Identification of the 5-methylthiopentosyl substituent in Mycobacterium tuberculosis lipoarabinomannan

Article abstract of DOI:10.1002/anie.200454119

An unexpected configuration of a novel carbohydrate structure in the cell wall of Mycobacterium tuberculosis was revealed by synthesizing the isomers of 5-deoxy-5-methylthio-D-pentofuranose as α- and β-methyl glycosides, and analyzing their NMR spectra (s

Full text of DOI:10.1002/anie.200454119

Communications
Cell-Wall Glycolipids
Identification of the 5-Methylthiopentosyl
Substituent in Mycobacterium tuberculosis
Lipoarabinomannan**
W. Bruce Turnbull, Kazumi Hiruma Shimizu,
Delphi Chatterjee, Steve W. Homans, and
Achim Treumann*
The search for new and improved drugs against tuberculosis
continues to focus on the biosynthesis of the Mycobacterium
tuberculosis (Mtb) cell wall.[1] Lipoarabinomannan (LAM)[2]
is a major component of this cell wall and facilitates entry of
Mtb into macrophages. Furthermore, LAM promotes the
intracellular survival of Mtb by down-regulating the immune
response and providing antioxidative protection.[2] LAM is a
lipid-anchored polysaccharide comprising dendritic arabinan
chains which emanate from a poly-a(1!6)-mannosyl core
bearing a(1!2)-mannosyl substituents (Figure 1). In Mtb, the
arabinan chains are capped with several further mannosyl
residues, which form the nonreducing termini. We recently
discovered[3] a novel substituent of these mannosyl caps,
which, through a combination of NMR spectroscopic and
[*] Dr. W. B. Turnbull,++ Dr. K. H. Shimizu, Prof. S. W. Homans,
Dr. A. Treumann+
Astbury Centre for Structural Molecular Biology
School of Biochemistry and Microbiology
University of Leeds
Leeds, LS29JT (UK)
E-mail: atreumann@rcsi.ie
Prof. D. Chatterjee
Department of Microbiology, Immunology, and Pathology
Colorado State University
Fort Collins, CO 80523-1682 (USA)
[+] Present address:
Royal College of Surgeons in Ireland
123 St Stephen’s Green, Dublin 2 (Ireland)
Fax: (+353)1-402-2274
[
++] Present Address:
School of Chemistry
University of Leeds
Leeds, LS29JT (UK)
[**] This work was supported in the UK by the Wellcome Trust and in the
USA by grants from the National Institutes of Health (NIAID
AI 37139 and AI 47197).
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DOI: 10.1002/anie.200454119
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Figure 1. Cartoon representation of LAM, a phosphatidylinositol-an-
chored polysaccharide from Mycobacterium tuberculosis. It is not known
how many arabinan side chains are attached to the branched mannan
core of LAM. The novel 5-deoxy-5-methylthiopentofuranosyl residue
was found to be attached to one of the short oligomannosyl chains
capping the arabinan side chains.
Scheme 1. Reagents: a) 1. TrCl/C5H5N; 2. BzCl/C5H5N; 3. 80% AcOH
~
(aq)/ ; 4. TsCl/C5H5N (48%); b) 1. MeSNa/[18]crown-6/DMF; 2.
NaOMe/MeOH (1: 94%, 2: 90%); c) 1. MeSNa/[18]crown-6/DMF; 2.
~
~
SrCl2/MeOH/TFA/ (3: 7%, 4: 41%); d) MeOH/TFA/ (5: 15%, 6:
40%). Tr=triphenylmethyl, Bz=benzoyl, Ts=4-toluenesulfonyl,
TFA=trifluoroacetic acid.
mass spectrometric techniques, was identified as having a 5-
deoxy-5-methylthiopentofuranosyl (MTP) structure. The
presence of an oxidized form of this residue, 5-deoxy-5-
methylsulfoxypentofuranosyl (MSP), was also observed. Such
monosaccharide residues have never previously been identi-
fied in an oligosaccharide. Herein, we use a combination of
chemical synthesis and NMR spectroscopy to reveal the
configuration of this novel component of the Mtb cell wall.
The fluctional conformations of furanose rings[4] make the
identification of their relative configuration from 3J coupling
constants more difficult than for pyranose sugars. Conse-
quently, we chose to synthesize all four isomers of 5-deoxy-5-
methylthio-d-pentofuranose, each as their a- and b-methyl
glycosides (see Figure 2, compounds 1–8). Only the xylo-
configured sugars 7 and 8 have been reported previously,
albeit as a mixture of a- and b-anomers.[5] Although it is not
possible to distinguish compounds 7 and 8 by thin layer
chromatography, they were separated readily by using an
anion exchange chromatography column (Dowex 1-X2, OHꢀ
form) and eluting with water. The a-arabino isomer 1 was
synthesized (Scheme 1) from the known[6] tosylate 11 by
reaction with MeSNa and subsequent removal of the benzoyl
groups under ZemplØn conditions. The analogous b isomer 2
was prepared in a similar fashion from methyl glycoside 10[7]
via tosylate 12.
substituents pointing toward the same face of the furanose
ring.[8] However, Angyal et al. discovered that the proportion
of the b-furanose isomer increased from trace quantities to
about 30% when the acid-catalyzed preparation of methyl
glycosides of lyxose was conducted in the presence of
strontium chloride.[9] Consequently, following the displace-
ment of the Ts group in compound 13[10] with MeSNa, the
acetonide protecting group was removed by acid-catalyzed
methanolysis in the presence of anhydrous SrCl2, with
concomitant anomerization. Isomers 3 and 4 were thus
obtained in 7 and 41% yield, respectively, after chromatog-
raphy on a Sr2+ ion exchange column (Dowex 50WX2-400)[9]
and eluting with water. Finally, the ribo-configured deriva-
tives 5 and 6 were prepared by methanolysis of 5’-methyl-
thioadenosine (14), which is commercially available. In this
case, the two isomers were found to be separable by careful
silica-gel chromatography. Analytical data for compounds 1–8
are presented in Table 1.
The HSQC spectra for compounds 1–8 are shown in
Figure 2.[11] The signals of the anomeric proton (Figure 2a)
are grouped together in two main clusters with 13C chemical
shifts of about d = 105 ppm for the 1,2-cis glycosides and
about d = 110 ppm for the 1,2-trans glycosides. These groups
are further divided into pairs of C3 epimers, with the
b anomers located upfield of the a anomers in the 1H
dimension. The remaining signals for the ring atoms are
more randomly spread, providing a fingerprint region for
comparison with the spectra of MTP-containing LAM.
Whereas the synthesis of a-lyxofuranose derivatives is
relatively straightforward, the b anomers are notoriously
difficult to prepare as a consequence of having all four
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Table 1: Chemical shifts[a] and optical rotations[b] for compounds 1–9 and MTP/MSP-LAM.
Compound
H-1
1
2
3
4
5
6
7
8
9
MTP[c]
MSP[c]
4.93
4.89
4.95
4.90
5.00
4.90
5.03
4.88
5.06
5.06
4.18
4.18
4.31
4.31
4.62
4.60
3.29
3.13
3.11
3.13
3.46
3.46
2.80
2.79
5.40
5.44
5.45
4.20
4.22
4.34
4.34
4.65
4.61
3.28
3.12
3.08
3.12
n.a.
H-2
4.06
3.95
4.14
2.80
2.88
3.41
2.18
4.13
4.05
3.98
2.77
2.86
3.42
2.17
4.17
4.26
4.34
2.74
2.82
3.46
2.17
4.23
4.22
4.26
2.74
2.86
3.40
2.18
4.20
4.02
4.22
2.73
2.83
3.44
2.17
4.08
4.19
4.13
2.74
2.88
3.41
2.19
4.17
4.24
4.37
2.69
2.79
3.46
2.17
4.14
4.17
4.45
2.76
2.85
3.40
2.19
4.21
4.26
4.38
2.68
2.80
n.a.[d]
2.21
105.2
79.4
78.3
80.5
35.8
n.a.
H-3
H-4
H-5
H-5’
OCH3
SCH3
C-1
2.84
2.83
105.4
105.4
79.4
79.3
78.5
78.5
76.5
75.6
55.6
57.1
n.a.
110.5
104.8
110.5
104.9
105.8
110.5
105.0
111.4
105.0
105.0
79.6
79.6
78.7
78.7
76.0
75.5
55.4
57.0
58.4
58.4
40.2
40.5
C-2
83.5
82.4
85.3
38.6
57.7
17.6
102.3
79.5
80.6
79.6
74.2
82.1
35.0
59.2
17.3
56.7
74.7
72.8
73.9
75.0
85.6
39.1
58.3
17.6
113.6
77.2
76.8
84.3
40.5
58.2
17.4
ꢀ5.4
79.6
78.4
80.4
35.8
58.7
17.4
119.6
82.5
77.8
C-3
C-4
83.1
82.4
84.3
C-5
40.6
36.7
35.9
OCH3
SCH3
[a]d
58.2
58.3
58.3
17.4
17.3
17.4
17.4
n.a.
39.9
40.2
n.a.
ꢀ60.1
ꢀ72.1
ꢀ38.2
n.d.[e]
[a] Chemical shifts [ppm] relative to 3-trimethylsilylpropionate in D2O. [b] Optical rotations recorded at the sodium D line at 228Cin H 2O have units of
degcm3 gꢀ1 dmꢀ1. [c] Values for MTP-LAM and MSP-LAM are taken from reference [3]; values in bold to aid comparison between 7 and MTP. [d] Not
applicable. [e] Not determined, as 9 is a mixture of diastereomers.
Similarly, the upfield region (Figure 2b) shows that the
methyl-group resonances have near identical chemical shifts
and that those for the methylene group attached to the
methylthio moiety are somewhat more dispersed.
downfield shift for the atoms adjacent to the sulfur atom,
following oxidation to 9, demonstrates that compounds 1–8
are free from oxidation products.
The discovery of a 5-deoxy-5-methylthio-a-xylofuranosyl
(MTX) structure in LAM is remarkable for two reasons.
Firstly, there is no precedent, to our knowledge, for a
methylthio sugar incorporated into an oligosaccharide. Sec-
ondly, there are very few other examples of xylo-configured
sugars outside the plant kingdom[12] and only one prior
example in a bacterial polysaccharide.[12c] Nevertheless, MTX
is widespread in strains of Mtb including the well-character-
ized laboratory strains H37Rv and H37Ra[3] and the clinical
isolates CSU20[3] and MT103.[13] Recently MTX was also
found in a clinical strain of the nontuberculous opportunistic
pathogen Mycobacterium kansasii,[14] in which it is attached to
the core mannan domain of the related lipomannan and of
LAM. This is distinct from LAM of Mtb wherein MTX is
attached to the mannosyl caps.
Comparison of these spectra with TOCSY (Figure 2c) and
HSQC (Figure 2d and e) spectra for LAM clearly demon-
strates that only the resonances for the a-xylo compound 7
are comparable to those for MTP. Indeed, the 13C resonances
all agree within Dd = 0.2ppm with those of MTP and the 1H
resonances within Dd = 0.04 ppm, with the exception of that
for the anomeric proton. It is perhaps not surprising that there
is disagreement between the anomeric signals when one
considers that MTP is attached in LAM to an underlying
mannosyl residue and not to a methyl group as in compounds
1–8. The extent of the downfield shift for the anomeric proton
(Dd = 0.4 ppm) is nevertheless significant and might indicate
steric effects constraining the conformation of the MTP–
mannosyl linkage. To seek further confirmation for the a-
xylo configuration, an aqueous solution of compound 7 was
treated with hydrogen peroxide to convert the thioether into a
1:1 diastereomeric mixture of sulfoxides 9. The HSQC
spectrum of compound 9 compares extremely well with that
for MSP-LAM (Figure 2d,e), again with the sole exception of
the anomeric proton resonance. Furthermore, the distinct
It would be reasonable to predict that MTX is derived
biosynthetically from 14, which is a ubiquitous by-product of
polyamine biosynthesis. We would thus tentatively assign the
d configuration to MTX, as this would require epimerization
of only one stereocenter in 14 (Scheme 2). Indeed, this
hypothesis is supported by the remarkable discovery of xylo-
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Figure 2. Structural formulas of the eight possible methyl glycosides of MTP (compounds 1–8) and their NMR spectra. a) Low- and b) high-field regions of
the HSQCspectra. c) A strip from the TOCSY spectrum of MTP-LAM showing resonances of nuclei correlating with the anomeric protons for MTP and MSP
(red and gray, respectively). d) Low- and e) high-field regions of the HSQCspectrum of MTP-LAM (in black) are presented for comparison with those of a-
xylo-configured compounds 7 and 9 (red and gray, respectively).
configured methylthioadenosine 15 in the mediterranean
nudibranch mollusc Doris verrucosa,[12d] wherein compound
15 is produced by epimerization at C3’.[15] Whether biosyn-
thesis of MTX-LAM (17) proceeds through direct glycosyla-
tion with 15, or, more likely, via a nucleoside diphosphate
activated sugar 16 remains to be demonstrated.
Although no biological activity has yet been observed for
15,[16] the extra biosynthetic effort that mycobacteria must
expend to make MTX-LAM and also its widespread occur-
rence[3,13,14] would suggest that MTX provides some biological
advantage to these pathogens. The relative stability of
compounds 1–8 would imply that the nonstereoselective
oxidation of MTX in LAM might occur as the result of
exposure to reactive oxygen species which would be found
within macrophage cells in vivo. Although the low concen-
tration of MTX in LAM detracts from the argument that
sulfide oxidation may give rise to the antioxidative properties
of LAM,[2] it is difficult to imagine how else a polysaccharide
could accomplish such protection chemically. Nevertheless,
the identification of an unusual xylo-configured sugar residue
in LAM opens new avenues for research into the role played
by LAM in Mtb pathogenesis. If enzymes involved in MTX-
LAM biosynthesis turn out to be essential for Mtb, they may
be potential novel targets for the future development of
antituberculosis drugs.
Scheme 2. Proposed biosynthesis of MTX-LAM (17).
Received: February 26, 2004 [Z54119]
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Keywords: carbohydrates · cell wall · NMR spectroscopy ·
.
structure elucidation · xylofuranose
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