Studies on n-octyl-5-(α-D-arabinofuranosyl)-β-D-galactofuranosides for mycobacterial glycosyltransferase activity

Article abstract of DOI:10.1016/S0968-0896(01)00343-1

The mycobacterial cell wall is a potential target for new drug development. Herein we report the preparation and activity of several n-octyl-5-(α-D-arabinofuranosyl)-β-D-galactofuranoside derivatives. A cell-free assay system has been utilized for determination of the ability of disaccharide analogues to act as arabinosyltransferase acceptors using [¹⁴C]-DPA as the glycosyl donor. In addition, 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). One of these disaccharides showed moderate activity against MTB. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing end showed better activity as compared to a totally deprotected disaccharide that more closely resembles the natural substrates in cell wall biosynthesis. Copyright

Full text of DOI:10.1016/S0968-0896(01)00343-1

Bioorganic & Medicinal Chemistry 10 (2002) 923–928
Studies on n-Octyl-5-(ꢀ-D-arabinofuranosyl)-ꢁ-D-
galactofuranosides for Mycobacterial Glycosyltransferase Activity
Ashish K. Pathak,a Vibha Pathak,a William J. Suling,b Sudagar S. Gurcha,c
Caroline B. Morehouse,c Gurdyal S. Besra,c Joseph A. Maddrya
and Robert C. Reynoldsa,*
aDepartment of Organic Chemistry, Southern Research Institute, PO Box 55305, Birmingham, AL 35255, USA
bDepartment of Biochemistry, Southern Research Institute, PO Box 55305, Birmingham, AL 35255, USA
cThe School of Microbiological, Immunological & Virological Sciences, The University of Newcastle upon Tyne,
The Medical School, Framlington Place, Newcastle upon Tyne NE24HH, UK
Received 12 September 2001; accepted 24 September 2001
Abstract—The mycobacterial cell wall is a potential target for new drug development. Herein we report the preparation and activity
of several n-octyl-5-(a-d-arabinofuranosyl)-b-d-galactofuranoside derivatives. A cell-free assay system has been utilized for deter-
mination of the ability of disaccharide analogues to act as arabinosyltransferase acceptors using [14C]-DPA as the glycosyl donor.
In addition, 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). One of these disaccharides showed moderate activity
against MTB. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing
end showed better activity as compared to a totally deprotected disaccharide that more closely resembles the natural substrates in
cell wall biosynthesis. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
enzymes offer targets for the development of new drugs
that will be highly active and selective for the tubercu-
losis bacillus.4
Tuberculosis (TB) has long been a cause of morbidity
and mortality worldwide. Over the past two decades,
there has been an increased interest in developing new
drugs to fight this deadly disease.1 This resurgence in
interest in fighting tuberculosis has resulted from several
factors including the growing incidence of multi-drug
resistant (MDR) forms of Mycobacterium tuberculosis
(MTB) as well as the prevalence of tuberculosis in the
AIDS community.2 The development of MDR forms of
tuberculosis and the publication of the TB genome has
focused attention on alternative potential targets in TB
that are unique to the bacterium. In particular, the
putative mechanisms of action of the existing drugs iso-
niazid (INH), ethambutol (EMB) and ethionamide has
focused target discovery efforts on new proteins
involved in the biogenesis of the mycobacterial cell
wall.3 In that regard, the unique polysaccharides of the
mycobacterial cell wall and their attendant preparative
M. tuberculosis is primarily an intracellular pathogen
which resides within the phagolysosomes of alveolar
macrophages. Perhaps the highly intricate features of
the mycobacterial cell wall have been developed as an
adaptation to the harsh environment and the require-
ments for existence within the intracellular milieu. The
polysaccharide structure of the MTB cell wall has been
extensively studied.5 These studies have shown the pre-
sence of arabinofuranose, galactofuranose, mannose
and rhamnose in specific linkages that make up the cri-
tical polysaccharide underpinning for the mycolate fatty
acids, all critical components for cell wall infrastructure
and integrity.5,6 The arabinogalactan (AG, Fig. 1) of the
mycobacterial cell wall consists of an Araf a(1!5) Galf
linkage that anchors the arabinan component to the
galactan substructure.
In continuation of our ongoing search for anti-
mycobacterial agents targeting biogenesis of the
*Corresponding author. Tel.: +1-205-581-2454; fax: +1-205-581-
2877; e-mail: reynolds@sri.org
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00343-1

924
A. K. Pathak et al. / Bioorg. Med. Chem. 10 (2002) 923–928
mycobacterial cell wall polysaccharides,7À9 we report
herein the synthesis and biological evaluation of some
n-octyl arabinofuranosyl-a(1!5)galactofuranosyl dis-
accharides.
5 were debenzoylated using 7 N NH3/MeOH giving
excellent yields of disaccharides 6 and 7, respectively.
The totally deblocked disaccharide 8 was obtained by
the hydrogenation of disaccharide 6 over Pd/C at room
temperature. The NMR, FABMS and, whenever neces-
sary, APT and 2-D NMR experiments were performed
to characterize all disaccharides. Based on NMR spec-
tral studies of the disaccharides, the a-interglycosidic
linkages were determined by coupling constants
between H-10 and H-20 and the coupling constants
Results and Discussion
Synthesis
0
0
The glycosyl acceptors octyl 2,3,6-tri-O-benzyl-b-d-
galactofuranoside (1) and octyl 2,3,6-tri-O-methyl-b-d-
galactofuranoside (2) were prepared as described earlier
from b-d-galactofuranose pentaacetate.8 2,3,5-Tri-O-
benzoyl-a-d-arabinofuranosyl trichloroacetimidate 3 as
a glycosyl donor was prepared as described earlier.9 All
acceptors and donor were purified by flash chromato-
graphy and characterized by spectral and elemental
analysis. The stereoselective synthesis of disaccharides 4
and 5 (Fig. 2) were carried out by glycosylation in the
presence of a Lewis acid. The trichloroacetimidate
donor (3) and the acceptor were reacted for 2–4 h using
the Lewis acid BF.3Et2O as promoter at 0 ꢀC under an
inert atmosphere in dry CH2Cl2 at room temperature
over powdered 4 A molecular sieves. After standard
workupand column chromatography on silica gel, the
pure disaccharides 4 and 5 were obtained in high yields
(86 and 79%, respectively). Further, disaccharides 4 and
(J1 ,2 ) were found to be ꢁ1 Hz as expected for this
configuration.10 Similarly, in the 13C NMR spectra the
signals for C-10 were observed in the range of 105–106
ppm, which also supported the a-linkage.
Biological activity
In vitro assay using whole cells. In vitro assays11 of dis-
accharides were done with Mycobacterium tuberculosis
H37Ra (ATCC 25177) and M. avium (NJ 168, NJ 211
and NJ 3404). The results are reported in Table 1.
Ethambutol was used as a positive control. The dis-
accharides 4, 5, 7, and 8 had MIC’s >128 mg/mL, the
highest concentration tested, whereas the disaccharide 6
had a MIC of 32 mg/mL for MTB H37Ra. Therefore,
only modest activity was seen against MTB for the
blocked disaccharide 6. None of the compounds were
active against M. avium.
Figure 1. Structure of arabinogalactan of Mycobacterium tuberculosis.
Table 1. Activity of octyl Araf a(1!5) Galf disaccharides against
Mycobacterium tuberculosis (MTB) H37Ra and Mycobacterium avium
complex (MAC)
Compd
MIC (mg/mL)
MTB H37Ra MAC NJ168 MAC NJ211 MAC NJ3404
4
5
6
7
>128
>128
32
>128
>128
4
Not done
>128
>128
>128
>128
8
>128
>128
>128
>128
>128
4
Not done
>128
>128
>128
>128
8
8
EMB
Figure 2. Structure of acceptors, donor sugars and disaccharides.
EMB, ethambutol.

A. K. Pathak et al. / Bioorg. Med. Chem. 10 (2002) 923–928
925
value of 5.7 mM and Vmax of 8.0 pmol/mg/min. Further
competition-based experiments established that dis-
accharides 6 and 7 were effective inhibitors of their
native acceptor 8 in the arabinosyltransferase assay
resulting in IC50 values of 1.16 mM and 3.20 mM for
disaccharides 6 and 7, respectively. Compound 6 was
also active against MTB H37Ra in vitro.
Experimental
Synthesis
General procedures. All reactions were performed under
a dry argon atmosphere and reaction temperatures were
measured externally. Anhydrous solvents from Aldrich
were used in the reactions without further processing.
Whenever necessary, compounds and starting materials
were dried by azeotropic removal of water with toluene
under reduced pressure. The reactions were monitored
by thin-layer chromatography (TLC) on precoated E.
Merck silica gel (60F254) plates (0.25 mm) and visualized
using UV light (254 nm) and/or heating after spray with
(NH4)2SO4 solution (150 g ammonium sulfate, 30 mL
H2SO4, 750 mL H2O). All solvents used for workup
and chromatography were reagent grade from Fisher
Scientific. Flash chromatography was carried out on
Figure. 3. An autoradiogram of reactions products produced through
the inclusion of SRI-9582, mycobacterial membranes and [14C]-DPA.
Lane 1, no acceptor; lane 2, 0.25 mM; lane 3, 0.5 mM; lane 4, 1.0 mM;
lane 5, 2.5 mM; lane 6, 5.0 mM; and lane 7, 10 mM. TLC/auto-
radiography was performed using chloroform/methanol/ammonium
hydroxide/water (65:25:0.4:3.6) and products revealed through expo-
sure to Kodak X-Omat film at À70 ꢀC for 3 days.
1
Fisher silica gel 60 (230–400 mesh). H and 13C NMR
spectra were recorded on Nicolet NT 300NB instrument
1
at 300 and 75 MHz, respectively. The H NMR spectra
at 600 MHz was recorded on a Bruker Advance 600
System. Coupling constants (J) are reported in Hz and
chemical shifts are in ppm (d) relative to residual solvent
peak or internal standard. Microanalyses were per-
formed on a Perkin Elmer 2400 CHN analyzer, and
FABMS data were recorded on a Varian/MAT 311A
double-focusing mass spectrometer by adding NBA
with or without LiCl.
Figure 4. Kinetic analysis of acceptor SRI-9582. The inset illustrates
the double reciprocal plot for SRI-9582 as a substrate for the myco-
bacterial arabinosyltransferase.
Octyl-2,3,6-tri-O-benzyl-5-(2,3,5-tri-O-benzoyl-ꢀ-D-ara-
binofuranosyl) - ꢁ - D - galactofuranoside (4). The tri-
chloroacetimidate donor 3 (647 mg, 1.07 mmol) and the
acceptor 1 (500 mg, 0.89 mmol) were dissolved in dry
CH2Cl2 (25 mL) and dry powdered 4A molecular seives
were added. The mixture was stirred for 15 min and
cooled to À20 ꢀC. The promoter BF3. Et2O (0.14 mL,
1.07 mmol) was dissolved in 1.0 mL of dry CH2Cl2, and
this solution was added to reaction mixture dropwise.
The reaction mixture was then stirred at room tem-
perature for 30 min and filtered. A cold, saturated
NaHCO3 solution (10 mL) was added, and the mixture
was extracted with chloroform (20 mL). The CHCl3
layer was washed with deionized water followed by a
brine solution, dried over Na2SO4 and concentrated.
The resulting syrupwas subjected to column chromato-
graphy on silica gel (cyclohexane/EtOAc, 5:1) afforded
the pure disaccharide 4 (769 mg, 86%) as a colorless
syrup. FABMS (LiCl) m/e 1013.6 [M+Li]+.
C61H66O13:1/2 H2O (found: C, 72.27; H, 6.71. requires
C, 72.12; H, 6.70). 1H NMR (300 MHz, CDCl3) d 8.07–
7.96, 7.57–7.19 (25H, each m, Aromatic), 5.54 (1H, s,
H-20), 5.53 (1H, m, H-30), 5.46 (1H, s, H-10), 5.01 (1H, s,
Arabinosyl transferase acceptor activity. Based on the
previous use of specific arabinose-based neoglycolipid
acceptors,12 compounds 6 (SRI-20249), 7 (SRI-20305)
and 8 (SRI-9582) were synthesized and compared as
potential acceptors of [14C]Araf from [14C]-DPA within
an arabinosyltransferase assay. Assays performed in the
presence of membranes resulted in [14C]Araf incorpora-
tion from [14C]-DPA for only the fully de-blocked Ara-
Gal acceptor 8, related blocked-compounds 6 and 7
possessed no detectable acceptor activity. TLC/auto-
radiography (Fig. 3) demonstrated the enzymatic con-
version of the Ara-Gal disaccharide
8
to its
corresponding trisaccharide product, formed by the
attachment of a [14C]Araf unit to the 50-OH of dis-
accharide 8 as reported previously for the mycobacterial
rabinosyltransferase assay.12 The compounds 6 and 7
were not recognized as substrates for the arabinosyl-
transferase enzyme(s), presumably due to the presence
of the methyl and benzyl ether protecting groups on the
reducing sugar. The calculation of kinetic constants
(Fig. 4) revealed that disaccharide 8 possessed a Km
0
H-1), 4.69 (1H, m, H-5a ), 4.66 (1H, m, H-40), 4.60–4.37

926
A. K. Pathak et al. / Bioorg. Med. Chem. 10 (2002) 923–928
0
(5H, m, 2ÂCH2–Aromatic, H-5b ), 4.20 (1H, m, H-4),
4.15 (1H, dd, J=5.8, 11.3 Hz, H-5), 4.02 (1H, m, H-3),
4.00 (1H, d, J=0.9 Hz, H-2), 3.67 (1H, m, OCH2), 3.63
(2H, d, J=5.4 Hz, H2-6), 3.34 (1H, m, OCH2), 1.55
(2H, m, CH2), 1.25 (10H, m, CH2), 0.87 (3H, m, CH3);
13C NMR (CDCl3) d 166.16, 165.77, 165.18 (3ÂC¼O),
138.11, 137.76, 137.60, 133.36, 133.32, 132.92, 129.98,
129.85, 129.73, 129.26,128.42, 128.39, 128.38, 128.28,
128.24, 127.95, 127.92, 127.79, 127.65, 127.48, 127.44
(Aromatic), 105.76 (C-10), 105.68 (C-1), 88.41 (C-2),
83.26 (C-3), 82.32 (C-20), 80.62 (C-4, C-40), 77.84 (C-30),
75.40 (C-5), 73.20, 71.90 (2CH2–Aromatic), 70.02 (C-6),
67.57 (OCH2), 63.60 (C-50), 31.82, 29.50, 29.34, 29.28,
26.14, 22.66 (6ÂCH2), 14.11 (CH3).
(300 MHz, CDCl3) d 7.36–7.19 (m, Aromatic), 4.59 (1H,
s, H-10), 4.55 (1H, s, H-1), 4.59–4.32 (4H, m, 2ÂCH2–
romatic), 4.17 (1H, ddd, J=2.0, 2.1, 3.4 Hz, H-40), 4.12
(1H, m, H-5), 4.02 (1H, m, H-4), 4.01 (1H, d, J=0.2 Hz,
H-20), 3.96 (1H, dd, J=1.0, 3.0 Hz, H-2), 3.94 (1H, 0t,
J=0.9 Hz, H-30), 3.82 (1H, dd, J=2.4, 11.6 Hz, H-5a ),
0
3.77 (1H, m, H-3), 3.73 (1H, dd, J=2.1, 11.6 Hz, H-5b ),
3.65 (1H, m, OCH2), 3.46 (1H, dd, J=8.1, 10.2 Hz, H-
6a), 3.41 (1H, dd, J=3.9, 10.2 Hz, H-6b), 3.35 (1H, m,
OCH2), 1.56 (2H, m, CH2), 1.27 (10H, m, CH2), 0.88
(3H, m, CH3); 13C NMR (CDCl3) d 137.34, 137.29,
137.19, 128.42, 128.34, 128.32, 128.03, 128.01, 127.96,
127.87, 127.81, 127.77 (aromatic), 107.01 (C-10), 105.61
(C-1), 87.89 (C-2), 87.32 (C-40), 83.92 (C-3), 80.83 (C-4),
78.59 (C-20), 78.05 (C-30), 73.21 (CH2–aromatic), 73.06
(C-5), 72.03, 71.90 (2ÂCH2–aromatic), 69.15 (C-6),
67.58 (OCH2), 61.70 (C-50), 31.77, 29.40, 29.31, 29.21,
26.08, 22.61 (6ÂCH2), 14.06 (CH3).
Octyl-2,3,6-tri-O-methyl-5-(2,3,5-tri-O-benzoyl-ꢀ-D-ara-
binofuranosyl) - ꢁ - D - galactofuranoside (5). The tri-
chloroacetimidate donor 3 (327 mg, 0.54 mmol) and the
acceptor 2 (150 mg, 0.45 mmol) were dissolved in dry
CH2Cl2 (15 mL), and dry powdered 4 A molecular
sieves were added under Ar atmosphere. The mixture
was stirred for 15 min and cooled to 0 ꢀC. The promoter
BF.3Et2O (68 mL, 0.54 mmol) was dissolved in 1.0 mL of
dry CH2Cl2 and added to the reaction mixture drop-
wise. The reaction mixture was then stirred at room
temperature for 30 min and filtered. A cold, saturated
NaHCO3 solution (10 mL) was added, and the mixture
was extracted with chloroform (20 mL). The CHCl3
layer was washed with deionized water followed by a
brine solution, dried over Na2SO4 and concentrated.
The resulting syrupwas subjected to column chromato-
graphy on silica gel (cyclohexane/EtOAc, 6:1) that
afforded the pure disaccharide 5 (276 mg, 79%) as a
colorless syrup. FABMS (LiCl) m/e 785.5 [M+Li]+.
C43H54O13:0.5 H2O (found: C, 65.32; H, 6.82. requires
C, 65.55; H, 7.03). 1H NMR (300 MHz, CDCl3) d 8.12–
7.99, 7.61–7.28 (m, Aromatic), 5.58 (1H, s, H-10, H-20,
H-30), 4.96 (1H, s, H-1), 4.81 (1H, dd, J=3.0, 11.2 Hz,
Octyl-2,3,6-tri-O-methyl-5-(ꢀ-D-arabinofuranosyl)-ꢁ-D-
galactofuranoside (7). The disaccharide 5 (100 mg, 0.13
mmol) was stirred with 10 mL of 7 N NH3/MeOH at
room temperature overnight. The reaction mixture was
concentrated in vacuo to give a syrupthat was subjected
to flash column chromatography on silica gel G
(CHCl3/MeOH 7:1) to give 7 as a colorless syrup(50
mg, 83%). FABMS (LiCl) m/e 473.6 [M+Li]+.
C22H42O10:1/3 H2O (found: C, 55.93; H, 8.81. requires
1
C, 55.99; H, 9.09). H NMR (300 MHz, CDCl3) d 4.82
(1H, s, H-10), 4.74 (1H, s, H-1), 4.59 (1H, m, 20-OH),
4.23 (1H, d, J=1.6 Hz, H-40), 4.13 (1H, m, H-5), 4.04
(4H, m, H-20, H-30, 30-OH, 50-OH), 3.96 (1H, t,0 J=6.6
Hz, H-4), 3.86 (1H, dd, J=1.8, 11.4 Hz, H-5a ), 3.78
0
(1H, dd, J=0.9, 11.4 Hz, H-5b ), 3.68 (1H, dd, J=0.7,
2.3 Hz, H-2), 3.65 (1H, m, OCH2), 3.52 (1H, dd, J=2.2,
6.8 Hz, H-3), 3.40 (3H, m, H2-6, OCH2), 3.39 (6H, s,
OCH3), 3.36 (3H, s, OCH3), 1.55 (2H, m, CH2), 1.27
(10H, m, CH2), 0.88 (3H, m, CH3); 13C NMR (CDCl3)
d 106.95 (C-10), 105.20 (C-1), 89.41 (C-2), 87.39 (C-40),
85.98 (C-3), 81.09 (C-4), 78.76 (C-20), 77.98 (C-30), 72.52
(C-5), 71.77 (C-6), 67.47 (OCH2), 61.61 (C-50), 31.67,
29.29, 29.21, 29.10, 25.95, 22.51 (6ÂCH2), 13.97 (CH3).
H-5a ), 4.73 (1H, m, H-40), 4.67 (1H, dd, J=4.7, 11.2
Hz, H-5b ), 4.08 (1H, m, H-4, H-5), 3.72 (1H, m, H-2,
0
0
H-3), 3.66 (1H, m, OCH2), 3.57 (2H, m, H2-6), 3.41,
3.39, 3.32 (3ÂOCH3), 3.36 (1H, m, OCH2), 1.56 (2H, m,
CH2), 1.25 (10H, m, CH2), 0.86 (3H, m, CH3); 13C
NMR (CDCl3) d 166.22, 165.84, 165.26 (3ÂC¼O),
133.62, 133.39, 133.34, 129.96, 129.83, 129.77, 129.24,
128.43, 128.39, 128.39, 128.25 (aromatic), 105.89 (C-10),
105.03 (C-1), 89.98 (C-2), 85.36 (C-3), 82.24 (C-20),
80.88 (C-4), 80.74 (C-40), 77.84 (C-30), 75.33 (C-5), 72.37
(C-6), 67.50 (OCH2), 63.83 (C-50), 58.92, 57.83, 57.38
(3OCH3), 31.80, 29.44, 29.35, 29.24, 26.07, 22.63
(6CH2), 14.08 (CH3).
Octyl-5-(ꢀ-D-arabinofuranosyl)-ꢁ-D-galactofuranoside
(8). To a methanol solution (5 mL) of disaccharide 6
(100 mg, 0.14 mmol) was added Pd/C (10%, 25 mg),
and the mixture was stirred at room temperature under
hydrogen (30 mL, 24 h). Filtration through a Celite pad
and concentration gave a viscous, colorless oil. Flash
column chromatography on silica gel (CHCl3/MeOH
3:1) afforded the pure disaccharide 8 (54 mg, 90%) as a
colorless syrup. FABMS (LiCl) m/e 431.3 [M+Li]+.
C19H36O10:H2O (found: C, 51.84; H, 8.45. requires C,
Octyl-2,3,6-tri-O-benzyl-5-(ꢀ-D-arabinofuranosyl)-ꢁ-D-
galactofuranoside (6). To a solution of disaccharide 4
(437 mg, 0.43 mmol) in dry methanol (15 mL) was
added 7 N NH3/MeOH (8 mL) dropwise, and the reac-
tion mixture was allowed to stir at room temperature
overnight. The reaction mixture was concentrated in
vacuo to give a syrupthat was subjected to flash column
chromatography on silica gel G (CHCl3/MeOH 95:5),
giving 6 as a colorless syrup(264 mg, 88%). FABMS
(LiCl) m/e 701.6 [M+Li]+. C40H54O10:0.5 H2O (found:
1
51.58; H, 8.59). H NMR (600 MHz, D2O) d 5.33 (1H,
s, H-10), 5.09 (1H, s, H-1), 4.30 (1H, dd, J=1.8, 3.6 Hz,
H-20), 4.26 (1H, ddd, J=3.6, 5.4, 6.0 Hz, H-40), 4.18
(3H, br m, H-2, H-3, H-4), 4.08 (1H, dd, J=3.6, 6.0 Hz,
H-30), 4.03 (1H, m, H-5), 3.95 (1H, dd, J=3.6, 12.1 Hz,
0
H-5a ), 3.91 (1H, dd, J=6.6, 12.0 Hz, H-6a), 3.87 (1H,
dd, J=4.8, 12.0 Hz, H-6b), 3.85 (1H, m, OCH2), 3.84
0
1.73 (2H, m, CH2), 1.43 (10H, m, CH2), 1.01 (3H, m,
(1H, dd, J=6.0, 12.1 Hz, H-5b ), 3.64 (1H, m, OCH2),
C, 68.27; H, 7.78 requires C, 68.25; H, 7.73). H NMR
CH3); 13C NMR (CDCl3) d 108.73 (C-10), 107.29 (C-1),
1

A. K. Pathak et al. / Bioorg. Med. Chem. 10 (2002) 923–928
927
84.15 (C-40), 82.18, 81.54, 81.42 (C-2, C-4, C-20), 78.23
(C-3), 76.80 (C-30), 76.69 (C-5), 68.71 (OCH2), 61.85 (C-
6), 61.52 (C-50), 31.56, 29.16, 28.97, 28.90, 25.69, 22.41
(6ÂCH2), 13.76 (CH3).
eluate was dried and the resulting products partitioned
between the two phases arising from a mixture of n-
butanol (3 mL) and H2O (3 mL). The resulting organic
phase was recovered following centrifugation at 3500g
and the aqueous phase was again extracted twice with 3
mL of n-butanol saturated water, the pooled extracts
were back-washed twice with water saturated with n-
butanol (3 mL). The n-butanol-saturated water fraction
was dried and re-suspended in 200 mL of n-butanol. The
total cpm of radiolabeled material extractable into the
n-butanol phase was measured by scintillation counting
using 10% of the labeled material and 10 mL of EcoS-
cintA (National Diagnostics, Atlanta, GA, USA). The
incorporation of [14C]Araf 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 in CHCl3/CH3OH/NH4OH/
H2O (65:25:0.5:3.6) on aluminium backed Silica Gel 60
F254 plates (E. Merck, Darmstadt, Germany). Auto-
radiograms were obtained by exposing TLCs to X-ray
film (Kodak X-Omat) for 3 days. Competition based
experiments were performed by mixing compounds
together at various concentrations (8, 0.4 mM with 7 or
6 at 1.0, 2.0 and 3.6 mM) followed by thin-layer chro-
matography/autoradiography as described earlier to
determine the extent of product formation.
Arabinosyltransferase assay
Bacterial strains and growth conditions. M. smegmatis
mc2155 was a generous gift from W. R. Jacobs, Albert
Einstein College of Medicine, Bronx, New York.13
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.
Preparation of membrane and cell wall enzyme fractions.
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-mer-
captoethanol and 10 mM MgCl2 at 4 ꢀC and subjected
to probe sonication (Soniprep 150, MSE Sanyo Gallen-
kamp, 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 and 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 and enzymatically active cell
wall (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 frac-
tions were obtained by centrifugation of the 27,000g
supernatant at 100,000g for 1 h at 4 ꢀC. The supernatant
was carefully 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-Beijerland, The Netherlands).
Acknowledgements
The authors are thankful to Dr. J. M. Riordan for
NMR and Mr. M. D. Richardson for FABMS. The
600 MHz NMR work was supported by NCI grant CA-
13148 (the University of Alabama at Birmingham UAB
shared NMR Core Facility). This work was supported
by NIH/NIAID grant R01AI45317. G.S.B., who is
currently a Lister Institute-Jenner Research Fellow,
acknowledges support from The Medical Research
Council (49343 and 49342). The M. avium strains were
kindly provided by Dr. Leonid Heifets, National Jewish
Center for Immunology and Respiratory Diseases,
Denver, CO, USA.
Transferase assay. Compounds 6, 7, and 8 at a range
of concentrations from 0.25 to 10.0 mM (prepared
from stored 100 mM ethanol stocks) and [14C]-DPA
(20,000 cpm, 9 mM, 10 mL [stored in chloroform/
methanol, 2:1]), were dried under a stream of argon in a
microcentrifuge tube (1.5 mL) and placed in a vacuum
desiccator for 15 min to remove any residual solvent.
The dried constituents of the assay were then resus-
pended in 8 mL 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 membranes (250 mg) were added to a
final reaction volume of 80 mL. The reaction mixtures
were then incubated at 37 ꢀC for 1 h. A CHCl3/CH3OH
(1:1, 533 mL) solution was then added to the incubation
tubes and the entire contents centrifuged at 18,000g.
The supernatant was recovered and dried under a
stream of argon and re-suspended in C2H5OH/H2O (1:1, 1
mL) and loaded onto a pre-equilibrated [C2H5OH/H2O
(1:1)] 1 mL Whatmann strong anion exchange (SAX)
cartridge which was washed with 3 mL of ethanol. The
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