
0
0
J3 ,4 =6.6 Hz, H-30), 3.93 (1H, dd, J1 ,2 =1.8 Hz,
0
0
0
0
0
0
0
J2 ,3 =4.2 Hz, H-2 ), 3.86 (2H, m, H-3, H-5), 3.74 (1H,
ddd, J4 ,5 =4.2 Hz, J5 ,6 a=4.2 Hz, J5 ,6 b=7.2 Hz, H-50),
3.71 (1H, dd, J5,6a=7.3 Hz, J6a,6b=11.8 Hz, H-6a), 3.64
(1H, dd, J5,6b=4.0 Hz, J6a,6b=11.8 Hz, H-6b), 3.63 (1H,
0
0
0
0
0
0
dd, J5 ,6 a=4.2 Hz, J6 a,6 b=11.6 Hz, H-60a), 3.58 (1H,
0
0
0
0
dd, J5 ,6 b=7.2 Hz, J6 a,6 b=11.6 Hz, H-60b), 3.56 (1H,
m, OCH2), 3.37 (1H, m, OCH2), 1.50 (2H, m, CH2),
1.21 (10H, m, 5ÂCH2), 0.78 (3H, m, CH3). 13C NMR
(75 MHz, D2O): 107.71 (C-10), 107.42 (C-1), 82.81 (C-
40), 81.68 (C-4), 81.58 (C-2, C-20), 76.74 (C-3, C-30),
76.15 (C-5), 70.76 (C-50), 68.61 (OCH2), 63.09 (C-6),
0
0
0
0
Galactosyltransferase assay. Compounds 18–20 and
24–26, at a range of concentrations from 0.25 to 4.0 mM
(which were stored as 100 mM ethanol stocks), were

3142
A. K. Pathak et al. / Bioorg. Med. Chem. 9 (2001) 3129–3143
dried under a stream of argon in a microcentrifuge tube
(1.5 mL) and placed in a vacuum desiccator for 15 min
to remove any residual solvent. The compounds were
then re-suspended with the remaining constituents of
the galactosyltransferase assay in buffer A. The reaction
mixtures for assessing [14C]Gal incorporation consisted
of UDP-[U-14C]Gal (Amersham Pharmacia Biotech,
327 mCi/mmol, 0.25 mCi, 10 mL), ATP (1 mM, 5 mL),
NADH (100 mM, 8 mL), membranes (250 mg, 12.5 mL)
and the cell wall fraction (250 mg, 25 mL) in a final reac-
tion volume of 80 mL. The reaction mixtures were then
incubated at 37 ꢀC for 1 h. A CHCl3/CH3OH (1:1,
533 mL) solution was then added to the incubation tubes
and the entire contents centrifuged at 18,000g. The
supernatant was recovered and dried under a stream of
argon and re-suspended in C2H5OH/H2O (1:1, 1 mL)
and loaded onto a pre-equilibrated [C2H5OH/H2O (1:1)]
1 mL Whatman strong anion exchange (SAX) cartridge
which was washed with 3 mL of ethanol. The eluate was
dried and the resulting products partitioned between the
two phases arising from a mixture of n-butanol (3 mL)
and H2O (3 mL). The resulting organic phase was
recovered following centrifugation at 3,500g and the
aqueous phase was again extracted twice with 3 mL of
n-butanol saturated water, the pooled extracts were
back-washed twice with water saturated with n-butanol
(3 mL). The n-butanol-saturated water fraction was
dried and re-suspended in 200 mL of n-butanol. The
total cpm of radiolabeled material extractable into the
n-butanol phase was measured by scintillation count-
ing using 10% of the labeled material and 10 mL of
EcoScintA (National Diagnostics, Atlanta, USA). 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
compounds). Another 10% of the labeled material
was subjected to thin-layer chromatography (TLC)
in CHCl3/CH3OH/NH4OH/H2O (65:25:0.5:3.6) on
aluminium-backed Silica Gel 60 F254 plates (E. Merck,
Darmstadt, Germany). The autoradiograms were
obtained by exposing TLCs to X-ray film (Kodak
X-Omat) for 4–5 days. Competition based experiments
were performed by mixing compounds together at
various concentrations (20, 0.5 mM with 18 or 19 at 1.0,
1.5, 4.0, and 6.0 mM; 26, 0.5 mM with 24 or 25 at
1.0, 2.0, 4.0 and 6.0 mM) followed by thin-layer chro-
matography/autoradiography (Fig. 5A and B) as
described earlier to determine the extent of product
formation.
References and Notes
1. (a) World Health Organization. In Anti-tuberculosis Drug
Resistance in the World. The WHO/IUATLD Global Project
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Acknowledgements
The authors are thankful to Dr. J. M. Riordan for
NMR and Mr. M. D. Richardson for FAB-MS. This
work was supported by NIH/NIID grant R01AI45317.
600 MHz NMR spectra were supported by NCI grant
CA-13148 (the University of Alabama at Birmingham
UAB shared NMR Core Facility). GSB who is currently
a Lister Institute-Jenner Research Fellow acknowledges
support from The Medical Research Council (49343
and 49342).
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