A convenient synthesis of glucose monomycolate

Article abstract of DOI:10.1016/j.carres.2011.11.007

An efficient synthesis of glucose monomycolate, an important lipidic antigen from Mycobacterium tuberculosis is described.

Full text of DOI:10.1016/j.carres.2011.11.007

Carbohydrate Research 347 (2012) 151–154
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Note
A convenient synthesis of glucose monomycolate
⇑
Jacques Prandi
CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne BP 64182, F-31077 Toulouse, France
Université de Toulouse, UPS, IPBS, F-31077 Toulouse, France
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient synthesis of glucose monomycolate, an important lipidic antigen from Mycobacterium tuber-
Received 21 September 2011
Received in revised form 4 November 2011
Accepted 11 November 2011
Available online 19 November 2011
culosis is described.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Mycobacteria
Tuberculosis
Glucose monomycolate
Glucose monomycolate (GMM, 6-O-mycoloyl-
D
-glucose, Fig. 1)
are characteristic of the species.7 The structure of mycolic acids
from M. tuberculosis is shown in Figure 1.
is a mycobacterial antigen isolated from various species of coryne-
bacteria and mycobacteria,1 including Mycobacterium tuberculosis,
the etiologic agent of human tuberculosis. GMM and other anti-
genic mycobacterial glycolipids are presented to T cells by the
CD1 family of proteins and the antigen–protein complexes mediate
T cells response in the human host.2,3 The CD1 proteins are a third
type of antigen-presenting molecules, related to MHC-encoded
class I and class II molecules and are also associated with the b2
microglobulin. In humans are found five non-polymorphic CD1
proteins, CD1a, CD1b, CD1c, and CD1d are expressed at the surface
of antigen-presenting cells (APC), while CD1e is never found at the
cell surface but is involved in the processing of antigens inside the
APCs.4
Interest in these lipidic antigens has been boosted when it was
demonstrated that an uncharacterized mixture of lipids from M.
tuberculosis, could be used as a vaccine preparation and improved
pulmonary pathology in a guinea pig model of infection by M.
tuberculosis.5 More recently, it had been established that GMM
can induce a memory T cell response, just as protein antigens do,
in an experimental vaccination of cattle.6
Although GMM had been isolated and characterized from
mycobacterial cultures1 only tiny amounts can be obtained from
natural sources after long and tedious purifications. Mentions of
synthetic preparation of GMM are scattered in the literature8,9
but neither protocols nor yields are reported. These synthesis in-
volve acidic hydrolysis of trehalose dimycolate (TDM, 6,60-O-dimy-
coloyl-a,a
-trehalose) by 2 M trifluoroacetic acid for 2 h at 121 °C,8
or coupling of a 3-O-TBDMS-protected C32 synthetic mycolic acid
on a 1,2,3,4-tetra-O-TBDMS-glucose derivative,9 a method derived
from a reported synthesis of TDM,10 which is an obvious model for
the preparation of GMM. Two strategies had been described for the
preparation of TDM. The first one uses the SN2 displacement of bro-
mide11 or tosyl groups12,13 on the 6 and 60 positions of a protected
trehalose derivative by mycolic acid salts. The second approach10,14
involves the direct coupling on the 6,60 hydroxyl groups of a suit-
ably protected a,a-trehalose. In these cases, the 3-hydroxyl group
of the mycolic acid must be protected and coupling reactions had
been described under Mitsunobu conditions14 or with DCC.10 Both
methods gave good yields of product and the latter one had been
recently used for the preparation of TDM containing chiral syn-
thetic mycolic acids.15
The structure of GMM is simple; it is a free glucose esterified on
the 6-position by the mycolic acids (MAs). MAs are a family of
a-
branched-b-hydroxylated long-chain carboxylic acids (length up
to 90 carbons), which are ubiquitous in the mycobacteria genus.
However, depending on the species, the meromycolic chain carries
variable functionalities in the proximal and distal positions, which
As we needed milligram quantities of GMM in the course of an
ongoing program on lipidic mycobacterial antigens, we tried to pre-
pare GMM according to the SN2 method used by Toubiana for the
synthesis of TDM11 and mentioned by Moody8 for the preparation
of GMM. This method avoids the need to protect the hydroxyl group
of the mycolic acids, which is mandatory in any DCC-type coupling.
Starting from 1,2,3,4,6-penta-O-trimethylsilyl-D-glucopyranose
(2) (Fig. 2),16 selective deprotection of the 6-O-silyl group with
potassium carbonate was attempted but no 1,2,3,4-tetra-O-
⇑
Tel.: +33 5 61 17 54 85; fax: +33 5 61 17 59 00.
E-mail address: jacques.prandi@ipbs.fr
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.11.007

152
J. Prandi / Carbohydrate Research 347 (2012) 151–154
distal proximal
S
S
S
OH
O
meromycolic chain
OH
O
S
S
O
OMe
O
HO
HO
α-chain
OH
1 GMM
OH
Figure 1.
trimethylsilyl-glucose derivative (3) could be isolated because of the
very fast removal of the anomeric trimethyl silyl group under the
reaction conditions. We then tried to use the more resistant tert-
butyldimethylsilyl ethers instead of the trimethylsilyl ethers and
The reaction was complete in 24 h and gave a high yield of GMM
after chromatographic purification (91%).
To establish the identity of synthetic GMM 1 with the product
isolated from natural sources, we first checked that no reductive
opening of the cyclopropane rings had occurred during the hydrog-
enolysis of the benzyl ethers. Although the catalytic conditions we
used are known to leave cyclopropane rings intact20 we quantified
by 1H NMR spectroscopy (400 MHz) the signals for the cis-cyclopro-
pane ring (found around ꢀ0.4 ppm) using the signal corresponding
to the CHOMe group of the meromycolic chain as an internal stan-
dard, this signal is found isolated around 2.9 ppm.21 An identical ra-
tio was found (3.87 for GMM 1 and 3.80 for Benzyl GMM 10, see
Supplementary data) showing that no detectable cyclopropane
cleavage occurred during the hydrogenolysis reaction.
The MALDI-TOF spectrum for synthetic GMM (Fig. 3) was then
recorded and gave a complex pattern, as expected from the heter-
ogeneity of the mycolic acid mixture. Observed peak values for
synthetic GMM were compared to the reference masses recorded
for GMM in the Colorado State University data base22 and with
the known composition of the starting mycolic acid mixture.23
prepared the per-tert-butyldimethylsilylated D-glucose derivative
4, using an excess of tert-butyldimethylsilyl trifluoromethane sulfo-
nate and pyridine in dichloromethane.17 Selective deprotection of
the 6-O-silyl group of 4 could be achieved, but isolated yields of
1,2,3,4-tetra-O-TBDMS-glucose 5 were poor (<20%); once again,
considerable concomitant deprotection of the anomeric silyl group
was observed during the reaction. Despite this disappointing yield,
alcohol 5 was tosylated to give 6 in good yield (81%) and submitted
to the SN2 displacement by MAs. After four days, the reaction of 6
with the cesium salts of MAs in HMPA at 80 °C12 afforded 7, which
was isolated in 37% yield. The formation of polar unidentified prod-
ucts was also observed during the reaction. Final deprotection of 7 to
GMM was also troublesome. Acidic hydrolysis (HCl in CH2Cl2–
MeOH) or fluoride deprotection (NBu4F, THF–CHCl3) could not be
brought to completion. Even if some minor amounts of GMM could
be recovered, this procedure was far from satisfactory.
We looked for an alternative to the silyl group in this synthesis
Glucosyl esters of a-mycolic acids with 76, 78, 80, and 82 car-
and chose to start from benzyl 2,3,4-tri-O-benzyl-b-
D
-glucopyrano-
bon atoms gave peaks at m/z = 1294.3, 1322.3, 1350.3, and
1378.4 and corresponding peaks were found at m/z = 1146.1,
1174.1, 1202.2, and 1230.2 in the mycolic acid methyl esters
(MAMes) spectrum.23 The major species, at m/z = 1466.5 corre-
sponds to a glucose esterified with a methoxy-mycolic acid with
a total number of 87 carbon atoms, this peak nicely correlates to
a peak at m/z = 1318.3 found in the MAMes spectrum.23 Peaks for
GMM substituted by other members of this methoxy-mycolic acids
side 8 (Fig. 2) as the anomeric benzyl group was expected to be
much more stable than any anomeric silyl group. Glycoside. 8 can
be easily obtained in three steps and a >60% yield from D
-glucose.18
Tosylation of 8 proceeded smoothly and gave the expected tos-
ylate 9 in good yield (82%). Tosylate 9 was then cleanly displaced
by the cesium salt of MAs in a THF–DMF mixture following a pro-
cedure used for the preparation of mycoloyl-arabinan fragments.19
After two and a half days of reaction at 70 °C, a rewarding 58% yield
(based on a mean molecular weight of 1240 g molꢀ1 for the en-
gaged MAs) of protected GMM 10 was obtained. Final deprotection
of the benzyl groups of 10 was done by catalytic hydrogenation
with carbon-supported palladium hydroxide as catalyst (H2,
Pd(OH)2–C, petroleum ether–ethyl acetate–methanol mixture).
family were found at m/z = 1410.4 (C83), 1438.4 (C85), 1494.4 (C89
)
and 1522.5 (C91), in full agreement with observed peaks at m/
z = 1262.2, 1290.3, 1346.3 and 1374.3 in the MAMes spectrum. Fi-
nally, derivatives of keto-mycolic acids, the third family of mycolic
acids produced by M. tuberculosis are visible at m/z = 1380.4,
1408.4, and 1436.4 (80, 82, and 84 carbon atoms, respectively, cor-
responding peaks for MAMes at m/z = 1232.2, 1260.2, and 1288.3).
The observed data for synthetic GMM are in full agreement with
expected values and clearly showed that the heterogeneity of syn-
thetic GMM is fully representative of the natural mixture of myco-
lic acids, which is produced by M. tuberculosis H37Rv strain.
In conclusion, GMM from M. tuberculosis has been prepared
OP
OH
O
O
PO
PO
PO
PO
OP
OP
PO
PO
from D-glucose by a six-step sequence (overall yield of 26%) and
2 P = Me3Si
4 P = tBuMe2Si
3 P = Me3Si, α, β mixture
5 P = tBuMe2Si, α, β mixture
8 P = Bn, β anomer only
an efficient incorporation of the engaged mycolic acids in the final
product. GMM from any other mycobacterial species might also be
prepared using this sequence. This procedure makes this important
mycobacterial antigen readily available by synthesis and opens the
way for further biological studies.
OX
O
OX
O
TBDMSO
TBDMSO
BnO
BnO
OBn
1. Experimental
OTBDMS
TBDMSO
BnO
1.1. General methods
6 X = Ts
7 X = MA
9
X = Ts
10 X = MA
All reactions were run in anhydrous solvents under a dry argon
atmosphere using commercial reagents used as received. THF was
Figure 2.

J. Prandi / Carbohydrate Research 347 (2012) 151–154
153
1466.5
100
100
90
80
70
60
1322.3
1438.4
1350.3
50
50
1380.4
1378.4
1494.5
40
30
20
10
0
1436.4
1410.4
1408.4
1294.3
1522.5
1250
1316
1382
1448
1514
1580
0
1250
1316
1382
1514
1580
1448
Mass (m/z)
Figure 3. MALDI-TOF mass spectrum in positive ion mode of synthetic GMM.
dried by distillation from sodium benzophenone, dichloromethane
from phosphorus pentoxide, and DMF was distilled from BaO at re-
duced pressure and kept on activated 4 Å molecular sieves. Mycolic
acids were obtained from M. tuberculosis H37Rv strain, by saponifi-
cation of purified arabinogalactan complex. Optical rotations were
measured on a Perkin–Elmer 241 polarimeter. 1H and 13C NMR
spectra were recorded on Bruker AV400 and DPX300 spectrometers
working at 400 MHz (100.6 MHz for 13C) and 300 MHz (75.5 MHz
for 13C) and chemical shifts (d) are expressed in part per million
(ppm) from tetramethylsilane. MALDI-TOF spectra were obtained
on a 4700 Proteomics Analyser from Applied Biosystems, using
dihydroxybenzoic acid as the matrix and a Nd:YAG laser pulse at
355 nm. Chromatographic purifications (flash chromatography)
were done using Silica 60 (35–70 nm) from SDS (Peypin France).
Elemental analysis were obtained from the analytical service of
the Laboratoire de Chimie de Coordination (Toulouse, France).
1.3. 6-O-Mycoloyl-D-glucopyranose (GMM) (1)
Mycolic acids from M. tuberculosis H37Rv (39 mg, 0.03 mmol)
were dissolved in a mixture of THF and DMF (THF–DMF 5:1,
2.4 mL). To this solution were added the tosylate 9 (34.2 mg,
0.054 mmol) and dry cesium hydrogen carbonate (64 mg,
0.33 mmol). The mixture was brought to 70 °C and left at this tem-
perature for 2.5 days. TLC analysis (petroleum ether–EtOAc, 7:1) of
the reaction mixture showed the starting tosylate 9 at Rf = 0.2, MAs
as a single spot at Rf = 0.29 and 10 as (at least) three very close
spots with Rf ranging from 0.44 to 0.48. The reaction mixture
was then cooled to room temperature, diluted with EtOAc, and
the organic phase was washed twice with water. Chromatography
of the residue (7:1 petroleum ether–EtOAc) and pooling the highly-
moving spots (Rf from 0.44 to 0.48) gave benzyl 2,3,4-tri-O-benzyl-
6-O-mycoloyl-b-D-glucopyranoside (10) as a white amorphous so-
lid (31.8 mg, 58%). 1H NMR (400 MHz, CDCl3, 25 °C): d ꢀ0.41, 0.47
1.2. Benzyl 2,3,4-tri-O-benzyl-6-O-tosyl-b-D-glucopyranoside (9)
and 0.57 (3 m, cis-cyclopropane, myco.), 0.78 (d, 3JH,H = 7.0 Hz, CH–
3
CH3 myco.), 0.81 (t, JH,H = 7.0 Hz, CH2–CH3 myco.), 1.11–1.34 (m,
Glycoside 8 (294 mg, 0.54 mmol) was dissolved in dichloro-
methane (3 mL) and the solution was cooled to 0 °C. Pyridine
(0.7 mL), DMAP (30 mg, 0.24 mmol) and tosyl chloride (425 mg,
2.2 mmol) were sequentially added at 0 °C. The mixture was
warmed to room temperature and was stirred overnight. The mix-
ture was diluted with EtOAc and hydrolyzed with 3 M HCl (3 mL).
The organic phase was decanted and washed twice with saturated
NaHCO3 and brine. Chromatography of the residue (95:5?80:20
petroleum ether–EtOAc) gave the title compound as a white solid
CH2 myco.), 2.30–2.45 (m, CH–CO myco.), 2.88 (m, CH-OMe,
myco.), 3.26 (s, OCH3 myco.), 3.39–3.49 (m, H-2, H-4, H-5), 3.59
2
3
(m, H-3, CH–OH myco.), 4.14 (dd, JH,H = 11.5 Hz, JH,H = 4.5 Hz, H-
3
2
6), 4.45 (d, JH,H = 7.5 Hz, H-1), 4.46 (br d, JH,H = 11.5 Hz, H-6),
4.50–4.89 (8d, CH2-Ph). 13C NMR (100.6 MHz, CDCl3, 25 °C): d
62.8 (C-6), 71.1, 74.9, 75.1, 75.7 (CH2-Ph), 72.7 (C-4), 77.8 (C-2),
82.0 (C-5), 84.5 (C-3), 85.4, 102.3 (C-1), 175.2 (CO myco.).
Glycoside 10 (31.8 mg, 0.019 mmol) was dissolved in a mixture
of petroleum ether, EtOAc and CH3OH (2 mL of a 5:4:1 v/v/v mix-
ture) and treated under a hydrogen atmosphere with a catalytic
amount of 20% palladium hydroxide on charcoal. The mixture
was stirred for one day at room temperature and centrifuged
(10 min, 1000g). The clear supernatant was removed and the black
solid residue was taken up in CHCl3–CH3OH, shaken and centri-
fuged as above two more times. After pooling the organic phase
and evaporation of the solvents, the residue was filtered on silica
(319 mg, 82%). ½a D25
ꢁ
ꢀ3.3 (c 1.46, chloroform). 1H NMR (300 MHz,
CDCl3, 25 °C): d 2.39 (s, 3H, CH3-tosyl), 3.37–3.51 (m, 3H, H-2, H-
2
3
4, H-5), 3.60 (m, 1H, H-3), 4.14 (dd, JH,H = 10.5 Hz, JH,H = 4.5 Hz,
2
3
1H, H-6), 4.26 (dd, JH,H = 10.5 Hz, JH,H = 1.5 Hz, 1H, H-6), 7.15–
7.20 and 7.76–7.80 (2 m, 2 ꢂ 2H, H-Ar tosyl). 13C NMR
(75.5 MHz, CDCl3, 25 °C): d 21.6 (CH3-tosyl), 68.7 (C-6), 71.0,
74.9, 75.0, 75.7 (CH2-Ph), 72.7 (C-5), 76.9 (C-4), 82.0 (C-2), 84.4
(C-3), 102.1 (C-1), 127.7, 127.7, 127.8, 127.9, 128.0, 128.0, 128.1,
128.1, 128.3, 128.4, 128.4, 128.5, 129.8, 137.1, 137.6, 138.2,
138.3, 144.8 (C-Ar). Anal. Calcd for C41H42O8S (694.8 g molꢀ1): C,
70.87; H, 6.09. Found: C, 71.14, H, 6.19.
(CH2Cl2–CH3OH 6:1) to give GMM 1 (
a,b mixture) as a white amor-
phous solid. (24.4 mg, 91%). 1H NMR (400 MHz, 4:1 CDCl3–CD3OD,
3
3
25 °C): d ꢀ0.41 (m, JH,H = 5.0 Hz, JH,H = 4.0 Hz, cis-cyclopropane,
3
3
3
myco.), 0.48 (ddd, JH,H = 9.0 Hz, JH,H = 7.5 Hz, JH,H = 4.0 Hz,

154
J. Prandi / Carbohydrate Research 347 (2012) 151–154
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cis-cyclopropane, myco.), 0.57 (m, cis-cyclopropane, myco.), 0.78
3
and 0.82 (2d, JH,H = 7.5 Hz, CH–CH3 myco.), 0.80 (br t,
3JH,H = 7.5 Hz, CH2–CH3 myco.), 1.15–1.34 (m, CH2 myco.), 2.35
(m, CH–COO and CH–CO myco.), 2.92 (m, CH–COCH3 myco.), 3.12
3
3
(dd, JH,H = 9.0 Hz, JH,H = 8.0 Hz, H-2b), 3.22–3.37 (m, H-2
a
, H-
3
3
a,b, H-4b), 3.27 (s, –C–O–CH3 myco.), 3.43 (ddd, JH,H = 9.5 Hz,
3JH,H = 6.0 Hz, JH,H = 2.5 Hz, H-5b), 3.58 (m, CH–OH myco.), 3.60
3
3
3
3
(dd, JH,H = 9.5 Hz, JH,H = 9.5 Hz, H-4
a
), 3.91 (ddd, JH,H = 9.5 Hz,
3JH,H = 6.5 Hz, JH,H = 2.5 Hz, H-5
a
), 4.19 (2 dd, H-6 ,b), 4.38 (2
a
3
3
3
dd, H-6
a
,b), 4.42 (d, JH,H = 8.0 Hz, H-1b), 5.07 (d, JH,H = 3.5 Hz,
H-1
a
). 13C NMR (100.6 MHz, 4:1 CDCl3–CD3OD, 25 °C): d 10.6,
13.7, 14.5, 15.5, 22.4, 22.4, 15.2, 25.2, 25.8, 27.2, 27.2, 28.5, 28.9,
29.1, 29.1, 29.2, 29.2, 29.3, 29.4, 29.4, 29.6, 29.7, 30.0, 30.3, 31.7,
31.7, 32.2, 34.7, 35.1, 52.5, 52.6, 57.4, 63.4 (C-6), 69.1, 70.2, 70.3,
72.0, 72.3, 73.4, 73.6, 85.5, 92.2 (C-1a), 96.5 (C-1b), 175.1 (CO
myco.). MALDI-TOF (positive mode) see Figure 3.
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Acknowledgements
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We thank Sophie Tannière and Baptiste Cottin for technical
assistance, and Luis-Fernando Garcia-Alles for a generous gift of
mycolic acids from M. tuberculosis H37Rv strain.
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Supplementary data
20. Nicolaou, K. C.; Sasmal, P. K.; Rassias, G.; Reddy, M. V.; Altmann, K.-H.;
Wartmann, M.; O’Brate, A.; Giannakakou, P. Angew. Chem., Int. Ed. 2003, 42,
3515–3520.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2011.11.007.
21. Uenishi, Y.; Fujita, Y.; Kusunose, N.; Yano, I.; Sunagawa, M. J. Microbiol. Method
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