Synthesis of 2-deoxy-2-C-alkylglucosides of myo-inositol as possible inhibitors of a N-deacetylase enzyme in the biosynthesis of mycothiol

Article abstract of DOI:10.1016/S0960-894X(03)00157-4

Two new analogues of 1-D-1-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-myo-inositol, a biosynthetic intermediate in the production of mycothiol in the Mycobacteria have been synthesized. Both the 2-deoxy-2-C-(2′-hydroxypropyl)-D-glucoside 5, and the 2-deox

Full text of DOI:10.1016/S0960-894X(03)00157-4

Bioorganic& Medicinal Chemistry Letters 13 (2003) 2045–2049
Synthesis of 2-Deoxy-2-C-Alkylglucosides of myo-Inositol
as Possible Inhibitors of a N-Deacetylase Enzyme in the
Biosynthesis of Mycothiol
David W. Gammon,a,* Roger Hunter,a
Daniel J. Steenkampb and Theophilus T. Mudzungaa
aDepartment of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
bDivision of Chemical Pathology, Department of Laboratory Medicine, University of Cape Town,
Observatory, 7925, South Africa
Received 25 September 2002; revised 7 January 2003; accepted 9 February 2003
Abstract—Two new analogues of 1-d-1-O-(2-acetamido-2-deoxy-a-d-glucopyranosyl)-myo-inositol, a biosyntheticintermediate in
the production of mycothiol in the Mycobacteria have been synthesized. Both the 2-deoxy-2-C-(20-hydroxypropyl)-d-glucoside 5,
and the 2-deoxy-2-C-(20-oxopropyl)-d-glucoside 6, are derived from fully benzylated 1-d-1-O-(2-C-allyl-2-deoxy)-d-glucopyranosyl)-
myo-inositol 20, readily assembled via a protected 2-C-allyl-2-deoxyglucosyl fluoride. Both 5 and 6 inhibit the incorporation of
[3H]inositol by whole cells of Mycobacterium smegmatis into a number of metabolites which contain inositol.
# 2003 Published by Elsevier Science Ltd.
Mycothiol (1-d-1-O-[2-(N-acetamido-l-cysteinamido)-
2-deoxy-a-d-glucopyranosyl]-myo-inositol) (4, Scheme
1) is a low-molecular weight thiol, produced only by
actinomycetes,1 and is of significance in that it appears
to play an analogous role to glutathione in maintaining
a reduced intracellular environment in these Gram-
positive bacteria. Given the increasing need for new
classes of antituberculars, mycothiol is of considerable
contemporary interest, because of the pronounced
structural differences between it and glutathione, and
the role played by these thiol compounds in the detoxi-
fication of alkylating agents and other noxious chemi-
cals.2 Mycothiol was first isolated as the disulfide from
Streptomyces sp. AJ94633 and as the bimane derivative
and free thiol from M. bovis, which allowed the
demonstration of a mycothioldisulfide reductase activ-
ity, by analogy to glutathione reductase.4 We recently
reported the chemical synthesis of 2 and its uptake by
enzymes in a fraction from cell-free extracts of Myco-
bacterium smegmatis in the biosynthesis of mycothiol, or
Scheme 1. Biosynthesis of mycothiol.
*Corresponding author. Tel.: +272-1650-2547; fax: +272-1689-7499; e-mail: gammondw@science.uct.ac.za
0960-894X/03/$ - see front matter # 2003 Published by Elsevier Science Ltd.
doi:10.1016/S0960-894X(03)00157-4

2046
D. W. Gammon et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2045–2049
its precursor 3 in the absence of SCoA.5 A total synth-
esis of mycothiol as its bimane derivative has also been
published recently, providing further proof of struc-
ture.6 The proposed biosyntheticpathway to mycothiol
is summarized in Scheme 1.
2). Esterification11 of alcohol 7 with camphanic acid
chloride gave in high yield a separable (SiO2, CH2Cl2/
Et2O, 99:1) mixture of two diastereomers 8a (mp: 140–
144 ꢀC) and 8b (mp: 162–164 ꢀC). Basichydrolysis
afforded the desired alcohols 9 ([a]D +7.1 (c 1.0, CHCl3)
and 10 ([a]D À8 (c 1.0, CHCl3)12 in excellent yield.
The identification of key pseudo-disaccharide inter-
mediates 1 and 27 in the biosynthesis has led to our
exploration of the possibility that analogues 5 and 6,
with modifications at C-2 of the glucosamine unit, may
act as inhibitors of the N-deacetylase enzyme, thereby
preventing the production of mycothiol and the survival
of M. tuberculosis. We report here on efficient synthetic
routes to these analogues, and results of preliminary
evaluation of their biological activity.
Attention then turned to preparation of suitable glyco-
syl donors. We reasoned that donors of the type C
(Y=O) shown above, with a carbonyl group at position
20 of the side chain would not be suitable because of the
potential for participation in the glycosylation reaction
leading to b-selectivity. Consequently, 2-C-alkenyl-gly-
cosyl fluorides (C, Y=CH2), obtainable from glycosides
D, were targeted starting with preparation of 1,3,4,6-
tetra-O-acetyl-2-C-methallyl-2-deoxy-b-d-glucopyranose
1113 (Scheme 3). Selective deacetylation14 afforded
alcohol 12 and subsequent fluorination with DAST15
gave desired glycosyl donor 13 as a mixture of anomers
(a:b=2:1, 98%). Coupling of donor 13 with l-inositol
16
.
acceptor 10 in the presence of BF3 etherate gave
pseudo-disaccharide 14 in 73% yield with desired a-
selectivity but undesired isomerisation of the side chain
double bond.
The failure to obtain the 2-C-methallylglucoside
prompted a modified approach utilizing the known
2-deoxy-2-C-allylglucoside 1517b which was converted
as before via anomericdeacetylation and fluorination to
the glycosyl fluoride 17 (Scheme 3). Treatment of
.
acceptor 9 with donor 17 and BF3 etherate as promotor
gave pseudo-disaccharide 18 as an inseparable mixture
of anomers (a:b=8:1 from NMR), and in an approxi-
mate yield of 60% after removal of unreacted acceptor.
Zemplen deacetylation of 18 allowed for separation and
recovery of a-glycoside 19 by careful silica column
chromatography in 36% overall yield from inositol
acceptor 9 (Scheme 4). Benzylation of 19 gave fully
protected pseudo-disaccharide 20 (94%, [a]D +39.4
(c 2.7, CHCl3), m/z (FABMS) 1171.3 (C71H74O10.85Rb
requires m/z 1171.4). Evidence for the a-glycosidic bond
included a signal at d 5.09 (d, J=3.3 Hz) for H-1 in the
300 MHz 1H NMR spectrum, and a signal at d 94.92 for
C-1 in the 13C NMR spectrum. Diagnostic signals for
the allyl group were also clearly identified in both the 1H
and 13C NMR spectra.
The syntheticapproach was based on selection of key
building blocks A through D as our initial targets. It
was envisaged that suitably protected myo-inositol deri-
vative A could be coupled with activated glycosyl
donors B or C, which in turn were derivable from
2-deoxy-2-C-alkenylglucoside D.
The most practical route to the oxygenated alkyl side-
chains proved to be via epoxidation of the olefin followed
by reduction to the secondary alcohol and oxidation as
required (Scheme 4). Treatment of 20 with MCPBA18
proceeded slowly and completion of reaction was
ensured by periodicaddition of further equivalents of
MCPBA over 24 h to give a mixture (1:1) of inseparable
diastereoisomers 21. Regioselective reductive opening19
of the epoxide 21 with LiAlH4 in THF gave secondary
alcohols 22, again inseparable, in an acceptable yield.
Global debenzylation afforded the target 2-deoxy-2-C-
(20-hydroxypropyl)-d-glucoside
5 as a solid; m/z
From the range of reported methods for preparation of
the enantiomerically pure derivative of myo-inositol8À10
we opted for resolution of d,l-1,2,4,5,6-penta-O-benzyl-
myo-inositol10 7 as its (S)-(À)-camphanate ester (Scheme
(FABMS) 384.1 (C15H28O11 requires m/z 384.4); dH
(400 MHz; CDCl3; Me4Si) 5.11 (H-1), 4.24 (H-20), 1.20
(CH3); dC (400 MHz; CDCl3; Me4Si) 95.46 (C-1), 22.50
(C-CH3).

D. W. Gammon et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2045–2049
2047
Scheme 2. Reactions and conditions: (a) Camphanic acid chloride (1.2 equiv), Et3N, DMAP, CH2Cl2, rt, 24 h, 99%; (b) KOH, EtOH, reflux, 2 h, 94%.
Scheme 3. Reactions and conditions: (a) Hydrazine acetate (2 equiv), DMF, 60 ꢀC, 30 min, 98% (12), 92% (16); (b) DAST (3 equiv), THF, rt, 30
.
.
min, 90% (a:b=2:1); (c) 10 (1 equiv), 13 (1.4 eq), BF3 Et2O (5 equiv), 4 A ms, rt, 2 h, 73% (a:b >99:1); (d) 9 (1 equiv), 17 (1.6 equiv), BF3 Et2O (5
equiv), 4A ms, rt, 2 h, (a:b=8:1).
Scheme 4. Reactions and conditions: (a) NaOMe, MeOH–CH2Cl2, rt, 1 h, 36% from 9; (b) NaH, BnBr, THF, reflux, 20 h, 94%; (c) MCPBA (70%)
(6 equiv), CH2Cl2, rt, 24 h, 83% (1:1);. (d) LAH (10 equiv), THF, 0 ꢀC, 1 h, 82% (1:1); (e) H2, 10% Pd/C, EtOAc–MeOH, rt, 3 days, >90%; (f)
TPAP (cat), NMO (1.5 equiv), CH2Cl2, ms, rt, 25 min, 90%.

2048
D. W. Gammon et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2045–2049
Figure 2. Comparison of the radiolabel recovered in the three major
HPLC peaks after incubation of M. smegmatis in the presence of two
different levels of 5 and 6. (MSH; mycothiol.)
Figure 1. Effect of 5 on the incorporation of [3H]inositol into acid
soluble metabolites of Mycobacterium smegmatis: M. smegmatis was
grown in Middle brook medium containing 5% glycerol. [3H]Inositol
was added when the culture reached an absorbance of 0.44 at 600 nm.
Cells were incubated for 3.5 h in the presence of two different amounts
of 5 and was then harvested and sonicated in 0.25M HClO4 to obtain
the acid soluble fraction, which was chromatographed on a Vydac
201HS54 octadecilesilane column. Following injection of the sample
the column was eluted for 5 min at 100%A, then for 25 mins with a
linear gradient to 70%B and for 5 min with a linear gradient to
100%B (A: 0.1% TFA, B: 0.1%TFA in a 6:4 (v/v) mixture of aceto-
nitrile and water. The flow rate was 0.8 mL/min and 0.5 min fractions
were collected and counted. The traces represent a radiolabel recov-
ered in the eluate for a control culture lacking inhibitor (solid line),
and for cultures labeled in the presence of 20 mg/mL (Broken line) or
200 mg/mL (dotted line) 5.
of 5 (Fig. 2). At this level of the inhibitor the transport
of radiolabel into the organisms was unaffected as
compared to a control which lacked inhibitors. It is
evident, therefore, that 5 and 6 inhibit the incorporation
of [3H]inositol into a number of metabolites which con-
tain inositol. The identification of these metabolites,
more detailed enzymological studies and an evaluation
of the effects of 5 and 6 on the susceptibility of myco-
bacteria to various stresses will be of much interest. In
addition, continuing work will exploit the availability
of crucial synthetic intermediates 20 and 21 which
provide a platform for the synthesis of a variety of
other carba-analogues of mycothiol and its biosyn-
theticprecursors.
Oxidation of 22 with TPAP using NMO as co-oxidant20
gave ketone 23 in excellent yield. ([a]D +38.5 (c 2.2,
CHCl3); nmax/cmÀ1 1712 (C¼O); dH (400 MHz; CDCl3;
Me4Si) 5.28 (d, J=3.6, H-1), 4.17 (H-20), 1.62 (CH3), dC
(400 MHz; CDCl3; Me4Si) 207.78 (CH2COCH3), 94.26
(C-1), 29.68 (CH3)). Global deprotection gave the target
2-deoxy-2-C-(20-oxopropyl)-d-glucoside 6 as a solid. (dH
(300 MHz; D2O; dioxane) 4.84 (d, J=3.3, H-1), 3.87 (t,
J=2.5, H-20), 2.02 (CH3), dC 214.47 (C¼O), 94.96 (C-1),
29.48 (CH3).)
Acknowledgements
This work was supported by grants from the Welcome
Trust (UK), the National Research Foundation (SA)
and the University of Cape Town. Financial assistance
for TTM from the Department of Labour (SA) is also
gratefully acknowledged.
Only limited quantities of 5 and 6 were available for
biological tests, but preliminary results suggest that
these compounds are biologically active. While com-
pounds 5 and 6 did not inhibit the growth of Myco-
bacterium smegmatis in vitro, a pronounced inhibition
of incorporation of [3H]inositol by whole cells into ino-
sitol containing metabolites was observed (Fig. 1).
While each of the major radiolabeled peaks in Fig. 1
probably comprise a mixture of [3H]inositol labeled
compounds the peak eluted at 4–5.5 min was found to
contain inositol monophosphate as determined by coe-
lution with an authenticstandard on a Partisil SAX10
anion exchange column, while the peak eluted at 7–9
min contain mycothiol as determined by reaction with
sulfhydryl reagents and coelution with authentic
mycothiol as the 7-diethylamino-3-(40-maleimidyl-
phenyl)-4-methylcoumarin derivative. The [3H]inositol
labeled compound(s) eluted at 15.5–17 min have not as
yet been characterised, but incorporation of radiolabel
into this fraction was maximally inhibited at 20 mg/mL
References and Notes
1. Newton, G. L.; Arnold, K.; Price, M. S.; Sherrill, C.; Del-
cardayere, S. T.; Aharonowitz, Y.; Cohen, G.; Davies, J.;
Fahey, R. C.; Davis, C. J. Bacteriol. 1996, 178, 1990. Newton,
G. L.; Av-Gay, Y.; Fahey, R. C. Biochemistry 2000, 39, 10739.
2. Rawat, M.; Newton, G. L.; Ko, M.; Martinez, G. J.;
Fahey, R. C.; Av-Gay, Y. Antimicrob. Agents Chemother.
2002, 46, 3348.
3. Sakuda, S.; Zhou, Z. Y.; Yamada, Y. Biosci. Biotech. Bio-
chem. 1994, 58, 1347.
4. Spies, H. S. C.; Steenkamp, D. J. Eur. J. Biochem. 1994,
224, 203.
5. Jardine, M. A.; Spies, H. S. C.; Nkambule, C. M.; Gam-
mon, D. W.; Steenkamp, D. J. Biorg. Med. Chem. 2002, 10,
875.
6. Nicholas, G. N.; Kovac, P.; Bewley, C. A. J. Am. Chem.
Soc. 2002, 124, 3492.
7. Bornemann, C.; Jardine, M. A.; Spies, H. S. C.; Steen-
kamp, D. J. Biochem. J. 1997, 325, 623.
8. (a) Lindberg, J.; Ohberg, L.; Garregg, P. J.; Konradsson, P.

D. W. Gammon et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2045–2049
2049
Tetrahedron 2002, 58, 1387. (b) Martın-Lomas, M.; Flores-
Mosquera, M.; Chiara, J. L. Eur. J. Org. Chem 2000, 1547. (c)
Madsen, R.; Udodong, U. E.; Roberts, C.; Mootoo, D. R.;
Konradsson, P.; Fraser-Reid, B. J. Am. Chem. Soc. 1995, 117,
1554.
15. Posner, G. H.; Haines, S. R. Tetrahedron Lett. 1985,
26, 5.
16. (a) Elie, C. J. J.; Dreef, C. E.; Verduyn, R.; Van der
Marel, G. A.; Van Boom, J. H. Tetrahedron 1989, 45, 3477.
(b) Kunz, H.; Sager, W. Helv. Chim. Acta 1985, 68, 283.
17. (a) Giese, B. Radical in Organic Synthesis: Formation of
Carbon-Carbon Bonds; Pergamon Press: Elmsford, 1986. (b)
Korth, H.; Sustmann, R.; Giese, B.; Rucker, B.; Groninger,
K. S. Chem. Ber. 1990, 123, 1891.
18. (a) Imuta, M.; Ziffer, H. J. Org. Chem. 1979, 44, 1351. (b)
Chiappe, C.; Crotti, P.; Menichetti, E. Tetrahedron: Asym-
metry 1998, 9, 4079.
˜
9. Pietrusiewicz, K. M.; Salamonczyk, G. M.; Bruzik, K. S.
Tetrahedron 1992, 48, 5523.
10. Massy, D. J.; Wyss, P. Helv. Chim. Acta 1990, 73, 1037.
11. Vacca, P. P.; deSolms, J.; Huff, J. R. J. Am. Chem. Soc.
1987, 109, 3478.
12. Shvets, V. I.; Klyashchitskii, B. A.; Stepanov, A. E.;
Evstigneeva, R. P. Tetrahedron 1973, 29, 331.
13. Hang, H. C.; Bertozzi, C. R. J. Am. Chem. Soc. 2001, 123,
1242.
19. Trevoy, L. W.; Brown, W. G. J. Am. Chem. Soc. 1949, 71,
1675.
14. Excoffier, G.; Gagnaire, D.; Utille, J. Carbohydr. Res.
1975, 39, 368.
20. Ley, S. V.; Norman, J.; Griffith, W. P.; Marsden, P.
Synthesis 1994, 639.