C O M M U N I C A T I O N S
Scheme 2 a
values of 190 ( 30 s-1 (see Supporting Information). Thus, we
have shown that the composition of mycothiol is 1-D-myo-inosityl
2-deoxy-2-(N-acetamido-L-cysteinamido)-R-D-glucopyranoside (1).
Acknowledgment. We thank Gerald Newton and Robert Fahey
for natural mycothiol bimane, Noel Whitaker for mass spectrometry,
Tarek Sammakia for the suggestion of recording NMR spectra on
a mixed sample of natural and synthetic MSmB, and Andrew
Phillips for many helpful discussions. This work was supported in
part by the Intramural AIDS Targeted Antiviral Program of the
Office of the Director, National Institutes of Health (C.A.B.).
Supporting Information Available: Details of enzyme kinetics;
a Reagents: (a) 3,4,6-tri-O-acetyl-2-azido-2-deoxy-R-D-glucopyrano-syl
chloride, AgOTf, 2,6-diisopropyl-4-methyl-pyridine, CH2Cl2, 44%. (b) 1.
ethylene glycol, (+)-camphor sulfonic acid, CH3CN. 2. Ac2O, pyridine 80%.
(c) Pd-C, H2, EtOAc, 86%.
of the syntheses and analytical and spectroscopic data for all intermedi-
1
ates; H and 13C NMR spectra for new compounds, and for natural,
synthetic and mixed samples of MSmB; and a complete table of NMR
data for MSmB (1) (PDF). This material is available free of charge via
Scheme 3 a
References
(1) (a) Sakuda, S.; Zhou, Z.; Yamada, Y. Biosci. Biotechnol. Biochem. 1994,
58, 1347-1348. (b) Spies, H. S. C.; Steenkamp, D. J. European Journal
of Biochemistry 1994, 224, 203-213. (c) Newton, G. L.; Av-Gay, Y.;
Fahey, R. C. Biochemistry 2000, 39, 10739-46.
(2) Fahey, R. C. Annu. ReV. Microbiol. 2001, 55, 333-356.
(3) Newton, G. L.; Av-Gay, Y.; Fahey, R. C. Biochemistry 2000, 39, 10739-
46.
(4) Newton, G. L.; Av-Gay, Y.; Fahey, R. C. J. Bacteriol. 2000, 182, 6958-
6963.
(5) Nicholas, G. M.; Newton, G. L.; Fahey, R. C.; Bewley, C. A. Org. Lett.
2001, 3, 1543-1545.
(6) Patel, M. P.; Blanchard, J. S. J. Am. Chem. Soc. 1998, 120, 11538-11539.
(7) Bornemann, C.; Jardine, M. A.; Spies, H. S. C.; Steenkamp, D. J. Biochem.
J. 1997, 325, 623-629.
a Reagents: (a) DEPC, iPr2EtN, DMF, 31%.; (b) Mg(OMe)2, MeOH
(dry), 75%.
(8) The synthesis of a mixture of 1-L- and 1-D-myo-inosityl-2-amido-2-deoxy-
R-D-glucopyranoside has been reported.7 The authors used the isomeric
mixture for glycosylation to give a 1:1 mixture of R- and â-linked products
of the 1-D/L-myo-inositol mixture. After separation, the myo-inositol portion
of the four products was assigned the 1-L- or 1-D-configuration by
comparison of 1H and 13C NMR data with intact mycothiol, a strategy
that does not permit unambiguous assignment of stereochemistry.
(9) The D configuration for myo-inositol was based on comparisons of the
CD spectra of the 1-O-acetyl-2,3,4,5,6-penta-O-methyl derivative with
those reported in the literature.1a
(10) The numbering scheme used throughout maintains C-1 of unsubstituted
myo-inositol as C-1 in derivitives thereof.
(11) Mayer, T. G.; Schmidt, R. R. Liebigs Ann./Recl. 1997, 859-863.
(12) We were unable to recrystallize the 1-D-myo-inositol fragment prior to
benzylation, as reported in ref 11.
(13) Reaction time of 4 h versus 5 days at 40 °C in K2CO3 in methanol.11
(14) We note that we have completed the synthesis and characterization of
D-GlcNAc-R-(1-1)-D-myo-inositol and D-GlcNAc-R-(1-1)-L-myo-inositol
(unpublished data), and determined that the deacetylase enzyme used in
the biosynthesis of MSH failed to deacetylate D-GlcNAc-R-(1-1)-L-myo-
inositol, but cleaved the amide bond of GlcNAc-R-(1-1)-D-myo-inositol
with identical kinetics as reported for the natural substrate.4 Because this
step forms a biosynthetic intermediate of mycothiol, we completed the
synthesis of MSmB using the protected GlcNAc-R-(1-1)-D-myo-inositol
derivitive.
Figure 2. Molar CD of natural and synthetic MSmB (1). Spectra were
recorded on 20 µM samples at 25 °C.
1
The H and 13C NMR data for synthetic mycothiol bimane (1)
were identical to those recorded for natural material isolated from
M. smegmatis1c (see Supporting Information). Moreover, the CD
spectra (Figure 2) of natural and synthetic MSmB are superimpos-
able and show negative (∆ꢀ -2.3) and positive (∆ꢀ +6.9) Cotton
effects at 220 and 185 nm, respectively.18
To confirm substrate specificity of MCA, we measured in parallel
the rates and extent of cleavage of the amide bond in natural and
synthetic samples of MSmB by recombinant M. tuberculosis MCA
using a fluorescence-detected HPLC assay.3 Both samples were
quantitatively cleaved within 15 min in the presence of 13 nM
MCA. Kinetic studies with MCA using natural and synthetic MSmB
yielded indistinguishable results with specific activities of 14 200
( 700 nmol min-1 mg-1, Km values of 500 ( 30 mM, and Kcat
(15) Pavliak, V.; Kova´cˇ, P. Carbohydr. Res. 1991, 210, 333-337.
(16) Takuma, S.; Hamada, Y.; Shioiri, T. Chem. Pharm. Bull. 1982, 30, 3147-
3153.
(17) Xu, Y. C.; Bizuneh, A.; Walker, C. J. Org. Chem. 1996, 61, 9086-9089.
(18) No specific rotation of MSmB (1) has been reported for comparison. Our
synthetic material showed a concentration-dependent sign of the specific
rotation ([R]20 +10 (c 0.5 , H2O), [R]20 -12 (c 0.1, H2O)), leading us
D
D
to record CD spectra for natural and synthetic MSmB. It is possible that
the presence of the fluorescent bimane contributes to the change in sign
since emission occurs through 590 nm, the wavelength of the sodium D
line. (For a discussion of concentration-dependent changes in specific
rotations, see Eliel, E. L. In Stereochemistry of Organic Compounds;
Wilen, S. H., Mander, L. N., Eds.; Wiley & Sons: New York, 1994; pp
1071 ff.)
JA017891A
9
J. AM. CHEM. SOC. VOL. 124, NO. 14, 2002 3493