Epimeric and amino disaccharide analogs as probes of an α-(1→6)mannosyltransferase involved in mycobacterial lipoarabinomannan biosynthesist

Article abstract of DOI:10.1039/b916580k

Mycobacterial lipoarabinomannan (LAM) is an important, immunologically active glycan found in the cell wall of mycobacteria, including the human pathogen Mycobacterium tuberculosis. At the core of LAM is a mannan domain comprised of α-(1→6)-linked-mannopyranose (Manp) residues. Previously, we and others have demonstrated that α-Manp-(1→6)- α-Manp disaccharides (e.g., Manp-(1→6)-α-ManpOctyl, 1) are the minimum acceptor substrates for enzymes involved in the assembly of the LAM mannan core. We report here the synthesis five epimeric and three amino analogs of 1, and their subsequent biochemical evaluation against an α-(1→6)-ManT activity present in a membrane preparation from M. smegmatis. Changing the manno- configuration of either residue of 1 to talo- or gluco- led to a reduction or loss of activity, thus confirming earlier work showing that the C-2 and C-4 hydroxyl groups of each monosaccharide were important for enzymatic recognition. Characterization of the products formed from these analogs was done using a combination of mass spectrometry and glycosidase digestion, and full substrate kinetics were also performed. The analogs in which the acceptor hydroxyl group had been replaced with an amino group were, as expected, not substrates for the enzyme, but were weak inhibitors. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b916580k

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Epimeric and amino disaccharide analogs as probes of an a-(1→6)-
mannosyltransferase involved in mycobacterial lipoarabinomannan
biosynthesis†
Pui Hang Tam and Todd L. Lowary
Received 12th August 2009, Accepted 8th October 2009
First published as an Advance Article on the web 16th November 2009
DOI: 10.1039/b916580k
Mycobacterial lipoarabinomannan (LAM) is an important, immunologically active glycan found in the
cell wall of mycobacteria, including the human pathogen Mycobacterium tuberculosis. At the core
of LAM is a mannan domain comprised of a-(1→6)-linked-mannopyranose (Manp) residues.
Previously, we and others have demonstrated that a-Manp-(1→6)-a-Manp disaccharides (e.g.,
Manp-(1→6)-a-ManpOctyl, 1) are the minimum acceptor substrates for enzymes involved in the
assembly of the LAM mannan core. We report here the synthesis five epimeric and three amino analogs
of 1, and their subsequent biochemical evaluation against an a-(1→6)-ManT activity present in a
membrane preparation from M. smegmatis. Changing the manno- configuration of either residue of 1 to
talo- or gluco- led to a reduction or loss of activity, thus confirming earlier work showing that the C-2
and C-4 hydroxyl groups of each monosaccharide were important for enzymatic recognition.
Characterization of the products formed from these analogs was done using a combination of mass
spectrometry and glycosidase digestion, and full substrate kinetics were also performed. The analogs in
which the acceptor hydroxyl group had been replaced with an amino group were, as expected, not
substrates for the enzyme, but were weak inhibitors.
Introduction
Lipoarabinomannan (LAM) is an important cell wall constituent
in mycobacteria, including those that cause the human diseases tu-
berculosis (Mycobacterium tuberculosis) and leprosy (M. leprae).1
This glycoconjugate, which contains both arabinofuranose (Araf )
and mannopyranose (Manp) residues, has been implicated in a
host of biochemical and immunological processes associated with
mycobacterial infection.2,3 There has consequently been significant
interest not only in obtaining a molecular-level understanding
of its immunomodulatory activity, but also in elucidating the
biosynthetic pathway by which LAM is assembled.2–6
All mycobacteria, and a number of related organisms, produce
LAMs, the structure of which varies from species to species.2,3
In mycobacterial LAM (Fig. 1), the molecule is built upon an
acylated phosphatidyl inositol anchor from which extends a chain
of Manp residues connected via a-(1→6) linkages. Approximately
half of these core mannan residues are further elaborated by
single a-(1→2)-linked Manp branching units, and this structure
serves as a scaffold to which an arabinan domain containing
a-(1→2)-, a-(1→3)- and a-(1→5)-linked Araf residues is
bound.1–3 Depending on the species, the arabinan can be modified
Fig.
diacylglycerol.
1
Composite structure of mycobacterial LAM; DAG
=
at its periphery by a number of capping groups such as short a-
(1→2)-linked Manp oligosaccharides,2,3,7 5-deoxy-5-thiomethyl-
xylofuranose residues8–11 or inositol phosphate moieties.12
The role of each structural domain on the immunomodulatory
function of LAM remains relatively poorly understood. For exam-
ple, although previous studies suggested the capping motifs were
important for virulence,2,3,7 more recent studies have suggested this
is not the case.13 Other recent investigations have demonstrated
that the ability of these glycans to bind to the toll-like receptor
2 (TLR-2) is influenced by mannan chain length14 and acylation
Alberta Ingenuity Centre for Carbohydrate Science and Department of
Chemistry, The University of Alberta, Gunning-Lemieux Chemistry Centre,
Edmonton, AB T6G 2G2, Canada. E-mail: tlowary@ualberta.ca
† Electronic supplementary information (ESI) available: Additional exper-
imental details and data for new compounds and 1H and 13C NMR spectra.
See DOI: 10.1039/b916580k
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Fig. 2 Reported assay for measuring PPM-dependent a-(1→6)-ManT activity in mycobacterial membrane preparations. PPM = polyprenolphoshoman-
nose; GDP = guanosine disphosphate.
state.15 Similarly, the recognition of LAM fragments by anti-LAM
antibodies has also been studied recently.16–18
strains,25 has provided evidence that more than one a-(1→6)-
ManT is required.
The model proposed more than a decade ago by Brennan and
coworkers19 for the biosynthesis of LAM has, for the most part,
been borne out through genetic and biochemical investigations
carried out since that time.4,5 Of particular relevance to the work
reported in this paper are a family of polyprenolphosphoman-
nose (PPM)-dependent mannosyltransferases (ManTs) that are
involved in the assembly of the mannan domain of this glycan.
Earlier studies reported the use of a membrane preparation from
M. smegmatis to assay for the a-(1→6)-ManT activity required
for the synthesis of the LAM mannan core.20,21 This assay, in
which the donor substrate for the enzyme, is generated in situ
from GDP-mannose (Fig. 2), has been used to screen potential
substrates and inhibitors of this enzymatic activity,22,23 and it has
been demonstrated that a-Manp-(1→6)-a-Manp disaccharides
(e.g., 1) are the minimum acceptor substrates.21,24 It is currently
unknown how many enzymes are involved in the assembly of the
mannan core. Although earlier investigations in this area assumed,
without substantial experimental support, that a single enzyme
was involved, recent work with mycobacterial gene knock-out
In previous papers, we reported the preparation26 and biochem-
ical evaluation24 of a panel of monomethoxy and monodeoxy
analogs of disaccharide acceptor 1 (Fig. 3) as potential sub-
strates and inhibitors for this a-(1→6)-ManT activity. Among
the important findings from these investigations were that the
enzyme appears to form critical hydrogen bonding interactions
with a number of hydroxyl groups on the substrate because
deoxygenation leads, in all but two cases (C-2¢ and C-4), to
essentially a total loss of activity. To extend our understanding
of the substrate specificity of this enzymatic activity further, we
describe here the synthesis five epimeric (3, 5, 7–9) and three amino
(2, 4 and 6) analogs of 1 (Fig. 3), and their subsequent biochemical
evaluation against the a-(1→6)-ManT activity present in the
previously mentioned M. smegmatis membrane preparation.
The epimers were selected as targets to determine if the manno-
configuration was absolutely required by the enzyme, or if disac-
charides with rings in the gluco- (3, 7, 9) or talo- (5, 8, 9) configura-
tion were also recognized. The results obtained from these analogs
would provide additional insight into the important steric and
Fig. 3 Synthetic disaccharide targets 2–9.
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Scheme 1 Reagents and conditions: a) t-BuPh2SiCl, imidazole, DMF, 45 ◦C, 94%; b) 2,2-dimethoxypropane, pTsOH, acetone, quant.; c) i: oxalyl chloride,
DMSO, -78 ◦C; then alcohol 19, warm to -60 ◦C; then Et3N, warm to rt.; ii: NaBH4, MeOH, 62%; d) i: 80% aq. AcOH, 50◦◦C; ii: Ac2O, DMAP, CH2Cl2,
˚
pyridine, 65% for 21 and 26% for 22; e) Ac2O, CH2Cl2, pyridine (1 : 1), 89%; f) octanol, NIS, TMSOTf, 4 A MS, CH2Cl2, 0 C, 89%; g) TBAF, THF, 74%.
hydrogen bonding interactions in the active site of these proteins.
In addition, the amino analogs were synthesized and evaluated as
potential inhibitors. We envisioned that the amino groups of 2, 4
and 6 would be protonated at physiological pH, and the resulting
ammonium derivatives would serve as inhibitors via an ionic
interaction with a negatively charged residue in the enzyme active
site.27,28 It has been shown previously that octyl oligosaccharides
are efficient substrates for glycosyltransferases from mycobacteria
and other organisms.21,27–30 Moreover, the attachment of this
hydrophobic group to an oligosaccharide allows for convenient
product isolation using reversed-phase chromatography.30 Thus,
the target compounds 2–9 were synthesized as octyl glycosides.
Thioglycoside 13 and octyl glycoside 14 were synthesized from
the known thioglycoside 1734 as illustrated in Scheme 1. First,
reaction of 17 with tert-butylchlorodiphenylsilane and imidazole
provided silyl ether 18 in 94% yield. Subsequent reaction of 18
with 2,2-dimethoxypropane in the presence of a catalytic amount
of p-TsOH gave a quantitative yield of alcohol 19.
Oxidation of the C-2 hydroxyl group under Swern conditions
and subsequent reduction with sodium borohydride gave an
anomeric mixture of thioglycoside 20 (62% yield of a 6 : 1 a/b
mixture of isomers, which could be separated by chromatography).
In the 1H NMR spectrum of the major isomer, the H-1 resonance
was shifted to 5.33 ppm, which is in good agreement with the
a-configuration at the anomeric center. Presumably, the basic
conditions of the oxidation reaction resulted in the anomerization
of the ketone product, as has been reported previously.35 However,
the J1,2 (7.4 Hz) of the product obtained after borohydride
reduction was larger than would be expected for thioglycosides
with either the a-galacto- or a-talo-configuration.
Results and discussion
Preparation of monosaccharide building blocks
We envisioned that disaccharides 2–9 could be assembled from
monosaccharide precursors 10–16 (Fig. 4). Previously reported
methods were used to prepare octyl glycoside 1031 and 16,32
as well as thioglycosides 1133 and 12.26 The other building
blocks, talopyranosides 13 and 14, and glucopyranoside 15, were
synthesized as outlined below.
We postulated that the larger than expected coupling constant
value was due to the distortion of the chair conformation of the
pyranose ring by the isopropylidene protecting group. Therefore,
to confirm the stereochemistry at C-2, 20 was hydrolyzed with
80% aqueous AcOH and then reacted with acetic anhydride in the
presence of pyridine and DMAP to afford a mixture of 21 and 22.
The J1,2 and J2,3 of 22 (1.3 and 3.7 Hz, respectively) are consistent
with the a-talo-configuration, in turn confirming the structure of
20 as that shown in Scheme 1.
Having determined that the oxidation–reduction sequence had
provided the correct product, 20 was treated with acetic anhydride
thus providing an 89% yield of 13. Glycosidation of 13 with
n-octanol gave octyl talopyranoside 23 in 89% yield. Finally,
treatment of 33 with tetra-n-butylammonium fluoride provided
a 74% yield of the expected alcohol 14.
As illustrated in Scheme 2, thioglycoside 15, was obtained
from the known thioglucoside 24.36 Reaction of 24 with
Scheme 2 Reagents and conditions: a) t-BuPh2SiCl, imidazole, DMF,
Fig. 4 Monosaccharide building blocks used for the synthesis of 2–9.
45 ◦C; b) NaH, BnBr, DMF, 95% two steps.
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Scheme 3 Reagents and conditions: a) NIS, TMSOTf, 4 A MS, CH2Cl2, 0 ◦C, 85%; b) HF·pyridine–pyridine–THF (1 : 2 : 20), 66%; c) TsCl, pyridine,
91%; d) NaN3, DMF, reflux, 98%; e) i: Pd(OH)2–C, pyridine, H2; ii: (CF3CO)2O, pyridine, 0 ◦C to rt.; iii: Pd(OH)2–C, MeOH, H2, 68% over three steps;
f) NaOMe, MeOH, 61%.
˚
tert-butylchlorodiphenylsilane and imidazole provided silyl ether
intermediate 25. Without purification, the remaining hydroxyl
groups were protected as benzyl ethers upon treatment with
sodium hydride and benzyl bromide to afford 15 in 95% yield
over the two steps.
benzyl groups by treatment of 29 with hydrogen and a palladium
catalyst failed. Thin layer chromatographic analysis of the reaction
mixture showed several products, presumably a series of partially-
debenzylated derivatives of 29. No further reduction was observed,
even after prolonged reaction times. Instead, the azido group of
29 was reduced to the amine by hydrogenation over Pd(OH)2–C
in pyridine, and the product was immediately converted to its N-
trifluoroacetamide using trifluoroacetic anhydride in pyridine.40
Subsequent hydrogenolysis of the benzyl groups using Pd(OH)2–
C catalyst was achieved without incident and provided triol 30 in
68% yield over three steps. Final deprotection of 30 using sodium
methoxide in methanol furnished a 61% yield of the desired amino
disaccharide 2.
As illustrated in Scheme 4, the synthesis of disaccharides 3 and
4 were achieved by first coupling thioglycoside 15 with alcohol
10,31 under NIS–TMSOTf activation. This reaction afforded an
inseparable diastereoisomeric mixture (~5 : 1 a : b ratio) of prod-
ucts. Subsequent desilylation using hydrogen fluoride followed by
chromatographic purification gave the a-linked disaccharide 31
in 50% yield over the two steps. Final deprotection of the benzyl
ethers afforded 3 in 82% yield.
The amino disaccharide 4 was synthesized via a route analogous
to 2, starting with 31. Thus, the primary hydroxyl group in 31
was first tosylated to provide 32, which upon heating in reflux in
DMF with sodium azide gave azidosugar 33 in 76% yield over
the two steps. As was seen for compound 29, the primary azido
functionality in 33 was evident in the 13C NMR spectrum, which
showed a resonance for C-6¢ at 51.2 ppm. The azido group of
33 was then converted to the corresponding N-trifluoroacetamide
derivative in two steps (azide reduction and trifluoroacetylation of
Synthesis of disaccharides
As illustrated in Scheme 3, the synthesis of amino disaccharide
2 was began with the NIS–TMSOTf activated37 glycosylation of
thioglycoside 1133 and alcohol 10,31 which afforded disaccharide
26 in 85% yield. For this reaction, and all other glycosylations
described in this paper using mannopyranoside and talopyra-
noside donors, the a-stereochemistry of the glycosidic linkages
was confirmed by measurement of the one-bond heteronuclear
coupling constant for the anomeric carbon atom (1JC-1,H-1). In all
cases, this value was between 167 and 176 Hz, clearly indicating
the a-stereochemistry.38
Next, disaccharide 26 was desilyated using hydrogen fluoride in
pyridine39 to give 27 in 66% yield. The resulting primary alcohol
in 27 was then tosylated with tosyl chloride in pyridine (91%);
treatment of the product, 28, with sodium azide gave azido-
disaccharide 29 in 98% yield. That the substitution had occurred
1
was confirmed from the H NMR spectrum of 29, in which the
protons on C-6¢ resonated between 3.38 and 3.48 ppm, as would
be expected for hydrogens adjacent to an azido functionality. In
addition, in the 13C NMR spectrum, a resonance at 51.2 ppm could
be assigned to C-6¢.
Similar to an earlier observation on related disaccharides,40
simultaneous reduction of the azide and deprotection of the
Scheme 4 Reagents and conditions: a) i: NIS, TMSOTf, 4 A MS, CH2Cl2, 0 ◦C, 5 : 1 of a : b isomers; ii: HF·pyridine–pyridine–THF (1 : 2 : 20), 50%;
b) Pd(OH)2–C, MeOH, H2, 82%; c) TsCl, pyridine, 89%; d) NaN3, DMF, reflux, 85%; e) i: Pd(OH)2–C, pyridine, H2; ii: (CF3CO)2O, pyridine, 0 ◦C to rt.;
iii: Pd(OH)2–C, MeOH, H2, 72% over three steps; f) NaOMe, MeOH, 79%.
˚
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◦
˚
Scheme 5 Reagents and conditions: a) NIS, TMSOTf, 4 A MS, CH2Cl2, 0 C, 90%; b) HF·pyridine–pyridine–THF (1 : 2 : 20), 77%; c) i: 80% aq. AcOH,
50 ◦C; ii: NaOMe, MeOH, 91%; d) i: TsCl, pyridine, 91%; ii: NaN3, DMF, reflux, 84%; e) i: 80% aq. AcOH, 50 ◦C; ii: NaOMe, MeOH; iii: Pd(OH)2–C,
MeOH, H2, 86% over three steps.
the product amine). Without purification, the crude intermediate
was hydrogenated to provide 34 in 72% yield over three steps. Final
deprotection by treatment with sodium methoxide furnished a 79%
yield of the target analog 4.
The synthesis of 4 and 5, starting from octyl talopyranoside 14,
is shown in Scheme 5. Coupling of this acceptor with thioglycoside
1133 under NIS–TMSOTf activation afforded the corresponding
protected disaccharide 35 in 90% yield. Deprotection of the silyl
ether proceeded under standard conditions to give a 77% yield
of 36. Cleavage of the isopropylidene acetal and treatment with
sodium methoxide in methanol gave 5 in 91% overall yield. To
prepare 6, alcohol 36 was converted to azido disaccharide 37 via
the two step sequence (91% and 84% yields, respectively) described
for the preparation of 29. Reduction of azide 37, following removal
of the isopropylidene acetal and acyl protecting groups, provided
aminosugar 6 in 86% yield.
The preparation of disaccharides 7–9 is illustrated in Scheme 6.
Glycosylation of alcohols 10,31 14, 1632 with thioglycosides 12,26
13, 15 using NIS–TMSOTf activation, gave disaccharides 38, 39,
and 40 in excellent yields (92%, 81% and 88%, respectively).
Debenzoylation and hydrogenolysis of 38 provided the desired
compound 7 (84% yield). To obtain 8 and 9, disaccharides 39
and 40 were subjected to a four-step sequence (desilylation,
deacetylation, acetal cleavage and hydrogenolysis) to give the
expected products in overall yields of 45% and 62%, respectively.
NMR analysis of the final compounds suggested that the modified
oligosaccharides have a conformation similar to the parent com-
pounds. For example, the chemical shifts and coupling constants
were consistent with all of the pyranose rings adopting a 4C1
conformation.
˚
Scheme 6 Reagents and conditions: a) NIS, TMSOTf, 4 A MS, CH2Cl2,
0
◦C, 92% for 38, 81% for 39, 88% for 40 (5 : 1 of a : b isomers); b) i:
NaOMe, MeOH; ii: Pd(OH)2–C, MeOH, H2, 84%; c) i: TBAF, THF; ii:
NaOMe, MeOH; iii: 80% aq. AcOH, 50 ◦C; iv: Pd(OH)2–C, MeOH, H2,
45% for 8; 62% for 9.
Screening analogs as substrates and inhibitors
Once synthesized, 2–9 were screened as potential substrates and
inhibitors of the PPM-dependent a-(1→6)-ManT activity, present
in the M. smegmatis membrane preparation.20,21 Each analog
was incubated with [3H]-labeled GDP-mannose (Fig. 2) and the
membrane extracts, the radiolabeled products were recovered by
solvent extraction and reversed-phase chromatography, and the
radioactivity was measured by scintillation counting.
The ability of 2–9 to act as acceptor substrates for ManT
were compared with the parent compound 1 and the results are
summarized in Table 1. The initial screening revealed that analogs
3 (a-Glcp-(1→6)-a-Manp) and 5 (a-Manp-(1→6)-a-Talp) are
moderate substrates of ManT with 41% and 57% activity, relative
to 1 (a-Manp-(1→6)-a-Manp). Our previous data suggested that
the hydroxyl groups at C-2¢ and C-4 of 1 were not crucial for
substrate–enzyme interaction, as the replacement with hydrogen
had no dramatic effect on ManT catalysis.24 However, it appears
that epimerization at either position, leading to analog 3 or 5,
significantly affects the efficiency to serve as a substrate. This
finding is further indicated by the apparent kinetic parameters
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Table 1 Summary of ManT activities using analogs 1–9
b
Apparent Vmax
/
Remaining ManT
activityc (%)
Mass of oligosaccharide
productd
Analog
Relative activitya (%)
Apparent KM b/mM
pmol mg min-1
1
2
3
4
5
6
7
8
9
100
4
41
0
57
<1
10
<1
10
0.09 0.01
–
1.80 0.71
—
1.20 0.24
—
3.04 0.69
—
2.27 0.12
0.37 0.005
—
0.34 0.071
—
0.40 0.031
—
0.10 0.014
—
—
21
—
9
—
30
—
—
—
639.2, 801.3
—
639.1, 801.1
—
639.2, 801.2
—
639.3, 801.3, 963.3
—
639.3, 801.3
e
0.11 0.003
a Relative activities were measured at 2.0 mM acceptor concentration with 0.2 mCi of [3H]GDP-Man and are expressed with respect to disaccharide 1.
100% activity corresponds to 29.8 pmol mg-1 h-1. b Kinetic parameters were determined using a range of acceptor concentrations by nonlinear regression
analysis of the Michaelis–Menten equation with the GraphPad Prism 4.0 program. c Compounds were screened at a concentration of 2.0 mM with 1
as the substrate at 0.2 mM. d The enzymatic products were isolated from larger-scale incubations and their masses were determined by MALDI mass
spectrometry. The found values correspond to the sodium adducts, which were in good agreement with the calculated values. e Not determined.
shown in Table 1. These acceptor analogs, have KM values of
1.8 mM (3) and 1.2 mM (5), which are 13- and 20-fold larger,
respectively, than that of 1 (0.09 mM). The Vmax values of 3 or 5
are comparable to that of the parent compound.
Characterization of enzymatic products by mass spectrometry
In addition to the radiochemical assays, milligram-scale enzymatic
incubations of 1, 3, 5, 7 and 9 with unlabelled GDP-Man
and the membrane fraction were carried out to determine the
structure of the oligosaccharide products that were produced
from these analogs. After the incubations, the enzymatic products
were purified and analyzed by MALDI mass spectrometry. As
shown in Fig. S1, these reactions all resulted in the formation
of the corresponding trisaccharides and tetrasaccharides as the
major and minor products, respectively. Contrary to the proposed
processive nature of this enzyme,20 a homologous series of products
was not observed. The absence of these larger oligosaccharides
is probably due to much lower acceptor concentrations of the
resulting tri- and tetrasaccharide products, resulting in slower rates
of the subsequent elongation reactions. It is also possible that
the larger enzymatic products (e.g., tetra- and pentasaccharide)
are degraded by an a-(1→6)-endo-mannosidase present in the
membrane preparation.24
The lower relative activity of 3 and 5 compared to 1 appears
to be due to the steric requirements of the ManT. For both
compounds, the hydroxyl group is placed in the opposite orienta-
tion in the parent compound (changed from axial to equatorial
in 3, or vice versa in 5). This change appears to lead to a
potential negative steric interaction with amino acid residues in
the active site thus resulting in an unfavorable enzyme–substrate
interaction and a higher KM. These observations suggest that
while the enzyme preferentially recognizes disaccharides in which
both residues are in the manno-configuration, it will also accept
analogs of different stereochemical configurations, but with lower
affinity.
This finding is further supported by the results obtained from
analogs 7–9, in which a-Talp-(1→6)-a-Manp analog 8 is not a
substrate, and both the a-Manp-(1→6)-a-Glcp 7 and a-Glcp-
(1→6)-a-Talp analogs 9 are poor substrates with only 10% relative
activity compared to 1. Consistent with the results for 3 and 5,
subsequent kinetic analysis provided larger apparent KM values
for 7 and 9, 3.04 mM and 2.27 mM, respectively. However, the
Vmax values for these substrates was only marginally less than the
native substrate, 1. The poor substrate activity of 7, and the lack
of activity of 8, is consistent with our previous work,24 which
indicated that the C-4¢ and C-2 hydroxyl groups are essential
for ManT catalysis. In 3 and 5, the orientations of the C-4¢ and
C-2 hydroxyl groups were the same as in the parent compound,
while the stereochemistry at one other centre was inverted (C-2¢
in 3 and C-4 in 5), and these compounds were weak substrates
for the enzyme. However, evaluation of an analog in which the
stereochemistry at both C-2¢ and C-4 were inverted, the a-Glcp-
(1→6)-a-Talp isomer 9, showed even weaker ManT substrate
activity.
Characterization of enzymatic products by glycosidase digestion
In addition to the PPM-dependent a-(1→6)-mannosyl-
transferases, the crude membrane extract of M. smegmatis used in
these assays also contains a-(1→2)-ManT’s involved in mannan
core branching and the capping of the arabinan domain.25,41–43 To
confirm that the observed addition of radiolabeled mannose to 3,
5, 7 and 9 arose from a-(1→6)-ManT activity, the radiochemical
assays were repeated on a larger scale with [3H]-labelled GDP-
Man. After the reaction, the [3H]Man-labeled enzymatic products
were purified, divided evenly and digested with glycosidases.
As illustrated in Fig. 5, there is no significant difference
in the radioactivities between the control experiments and the
samples that have undergone treatment with the a-Man-(1→2)-a-
Man-specific Aspergillus saitoi a-(1→2)-mannosidase (AS). These
results demonstrate that none of the mannosylated products
contained an a-Man-(1→2)-a-Man linkage. On the other hand,
digestions of the radiolabeled products using a-mannosidases
from jack bean (JB, a-(1→2,3,6)-specific) and Xanthomonas mani-
hotis (XM, unbranched a-(1→6)-specific) removed essentially
In summary, the change of the configuration of the disaccharide
motif, regardless of whether the change was at the reducing or
non-reducing end, did not enhance but rather reduced the ManT
activity. This finding indicates that the enzyme requires a manno-
configuration of both rings for optimal activity.
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Inhibition effects of amino analogs
As expected, analogs 2, 4 and 6, which lack a hydroxyl group at
C-6¢, are not substrates of ManT; all compounds show relative
activities below 5% (Table 1). These amino derivatives were next
tested as inhibitors against ManT using 1 as the substrate. In
these studies, analogs 2, 4 and 6 were screened at a concentration
of 2.0 mM with the parent compound 1 at 0.2 mM. As shown in
Table 1, disaccharides 2, 4 and 6 inhibit the mannosylation of 1
by 79, 91 and 70%, respectively. These compounds are thus very
weak inhibitors (at 10-fold excess of 1) and not comparable to
other aminosugar-containing acceptor analogs, which have been
demonstrated to be very potent glycosyltransferase inhibitors.28
To examine if the inhibition was proportional to an increase in
inhibitor concentration, we studied the mannosylation of 1 (fixed
concentration of 0.2 mM) by the a-(1→6)-ManT in the presence
of these amino analogs at concentrations up to 4 mM. In all
cases, 50% inhibition was observed at about 1 mM under these
conditions, as shown in Fig. 7.
Fig. 5 Mannosidase digestion of products formed from 3, 5, 7 and 9.
Each acceptor at 2 mM was incubated with [3H] GDP-Man under the
assay conditions as described in the experimental section. The radiolabeled
enzymatic products were divided evenly after purification and treated
with exo-mannosidases including Aspergillus saitoi a-(1→2)-mannosidase
(AS), jack bean a-(1→2,3,6)-mannosidase (JB) and Xanthomonas maniho-
tis a-(1→6)-mannosidase (XM). The mannosidase-digested samples were
purified using C18 reversed-phase column and the radioactivities were
compared with the controls (without any mannosidase treatment).
all the [3H]-labeled mannose units. The combination of these
mannosidases enabled the relative proportions of the a-(1→6)-
and a-(1→3/4)-linkages to be determined. These results further
suggested that the observed mannosylations, in all cases except 3,
resulted exclusively from the action of an a-(1→6)-specific ManT
in the membrane preparation.
In the case of 3, the small difference of the residual radioactivity
between the initial product and that treated with mannosidases
from jack bean and X. manihotis suggests that a small amount of
the enzymatic products from 3 might contain a-(1→3/4)-linkages.
Besra and co-workers have recently demonstrated that in M.
tuberculosis an enzyme, MgtA is able to utilize GlcpAGroAc2 (1,2-
di-O-C16/C18 : 1-a-D-glucopyranosyluronic acid-(1→3)-glycerol as
a substrate to form a-Manp-(1→4)-GlcpAGroAc2 (Fig. 6).44
Fig. 7 Inhibition effects of amino analogs 2, 4 and 6 on ManT activity.
The activities were determined using aminosugar analog concentrations
up to 4.0 mM with 1 as the acceptor substrate at 0.2 mM. All other
reaction conditions were identical to the cell-free assay as described in the
experimental section.
Based on the data shown in Table 1, the substrate preference
for ManT, based on the relative activity of the epimeric substrate
analogs, appears to follow the order of a-Manp-(1→6)-a-Manp
1 (100%) > a-Manp-(1→6)-a-Talp 5 (57%) > a-Glcp-(1→6)-a-
Manp 3 (41%). We were therefore surprised to see that the 6-
amino-a-Glcp-(1→6)-a-Manp analog 4, rather than 6-amino-a-
Manp-(1→6)-a-Manp analog 2 is a comparably better inhibitor.
One explanation for this result is that these amino analogs
may not be competing with 1 in the active site, but rather act
in a noncompetitive or uncompetitive fashion. However, given
that these analogs are at best modest inhibitors of a-(1→6)-
mannosylation, we chose not to further probe the mechanism by
which they inhibit the enzyme.
Fig. 6 Reaction catalyzed by MgtA. DAG = diacylglycerol.
It is therefore plausible to speculate that MgtA has a re-
laxed substrate specificity, which allows it to recognize both
disaccharides such as 3 as well as GlcAGroAc2. With regard
to this proposal, it is important to note that the non-reducing
residue in 3, is a Glcp residue and this may be a reasonable
substrate for this newly discovered mannosyltransferase activity.
It is also important to consider that an MgtA-like enzymatic
activity has not, to date, been identified in M. smegmatis, the
source of the membrane fraction used in these investigations.
Although disaccharide 9 also carries a Glcp residue at its non-
reducing terminus, no a-(1→3/4)-linkage was detected from the
exo-mannosidase treatments. Unfortunately, we were unable to
carry out any further investigation due to the small turnover of
3, and the negligible amount of the observed a-(1→3/4)-linked
products obtained in the assay.
Conclusions
In this paper we have described the synthesis of a panel of epimeric
and amino analogs of disaccharide 1 and their evaluation as
potential substrates and inhibitors of a PPM-dependent a-(1→6)-
ManT involved in LAM/LM biosynthesis. The results presented
here are in agreement with our previous findings that the hydroxyl
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groups at C-4¢ and C-2 positions in 1 are important for both
the polar interactions and steric requirements. In particular, a
change of the manno- configuration of the parent substrate 1, at
either the reducing or non-reducing end, to the gluco- or talo-
configuration, results in poor turnover by the ManT, with the
most pronounced loss of activity being observed for a compound
in which the stereochemistry at C-4¢ and C-2 are both inverted.
Analogs 2, 4 and 6, in which the OH group at C-6¢ has been
substituted with an amino group, are weak inhibitors against
ManT. Although the observed inhibition may result from an ionic
interaction of the protonated amino groups of 2, 4 and 6 with a
negatively charged residue in the enzyme active site, the substrate
specificity trends observed in the epimeric substrates are different
from those seen with the aminosugar analogs. This suggests that
the mode of inhibition may not be competitive. Regardless of the
mode of action, the a-Manp-(1→6)-a-Manp (2), a-Glcp-(1→6)-
a-Manp (4), and a-Manp-(1→6)-a-Talp (6) analogs all show 50%
inhibition at a concentration of ~1 mM. A summary of these
results in presented graphically in Fig. 8.
were recorded at 400, 500 or 600 MHz, and chemical shifts are
referenced to either TMS (0.0, CDCl3), or HOD (4.78, D2O and
CD3OD). 13C NMR spectra were recorded at 100 or 125 MHz and
chemical shifts are referenced to internal CDCl3 (77.23, CDCl3),
or CD3OD (48.9, CD3OD). Assignments of NMR spectra were
made based on two-dimensional (1H–1H COSY and HMQC)
experiments. The stereochemistry at the anomeric centres of the
mannopyranose and talopyranose rings were proven by measuring
7
the 1JC1-H1
.
19F spectra were recorded at 376 MHz, and chemical
shifts were referenced to external CFCl3. Samples were prepared
by the cast film method and infrared spectra were measured on a
FT-IR spectrometer. Electrospray mass spectra were recorded on
samples suspended in mixtures of THF with MeOH and added
NaCl.
Octyl
6-amino-6-deoxy-a-D-mannopyranosyl-(1→6)-a-D-
mannopyranoside (2). Disaccharide 30 (17 mg, 0.020 mmol)
was dissolved in MeOH (4 mL) and 1 M NaOMe (1 mL) was
added. After stirring overnight, the solution was neutralized with
Amberlite 120 resin (H+ form), filtered and concentrated. The
crude was dissolved in satd aq NaHCO3 (5 mL) and extracted
with EtOAc (5 mL). The aqueous layer was purified by SepPak C18
reversed-phase column to give 2 (6 mg, 61%), after lyophilization,
as white solid; [a]D = +75.0 (c 0.3, CH3OH); 1H NMR (500 MHz,
CD3OD) dH 4.78 (d, 1H, J = 1.7 Hz, H-1¢), 4.69 (d, 1H, J =
1.7 Hz, H-1), 3.87–3.93 (m, 1H, H-5¢), 3.83 (dd, 1H, J = 3.5,
1.7 Hz, H-2¢), 3.76 (dd, 1H, J = 2.9, 1.7 Hz, H-2), 3.58–3.74 (m,
6H, H-3¢, H-4, H-5, H-6a, H-6b, octyl OCH2), 3.57 (dd, 1H, J =
7.2, 2.9 Hz, H-3), 3.50 (dd, 1H, J = 9.5, 9.5 Hz, H-4¢), 3.39 (dt,
1H, J = 9.5, 6.3 Hz, octyl OCH2), 2.97 (dd, 1H, J = 13.4, 2.7 Hz,
H-6a¢), 2.74 (dd, 1H, J = 13.4, 7.2 Hz, H-6b¢), 1.52–1.64 (m, 2H,
octyl OCH2CH2), 1.22–1.44 (m, 10H, octyl CH2), 0.89 (t, 3H,
J = 6.9 Hz, octyl CH3); 13C NMR (125 MHz, CD3OD) dC 101.7
(C-1¢), 101.4 (C-1), 73.1 (2C, C-5¢, C-5), 72.9 (C-3), 72.5 (C-3¢),
72.2 (C-2¢/C-2), 72.1 (C-2¢/C-2), 70.1 (C-4¢), 68.7 (octyl OCH2),
68.6 (C-4), 67.4 (C-6), 43.2 (C-6¢), 33.0 (octyl CH2), 30.6 (octyl
CH2), 30.5 (octyl CH2), 30.4 (octyl CH2), 27.4 (octyl CH2), 23.7
(octyl CH2), 14.4 (octyl CH3). HRMS (ESI) calcd. for (M + H)
C20H40NO10: 454.2647. Found: 454.2645.
Fig.
8 Graphical summary of activity of analogs 1–9 with
a-(1→6)-ManT.
Experimental section
Octyl a-D-glucopyranosyl-(1→6)-a-D-mannopyranoside (3).
Disaccharide 31 (37 mg, 0.036 mmol) was dissolved in CH3OH
(4 mL) and 20% Pd(OH)2–C (15 mg) was added. The mixture
was stirred overnight under a H2 atmosphere and the catalyst was
separated by filtration through a short pad of Celite. The crude
product was purified by chromatography on Iatrobeads (4 : 1
CH2Cl2–CH3OH) to give 3 (14 mg, 82%) as clear glass. Rf 0.54
General methods for chemical synthesis
All reagents used were purchased from commercial sources and
were used without further purification unless noted. Solvents
used in reactions were purified by successive passage through
columns of alumina and copper under nitrogen. Unless indicated
otherwise, all reactions were performed at room temperature
(rt) and under a positive pressure of argon. The reactions were
monitored by analytical TLC on silica gel 60-F254 (0.25 mm,
Silicycle) and spots were detected under UV light or by charring
with acidified anisaldehyde solution in ethanol. Organic solvents
were evaporated under reduced pressure at <40 ◦C. Products
were purified by chromatography using silica gel (40–60 mM),
Iatrobeads (Iatron Laboratories, Tokyo) or SepPak C18 reversed-
phase cartridges (Waters). Before use, the SepPak cartridges were
prewashed with 10 mL of MeOH followed by 10 mL of H2O. The
crude residue in water was loaded onto the prewashed column.
After washing the column with water (10 mL), the desired product
was eluted with MeOH (4 mL). Optical rotations were measured at
22 2 ◦C and are in units of degrees mL/(g dm). 1H NMR spectra
1
(4 : 1 CH2Cl2–MeOH); [a]D = +99.1 (c 0.3, CH3OH); H NMR
(500 MHz, CD3OD) dH 4.82 (d, 1H, J = 3.7 Hz, H-1¢), 4.70 (d,
1H, J = 1.7 Hz, H-1), 4.00 (dd, 1H, J = 10.6, 4.0 Hz, H-6a),
3.61–3.82 (m, 9H, H-3¢, H-5¢, H-6a¢, H-6b¢, H-2, H-3, H-4, H-5,
octyl OCH2), 3.60 (dd, 1H, J = 10.6, 2.3 Hz, H-6b), 3.39 (dt, 1H,
J = 9.6, 6.3 Hz, octyl OCH2), 3.35 (dd, 1H, J = 9.6, 3.7 Hz, H-2¢),
3.30 (overlap with residual CD2HOD peak, 1H, H-4¢), 1.52–1.62
(m, 2H, octyl OCH2CH2), 1.23–1.42 (m, 10H, octyl CH2), 0.89
(t, 3H, J = 7.0 Hz, octyl CH3); 13C NMR (125 MHz, CD3OD)
dC 101.8 (C-1), 100.0 (C-1¢), 74.3 (C-5¢/C-5), 73.9 (C-2¢), 73.5
(C-5¢/C-5), 72.8 (C-3¢, C-3), 72.3 (C-2), 71.8 (C-4¢), 68.8 (octyl
OCH2), 68.4 (C-4), 67.3 (C-6), 62.6 (C-6¢), 33.0 (octyl CH2),
30.6(3) (octyl CH2), 30.5(6) (octyl CH2), 30.4 (octyl CH2), 27.4
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(octyl CH2), 23.7 (octyl CH2), 14.4 (octyl CH3). HRMS (ESI)
calcd. for (M + Na) C20H38O11: 477.2306. Found: 477.2305.
6 h. The mixture was cooled, diluted with EtOAc (20 mL),
washed with satd aq NaHCO3 (5 mL), dried (Na2SO4), and
concentrated. The crude product was subsequently dissolved in
MeOH (4 mL) and 1 M NaOMe (1 mL) was added. After stirring
overnight, the solution was neutralized with Amberlite 120 resin
(H+ form), filtered and concentrated. The azidosugar intermediate
was dissolved in CH3OH (8 mL) and 20% Pd(OH)2–C (50 mg) was
added. The mixture was stirred overnight under a H2 atmosphere
and the catalyst was separated by filtration through a short pad
of Celite. The eluant was concentrated, redissolved in satd aq
NaHCO3 (5 mL) and extracted with EtOAc (5 mL). The aqueous
layer was purified by on a SepPak C18 reversed-phase cartridge to
Octyl 6-amino-6-deoxy-a-D-glucopyranosyl-(1→6)-a-D-manno-
pyranoside (4). Disaccharide 34 (61 mg, 0.062 mmol) was
dissolved in MeOH (4.5 mL) and 1 M NaOMe (1 mL) was
added. After stirring overnight, the solution was neutralized with
Amberlite 120 resin (H+ form), filtered and concentrated. The
crude was dissolved in satd aq NaHCO3 (5 mL) and extracted
with EtOAc (5 mL). The aqueous layer was purified by SepPak C18
reversed-phase column to give 4 (14 mg, 79%), after lyophilization,
as white solid; [a]D = +149.2 (c 0.1, CH3OH); 1H NMR (500 MHz,
CD3OD) dH 4.81 (d, 1H, J = 3.8 Hz, H-1¢), 4.71 (d, 1H, J = 1.8 Hz,
H-1), 4.01 (dd, 1H, J = 10.5, 3.9 Hz, H-6a), 3.77 (dd, 1H, J = 9.6,
9.6 Hz, H-4), 3.77 (dd, 1H, J = 3.2, 1.8 Hz, H-2), 3.58–3.74 (m,
6H, H-3¢, H-5¢, H-3, H-5, H-6b, octyl OCH2), 3.40 (dt, 1H, J =
9.6, 6.3 Hz, octyl OCH2), 3.35 (dd, 1H, J = 9.7, 3.8 Hz, H-2¢), 3.13
(dd, 1H, J = 9.8, 8.9 Hz, H-4¢), 3.00 (dd, 1H, J = 13.4, 2.8 Hz,
H-6a¢), 2.68 (dd, 1H, J = 13.4, 7.7 Hz, H-6b¢), 1.52–1.64 (m, 2H,
octyl OCH2CH2), 1.23–1.42 (m, 10H, octyl CH2), 0.88 (t, 3H,
J = 7.0 Hz, octyl CH3); 13C NMR (125 MHz, CD3OD) dC 101.9
(C-1), 99.8 (C-1¢), 75.3 (C-5¢/C-5), 73.9 (C-5¢/C-5), 73.6 (C-2¢),
73.5 (C-4¢), 72.9, 72.8 (C-3¢, C-3), 72.2 (C-2), 68.9 (octyl OCH2),
68.2 (C-4), 67.1 (C-6), 43.8 (C-6¢), 33.0 (octyl CH2), 30.6(3) (octyl
CH2), 30.5(5) (octyl CH2), 30.4 (octyl CH2), 27.4 (octyl CH2), 23.7
(octyl CH2), 14.4 (octyl CH3). HRMS (ESI) calcd. for (M + H)
C20H40NO10: 454.2647. Found: 454.2649.
give 6 (24 mg, 86%), after lyophilization, as white solid; [a]D
=
1
+100.0 (c 0.2, CH3OH); H NMR (500 MHz, CD3OD) dH 4.81
(br s, 1H, H-1), 4.79 (d, 1H, J = 1.6 Hz, H-1¢), 3.86–3.94 (m, 2H,
H-5, H-6a), 3.80 (dd, 1H, J = 3.3, 1.8 Hz, H-2¢), 3.77–3.81 (m, 1H,
H-2), 3.58–3.75 (m, 6H, H-3¢, H-5¢, H-3, H-4, H-6b, octyl OCH2),
3.52 (dd, 1H, J = 9.5, 9.5 Hz, H-4¢), 3.42 (dt, 1H, J = 9.6, 6.3 Hz,
octyl OCH2), 3.05 (dd, 1H, J = 13.5, 2.9 Hz, H-6a¢), 2.83 (dd,
1H, J = 13.5, 7.0 Hz, H-6b¢), 1.52–1.64 (m, 2H, octyl OCH2CH2),
1.23–1.42 (m, 10H, octyl CH2), 0.90 (t, 3H, J = 6.9 Hz, octyl
CH3); 13C NMR (125 MHz, CD3OD) dC 102.2 (C-1), 101.6 (C-
1¢), 73.7 (C-5¢), 72.6, 72.5 (C-3¢, C-3), 72.1 (C-2¢), 71.8 (C-2), 71.1
(C-5), 69.8 (C-4¢), 68.9 (octyl OCH2), 68.2 (C-6), 67.2 (C-4), 43.4
(C-6¢), 33.0 (octyl CH2), 30.6 (octyl CH2), 30.5 (octyl CH2), 30.4
(octyl CH2), 27.4 (octyl CH2), 23.7 (octyl CH2), 14.4 (octyl CH3).
HRMS (ESI) calcd. for (M + H) C20H40NO10: 454.2647. Found:
454.2649.
Octyl a-D-mannopyranosyl-(1→6)-a-D-talopyranoside (5).
Protected disaccharide 36 (84 mg, 0.077 mmol) was dissolved in
THF (5 mL) and AcOH (12 mL, 0.46 mmol) and 1.0 M tetra-
n-butylammonium fluoride in THF (0.15 mL, 0.15 mmol) were
added. After stirring overnight, the mixture was filtered through
a short pad of silica gel and concentrated. The crude product
Octyl a-D-mannopyranosyl-(1→6)-a-D-glucopyranoside (7).
Disaccharide 38 (45 mg, 0.041 mmol) was dissolved in MeOH
(6 mL) and NaOMe (32 mg) was added. After stirring overnight,
the solution was neutralized with acetic acid, concentrated, and
the residue was purified by chromatography (10 : 1 CH2Cl2–
CH3OH). The pure fractions were collected, concentrated, and
redissolved in CH3OH (8 mL) and 20% Pd(OH)2–C (30 mg) was
added. The mixture was stirred overnight under a H2 atmosphere
and the catalyst was separated by filtration through a short pad
of Celite. The filtrate was concentrated to give 7 (16 mg, 84%) as
clear glass. Rf 0.36 (4 : 1 CH2Cl2–MeOH); [a]D = +95.7 (c 0.5,
◦
was redissolved in 4 : 1 AcOH–H2O (5 mL) and heated at 50 C
for 3 h. The mixture was diluted with EtOAc (20 mL), washed
with satd aq NaHCO3 (5 mL), dried (Na2SO4), and concentrated.
The resulting product was subsequently dissolved in MeOH
(5 mL) and NaOMe (54 mg) was added. After 2 h, the solution
was neutralized with AcOH and, following concentration, the
crude product was purified by chromatography on Iatrobeads
(4 : 1 CH2Cl2–CH3OH) to give 5 (21 mg, 91% over three steps) as
clear glass. Rf 0.54 (4 : 1 CH2Cl2–MeOH); [a]D = +78.2 (c 0.4,
CH3OH); 1H NMR (600 MHz, CD3OD) dH 4.80 (br s, 1H, H-1),
4.79 (d, 1H, J = 1.8 Hz, H-1¢), 3.60–3.74 (m, 2H, H-5, H-6a), 3.82
(dd, 1H, J = 11.8, 2.3 Hz, H-6a¢), 3.80 (dd, 1H, J = 3.3, 1.8 Hz,
H-2¢), 3.58–3.74 (m, 9H, H-3¢, H-4¢, H-5¢, H-6b¢, H-2, H-3, H-4,
H-6b, octyl OCH2), 3.43 (dt, 1H, J = 9.7, 6.3 Hz, octyl OCH2),
1.52–1.62 (m, 2H, octyl OCH2CH2), 1.24–1.41 (m, 10H, octyl
CH2), 0.89 (t, 3H, J = 7.0 Hz, octyl CH3); 13C NMR (125 MHz,
CD3OD) dC 102.2 (C-1), 101.6 (C-1¢), 74.6 (C-5¢), 72.7, 72.6 (C-3¢,
C-3), 72.2 (C-2), 71.7 (C-2¢), 71.0 (C-5), 68.9 (octyl OCH2), 68.5
(C-4¢), 67.9 (C-6), 67.2 (C-4), 62.9 (C-6¢), 33.0 (octyl CH2), 30.6
(octyl CH2), 30.5 (octyl CH2), 30.4 (octyl CH2), 27.4 (octyl CH2),
23.7 (octyl CH2), 14.4 (octyl CH3). HRMS (ESI) calcd. for (M +
Na) C20H38O11: 477.2306. Found: 477.2307.
1
CH3OH); H NMR (600 MHz, CD3OD) dH 4.80 (d, 1H, J =
1.7 Hz, H-1¢), 4.74 (d, 1H, J = 3.9 Hz, H-1), 3.89 (dd, 1H, J =
11.1, 5.3 Hz, H-6a), 3.78–3.84 (m, 2H, H-2¢, H-6a¢), 3.65–3.74
(m, 5H, H-3¢, H-6b¢, H-5, H-6b, octyl OCH2), 3.58–3.65 (m, 3H,
H-4¢, H-5¢, H-3), 3.43 (dt, 1H, J = 9.6, 6.3 Hz, octyl OCH2),
3.37 (dd, 1H, J = 9.7, 3.9 Hz, H-2), 3.33 (dd, 1H, J = 10.0,
9.0 Hz, H-4), 1.56–1.69 (m, 2H, octyl OCH2CH2), 1.24–1.46 (m,
10H, octyl CH2), 0.89 (t, 3H, J = 6.6 Hz, octyl CH3); 13C NMR
(125 MHz, CD3OD) dC 101.5 (C-1¢), 100.2 (C-1), 75.3 (C-3), 74.4
(C-2), 73.6, 72.7, 72.2, 72.1 (4C, C-2¢, C-3¢, C-5¢, C-5), 71.7 (C-4),
69.3 (octyl OCH2), 68.6 (C-4¢), 67.1 (C-6), 62.9 (C-6¢), 33.0 (octyl
CH2), 30.7 (octyl CH2), 30.6 (octyl CH2), 30.4 (octyl CH2), 27.4
(octyl CH2), 23.7 (octyl CH2), 14.4 (octyl CH3). HRMS (ESI)
calcd. for (M + Na) C20H38O11: 477.2306. Found: 477.2305.
Octyl a-D-talopyranosyl-(1→6)-a-D-mannopyranoside (8).
Disaccharide 39 (39 mg, 0.037 mmol) was dissolved in THF
(3 mL) and 1.0 M tetra-n-butylammonium fluoride in THF
(0.15 mL, 0.15 mmol) was added and the solution was stirred
at rt overnight. The reaction mixture was diluted with CH2Cl2
Octyl 6-amino-6-deoxy-a-D-mannopyranosyl-(1→6)-a-D-talo-
pyranoside (6). Disaccharide 37 (61 mg, 0.062 mmol) was
dissolved in 4 : 1 AcOH–H2O (5 mL) and heated at 50 ◦C for
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(15 mL), washed with H2O (5 mL), and the organic phase
was dried (Na2SO4) and concentrated. The resulting crude
product was redissolved in MeOH (4 mL) and NaOMe was
added (22 mg). The reaction mixture was stirred for 3 h and
neutralized with AcOH. The solution was then diluted with
CH2Cl2 (15 mL), washed with H2O (5 mL), and the organic
phase was concentrated. The partially deprotected intermediate
was dissolved and stirred in 4 : 1 HOAc–H2O (5 mL) at 50 ◦C
overnight. The mixture was diluted with CH2Cl2 (15 mL), washed
with satd aq NaHCO3 (5 mL ¥ 2), and the organic phase was dried
(Na2SO4) and concentrated. The resulting crude product was
redissolved in CH3OH (8 mL) and 20% Pd(OH)2–C (15 mg) was
added. The mixture was stirred overnight under a H2 atmosphere
and the catalyst was separated by filtration through a short pad
of Celite. The filtrate was concentrated and the residue purified
by chromatography on Iatrobeads (4 : 1 CH2Cl2–MeOH) to give 8
octyl OCH2CH2), 1.24–1.42 (m, 10H, octyl CH2), 0.89 (t, 3H, J =
7.2 Hz, octyl CH3); 13C NMR (125 MHz, CD3OD) dC 102.3 (C-1),
100.2 (C-1¢), 75.2 (C-3¢), 73.8 (C-5¢), 73.6 (C-2¢), 72.7 (C-2), 71.7
(C-4¢), 71.4 (C-3), 70.8 (C-5), 69.0 (octyl OCH2), 68.0 (C-6), 67.1
(C-4), 62.6 (C-6¢), 33.0 (octyl CH2), 30.6 (octyl CH2), 30.4 (2C,
octyl CH2), 27.4 (octyl CH2), 23.7 (octyl CH2), 14.5 (octyl CH3).
HRMS (ESI) calcd. for (M + Na) C20H38O11: 477.2306. Found:
477.2299.
Bacterial strains and growth conditions
M. smegmatis mc2155 was a generous gift from Professor William
R. Jacobs, Jr. at the Albert Einstein College of Medicine. The
bacteria were grown at 37 ◦C in 100 mL of Luria Bertoni (LB)
broth medium containing 0.05% Tween 80 to an A600nm of < 1.0
(~two days from a frozen bacterial stock). 50 mL liquid cultures
were then transferred to 2 ¥ 1 L of fresh media and cultured further
for 24 h at 37 ◦C. Cells were harvested by centrifugation,◦washed
with phosphate buffered saline (PBS) and stored at -20 C until
use.
(8 mg, 45%) as clear glass. Rf 0.38 (4 : 1 CH2Cl2–MeOH); [a]D
=
+83.5 (c 0.3, CH3OH); 1H NMR (600 MHz, CD3OD) dH 4.90 (d,
1H, J = 1.5 Hz, H-1¢), 4.70 (d, 1H, J = 1.7 Hz, H-1), 3.80–3.93
(m, 3H, H-3¢, H-4¢, H-6a), 3.77 (dd, 1H, J = 3.2, 1.7 Hz, H-2),
3.71–3.77 (m, 5H, H-2¢, H-5¢, H-6a¢, H-6b¢, H-6b), 3.60–3.69 (m,
4H, H-3, H-4, H-5, octyl OCH2), 3.40 (dt, 1H, J = 9.6, 6.4 Hz,
octyl OCH2), 1.52–1.62 (m, 2H, octyl OCH2CH2), 1.24–1.42 (m,
10H, octyl CH2), 0.90 (t, 3H, J = 7.2 Hz, octyl CH3); 13C NMR
(125 MHz, CD3OD) dC 102.0 (C-1¢), 101.7 (C-1), 73.1, 72.8,
72.5, 72.2 (5C, C-2¢, C-3¢, C-2, C-3, C-4), 71.8 (C-4¢), 68.6 (octyl
OCH2), 68.6 (C-5), 67.5 (C-6), 67.3 (C-5¢), 62.9 (C-6¢), 33.0 (octyl
CH2), 30.6 (octyl CH2), 30.5 (octyl CH2), 30.4 (octyl CH2), 27.4
(octyl CH2), 23.7 (octyl CH2), 14.4 (octyl CH3). HRMS (ESI)
calcd. for (M + Na) C20H38O11: 477.2306. Found: 477.2303.
Preparation of membrane fractions from M. smegmatis
The M. smegmatis cell pellet (~10
g wet weight) was
washed and resuspended in 100 mL of 50 mM 3-(N-
morpholino)propanesulfonic acid (MOPS) buffer (adjusted to
pH 7.9 with KOH) containing 5 mM b-mercaptoethanol and
10 mM MgCl2 supplemented with Complete Protease Inhibitor
Cocktail Tablets (Roche) at 4 ◦C. The cells were subjected to
two passes through a Thermo Spectronic French Pressure Cell
Press at 20 000 psi. The cell lysate was centrifuged at 600 ¥ g
for 15 min and then at 27 000 ¥ g for 20 min. The resulting
supernatant was centrifuged at 100 000 ¥ g for 60 min. The
supernatant was carefully removed and the membrane pellets
were gently resuspended in 1 mL of 50 mM MOPS buffer,
pH 7.9, containing 5 mM b-mercaptoethanol and 10 mM MgCl2.
Protein concentration (31.6 mg mL-1) was determined by the
BCA(tm) Protein Assay (Pierce) using bovine serum albumin as
the standard.
Octyl a-D-glucopyranosyl-(1→6)-a-D-talopyranoside (9). Dis-
accharide 40 (85 mg, 0.081 mmol) was dissolved in THF (7 mL)
and 1.0 M tetra-n-butylammonium fluoride in THF (0.32 mL,
0.32 mmol) was added and the solution was stirred at rt overnight.
The reaction mixture was diluted with CH2Cl2 (15 mL), washed
with H2O (5 mL), and the organic phase was dried (Na2SO4)
and concentrated. The resulting crude product was redissolved in
MeOH (8 mL) and NaOMe was added (43 mg). The solution was
stirred for 2 h and neutralized with AcOH. The mixture was diluted
with CH2Cl2 (15 mL), washed with H2O (5 mL), and the organic
phase was concentrated. The partially deprotected intermediate
was dissolved and stirred in 4 : 1 HOAc–H2O (5 mL) at 50 ◦C
overnight. The mixture was diluted with CH2Cl2 (15 mL), washed
with sat. aq. NaHCO3 (5 mL x 2), and the organic phase was
dried (Na2SO4) and concentrated. The resulting crude product was
redissolved in CH3OH (8 mL) and 20% Pd(OH)2–C (20 mg) was
added. The mixture was stirred overnight under a H2 atmosphere
and the catalyst was separated by filtration through a short pad of
Celite. The filtrate was concentrated and the residue was purified
by chromatography on Iatrobeads (4 : 1 CH2Cl2–MeOH) to give 9
(23 mg, 62%) as clear glass. Rf 0.33 (4 : 1 CH2Cl2–MeOH); [a]D =
Radiochemical activity assays
The ManT enzyme activity was determined using the previously
established cell-free system.21 Unless indicated otherwise, the
synthetic acceptor analogs at a concentration of 2.0 mM were
incubated with 0.20 mCi of guanosine diphosphate mannopyra-
nose (GDP-mannose), [mannose-2-3H] (American Radiolabeled
Chemicals, Inc., 20 Ci mmol-1) in 50 mM MOPS buffer, pH 7.9,
containing 1 mM ATP, 10 mM MgCl2, 5 mM b-mercaptoethanol,
and membrane fraction (94.8 mg of protein) in a total volume of
80 mL. All assays were performed in duplicate and control assays
without acceptor were also performed in parallel to correct for
the presence of endogenous acceptor. The enzymatic activities
were determined using ◦radiochemical SepPak C18 assays.30 Briefly,
after incubation at 37 C for 1 h, the reactions were stopped by
adding 100 mL of CHCl3–MeOH (2 : 1 v/v) and the mixtures were
centrifuged. The supernatants were recovered and further diluted
with H2O before loading onto SepPak C18 cartridges (Waters). The
unreacted donor was removed by washing the cartridges with H2O
(50 mL) and the radiolabeled products were eluted with MeOH
1
+102.9 (c 1.3, CH3OH); H NMR (500 MHz, CD3OD) dH 4.82
(d, 1H, J = 3.7 Hz, H-1¢), 4.80 (d, 1H, J = 1.5 Hz, H-1), 3.93
(dd, 1H, J = 6.3, 6.3 Hz, H-5), 3.84–3.90 (m, 2H, H-4, H-6a), 3.79
(dd, 1H, J = 11.9, 2.4 Hz, H-6a¢), 3.66–3.76 (m, 5H, H-6b¢, H-2,
H-3, H-6b, octyl OCH2), 3.59–3.66 (m, 2H, H-3¢, H-5¢), 3.41 (dt,
1H, J = 9.7, 6.4 Hz, octyl OCH2), 3.40 (dd, 1H, J = 9.8, 3.7 Hz,
H-2¢), 3.31 (dd, 1H, J = 9.8, 8.9 Hz, H-4¢), 1.52–1.62 (m, 2H,
190 | Org. Biomol. Chem., 2010, 8, 181–192
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(4.0 mL). The isolated products in the eluants were quantified by
liquid scintillation counting on a Beckman LS6500 Scintillation
Counter using 10 mL of Ecolite cocktail. For kinetic analysis,
the ManT activities were determined using a range of acceptor
concentrations. All other reaction conditions were identical to the
cell-free assay as described above. Assays were performed under
the conditions in which the formations of radiolabeled products
were linear for both time and protein concentration. The kinetic
parameters KM and Vmax were obtained by nonlinear regression
analysis using the Michaelis–Menten equation with the GraphPad
Prism 4.0 program (GraphPad Software, San Diego, CA).
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Acknowledgements
This work was supported by the Alberta Ingenuity Centre
for Carbohydrate Science, the University of Alberta and the
Natural Sciences and Engineering Research Council of Canada.
PHT thanks Alberta Ingenuity for an Ingenuity PhD Student
Scholarship.
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