Regioselective mannosylation routes to the antigenic myo-inositol component of Mycobacterium tuberculosis

Article abstract of DOI:10.1016/S0040-4020(00)00156-3

A differentially protected derivative of myo-inositol with free hydroxyl groups at C6 and C2 is regioselectively mannosylated at C2, and subsequently at C6 with the same or a different donor to give the dimannosylated inositol antigenic core of Mycobacterium tuberculosis. Deacetylation now frees C1 for phosphorylation. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00156-3

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 1993±1997
Regioselective Mannosylation Routes to the Antigenic
myo-Inositol Component of Mycobacterium tuberculosis
*
G. Anilkumar, Mark R. Gilbert and Bert Fraser-Reid
²
Natural Products and Glycotechnology Research Institute, Inc. , 4118 Swarthmore Road, Durham, NC 27707, USA
Received 12 November 1999; accepted 16 February 2000
AbstractÐA differentially protected derivative of myo-inositol with free hydroxyl groups at C6 and C2 is regioselectively mannosylated at
C2, and subsequently at C6 with the same or a different donor to give the dimannosylated inositol antigenic core of Mycobacterium
tuberculosis. Deacetylation now frees C1 for phosphorylation. q 2000 Elsevier Science Ltd. All rights reserved.
The emergence of multiple drug resistant tuberculosis (TB)
is of major concern, as is readily apparent from publications
in technical journals,1 as well as prestigious newspapers.2
The probability of contracting the disease in crowded
airplanes, auditoriums, and jails invoke frightening
scenarios for hitherto `safe' countries; but for `third-
world', mainly tropical nations, the already staggering
statistics of one death every 10 s from TB, implies an
even more dismal prospect. The threat to the HIV-compro-
mised population is of particular concern, since infection by
the drug resistant strain is often not detected by normal
pulmonary tuberculosis screening protocols.3,4
mycobacter species,10,11 and hence is of interest as a
serological marker for screening of tuberculosis patients
and for the development of an anti-tuberculosis vaccine
(Scheme 1).12
Our interest in this chemistry was triggered by our work13 on
glycosylphosphatidylinositols (GPIs), summarized as 2. The
inositol moieties of 1 and 2 are seen to be functionalized at
three contiguous hydroxyl groups, C6, C1 and C2. However,
in 1, the C6 and C2 substituents are mannoses,5,6 while in 2
they are glucosamine and (sometimes) fatty acyl residues.14
Recent work in our laboratory has shown that exquisite
differentiation between C6, C1, and C2 sites can be
achieved15 (see below).
The major antigenic component of the causative agent
Mycobacterium tuberculosis is a complex glycoconjugate
known as lipoarabinomannan (LAM).5,6 Brennan and co-
workers have proposed the construct 1a,7 while Puzo and
colleagues have recently identi®ed multi-acylated modi®ca-
tions, summarized as 1b.8 The pseudo-trisaccharide
motif highlighted in 1, identi®ed 30 years ago by Lee and
Ballou,9 is a critical component for biosynthesis of several
In this connection, we recall the unusual reactivity
differences encountered by van Boom and co-workers ten
years ago, as summarized in Scheme 2. Thus, in an
elegant study directed towards the pseudo-trisaccharide
core of 1, the inositol derivative 3 was mannosylated at
C6, and the product, 4, transformed into 5 as a candidate
Scheme 1.
Keywords: myo-inositol; mannosylation; Mycobacterium tuberculosis.
* Corresponding author. Tel.: 11-919-493-6113; fax: 11-919-493-6113; e-mail: dglucose@aol.com
A non-pro®t organisation with laboratories at Centennial Campus (NC State University), Raleigh, NC
²
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00156-3

1994
G. Anilkumar et al. / Tetrahedron 56 (2000) 1993±1997
Scheme 2.
for C2 mannosylation. However, attempts with donors 6a
and 6b failed to produce 7 (R1ˆBz).16 In an alternative
approach, the C6±OH of 3 was protected in 8, allowing
the mannosylation at C2 to give 9. Addition of the second
mannose then was found to succeed, giving 7 in 84%
yield.17
direct alkylation with benzyl bromide gave diol 1320 in
82% yield from 11b. This material was treated with
1.3 equiv. of the known n-pentenyl mannoside 1421 at
room temperature under standard conditions, and after
10 min, the reaction was quenched by addition of sodium
thiosulfate. Flash chromatography afforded the major
product in 49% yield, which was easily shown to be 15a.
Thus, benzoylation afforded material whose 1H NMR spec-
trum displayed a telling low ®eld double doublet at
5.76 ppm with splittings of 9.6 and 10 Hz, assignable to
the proton a to the benzoate group in 15b. The minor
product 16a, obtained in 16% yield, was characterized by
acetylation to 16b, which showed a low ®eld double doublet
at 5.85 ppm with splittings of 2.8 and 2.4 Hz (Scheme 3).
van Boom's results clearly indicated that the order for
mannosylation had to be C2 before C6. This observation,
con¯ated with our own on the regioselective alkylation at
the two sites in question, encouraged us to determine
whether the regioselectivity could be extended for man-
nosylation reactions.
We report herein upon this study (Scheme 2).
The encouraging results in Scheme 3 would be more attrac-
tive if the Bender±Budhu product 11a could have been used
directly, thereby avoiding the steps for replacing acetyl with
benzyl in 13. In the event, reaction of 11a with pentenyl
glycoside 17 gave the 2-O-mannosylated product 18a in
Glycoside 10 was converted into the myo-inositol derivative
11a by the ef®cient procedure of Bender and Budhu,18 and
deacetylation afforded triol 11b. Treatment with dibutyltin
oxide gave the stannylene acetal19 designated as 12, and
Scheme 3. Reaction conditions: (i) NaOMe, MeOH, RT, 65%; (ii) Bu2SnO, PhH, re¯ux; (iii) BnBr (3 equiv.), 708C, 2 h, 82%; (iv) 14 (1.3 equiv.), NIS
(1.3 equiv.), TESOTf (cat), CH2Cl2, RT, 10 min, 65% (3:1); (v) BzCl, DMAP, Pyr, 08C±RT, 14 h, 94%; (vi) Ac2O, DMAP, Pyr, 08C±RT, 3 h, 59%.

G. Anilkumar et al. / Tetrahedron 56 (2000) 1993±1997
1995
Scheme 4. Reaction conditions: (i) 17 (1.3 equiv.), NIS (1.3 equiv.), BF3´Et2O (cat), CH2Cl2, RT, 30 min, 67% (major pdt); (ii) (ClAc)2O, DMAP, Pyr, 08C±
RT, 14 h, 45%; (iii) 17 (1.3 equiv.), NIS (1.3 equiv.), TBDMSOTf (cat), CH2Cl2, RT, 20 min, 70% (major pdt); (iv) NaOMe, MeOH/CH2Cl2, RT, 14 h, 83%.
67% yield with N-iodosuccinimide and borontri¯uoride
etherate as promoters. Notably, when triethylsilyltri¯ate
was used as Lewis acid, a much lower yield of 49% was
obtained. To verify regioselectivity, the material was
chloroacetylated to 18b, which showed a con®rmatory
signal at 5.40 ppm with splittings of 10 and 10 Hz. Further
mannosylation of 18a with 17 then gave 19a in 70%
yield, although with recovery of substantial amounts of
stirring for 3 min TESOTf (7 mL, 0.027 mmol) was added.
The reaction mixture was quenched after 10 min with 10%
aq. sodium thiosulphate and saturated aq. sodium bicarbo-
nate, and extracted with CH2Cl2. The organic layer was
separated, dried, and the solvent was removed under
reduced pressure. The crude residue on ¯ash column
chromatography (1:4 EtOAc±Hexane) afforded 15a
(44 mg, 0.036 mmol, 49%) as the major product. Rf [1:2
EtOAc±Hexane] 0.5, and 16a (15 mg, 0.012 mmol, 16%)
as the minor product. Rf [1:2 EtOAc±Hexane] 0.55 yield
based on the recovered diol 13 (10 mg, 0.018 mmol).
1
17. The H NMR spectrum of the deacetylated material,
19b, ®t completely with the published data of van Boom
(Scheme 4).
The direct, one-pot, double mannosylation of 11a was an
attractive prospect but, unfortunately, treatment of 11a with
3 equiv. of donor 17 did not produce appreciable amounts of
19a. In spite of this defect, the procedure 11a!18a!19a
makes ef®cient use of the readily prepared Bender diol for
assembly of libraries containing the same or different
mannoses at C2 and C6, without multiple protecting group
adjustments. Particularly signi®cant is the fact that these
operations can be carried out with an acetyl protection at
C1, readily removed for future phosphorylation.
Data for 15a: 1H NMR (400 MHz, CDCl3): d 7.75±6.92 (m,
45H, Ar), 5.35 (d, 1H, H-1, Jˆ1.2 Hz), 4.95±4.41 (m, 14H,
Bn), 4.37 (t, Jˆ2.4 Hz, 1H), 4.28±4.23 (dd, Jˆ10, 9.6 Hz,
1H), 3.98±3.85 (m, 3H), 3.79±3.71 (m, 3H), 3.58±3.56 (d,
Jˆ11.2 Hz, 1H), 3.32±3.25 (m, 2H), 3.19±3.16 (dd, Jˆ9.6,
2 Hz, 1H), 2.39 (bs, 1H, OH) 1.02 (s, 9H, t-Bu). 13C NMR: d
(138.99, 138.67, 138.57, 137.73, 137.43, 136.06, 135.64,
133.91, 133.61, 129.42, 129.36, 128.62, 128.47, 128.34,
128.29, 128.22, 128.18, 128.13, 128.06, 128.02, 127.96,
127.74, 127.56, 127.47, 127.36, 127.33, 127.17, 127.11,
127.06, 98.00 (C-1), 83.22, 80.85, 80.47, 79.38, 79.15,
75.78, 75.61, 75.21, 74.43, 72.90, 72.71, 72.16, 72.10,
70.08, 62.70, 26.80, 19.33. FABMS: m/z 1209.6 (M121).
Experimental
Data for 16a: 1H NMR (400 MHz, CDCl3): d 7.68±6.85 (m,
Ar), 5.48 (bs, 1H), 4.88±4.36 (m, 14H), 4.21±4.09 (m, 3H),
3.93±3.76 (m, 4H), 3.56±3.48 (m, 2H), 3.33±3.30 (dd,
Jˆ10, 2.8 Hz, 1H), 3.27±3.19 (m, 2H), 2.32 (s, 1H,
±OH), 0.96 (s, 9H). FABMS: m/z 1209.6 (M121), 1250
(M1K1), 1243.5 (M1Cs1).
General methods
All reactions were conducted under an inert argon
atmosphere. TLC plates (Riedel-de Haen, coated with silica
gel 60 F 254) were detected by UV. Silica gel (Spectrum
SIL 58, 230±400 mesh, grade 60) was used for column
chromatography. All NMR spectra were recorded at 258C
at 400 MHz (1H) or 100 MHz (13C), and chemical shifts are
reported relative to internal TMS. Accurate mass measure-
ments were made using FAB at 10 K resolution, and
elemental analyses were conducted by Atlantic Microlab,
Norcross, GA.
1,3,4,5-Tetra-O-benzyl-6-O-benzoyl-2-O-(2,3,4-tri-O-
benzyl-6-O-[t-butyldiphenylsilyl]-a-d-mannopyrano-
syl)-d-myo-inositol (15b). The mannoinositol 15a (30 mg,
0.025 mmol) was dissolved in pyridine (2 mL) at 08C. To
the solution was added DMAP (3 mg, 0.025 mmol)
followed by benzoylchloride (20 mL, 0.25 mmol) and stir-
red for 14 h at room temperature. The reaction was
quenched with drops of water and solvent was removed
under reduced pressure. The residue was ¯ash chromato-
graphed (1:4 EtOAc±Hexane) to afford 15b (31 mg,
0.0235 mmol, 94%) as a colorless paste. Rf (1:4 EtOAc±
Hexane) 0.5. 1H NMR (400 MHz, CDCl3): d 8.17±6.95 (m,
Ar), 5.79±5.74 (dd, Jˆ9.6 Hz, 10, 1H), 5.40 (bs, 1H), 4.99±
4.41 (m, 15H), 4.30±4.25 (dd, Jˆ10 Hz, 10, 1H), 4.02±3.86
(m, 4H), 3.79±3.76 (dd, Jˆ11.2 Hz, 2.8, 1H), 3.60±3.51 (m,
2H), 3.44±3.41 (dd, Jˆ10, 2 Hz, 1H), 3.32±3.29 (dd, Jˆ10,
2.4 Hz, 1H), 1.03 (s, 9H). FABMS: m/z 1313.45 (M121),
1,3,4,5-Tetra-O-benzyl-2-O-(2,3,4-tri-O-benzyl-6-O-[t-
butyldiphenylsilyl]-a-d-mannopyranosyl)-d-myo-ino-
sitol (15a) and 1,3,4,5-tetra-O-benzyl-6-O-(2,3,4-tri-O-
benzyl-6-O-[t-butyldiphenylsilyl]-a-d-mannopyrano-
syl)-d-myo-inositol (16a). The diol 1320 (50 mg, 0.092
mmol) and glycosyl donor 1421 (91 mg, 0.119 mmol) were
together taken up in a small quantity of toluene, azeotroped
to remove traces of water and then placed under high
vacuum overnight. The mixture was dissolved in dry
CH2Cl2 (2 mL) under Argon atm. N-Iodosuccinimide
(27 mg, 0.119 mmol) was added to the solution, and after

1996
G. Anilkumar et al. / Tetrahedron 56 (2000) 1993±1997
1354.40 (M11K), 1447.42 (M11Cs). Anal. calcd for
C48H86O12Si: C, 76.68, H, 6.59. Found; C,76.76, H, 6.63.
NMR (400 MHz, CDCl3): d 7.36±7.10 (m, Ar), 5.42±5.37
(dd, Jˆ10, 10 Hz, 1H), 5.15±5.14 (d, Jˆ2 Hz, 1H), 4.88±
4.26 (m, 16H), 4.06±4.04 (m, 2H), 3.93±3.91 (dd, Jˆ2 Hz,
1H), 3.85±3.78 (m, 2H), 3.70±3.59 (q, Jˆ14.4 Hz, 2H),
3.48±3.43 (m, 2H), 3.41±3.38 (dd, Jˆ9.6, 2.4 Hz, 1H),
3.28±3.26 (m, 1H), 1.89 (s, 3H). FABMS: m/z 1091.4
(MH1), 1089.4 (M121).
2-O-Acetyl-1,3,4,5-tetra-O-benzyl-6-O-(2,3,4-tri-O-benzyl-
6-O-[t-butyldiphenylsilyl]-a-d-mannopyranosyl)-d-myo-
inositol (16b). The mannoinositol 16a (10 mg,
0.0082 mmol) was dissolved in pyridine (1 mL) at 08C. To
the solution was added DMAP (1 mg, 0.0082 mmol)
followed by acetic anhydride (3 mL, 0.033 mmol) and stir-
red for 3 h, the temperature being slowly increased to room
temperature. The reaction was quenched with drops of water
and the solvent was removed under reduced pressure. The
residue was ¯ash column chromatographed to afford 16b
(6 mg, 0.0048 mmol, 59%) as a colorless paste. Rf (1:4
EtOAc±Hexane) 0.4. 1H NMR (400 MHz, CDCl3): d
7.71±6.88 (m, Ar), 5.85±5.84 (dd, Jˆ2.8, 2.4 Hz, 1H),
5.496±5.493 (d, Jˆ1.2 Hz, 1H), 4.91±4.32 (m, 14H),
4.21±4.16 (dd, Jˆ10, 10 Hz, 1H), 4.09±4.04 (dd, Jˆ10,
9.6 Hz, 1H), 3.92±3.76 (m, 4H), 3.59±3.57 (m, 2H) 3.46±
3.43 (dd, Jˆ10, 2.8 Hz, 1H), 3.39±3.36 (dd, Jˆ9.6, 2.4 Hz,
1H), 3.31±3.26 (dd, Jˆ9.6, 9.6 Hz, 1H), 2.12 (s, 3H), 0.99
(s, 9H). FABMS: m/z 1251.56 (M121).
3,4,5-Tri-O-benzyl-2-O-(2,3,4,6-tetra-O-benzyl-a-d-man-
nopyranosyl)-6-O-(2,3,4,6-tetra-O-benzyl-a-d-manno-
pyranosyl)-d-myo-inositol (19b). The mannoinositol 18a
(90 mg, 0.09 mmol) and glycosyl donor 17 (71 mg,
0.12 mmol) were together taken up in a small quantity of
toluene, azeotroped to remove traces of water and then
placed under high vaccum overnight. The mixture was
dissolved in CH2Cl2 (3 mL) under Argon atmosphere. NIS
(27 mg, 0.12 mmol) was added and after stirring for 3 min,
TBDMSOTf (7 mL, 0.027 mmol) was added. The reaction
mixture was quenched after 20 min with sodium thiosulfate
and washed with saturated sodium bicarbonate solution. The
aqueous phase was extracted with CH2Cl2. The organic
layer was separated, dried and the solvent was removed
under reduced pressure. The residue on ¯ash column chro-
matography (1:4 EtOAc±Hexane) afforded 19a (33 mg,
0.021 mmol, 70 %)(Rf (1:4 EtOAc±Hexane) 0.3) as the
major product based on recovered disaccharide 18a
(60 mg, 0.06 mmol). The trisaccharide 19a (25 mg,
0.016 mmol) was dissolved in MeOH/CH2Cl2 (2 mL/
1 mL) solvent mixture, and stirred with NaOMe (10 mg,
excess) for 14 h. The solvent was evaporated under reduced
pressure and the residue on ¯ash column chromatography
(1:3 EtOAc±Hexane) afforded 19b (20 mg, 0.013 mmol,
83%) as a colorless paste (Rf (1:2 EtOAc±Hexane) 0.4)
(spectral data were identical to that reported in Ref. 17).
1-O-Acetyl-3,4,5-tri-O-benzyl-2-O-(2,3,4,6-tetra-O-benzyl-
a-d-mannopyranosyl)-d-myo-inositol (18a). The diol 11a
(50 mg, 0.1 mmol) and glycosyl donor 17 (79 mg,
0.13 mmol) were together taken up in a small quantity of
toluene, azeotroped to remove traces of water and then
placed under high vacuum overnight. The mixture was
dissolved in CH2Cl2 (3 mL) under Argon atm. NIS
(30 mg, 0.13 mmol) was added to the solution, and after
stirring for 3 min at room temperature BF3´OEt2 (4 mL,
0.03 mmol) was also added. The reaction mixture was
quenched after 30 min with 10% sodium thiosulphate and
saturated aq. sodium bicarbonate and then extracted with
CH2Cl2. The organic layer was separated, dried, and solvent
was removed under reduced pressure. The crude residue on
¯ash column chromatography afforded 18a (48 mg,
0.047 mmol, 67%) as the major product (Rf (2:3 EtOAc±
Hexane) 0.5), along with other minor products (yield is
Acknowledgements
This work is supported by grants from the NIH (GM40171)
and the Research in Tropical Diseases (TDR) program of
World Health Organization.
³
based on the recovered diol 11a (15 mg, 0.030 mmol)).
1H NMR (400 MHz, CDCl3): d 7.35±7.04 (m, Ar), 5.28
(d, Jˆ,1.2 Hz, 1H), 4.97±4.42 (m, 16H), 4.34±4.23 (m,
2H), 4.10±4.05 (dd, Jˆ8.8, 9.6 Hz, 1H), 3.96±3.72 (m,
4H), 3.50±3.52 (dd, Jˆ9.6, 2.8 Hz, 1H), 3.40±3.25 (m,
2H), 2.35 (bs, 1H), 1.99 (s, 3H). FABMS: m/z 1013.4
(M121).
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room temperature. The reaction was quenched with drops of
water and the solvent was evaporated under reduced pres-
sure. The residue on ¯ash column chromatography (1:4
EtOAc±Hexane) afforded 18b (15 mg, 0.013 mmol, 45%)
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³
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