Desymmetrization of 2,4,5,6-tetra-o-benzyl-d-myo-inositol for the synthesis of mycothiol

Article abstract of DOI:10.1021/ol202218n

An efficient chemical synthesis of mycothiol involving the regioselective ketopinyl desymmetrization of 2,4,5,6-tetrabenzylated d-myo-inositol as the key step is described. Together with a highly α-stereoselective d-glucosaminylation, the whole procedure was accomplished in eight steps with an overall yield of 40%.

Full text of DOI:10.1021/ol202218n

ORGANIC
LETTERS
2011
Vol. 13, No. 20
5496–5499
Desymmetrization of 2,4,5,6-Tetra-O-
benzyl-D-myo-inositol for the
Synthesis of Mycothiol
Chuan-Chung Chung,† Medel Manuel L. Zulueta,† Laxmansingh T. Padiyar,† and
Shang-Cheng Hung*,†,‡
Genomics Research Center, Academia Sinica, 128 Sec.2 Academia Road, Taipei 115,
Taiwan, and Department of Applied Chemistry, National Chiao Tung University, 1001
Ta-Hsueh Road, Hsinchu 300, Taiwan
schung@gate.sinica.edu.tw
Received August 16, 2011
ABSTRACT
An efficient chemical synthesis of mycothiol involving the regioselective ketopinyl desymmetrization of 2,4,5,6-tetrabenzylated D-myo-inositol as
the key step is described. Together with a highly R-stereoselective D-glucosaminylation, the whole procedure was accomplished in eight steps
with an overall yield of 40%.
Low molecular weight thiols safeguard various organisms
against toxic oxidants and offer a reducing environment
necessary for effective cellular operation.1 Mycothiol is the
principal low molecular weight thiol in actinobacteria,2 a
diverse group of Gram-positive, high-GþC microorgan-
isms that include notable genera as Mycobacterium and
Streptomyces. Similar to glutathione, its functional analog
in eukaryotes and Gram-negative bacteria, the sulfhydryl
group of mycothiol is acquired from cysteine of which
the carboxyl and amino groups are blocked to avoid
autoxidation.3 In mycothiol (1), the cysteinyl moiety is N-
acetylated and is attached as an amide to D-glucosamine
(GlcN), which, in turn, is R10f1-linked to D-myo-inositol
(Ins) (Figure 1). Mycothiol disulfide (2) is produced upon
reaction with oxidants but is rapidly recycled back by
mycothiol disulfide reductase to 1 preserving a high
thiol/disulfide ratio within the confines of the cell.4 The
sulfhydryl group could also attack electrophilic xenobio-
tics leading to an S-conjugated mycothiol (exemplified by
the bimane derivative 3), which is further cleaved at the
D-glucosaminyl amide bond by mycothiol-S-conjugate
amidase to form the cysteine-bound toxin that is excreted
out of the cell and the GlcN-Ins pseudodisaccharide that is
reused as a substrate for mycothiol biosynthesis.5 Myco-
bacterial strains depleted of mycothiol have shown a
dramatic increase in susceptibility to oxidative stress and
some antitubercular agents.2a,6 As tuberculosis necessi-
tates extensive treatment coupled with its lethal combina-
tion with HIV infection and the emergence of multidrug-
resistant strains of its causative agent, M. tuberculosis,
exploration of mycothiol as an avenue for rational drug
design may be critical in fighting the disease.
† Genomics Research Center, Academia Sinica.
‡ Department of Applied Chemistry, National Chiao Tung University.
(1) (a) Moran, L. K.; Gutteridge, J. M. C.; Quinlan, G. J. Curr. Med.
Chem. 2001, 8, 763–772. (b) Hand, C. E.; Honek, J. F. J. Nat. Prod. 2005,
68, 293–308.
Mycothiol-based studies would benefit from the in-
creased availability of this compound that is currently
(2) Recent reviews: (a) Newton, G. L.; Buchmeier, N.; Fahey, R. C.
Microbiol. Mol. Biol. Rev. 2008, 72, 471–494. (b) Jothivasan, V. K.;
Hamilton, C. J. Nat. Prod. Rep. 2008, 25, 1091–1117.
(3) 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.
(4) Patel, M. P.; Blanchard, J. S. Biochemistry 1999, 38, 11827–11833.
(5) Newton, G. L.; Av-Gay, Y.; Fahey, R. C. Biochemistry 2000, 39,
10739–10746.
r
10.1021/ol202218n
Published on Web 09/15/2011
2011 American Chemical Society

Table 1. Desymmetrization of the Meso 1,3-Diol 7
yield (%)
Figure 1. Structures of mycothiol (1), mycothiol disulfide (2),
and mycothiol bimane (3).
temp time
entry
Xa
reagents
solvent (°C) (h)
6
8
9
1
2
3
4
5
6
7
8
9
ketopinate ꢀ
pyr.
4
4
4
4
4
4
4
4
4
4
35 19 10b
27 23 7c
32 12 8d
acquired routinely in e1.5 mg for every liter of the M.
smegmatis cell culture.7 Chemical synthesis,8 on the other
hand, is hampered by difficulties in the regioselective
protection and desymmetrization of myo-inositol, the R-
stereoselective glycosidic bond formation, and the epimer-
ization-prone cysteine introduction. Given our experience
with regioselective inositol desymmetrization9 as well
as the R-D-glucosamine formation for heparan sulfate
synthesis,10 we decided to apply these related approaches
in mycothiol preparation. Scheme 1 illustrates our retro-
synthetic plan. As a direct precursor of 1, we envisioned
that the pseudodisaccharide 4 could be generated from the
thioglycoside 5 and the 3-ketopinate 6. The protecting
group combination of the D-glucosamine-derived 5 is
OH
OH
Cl
EDC, DMAP
EDC, DMAP
Et3N
THF
CH2Cl2
CH2Cl2
63 22
0
Cl
Et3N, DMAP CH2Cl2
35 15 28e
Cl
Et3N, DMAP CH2Cl2 ꢀ40 24 65 26
0
0
0
0
0
Cl
ꢀ
pyr.
4
4
66 21
ꢀ40 24 69 24
60 32
Cl
DMAP
DIPEA
pyr.
Cl
CH2Cl2
4
4
10 Cl
DIPEA, DMAP CH2Cl2 ꢀ40 24 63 31
Et3N, DMAP CH3CN
Et3N, DMAP CH3CN ꢀ40 24 26 17
11 Cl
12 Cl
4
4
30 27 11
0
a 1.1 equiv of the (1S)-ketopinyl sources were used. b 30% of 7
was recovered. c 38% of 7 was recovered. d 44% of 7 was recovered.
e 13% of 7 was recovered. EDC: 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide.
instrumental in assuring exclusive R-linkage in our pre-
paration of heparan sulfate derivatives.10b Conversely, the
3-ketopinate 6 would be achievable by desymmetrization
of the meso 1,3-diol 7, which could be generated from the
commercially available myo-inositol 1,3,5-orthoformate
(Kishi’s triol) in four known steps.11 (1S)-Ketopinate
was chosen from other chiral derivatizing agents (e.g.,
camphanic acid, N-tosyl-L-proline, and Mosher’s acid)
because its monoesters with 7 could be effectively sepa-
rated by silica gel column chromatography.
Scheme 1. Retrosynthetic Analysis of Mycothiol (1)a
We first examined various conditions for the desym-
metrization of the 1,3-diol 7 (Table 1). The favorable
formation of the 3-O-ketopinyl 6 over its 1-O counterpart
(compound 8) was apparent from the use of (1S)-ketopinic
a 2-NAP: 2-naphthylmethyl, p-BrBn: p-bromobenzyl, Tol: toluenyl,
TBDPS: tert-butyldiphenylsilyl.
(9) (a) Patil, P. S.; Hung, S.-C. Chem.;Eur. J. 2009, 15, 1091–1094.
(b) Patil, P. S.; Hung, S.-C. Org. Lett. 2010, 12, 2618–2621. (c) Padiyar,
L. T.; Wen, Y.-S.; Hung, S.-C. Chem. Commun. 2010, 46, 5524–5526.
(10) (a) Lu, L.-D.; Shie, C.-R.; Kulkarni, S. S.; Pan, G.-R.; Lu, X.-A.;
Hung, S.-C. Org. Lett. 2006, 8, 5995–5998. (b) Hu, Y.-P.; Lin, S.-Y.;
Huang, C.-Y.; Zulueta, M. M. L.; Liu, J.-Y.; Chang, W.; Hung, S.-C.
Nat. Chem. 2011, 3, 557–563.
(11) Conway, S. J.; Gardiner, J.; Grove, S. J. A.; Johns, M. K.; Lim,
Z.-Y.; Painter, G. F.; Robinson, D. E. J. E.; Schieber, C.; Thuring, J. W.;
Wong, L. S.-M.; Yin, M.-X.; Burgess, A. W.; Catimel, B.; Hawkins,
P. T.; Ktistakis, N. T.; Stephens, L. R.; Holmes, A. B. Org. Biomol.
Chem. 2010, 8, 66–76.
(6) (a) Rawat, M.; Newton, G. L.; Ko, M.; Martinez, G. L.; Fahey,
R. C.; Av-Gay, Y. Antimicrob. Agents Chemother. 2002, 46, 3348–3355.
(b) Ung, K. S. E.; Av-Gay, Y. FEBS Lett. 2006, 580, 2712–2716.
(7) Steenkamp, D. J.; Vogt, R. N. Anal. Biochem. 2004, 325, 21–27.
(8) (a) Jardine, M. A.; Spies, H. S. C.; Nkambule, C. M.; Gammon,
D. W.; Steenkamp, D. J. Bioorg. Med. Chem. 2002, 10, 875–881. (b)
ꢀꢁ
Nicholas, G. M.; Kovac, P.; Bewley, C. A. J. Am. Chem. Soc. 2002, 124,
3492–3493. (c) Lee, S.; Rosazza, J. P. N. Org. Lett. 2004, 6, 365–368. (d)
Stewart, M. J. G.; Jothivasan, V. K.; Rowan, A. S.; Wagg, J.; Hamilton,
C. J. Org. Biomol. Chem. 2008, 6, 385–390. (e) Ajayi, K.; Thakur, V. V.;
Lapo, R. C.; Knapp, S. Org. Lett. 2010, 12, 2630–2633.
Org. Lett., Vol. 13, No. 20, 2011
5497

Scheme 2. Synthesis of the Pseudodisaccharide 11
Figure 2. ORTEP drawing of compound 6.
Table 2. Coupling of the D-Glucosamine-Derived Thioglycoside
5 with the Meso 1,3-Diol 7
anhydride, (1S)-ketopinic acid, or (1S)-ketopinyl chloride
as the acyl group source. The anhydride (entry 1) and the
acid (entries 2 and 3), however, both gave low yields and
modest regioselectivities for the desired compound 6 after
4 h of reaction at 4 °C. The regioselectivity improved
remarkably upon the use of (1S)-ketopinyl chloride at 4 °C
in the presence of pyridine assolvent (entry 7) or CH2Cl2 as
solvent and either triethylamine (entry 4) or diisopropyl-
ethylamine (DIPEA) (entry 9) as base. The addition of
catalytic DMAP reduced the regioselectivity of the 3-O-
esterification at 4 °C (entry 5) but was effective in a slow
reaction (entry 6, ꢀ40 °C, 24 h). Use of CH3CN as solvent
did not give good results. Overall, the best yield (69%,
entry 8) and regioselectivity [3.1/1 (6/8), entry 7] leading to
compound 6 were observed when pyridine was used as
both the base and solvent. The absolute structure of 6 was
confirmed by X-ray crystallographic analysis (Figure 2).
With compound 6 in hand, we proceeded to generate the
pseudodisaccharide backbone of mycothiol (Scheme 2).
Condensation of the alcohol 6 with the D-glucosaminyl
donor 5 employing NIS and TfOH as promoters led
yield (%)
temp
time
(h)
entrya
activator
(°C)
11
12
58
1
2
3
4
5
NIS, TfOH
NIS, TfOH
NIS, TMSOTf
NIS, AgOTf
BF3•Et2O
ꢀ60
3
3
3
6
6
30
41
35
33
29
ꢀ60 f ꢀ20
ꢀ60
49
57
37b
34c
ꢀ60
ꢀ78 f ꢀ20
a Using 1.5 equivof donor 5. b 22% of 7 was recovered. c 20% of 7 was
recovered.
exclusively to the R-glycoside 10 (3J1 ,2 = 3.6 Hz) in
84% yield. This outcome affirms our recent report10b that
the protecting group combination in 5 enables full R-
stereoselectivity in glycosylation. Further NaOH-mediated
cleavage of the ketopinyl group in 10 provided the 3-alco-
hol 11 in 95% yield.
0
0
activated by NIS in tandem with TfOH (entry 1) or
TMSOTf (entry 3). As expected, the regioselectivity dis-
appeared when the temperature was raised gradually to
ꢀ20 °C (entry 2) providing the best yield of 41% for the
desired 11. Other activators, such as NIS/AgOTf (entry 4)
and BF3•Et2O (entry 5), were also used, but no improve-
ments were observed.
Scheme 3 displays the remaining steps toward the syn-
thesis of mycothiol. Cleavage of the silyl group in the
3-alcohol 11 was carried out using TBAF (95%). Subse-
quent global hydrogenolysis with concomitant azido re-
duction [Pd(OH)2, H2] generated the pseudodisaccharide 4
in 98% yield. Coupling of compound 4 with the cysteine
derivative 13 using O-(7-azabenzotriazol-1-yl)-N,N,N0,
We demonstrated regioselective D-mannosylation of a
meso myo-inositol-derived 4,6-diol in our preparation of
phosphatidylinositol dimannosides.9a,b In the present case,
a direct glycosylation of diol 7 with donor 5 leading to
compound 11 would shorten the synthetic process. The
plausibility of this approach has been evaluated, and
the results are outlined in Table 2. Unfortunately, the
glycosylation revealed a higher preference for the 3-O
position of the inositol moiety similar to the ketopinyl
ester formation. The regioselective formation of the isomer
12 is more pronounced at low temperature (ꢀ60 °C) when
5498
Org. Lett., Vol. 13, No. 20, 2011

acidmediateddeprotection of the aminegroupfollowed by
pyridine treatment triggering SfN acetyl migration fur-
nished the target compound 1 in a quantitative yield over
two steps. The H NMR spectrum of 1 conforms with
previous reports.8c,e
Scheme 3. Synthesis of Mycothiol (1)
1
In conclusion, we have developed an efficient protocol
for the chemical synthesis of mycothiol. Our strategy relied
on the regioselective ketopinyl desymmetrization of the
meso inositol-derived 1,3-diol 7 and the fully R-stereose-
lective glycosylation with the D-glucosaminyl donor 5.
Starting from compound 7, the procedure involved eight
steps inanoverallyield of 40%. Application ofthismethod
could ease the acquisition of mycothiol and mycothiol-
related compounds for biological studies.
Acknowledgment. This work was supported by the
National Science Council (NSC 97-2113-M-001-033-MY3,
NSC 98-2119-M-001-008-MY2) and Academia Sinica.
Supporting Information Available. Experimental pro-
cedures, NMR (1H and 13C) spectra and crystallographic
data for compound 6. This material is available free of
charge via the Internet at http://pubs.acs.org.
N0-tetramethyluronium hexafluorophosphate (HATU) and
DIPEA8e supplied the amide 14 (78%). Trifluoroacetic
Org. Lett., Vol. 13, No. 20, 2011
5499