Shortcut to mycothiol analogues

Article abstract of DOI:10.1021/ol0269796

(equation presented) The synthesis of a simplified thioglycosidic analogue (2) of mycothiol (1) is described. Evaluation of 2 against mycothiol S-conjugate amidase from Mycobacterium tuberculosis reveals good specific activity (7500 nmol min^-1

Full text of DOI:10.1021/ol0269796

ORGANIC
LETTERS
2002
Vol. 4, No. 24
4337-4339
Shortcut to Mycothiol Analogues
Spencer Knapp,* Silvia Gonzalez, David S. Myers, Lisa L. Eckman,† and
Carole A. Bewley†
Department of Chemistry and Chemical Biology, Rutgers The State UniVersity of
New Jersey, 610 Taylor Rd., Piscataway, New Jersey 08854-8087, and
Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health,
Bethesda, Maryland 20892-0820
knapp@rutchem.rutgers.edu
Received September 27, 2002
ABSTRACT
The synthesis of a simplified thioglycosidic analogue (2) of mycothiol (1) is described. Evaluation of 2 against mycothiol S-conjugate amidase
from Mycobacterium tuberculosis reveals good specific activity (7500 nmol min-1 mg-protein-1, vs 14 200 for 1), indicating that 2 can serve
as a starting point for antitubercular drug design.
Mycothiol1 (1) is the major low molecular weight thiol found
in actinomycetes, including Mycobacterium tuberculosis.2-5
It is thought to protect these organisms against oxidative
stress6-10 and function in the removal of exogenous elec-
trophilic agents.11-15 The biosynthesis of 1 (Scheme 1)
proceeds by way of 1-O(2′-acetamido-2′-deoxy-R-D-gluco-
pyranosyl)-D-myo-inositol 3, first by deacetylation to give
4,16 and then acylation with L-cysteine under the influence
of a ligase17,18 to provide 5. A transacetylase17,19 converts 5
to 1. Two further pathways involving 1 have been elucidated
(Scheme 1). A reductase6-8 regenerates 1 from the corre-
sponding disulfide, mycothione (6), thus maintaining the
reducing intracellular environment. Upon reaction of 1 with
electrophiles E+, the resulting conjugate 7 is cleaved by
mycothiol S-conjugate amidase14,15 into 4 and an N-acetyl-
cysteine adduct 8 that is exported from the cell.
† National Institutes of Health.
(1) Systematic name: 1-O(2′[N-acetyl-L-cysteinyl]amido-2′-deoxy-R-D-
glucopyranosyl)-D-myo-inositol.
(2) Sakuda, S.; Zhou, Z.-Y.; Yamada, Y. Biosci. Biotech. Biochem. 1994,
58, 1347-1348.
(3) Spies, H. S. C.; Steenkamp, D. J. Eur. J. Biochem. 1994, 224, 203-
213.
(4) Newton, G. L.; Arnold, K.; Price, M. S.; Sherrill, C.; delCardayre,
S. B.; Aharonowitz, Y.; Cohen, G.; Davies, J.; Fahey, R. C.; Davis, C. J.
Bacteriol. 1996, 178, 1990-1995.
(5) Fahey, R. C. Annu. ReV. Microbiol. 2001, 55, 333-356.
(6) Blanchard, J. S.; Patel, M. P. J. Am. Chem. Soc. 1998, 120, 11538-
11539.
(7) Patel, M. P.; Blanchard, J. S. Biochemistry 1999, 38, 11827-11833.
(8) Patel, M. P.; Blanchard, J. S. Biochemistry 2001, 40, 5119-5126.
(9) Newton, G. L.; Bewley, C. A.; Dwyer, T. J.; Horn, R.; Aharonowitz,
Y.; Cohen, G.; Davies, J.; Faulkner, D. J.; Fahey, R. C. Eur. J. Biochem.
1995, 230, 821-825.
(10) Newton, G. L.; Unson, M.; Anderberg, S.; Aguilera, J. A.; Oh, N.
N.; delCardayre, S.; Davies, J.; Av-Gay, Y.; Fahey, R. C. Biochem. Biophys.
Res. Commun. 1999, 255, 239-244.
(11) Norin, A.; van Ophem, P. W.; Piersma, S. R.; Persson, B.; Duine,
J. A.; Jornvall, H. Eur. J. Biochem. 1997, 248, 282-289.
(12) Misset-Smits, M.; van Ophem, P. W.; Sakuda, S.; Duine, J. A. FEBS
Lett. 1997, 409, 221-222.
Drug-resistant tuberculosis now threatens a large portion
of earth’s population,20 and the development of new treat-
ments for tuberculosis infection has become a national21 and
international22 priority. The disruption of enzymatic pathways
of mycothiol biosynthesis and/or mycothiol-based detoxifi-
10.1021/ol0269796 CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/02/2002

version of the inositol ring could be used as a component of
inhibitors. We recently found that a variety of R-GlcNAc
thioconjugates can be prepared stereoselectively and in good
yield by S-derivatization of an R-GlcNAc mercaptan.24,25
Thioglycosides are generally more resistant to degradation
by glycosidases than O-glycosides,26-28 so this approach to
inhibitor design combines several possible advantages.
Scheme 1. Enzyme-Mediated Mycothiol Pathways
Commercially available 2-acetamido-2-deoxy-â-D-gluco-
pyranose tetraacetate (9, Scheme 2) was treated with Lawes-
Scheme 2. Synthesis of Simplified Mycothiol Analogue 2
cation could leave M. tuberculosis vulnerable to drugs,
oxygen, and other stress factors. The enzymes shown in
Scheme 1 accept substrates or produce products that are
N-acylated 1-O(2′-amino-2′-deoxy-R-D-glucopyranosyl)-D-
myo-inositols. For this reason, compounds based on the
GlcN-Ins substructure that additionally bear groups on N
that resemble those of the respective transition states are
potential inhibitors for any one, or more than one, of these
enzymes. In previous synthetic studies on 1 and related
compounds,6,15,17,23 the preparation of a protected D-myo-
inositol glycosylation acceptor has required several steps and
a resolution, and both the inositol R-glycosylation and
N-acylation steps have been problematic. Some of these
synthetic difficulties could be dodged if a stripped-down
son’s reagent as described previously,24,26 and the resulting
thiazoline 10 was then hydrolyzed to the acetamido mer-
captan 11.24 Reaction of 11 with cyclohexene under condi-
tions for free radical addition of anomeric mercaptans to
alkenes [chloroform as a cosolvent, azobis(isobutyronitrile)
as a radical initiator]25 afforded the cyclohexyl thioglycoside
12 with no trace of the corresponding â-isomer. Hydrazin-
olysis29 of the four acetyls provided aminotriol 13, and then
coupling with S-acetyl-N-Boc-L-cysteine30 gave 14 in good
(13) Duine, J. A. Biofactors 1999, 10, 201-206.
(14) Newton, G. L.; Av-Gay, Y.; Fahey, R. C. Biochemistry 2000, 39,
10739-10746.
(15) Nicholas, G. M.; Kova´c, P.; Bewley, C. A. J. Am. Chem. Soc. 2002,
124, 3492-3493.
(16) Newton, G. L.; Av-Gay, Y.; Fahey, R. C. J. Bacteriol. 2000, 182,
6958-6963.
(17) Bornemann, C.; Jardine, M. A.; Spies, H. S. C.; Steenkamp, D. J.
Biochem. J. 1997, 325, 623-629.
(24) Knapp, S.; Myers, D. S. J. Org. Chem. 2001, 66, 3636-3638.
(25) Knapp, S.; Myers, D. S. J. Org. Chem. 2002, 67, 2995-2999.
(26) Knapp, S.; Vocadlo, D.; Gao, Z.; Kirk, B.; Lou, J.; Withers, S. G.
J. Am. Chem. Soc. 1996, 118, 6804-6805.
(27) Bousquet, E.; Spadaro, A.; Pappalardo, M. S.; Bernardini, R.;
Romeo, R.; Panza, L.; Ronisvalle, G. J. Carbohydr. Chem. 2000, 19, 527-
541.
(28) Cohen, S. B.; Halcomb, R. L. J. Org. Chem. 2000, 65, 6145-6152.
(29) Fujinaga, M.; Matsushima, Y. Bull. Chem. Soc. Jpn. 1966, 39, 185-
190.
(30) S-Acetyl-N-Boc-L-cysteine was prepared from commercially avail-
able N,N′-bis(Boc)-L-cystine by zinc reduction and then in situ S-acetylation.
Zahn, H.; Hammerstro¨m, K. Chem. Ber. 1969, 102, 1048-1052.
(18) Sareen, D.; Steffek, M.; Newton, G. L.; Fahey, R. C. Biochemistry
2002, 41, 6885-6890.
(19) Vetting, M. W.; Hegde, S. S.; Javid-Majd, F.; Blanchard, J. S.;
Roderick, S. L. Nature Struct. Biol. 2002, 9, 653-658.
(20) Bloom, B. R.; Murray, C. J. L. Science 1992, 257, 1055-1064.
(21) For example, see updated web information at: http://www.
niaid.nih.gov/publications/tb.htm.
(22) For example, see updated web information at: http://www.
who.int/health-topics/tb.htm.
(23) Jardine, M. A.; Spies, H. S. C.; Nkambule, C. M.; Gammon, D.
W.; Steenkamp, D. J. Bioorg. Med. Chem. 2002, 10, 875-881.
4338
Org. Lett., Vol. 4, No. 24, 2002

yield and high isomeric purity. The N-Boc protecting group
was removed by treatment of 14 with neat trifluoroacetic
acid, leading to ammonium salt 15, and then basification with
pyridine in the same pot gave the simplified mycothiol
analogue 2 as the result of a spontaneous and convenient
intramolecular S-to-N acetyl migration.31
Evaluation of 2 as a substrate for M. tuberculosis myco-
thiol S-conjugate amidase14,15 was carried out by prefatory
S-alkylation with bromobimane (16, Scheme 3) under mildly
cysteine-S-bimane product (see 8) by fluorescence-detected
HPLC assay.15 Specific activities for 18 and 17 are 7500
and 14 200 nmol min-1 mg-protein-1, respectively, establish-
ing 18 as a good substrate for this amidase. Neither the
inositol hydroxyls nor the glycosidic linking atom (O vs S)
plays a major role in enzyme binding. An earlier study6 had
indicated that the inositol ring is not required for reduction
of disulfides (see 6) by the M. tuberculosis mycothione
reductase. The accumulated information thus suggests that
2, which dispenses with the inositol hydroxyls and the linking
oxygen atom, can serve as a suitable foundation upon which
to base inhibitor design.
Scheme 3. Preparation of Bimane Derivatives
Acknowledgment. We thank Merck & Co. and Wyeth-
Ayerst Research for financial support and Dr. Gregory J.
Morriello and Dr. George A. Doss for assistance with NMR
spectroscopy. S.G. was a Johnson & Johnson and Henry
Rutgers Undergraduate Research Fellow, and D.S.M. was a
Rutgers University Graduate Fellow. The work at NIH was
supported in part by the Intramural AIDS Targeted Antiviral
Program of the Office of the Director, National Institutes of
Health.
basic conditions.15 The resulting bimane derivative 18 was
subjected to cleavage by the amidase in parallel with
mycothiol-bimane 17, while monitoring formation of the
Supporting Information Available: Experimental details
and spectral characterization for all new compounds and
description of the enzyme assay. This material is available
free of charge via the Internet at http://pubs.acs.org.
(31) Meyer zu Reckendorf, W.; Bonner, W. A. J. Org. Chem. 1961, 26,
4596-4599.
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