First Total Synthesis of Mycothiol and Mycothiol Disulfide

Article abstract of DOI:10.1021/ol0362008

(Matrix presented) The first total synthesis of mycothiol and mycothiol disulfide was achieved by linking D-2,3,4,5,6-penta-O-acetyl-myo-inositol, O-(3,4,6-tri-O-acetyl)-2-azido-2-deoxy-α,β-D-glucopyranosyl) trichloroacetimidate, and N,S-diacetyl-L-cysteine and deprotecting peracetylated mycothiol. The first full spectral characterization is reported for underivatized mycothiol. The structure of mycothiol was confirmed by spectral analysis of the known bimane derivative.

Full text of DOI:10.1021/ol0362008

ORGANIC
LETTERS
2004
Vol. 6, No. 3
365-368
First Total Synthesis of Mycothiol and
Mycothiol Disulfide
Sungwon Lee and John P. N. Rosazza*
DiVision of Medicinal and Natural Products Chemistry, College of Pharmacy, and
Center for Biocatalysis and Bioprocessing, The UniVersity of Iowa,
Iowa City, Iowa 52242
john-rosazza@uiowa.edu
Received November 10, 2003
ABSTRACT
The first total synthesis of mycothiol and mycothiol disulfide was achieved by linking D-2,3,4,5,6-penta-O-acetyl-myo-inositol, O-(3,4,6-tri-O-
acetyl)-2-azido-2-deoxy-r,â-D-glucopyranosyl) trichloroacetimidate, and N,S-diacetyl-L-cysteine and deprotecting peracetylated mycothiol. The
first full spectral characterization is reported for underivatized mycothiol. The structure of mycothiol was confirmed by spectral analysis of the
known bimane derivative.
Mycothiol1 is the major low molecular weight thiol found
in most actinomycetes including mycobacteria and strepto-
mycetes.2 MSH has functional similarities to glutathione, the
major thiol found in eukaryotes. MSH appears to maintain
a reducing intracellular environment and serves as an
antioxidant in MSH-producing organisms.3
deacetylase (mshB),5 ATP-dependent Cys:GlcN-Ins ligase
(mshC),6 and acetyltransferase (mshD)7 have been identified
in Mycobacterium smegmatis and Mycobacterium tubercu-
losis. Three MSH-dependent enzymes have been identified
so far. These include MSH-dependent formaldehyde dehy-
drogenase, which catalyzes dissimilatory conversion of
formaldehyde to formic acid, from Amycolatopsis methan-
olica.8 MSH disulfide reductase with high homology to
glutathione reductase was identified from the M. tuberculosis
genome.9 MSH S-conjugate amidase purified from M.
smegmatis plays a significant role in the detoxification of
The biosynthesis and metabolism of MSH have received
considerable recent attention as a means of identifying
potential new targets for antitubercular drug development.2
The genes encoding for MSH biosynthetically relevant
enzymes including glycosyltransferase (mshA),4 GlcNAc-Ins
(1) Mycothiol: 1-D-myo-inosityl 2-deoxy-2-(N-acetamido-L-cysteina-
mido)-R-D-glucopyranoside, MSH, or AcCys-GlcN-Ins.
(2) 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.
(3) Newton, G. L.; Fahey, R. C. Arch. Microbiol. 2002, 178, 388.
(4) Newton, G. L.; Koledin, T.; Gorovitz, B.; Rawat, M.; Fahey, R. C.;
Av-Gay, Y. J. Bacteriol. 2003, 185, 3476.
(5) Newton, G. L.; Av-Gay, Y.; Fahey, R. C. J. Bacteriol. 2000, 182,
6958.
(6) Sareen, D.; Steffek, M.; Newton, G. L.; Fahey, R. C. Biochemistry
2002, 41, 6885.
(7) Koledin, T.; Newton, G. L.; Fahey, R. C. Arch. Microbiol. 2002,
178, 331.
(8) Norin, A.; VanOphem, P. W.; Piersma, S. R.; Persson, B.; Duine, J.
A.; Jornvall, H. Eur. J. Biochem. 1997, 248, 282.
10.1021/ol0362008 CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/03/2004

alkylating agents and antibiotics.10 This enzyme cleaves
amide bonds of MSH thiol conjugates formed by reactions
between electrophiles with the free sulfhydryl group of MSH.
Subsequently, cysteine S-conjugates are excreted from the
cells leaving behind GlcN-Ins that is recycled for MSH
biosynthesis.
inositol 6 with O-(3,4,6-tri-O-acetyl)-2-azido-2-deoxy-R,â-
D-glucopyranosyl) trichloroacetimidate 7.19
Synthesis of 6 was pursued from D/L-myo-inositol via
literature procedures with minor modifications (Scheme
1).16,20,21
Scheme 1. Synthesis of D-2,3,4,5,6-Penta-O-acetyl-myo-inositol
Figure 1. Mycothiol (MSH), the mycothiol disulfide (MSSM), and
mycothiol bimane (MSmB).
MSH has been isolated as the disulfide (MSSM) or as the
bimane derivative (MSmB) from Streptomyces sp. and
Mycobacterium BoVis.11-13 We also quantified and isolated
MSH as the bimane derivative from Nocardia NRRL 5646
and confirmed the structure by NMR and mass spectrom-
etry.14 To date, efforts to isolate or synthesize free MSH have
been elusive. Previous attempts to synthesize MSH and its
analogues15-18 used extensive protection and deprotection
strategies. Moreover, R-glycosylation of D-myo-inositol and
linkage of N-acetylcysteine to the resulting inositol-glu-
cosamine pseudodisaccharide and deprotection to MSH have
been problematic. Herein, we report the first total synthesis
of MSH and MSSM including full spectral characterization
of underivatized MSH.
Treatment of 1 with 1-ethoxycyclohexene gave mixed,
myo-inositol biscyclohexene ketals. The desired racemic 1,2:
4,5-dicyclohexylidene-myo-inositol 2 was obtained by crys-
tallization. Benzylation of 2 gave (()-3-benzyl-1,2:4,5-
dicyclohexylidiene-myo-inositol 3. Benzylated D/L isomers
of myo-inositol were separated as their diasteroisomeric
camphanate esters. Confusion of identities of these isomers
appears in earlier literature.20,21 The identities of each isomer
were unambiguously established as reported later.22,23
The more polar L-isomer was obtained directly by crystal-
lization, whereas 4, the less polar isomer, was chromato-
graphically separated from mother liquors. Basic hydrolysis
of the camphanate ester and ketal cleavage with acetic acid
Our approach to the GlcN-Ins component of MSH entailed
coupling of the 1-OH of D-2,3,4,5,6-penta-O-acetyl-myo-
(9) Patel, M. P.; Blanchard, J. S. Biochemistry. 1999, 38, 11827.
(10) Newton, G. L.; Av-Gay, Y.; Fahey, R. C. Biochemistry 2000, 39,
10739.
(11) Sakuda, S.; Zhou, Z. Y.; Yamada, Y. Biosci. Biotechnol. Biochem.
1994, 58, 1347.
(12) Spies, H. S. C.; Steenkamp, D. J. Eur. J. Biochem. 1994, 224, 203.
(13) 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.
(14) Unpublished data: we have quantified and isolated MSH as the
bimane derivative form Nocardia sp. NRRL 5646.
(15) Patel, M. P.; Blanchard, J. S. J. Am. Chem. Soc. 1998, 120, 11538.
(16) Jardine, M. A.; Spies, H. S. C.; Nkambule, C. M.; Gammon, D.
W.; Steenkamp, D. J. Bioorg. Med. Chem. 2002, 10, 875.
(17) Nicholas, G. M.; Kovac, P.; Bewley, C. A. J. Am. Chem. Soc. 2002,
124, 3492.
(19) Grundler, G.; Schmidt, R. R. Liebigs Ann. Chem. 1984, 11, 1826-
1847.
(20) Vacca, J. P.; Desolms, S. J.; Huff, J. R.; Billington, D. C.; Baker,
R.; Kulagowski, J. J.; Mawer, I. M. Tetrahedron 1989, 45, 5679.
(21) Vacca, J. P. Tetrahedron 1991, 47, 907.
(22) Cottaz, S.; Brimacombe, J. S.; Ferguson, M. A. J. J. Chem. Soc.,
Perkin. Trans. 1 1993, 23, 2945.
(18) Knapp, S.; Gonzalez, S.; Myers, D. S.; Eckman, L. L.; Bewley, C.
A. Org. Lett. 2002, 4, 4337.
(23) Aneja, R.; Aneha, S. G.; Parra, A. Tetrahedron: Asymmetry 1995,
6, 17.
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Org. Lett., Vol. 6, No. 3, 2004

followed by acetylation gave D-1-benzyl-2,3,4,5,6-penta-O-
acetyl-myo-inositol 5. Debenzylation of 5 afforded D-2,3,4,5,6-
penta-O-acetyl-myo-inositol 6.
Scheme 3. Synthesis of MSH, MSSM, and MSmB
Glycosidic linkage of 6 to 7 with trimethylsilyl trifluo-
romethanesulfonate (TMSOTf) as a promoter gave an
excellent 9:1 ratio of R and â anomers in 56% and 9% yields,
respectively (Scheme 2). Reported synthesis of protected
Scheme 2. Conjugation of Protected D-myo-Inositol,
D-Glucosamine, and L-Cysteine
MSSM was quantitatively reduced to MSH by treatment
with bis(2-mercaptoethyl)sulfone (BMS) 12 in water for 5
days (Scheme 3).27 Advantages of BMS in disulfide reduc-
tions versus use of Zn/acid, NaBH4, or triphenylphospine/
HCl include its facile use in water at room temperature
without acid and the ready removal of unreacted and oxidized
BMS from reactions by EtOAc extraction. The structure of
GlcN-Ins using 3,4,6-tri-O-acetyl-2-deoxy-2-(2,4-dinitophe-
nyl-amino)-R-D-glucopyranosyl bromide or 3,4,6-tri-O-acetyl-
2-azido-2-deoxy-R-D-glucopyranosyl chloride in the presence
of silver triflate with different glycosylation acceptors gave
lower yields of the desired R-anomer and less R,â anomeric
selectivity compared with use of 7 as a glycosylation
donor.16,17 The reduction of 8 with Pd/C in the presence of
HCl under atmospheric pressure gave protected amine
hydrochloride 9 in 81% yield.
L-Cysteine hydrochloride was treated with Ac2O to obtain
N,S-diacetyl-L-cysteine 10.24 Reaction of 9 and 10 using
O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HATU) with 1-hydroxy-7-azabenzo-
triazole (HOAt) and collidine gave protected MSH 11
(Scheme 2).25 Carbodiimides such as EDC and DCC were
used for amidation of 9 with 10. However, they caused
extensive epimerization at position 2′′ of N-acetyl-L-cysteine
with lower yields.
1
MSH was confirmed directly (MS, H, 13C, 2D NMR, and
CD) and by matching spectral characteristics of the MSmB
with the same compound isolated from Nocardia sp. and
also those in the literature.14,17,28 The major differences in
NMR spectra between MSH and MSSM are observed in the
1H-coupling constant (J) and 13C-chemical shift values of
the 2′′ and 3′′ positions of N-acetyl-L-cysteine (Supporting
Information).
In conclusion, we have achieved the first total synthesis
of MSH and MSSM by the linkage of the three moieties
D-2,3,4,5,6-penta-O-acetyl-myo-inositol, O-(3,4,6-tri-O-acetyl)-
2-azido-2-deoxy-R,â-D-glucopyranosyl) trichloroacetimidate,
and N,S-diacetyl-L-cysteine and the subsequent deprotection
of peracetylated MSH. Furthermore, the first full spectral
characterization is reported for underivatized MSH and its
structure was confirmed by making the known MSH deriva-
Although Mg(OMe)2 has not been used to selectively
deacylate esters and thioesters while leaving amides intact,26
this reagent was used in MeOH to deprotect 11 giving MSH
and MSSM in 40% and 29% yields, respectively (Scheme
3).
(27) Lamoureux, G. V.; Whitesides, G. M. J. Org. Chem. 1993, 58, 633.
(28) All spectral data (1H, 13C NMR MS and CD) for synthetic MSmB
were identical with natural MSmB from Nocardia NRRL 5646 purified by
our laboratory.14 Reported specific rotations of MSmB17 were concentration-
dependent and variable ([R]20D ) +10 (c ) 0.5 in H2O), [R]20D ) -12 (c
(24) Watzig, H.; Dette, C.; Aigner, A.; Wilschowitz, L. Pharmazie 1994,
49, 249.
(25) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.
(26) Xu, Y. C.; Bizuneh, A.; Walker, C. Tetrahedron Lett. 1996, 37,
455.
) 0.1 in H2O)). Our synthetic and natural MSmB both had [R]25 ) +26
D
(c ) 0.1 in H2O).
Org. Lett., Vol. 6, No. 3, 2004
367

tive, MSmB. We also have shown that MSSM can be
conveniently reduced to MSH with BMS. The synthetic MSH
and MSSM could help in better understanding of the roles
of MSH in organisms producing it.
California, San Diego, for an authentic sample of MSH-
bimane.
Supporting Information Available: Experimental pro-
cedures and spectroscopic data of the key intermediates
including MSH, MSSM, and synthetic MSmB. This material
is available free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. S.L. thanks the Center for Biocatalysis
and Bioprocessing at the University of Iowa for a predoctoral
fellowship. We thank Prof. Fahey of the University of
OL0362008
368
Org. Lett., Vol. 6, No. 3, 2004