Synthesis of a key Mycobacterium tuberculosis biosynthetic phosphoinositide intermediate

Article abstract of DOI:10.1016/j.bmcl.2004.04.103

Regioselective mannosylations of a myoinositol acceptor diol are readily achieved by Lewis acid mediated iodinolysis of n-pentenyl ortho-esters. The procedure affords a psuedotrisaccharide to which the phosphoglyceryl and other lipid residues are added le

Full text of DOI:10.1016/j.bmcl.2004.04.103

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3815–3819
Synthesis of a key Mycobacterium tuberculosis biosynthetic
phosphoinositide intermediateq
K. N. Jayaprakash, Jun Lu and Bert Fraser-Reid*
Natural Products and Glycotechnology Research Institute, Inc., (NPG), 4118 Swarthmore Road, Durham, NC 27706, USA
Received 22 March 2004; revised 21 April 2004; accepted 23 April 2004
Available online 2 June 2004
Abstract—Regioselective mannosylations of a myoinositol acceptor diol are readily achieved by Lewis acid mediated iodinolysis of
n-pentenyl ortho-esters. The procedure affords a psuedotrisaccharide to which the phosphoglyceryl and other lipid residues are
added leading to the key biosynthetic intermediate of Mycobacterium species.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Recent reviews1 draw attention to the protracted rav-
ages of tuberculosis. Estimated to infect eight million
new subjects per year, the disease has now spread to
one-third of the world’s population. Primary tubercu-
losis develops two years after initial infection, and
eventually results in two million deaths annually.2
Equally devastating are the challenges to the patients’
immune status,3 this being a major concern in the case of
HIV infected patients.4 The persistence factors, which
are varied and devious, are tied to the pathogen’s dis-
tinctive ability to survive, and indeed flourish inside
macrophages,5 thereby interfering with intercellular
trafficking. The depredation caused by drug-resistant
strains has focussed attention on the causative parasite,
Mycobacterium tuberculosis, with the result that several
elegant structure–activity studies have recently
emerged.6
which has been deduced in several laboratories.9
Impressive advances have been made in elucidating
LAM biosynthesis,10 and these studies will profit from
the recent decoding of the parasite’s complete genome
sequence.11
The chronology PI fi [PIMs] fi LM fi LAM has been
recently established.12 The abbreviation [PIMs] refers to
a sub-set of intermediates shown within the brackets in
Scheme 1. Biosynthesis begins with the phosphatidyl
inositol (PI), 1, which undergoes mannosylation at O2
to give the phosphoinositide, 2a, (Ac2PIM1). Acylation
of the primary-OH to 2b (Ac3PIM1) follows as a man-
datory step, which precedes further mannosylation at
O6 to give the pseudotrisaccharide, 3, (Ac3PIM2).
Elaboration of the mannan array then begins, leading to
4 and thence to the linear mannan (LM), 5, the value of
n being approximately 10. Addition of the arabinan
complex then proceeds to afford the lipoarabinomannan
(LAM).
Of special interest is the parasite’s cell wall, which, in
spite of its formidable intricacy, has yielded discrete foci
of attention. Primary among these is the lipoarabino-
mannan (LAM) component,7;8 the covalent structure of
However, timely investigations by McConville and
co-workers13 involving cell-free experiments with
M. smegmatis, now indicate that addition of the third
mannose, that is 3 fi 4, is ‘an important step in regu-
lating the size of’ the PIMs, and that the ‘mannosyl-
transferase that catalyzes this step may be more labile––
or––present at very low levels’. In view of this specula-
tion, the heavily lipidated dimannosylated phosphoino-
sitide, 3, is a crucial intermediate, and its identification,
in two independent studies,12;13 as a mass spectral
Keywords: Mycobacterium tuberculosis; Selective glycosidation; Phos-
phoinositide; Glycerolipid.
q Supplementary data associated with this article can be found, in the
online version, at doi:10.1016/j.bmcl.2004.04.103
* Corresponding author. Tel./fax: +1-919-493-6113; e-mail: dglucose@
aol.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.103

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K. N. Jayaprakash et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3815–3819
Scheme 1.
fragment in M. smegmatis extracts, enhances its signifi-
cance. In this manuscript we report the first laboratory
preparation of this crucial biosynthetic intermediate.
attractive approach. The starting material was the diol 9
(Scheme 2b), which is readily available16 from methyl a-
D
-glucopyranoside by the procedure of Bender and
Budhu.17
Recent reports from our laboratory14 have revisited
Paulsen’s concept of ‘matching’ donors with acceptors
in order to optimize saccharide coupling under
nonforcing conditions. This process is facilitated by
chemoselective activation of n-pentenyl donors by Lewis
acid-controlled decomposition of N-iodosuccinimide.15
For example, ytterbium triflate (Yb(OTf)3) activates
n-pentenyl ortho-esters (NPOEs), for example, 6, which
in the absence of an acceptor are quantitatively
rearranged to disarmed n-pentenyl glycosides, for
example, 7. The armed counterparts, for example, 8
are then easily obtained by routine transformations
(Scheme 2a).
Glycosidation with various n-pentenyl ortho-esters
(NPOEs)18 was tested using a variety of Lewis acids to
activate N-iodosuccinimide. The most remarkable result
in Table 1 is the formation of only one monoman-
nosylation product, regardless of the type, or amount, of
Lewis acid used. The structure of the product was
1
unequivocally established by acetylation and H NMR
analysis. In view of the parameters of the downshifted
proton (5.8 ppm; J ¼ 2:7 and 2.4 Hz), the material was
identified as 11 rather than 10 in keeping with prece-
dents.14
In light of the foregoing, it is further remarkable that,
whereas the Yb(OTf)3 catalyzed reactions were exquis-
itively selective, those with scandium triflate (Sc(OTf)3)
and tert-butyldimethylsilyltriflate (TBDMSOTf) in-
duced formation of minor amounts of double glycosi-
dation products, 12 (entries i fi vi). These data support
previous findings in van Boom’s19 laboratory and ours,20
Notably, Yb(OTf)3 does not activate 7 or 8, thereby
permitting use of this salt specifically for NPOEs. By
contrast, scandium triflate (Sc(OTf)3) and tert-butyl-
dimethysilyltriflate (TBDMSOTf) activate armed and
disarmed donors.15 Application of these glycosylation
strategies to an appropriate inositol acceptor was an

K. N. Jayaprakash et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3815–3819
3817
Scheme 2.
Table 1. Effect of Lewis acid promoters on NIS-induced mannosylation of acceptor 9 with n-pentenyl ortho-estersa
Entry
Lewis acid
NPOE
Donor (equiv)
Glycosidation products (%)
Yield (%)a
10a
11a
12a
i
Sc(OTf)3
6a
6a
6c
6a
6b
6c
6a
6b
6c
3
3
3
3
3
3
3
3
3
0
0
0
0
0
0
0
0
0
65
25
23
26
26
21
27
0
87
82
85
82
62
84
92
97
96
ii
70
68
iii
iv
v
TBDMSOTf
Yb(OTf)3
67
72
vi
vii
viii
ix
75
100
100
100
0
0
a Yields and selectivity were given as an average value of two set of reactions.
about the difficulty of introducing an O2 mannosyl
group into a psuedodisaccharide acceptor such as 11––
particularly when a disarmed donor19 or NPOE20 is
used.
catalyzed desilylation solved the problem, in that two
compounds could be separated. Identification of each
was based on the 13C NMR chemical shifts of the
anomeric carbons in keeping with established trends.22
Thus one showed well separated signals at 99.339 and
101.951, while the other showed closely related signals at
98.908 and 99.363. The former could therefore be
assigned as the a=b diastereomer 15, and the latter
as the a=a counterpart 14. Acylation with palmitic acid
in the presence of DCC and DMAP followed by
deallylation gave 16 in 54% yield over two steps
(Scheme 3).
An armed donor is the proper ‘match’ for mannosyl-
ation at O2.21 The desired candidate had to provide for
future acylation of the primary-OH and, at the same
time, minimize b-coupling. Several experiments were
necessary to determine the best choice. The tert-butyl-
diphenylsilylated acceptor 11a yielded products only in
the 20% range regardless of the Lewis Acid (Sc(OTf)3 or
TBDMSOTf) that was used to promote iodonium
induced reaction of donor 8a. The tritylated analogue
11b, gave acceptable yields of pseudotrisaccharide 13a;
however removal of the trityl group under mild acidic
conditions was problematic, because desilylation also
occurred.
The phosphoglycerolipid moiety was attached by use of
the phosphoamidite and 1H tetrazole, followed by
oxidation with MCPBA23 to provide psuedotrisacchar-
ide 17 in 70% yield. Cleavage of the benzyl groups was
effected by atmospheric hydrogenolysis over 5% palla-
dium on carbon in methanol/chloroform for 12 h,
followed by addition of water, then continuing for
The best choice involved reaction of acceptor 11c with
armed donor 8d, which gave compound 13b. The a=b
isomers could not be separated at this stage, nor could
the products from saponification and subsequent
benzylation, 13c and 13d, respectively. However, acid
a further 20 h. Compound 3 was identified by
MALDI (1473.9 M+Naþ; 1489.6 M+Kþ), and by
600 MHz H NMR in DMSO, which showed, among
other things, anomeric protons at 4.97 and 4.81 ppm,
1

3818
K. N. Jayaprakash et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3815–3819
Scheme 3. Reagents and conditions: (i) 8b, Yb(OTf)3/NIS, CH2Cl2, 92%; (ii) NaOMe, CH2Cl2/MeOH, 82%; (iii) BnBr, NaH, DMF, 95%; (iv) TsOH,
MeOH/CH2Cl2, 72%; (v) a. CH3(CH2)14COOH, DMAP, DCC, CH2Cl2, 24 h; b. HOAc/H2O, PdCl2, NaOAc, 2 days, 54%; (vi) C17H35CO-
OCH2CH(OCOC17H35)CH2OP(OBn)N(i-Pr)2/1H-tetrazole, )20 ꢁC/MCPBA, 70%; (vii) 5% Pd/C, MeOH (1 mL)/CHCl3 (1 mL), H2 (balloon), 12 h,
H2O (0.3 mL), 20 h, 40%.
3. Ernst, W. A.; Maher, J.; Cho, S.; Niazi, K. R.; Chatterjee,
D.; Moody, D. B.; Besra, G. S.; Watanabe, Y.; Jensen, P.
E.; Porcelli, S. A. Immunity 1998, 8, 331; Fratti, R. A.;
Chua, J.; Vergne, I.; Deretic, V. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 5437; Russell, D. G.; Mwandumba, H.
C.; Rhoades, E. E. J. Cell Biol. 2002, 158, 421.
and 6a methyl protons at 2.34–2.21 ppm. Biological
studies with 3 will be undertaken and reported in due
course.
4. Selwyn, P. A.; Harter, D.; Lewis, V. A.; Schoenbaum,
E. E.; Vermund, S. H.; Klein, R. S.; Walker, A. T.;
Friedland, G. H. New Engl. J. Med. 1989, 320, 545.
5. Knutson, K. L.; Hmama, Z.; Herrera-Velit, P.; Rochford,
R.; Reiner, N. E. J. Biol. Chem. 1998, 273, 645; Venisse,
A.; Fournie, T. J.; Puzo, G. Eur. J. Biochem. 1995, 231,
440; Schlesinger, L. S.; Kaufman, T. M.; Iyer, S. R.;
Marchiando, L. K. J. Immunol. 1996, 157, 4568; Kauf-
mann, S. H. Nat. Rev. Immunol. 2001, 1, 20.
6. Brennan, P. J. Tuberculosis 2003, 83, 1; Gilleron, M.;
Nigou, J.; Cahuzac, B.; Puzo, G. J. Mol. Biol. 1999, 285,
2147; Apostolou, I.; Takahama, Y.; Belmant, C.; Kawano,
T.; Huerre, M.; Marchal, G.; Cul, J.; Taniguchi, M.;
Nakauchi, H.; Fournie, J. J.; Kourilsky, P.; Gachelin, G.
Proc. Natl. Acad. Sci. U.S.A. 1999, 98, 5141; Gilleron, M.;
Ronet, C.; Mempel, M.; Monsarrat, B.; Gachelin, G.;
Puzo, G. J. Biol. Chem. 2001, 276, 34896.
Acknowledgements
We are grateful to the World Health Organization, the
Human Frontier Science Program Organization, the
National Science Foundation, and the Mizutani Foun-
dation for support. We thank Dr. George Dubay,
Director of Instrument Operations in the Department of
Chemistry, Duke University for the mass spectroscopic
and MALDI measurements, and Professor Marcus
Germann of Georgia State University for the 600 MHz
spectrum of 3.
References and notes
7. Chatterjee, D. Curr. Opin. Biol. 1997, 1, 579.
1. Stewart, G. R.; Robertson, B. D.; Young, D. B. Nat. Rev.
Microbiol. 2003, 1997; Raviglione, M. C. Tuberculosis
2003, 83, 4.
8. Lowary, T. L. In Glycoscience: Chemistry and Biology;
Fraser-Reid, B., Tatsuta, K., Thiem, J., Eds.; Springer
Heildelberg, 2001; p 2005.
2. World Health Organization, tuberculosis, Fact Sheet no
104, World Health Organization, Geneva, Available from
http://www.who.int/me/diacentre/factsheets/who104/en/.
9. See for example: Chatterjee, D.; Khoo, K. H. Glycobiology
1998, 8, 113; Vercellone, A.; Nigou, J.; Puzo, G.
Front. Biosci. 1998, 3, c149; Nigou, J.; Gilleron, M.;

K. N. Jayaprakash et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3815–3819
3819
Rojas, M.; Garcia, L. F.; Thurnher, M.; Puzo, G.
Microbes Infect. 2002, 4, 945; Guerardel, Y.; Maes, E.;
Elass, E.; Leroy, Y.; Timmerman, P.; Besra, G. S.; Locht,
C.; Strecker, G.; Kremer, L. J. Biol. Chem. 2002, 277,
30635.
14. Fraser-Reid, B.; Lopez, J. C.; Radhakrishnan, K. V.;
Mach, M.; Schlueter, U.; Gomez, A.; Uriel, C. Can. J.
Chem. 2002, 80, 1075; Uriel, C.; Gomez, A.; Lopez, J. C.;
Fraser-Reid, B. Synlett 2003, 2203; Fraser-Reid, B.;
Lopez, J. C.; Gomez, A. M.; Uriel, C. Eur. J. Org. Chem.
2004, 1387.
15. Jayaprakash, K. N.; Radhakrishnan, K. V.; Fraser-Reid,
B. Tetrahedron Lett. 2002, 43, 6953; Jayaprakash, K. N.;
Fraser-Reid, B. Synlett 2004, 301.
16. Jia, Z. J.; Olsson, L.; Fraser-Reid, B. J. Chem. Soc., Perkin
Trans. 1 1998, 631; Anilkumar, G.; Jia, Z. J.; Kraehmer,
R.; Fraser-Reid, B. J. Chem. Soc., Perkin Trans. 1 1999,
3591.
17. Bender, S. L.; Budhu, R. J. J. Am. Chem. Soc. 1991, 113,
9883.
10. See for example: Kordulakova, J.; Gilleron, M.; Puzo, G.;
Brennan, P. J.; Gicquel, B.; Mikusova, K.; Jackson, M.
J. Biol. Chem. 2003, 278, 36285; Patterson, J. H.; Waller,
R. F.; Jeevarajan, D.; Billman-Jacobe, H.; McConville,
M. J. Biochem. J. 2003, 372, 77; Nigou, J.; Gilleron, M.;
Puzo, G. Biochimie 2003, 85, 153; Schaeffer, M. L.; Khoo,
K. H.; Besra, G. S.; Chatterjee, D.; Brennan, P. J.; Belisle,
J. T.; Inamine, J. M. J. Biol. Chem. 1999, 274, 31625;
Brennan, P. J.; Ballou, C. E. J. Biol. Chem. 1968, 243,
2975.
11. Cole, S. T.; Brosch, R.; Parkhill, J.; Garnier, T.; Churcher,
C.; Harris, D.; Gordon, S. V.; Eiglmeier, K.; Gas, S.;
Barry, C. E.; Tekala, F.; Badcock, K.; Basham, D.;
Brown, D.; Chillingworth, T.; Connor, R.; Davies, R.;
Devlin, K.; Feltwell, T.; Gentles, S.; Hamlin, N.; Holroyd,
S.; Hornsby, T.; Jagels, K.; Krogh, A.; McLean, J.;
Moule, S.; Murphy, L.; Oliver, K.; Osbourne, J.; Quail,
M. A.; Rajandream, M.-A.; Rogers, J.; Rutter, S.; Seeger,
K.; Skelton, J.; Squares, R.; Squares, S.; Sulston, J. E.;
Taylor, K.; Whitehead, S.; Barrell, B. G. Nature 1998, 393,
537.
18. Mach, M.; Schlueter, U.; Mathew, F.; Fraser-Reid, B.;
Hazen, K. C. Tetrahedron 2002, 58, 7345.
19. Elie, C. J. J.; Verduyn, R.; Dreff, C. E.; Brounts, D. M.;
van der Marel, G. A.; van Boom, J. H. Tetrahedron 1990,
46, 8243; Elie, C. J. J.; Verduyn, R.; Dreff, C. E.; van der
Marel, G. A.; van Boom, J. H. J. Carbohydr. Chem. 1992,
11, 715.
20. Anilkumar, G.; Gilbert, M. R.; Fraser-Reid, B. Tetra-
hedron 2000, 56, 1993.
21. Anilkumar, G.; Nair, L. G.; Fraser-Reid, B. Org. Lett.
2000, 2, 2587.
12. Kremer, L.; Gurcha, S. S.; Bifani, P.; Hitchen, P. G.;
Baulard, A.; Morris, H. R.; Dell, A.; Brennan, P. J.; Besra,
G. S. Biochem. J. 2002, 363, 437.
22. Bock, K.; Pedersen, C. Adv. Carbohydr. Chem. Biochem.
1983, 41, 27; Bock, K.; Pedersen, C.; Pedersen, H. Adv.
Carbohydr. Chem. Biochem. 1984, 42, 193.
13. Morita, Y. S.; Patterson, J. H.; Billman-Jacobe, H.;
McConville, M. J. Biochem. J. 2004, 378, 589.
23. Campbell, A. S.; Fraser-Reid, B. Bioorg. Med. Chem.
1994, 2, 1209.