Synthesis of mycothiol, 1D-1-O-(2-[N-acetyl-L-cysteinyl]amino-2-deoxy-α-D-glucopyranosyl)-myo-inositol, principal low molecular mass thiol in the actinomycetes

Article abstract of DOI:10.1016/S0968-0896(01)00383-2

Members of the actinomycetes produce 1D-1-O-(2-[N-acetyl-l-cysteinyl]amino-2-deoxy-α-D-glucopyranosyl)-myo-inositol or mycothiol 1 as principal low molecular mass thiol. Chemical synthesis of a biosynthetic precursor of mycothiol, the pseudo-disaccharide

Full text of DOI:10.1016/S0968-0896(01)00383-2

Bioorganic & Medicinal Chemistry 10 (2002) 875–881
Synthesis of Mycothiol, 1D-1-O-(2-[N-Acetyl-L-cysteinyl]amino-2-
deoxy-ꢀ-D-glucopyranosyl)-myo-inositol, Principal Low
Molecular Mass Thiol in the Actinomycetes
M. Anwar Jardine,a Hendrik S.C. Spies,b,, Comfort M. Nkambule,c
David W. Gammonc and Daniel J. Steenkampa,*
aDepartment of Chemical Pathology, University of Cape Town Medical School, Observatory 7925, South Africa
bNMR Laboratory, University of Stellenbosch, Matieland 7602, South Africa
cDepartment of Chemistry, University of Cape Town, Rondebosch, South Africa
Received 17 June 1999; accepted 5 September 2001
Abstract—Members of the actinomycetes produce 1D-1-O-(2-[N-acetyl-l-cysteinyl]amino-2-deoxy-a-d-glucopyranosyl)-myo-inosi-
tol or mycothiol 1 as principal low molecular mass thiol. Chemical synthesis of a biosynthetic precursor of mycothiol, the pseudo-
disaccharide 1D-1-O-(2-amino-2-deoxy-a-d-glucopyranosyl)-myo-inositol 13 was achieved by the following steps: (1)
Enantioselective synthesis gave the glycosyl acceptors (ꢀ)-2,3,4,5,6-penta-O-acetyl-d-myo-inositol D-7 and the corresponding l-
isomer L-7. (2) Condensation of D-7 and L-7 with the glycosyl donor 3,4,6-tri-O-acetyl-2-deoxy-2-(2,4-dinitrophenylamino)-a-d-
glucopyranosylbromide afforded the corresponding a and b anomeric products, which could be resolved by silica gel chromato-
graphy. (3) Deprotection of these by hydrolysis using an anion exchange resin gave 1D- and 1L-1-O-(2-amino-2-deoxy-a-d-gluco-
pyranosyl)-myo-inositol 13 and 15 and the corresponding b-coupled anomers 14 and 16. Only 13, and to a much lesser extent 15,
were used by enzymes present in an ammonium sulphate fraction of a cellfree extract of Mycobacterium smegmatis for the enzy-
matic synthesis of mycothiol. In the absence of acetyl-SCoA, the immediate biosynthetic precursor of 1, desacetylmycothiol, was
the major product. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
nificant differences between gram negative and gram
All organisms capable of growth in an aerobic environ-
ment produce one or more low molecular mass thiols as
a major metabolite. Studies on the distribution of these
compounds in procaryotes1,2 have brought to light sig-
positive bacteria. As in the vast majority of eukaryotes,
glutathione is the principal antioxidant thiol in gram
negative bacteria. Fahey et al.2,3 proposed that the need
to incorporate cysteine into the form of this proteolyt-
ically stable tripeptide stemmed from the rapid rate of
autooxidation of free cysteine, which generates peroxide
as a harmful product. Many gram positive bacteria,
however, lack glutathione and, instead, produce a
*Corresponding author. Tel.: +27-21-406-6105; fax: +27-21-406-
6153; e-mail: daan@chempath.uct.ac.za
,Deceased.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00383-2

876
M. A. Jardine et al. / Bioorg. Med. Chem. 10 (2002) 875–881
number of alternate thiols. Most Actinomycetes produce
mycothiol
In this paper, we describe the synthesis of a-d-GI and its
enzymatic conversion to mycothiol, which allowed an
elucidation of the mycothiol biosynthetic pathway, as
reported in a previous paper.12 Our synthetic plan gave
us access to enantiomeric and stereoisomeric analogues
of a-d-GI, which were used to investigate the substrate
specificity of the a-d-GI-L-cysteine ligase.12
(1D-1-O-(2-[N-acetyl-l-cysteinyl]amino-2-
deoxy-a-d-glucopyranosyl-myo-inositol) (Scheme 1) as
their principal low molecular mass thiol.4ꢀ6
Interest in mycothiol derives from several considera-
tions. In its functions, mycothiol probably substitutes
for glutathione as in the gram-positive bacteria, Amy-
colatopsis methanolica and Rhodococcus erythropolis,
which use mycothiol, instead of glutathione, as the fac-
tor in the NAD/factor-dependent formaldehyde dehy-
drogenase.7 The role of glutathione in bacteria is mainly
a protective one and mutants defective in its synthesis
show significantly enhanced sensitivity to oxidative
stress and alkylating reagents. Glutathione also plays an
important role in the metabolism of drugs. In the enteric
bacteria the expression of several antioxidant enzymes,
including glutathione reductase, g-glutamylcysteine
synthetase, catalase/peroxidase and alkylhydroperox-
idase, is governed by the oxidation state of the oxyR
transcription factor.8 Inactivating mutations with
respect to oxyR in the enterobacteria results in sensitiv-
ity to isoniazid,9 a frontline drug for the treatment of
tuberculosis. Since Mycobacterium tuberculosis WT car-
ries an inactive oxyR gene10 this phenomenon is prob-
ably a contributing factor in the exceptional sensitivity
of M. tuberculosis to isoniazid. The relationship between
the levels of expression of the antioxidant enzymes and
isoniazid sensitivity, however, seems to be a complex
one which is not entirely understood.10 The recent
observation that a mycothiol deficient mutant of M.
smegmatis is considerably more resistant to isoniazid
than the wildtype, can, for instance, not readily be
rationalized in terms of current knowledge concerning
mechanisms of resistance against the drug11 and points
to the need for more detailed studies on the metabolism
and functions of mycothiol.
Results and Discussion
The pseudo-disaccharide moiety of mycothiol con-
sists of d-glucosamine glycosidically coupled (a1-10)
to d-myo-inositol.4 Such a coupling can be achieved
synthetically, using an appropriately protected glycosyl
donor and a myo-inositol acceptor. This synthetic route
The biosynthesis of mycothiol proceeds with the early
formation of the pseudodisaccharide, 1D-1-O-{2-N-
acetylamino-2-deoxy-a-d-glucopyranosyl)-myo-inositol
(N-acetyl-a-d-GI), which is deacetylated and further
metabolized by the sequential addition of first cysteine
and then acetate, from acetyl-SCoA, to form mycothiol
(Scheme 2).12,13 The steps involved in the formation of
N-acetyl-a-d-GI have not yet been characterized. Myco-
thiol is maintained in the reduced form by a NAD(P)H
dependent disulfide reductase4 encoded by gorA in the
mycobacterial genome.14,15 Mycothiol and its biosyn-
thetic intermediates are required for the further char-
acterization of the enzymes involved in its synthesis and
of the newly discovered disulfide reductase.
Scheme 1. Structure of mycothiol.
Scheme 2. Biosynthesis of mycothiol.

M. A. Jardine et al. / Bioorg. Med. Chem. 10 (2002) 875–881
877
necessitated the selective protection of myo-inositol in
such a manner that it renders the 10-OH available for
glycosidic coupling. Commercially available myo-ino-
sitol (1; Scheme 3) was converted into racemic 1,2:4,5-
dicyclohexylidene-myo-inositol (DL-2) (26%).16 The
protected myo-inositol (DL-2) was subsequently selec-
tively benzylated to give the 3-benzylated product (DL-
3) (60%).17 The 3-benzylated products (DL-3) were
resolved as the camphanate esters, using known litera-
ture procedures to give (D-4) and (L-4), respectively.17
Acid hydrolysis secured the removal of the cyclohex-
ylidene groups to give the monobenzylated myo-inositol
(D-5 and L-5), which was subsequently acetylated to
give D/L-1-O-benzyl-2,3,4,5,6-penta-O-acetyl-myo-ino-
sitol (D-6 and L-6). Reduction of the benzyl group gave
the D/L-2,3,4,5,6-penta-O-acetyl-myo-inositol (D-7 and
L-7). Koenigs–Knorr coupling of products D-7 and L-7
The a-coupled D,D (9) and D,L (11) diastereomers were
separated, with great effort, using thin-layer chromato-
graphy, whereas the b-coupled products D,D (10) and
D,L (12), were inseparable. Due to these purification
difficulties, the enantioselective synthesis of a-D-GI (13)
seemed a more practical route to follow.
Our synthetic plan gave us access to stereoisomeric
analogues of the mycothiol precursor, a-D-GI (13),
namely compounds 14–16. These analogues where used
to investigate the substrate specificity of the a-d-GI-L-
cysteine ligase.12
The final step in mycothiol synthesis, namely the cou-
pling of cysteine, was achieved enzymatically (Scheme
2). A crude, undialysed, cell-free extract of M. smegmatis
catalysed the conversion of a-D-GI (13) into a mixture of
desacetyl mycothiol and mycothiol (1:4, 40% conver-
sion).12 The products were quantified by [35S]-cysteine
incorporation, and characterised as the bimane deriva-
tive.4 The a-d-GI-L-cysteine ligase produced mycothiol
from a-D-GI (13) [Vmax (app)=0.92Æ0.02 nmol/min/
with
3,4,6-tri-O-acetyl-2-deoxy-2-(2,4-dinitrophenyl-
amino)-a-d-glucopyranosyl bromide (8),18 respectively,
gave a mixture of a- and b-linked pseudo-disaccharides
(a/b 1:1, w/w) (60%) (9 and 11) and (10 and 12),
respectively (Scheme 4). Treatment of the protected
pseudo-disaccharides (9–12), with an anion exchange
resin (IR400-OH), gave pseudo-disaccharides (13–16).
The synthesis of a-d-GI (13), without the resolution of
the protected myo-inositol (D,L-3) was also attempted.
The racemic D,L-2,3,4,5,6-penta-O-acetyl-myo-inositol
(D,L-7) were synthesised, using the same reaction
sequence as before. Glycosidic coupling of 3,4,6-tri-O-
acetyl-2-deoxy-2-(2,4-dinitrophenylamino)-a-d-gluco-
pyranosyl bromide (8) with (D,L-7), gave a racemic
mixture of a- and b-linked pseudo-disaccharides (9–12).
Scheme 4. Reagents: (i) 1-ethoxycyclohexane, p-toluenesulphonic acid
and dimethylformamide; (ii) NaH, BnBr and toluene; (iii) camphanic
acid chloride and Et3N in CH2Cl2; (iv) (a) KOH in ethanol; (b) 80%
acetic acid (v/v); (v) acetic anhydride and pyridine; (vi) H2 and Pd/C;
(vii) AgOTf, 2,6-tert-dibutylpyridine and CH2Cl2; (viii) Amberlite
IR400 (OH).
Scheme 3.

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M. A. Jardine et al. / Bioorg. Med. Chem. 10 (2002) 875–881
mg, Km (app)=140Æ9 mM], whereas a-l-GI (15) dis-
played poor binding [Vmax (app)=0.51Æ0.22 nmol/min/
mg, Km (app)=2.69Æ1.34 mM]. The b-coupled pro-
ducts (14 and 16), were inactive. The synthetic coupling
of N-acetyl-cysteine to a-d-GI (13), using a variety of
water soluble coupling and activating reagents was
hitherto unsuccessful. This could be due to a combina-
tion of insolubility and steric demand of the various
reactants. An alternative synthetic approach to myco-
thiol is currently in progress. This involves the synth-
esis of an appropriately protected a-d-GI (13), with the
2-amino group available for coupling with cysteine.
Coupling of a S-protected N-acetyl-cysteine to the
aforementioned protected a-d-GI (13), may be success-
ful in an organic solvent. All products were charac-
terised by NMR and electron spray mass spectrometry.
shifts in CDCl3 were obtained relative to tetra-
methylsilane (d 0.0 ppm) as internal reference. 13C che-
mical shifts in D2O were measured using 2.5% dioxane
(d 67.3 ppm) as external reference. 13C multiplicities
were determined using the APT19 technique. Two-
dimensional 1H–1H correlation spectra (COSY) were
obtained using the pulse sequence: D1–90ꢁ–(t1+D3)–
60ꢁ–AQ,20 where D3=0.25 s was used to enhance long-
1
range coupling effects. Two-dimensional H–13C corre-
lation spectra (HETCOR) were obtained by means of
the pulse sequence described previously.21
NMR spectral assignments for compounds L-6 to 16 are
presented in tabular form (Tables 1–6).
Synthetic procedures
(+)-1-O-Benzyl-2,3,4,5,6-penta-O-acetyl-D-myo-inositol
(D-6). The (ꢀ)-3-benzyl-1,2:4,5-dicyclohexylidene-d-
myo-inositol (D-4)17 (1.68 g, 3.91 mmol) was treated
with a solution of KOH (2.24 g, 40 mmol) in ethanol for
16 h at 20 ꢁC. The solvent was evaporated, followed by
the addition of 80% aqueous acetic acid (89 mL). The
reaction mixture was allowed to stir at 95 ꢁC for 1 h.
Evaporation of the solvents, gave the crude 1-O-benzyl-
d-myo-inositol (D-5). The latter compound was dis-
solved in pyridine (20 mL) and acetic anhydride (20 mL)
and allowed to stir at 25 ꢁC for 16 h. The reaction mix-
ture was subsequently poured on ice, resulting in the
precipitation of the crude white crystalline product (D-
6), which was filtered and dried (1.7 g, 91%); mp 185–
187 ꢁC (ethyl acetate–hexane); [a]D +30.7ꢁ (Æ0.6ꢁ) (c
0.44, CHCl3).
Experimental
General methods
All reactions were carried out under a nitrogen atmo-
sphere, unless otherwise specified. Reactions were mon-
itored by TLC using Merck F254 precoated silica gel
plates. Detection was by means of a UV lamp (wave-
length 254 nm) and by heating the plate at 200 ꢁC after
spraying with a solution of 5% vanillin/90% ethanol/
5%H2SO4. All combined organic reactions were dried
over anhydrous magnesium sulphate before evaporation
on a Buchi rotary evaporator with bath temperature of
50 ꢁC or below. Column chromatography was carried
out on silica gel and the eluent mixture used is specified
in each experiment. Commonly used solvents were pur-
ified and dried as follows: Methanol was distilled from
magnesium powder under a nitrogen atmosphere and
stored over molecular sieves type 3 A. Triethylamine
and pyridine was distilled from potassium hydroxide
under a nitrogen atmosphere and stored over potassium
hydroxide pellets. Dichloromethane was distilled from
phosphorus pentoxide and stored over molecular sieves
type 4 A. Diethyl ether was distilled from sodium wire
under a nitrogen atmosphere immediately before use.
Ethyl acetate was washed with saturated aqueous
sodium carbonate and brine, dried over sodium sulfate
and distilled. N,N-Dimethylformamide was distilled
from calcium hydride under reduced pressure and stored
over molecular sieves type 4 A. All melting points were
determined in open capillary tubes and are uncorrected.
Specific rotations ([a]D) were determined at 20 ꢁC, unless
otherwise specified, using a Perkin-Elmer 141 polari-
(ꢀ)-1-O-Benzyl-2,3,4,5,6-penta-O-acetyl-L-myo-inositol
(L-6). Repetition of the above procedure, using (+)-3-
benzyl-1,2:4,5-dicyclohexylidene-l-myo-inositol (L-4)17
Table 1. 13C NMR data for compounds 6 and 7 (d, ppm) at 25 ꢁC in
CDCl3
D-6 or L-6
D-7 or L-7
Inositola
71.84
71.21
70.93
69.53
69.17
66.69
72.50
70.77
70.49
69.47
69.20
68.85
Acetyl
CH3
20.87
20.69
20.83
20.76
20.56
20.52Â2
171.02
170.27
169.91
169.81
169.70
20.57Â2
ꢁ
meter and are recorded in units of 10ꢀ1 cm2 gꢀ1. Mass
spectra were recorded on a VG Autospec 4600.
20.53
CO
170.10
169.94
169.90
169.70
169.63
NMR spectroscopy
NMR spectra were obtained on a Varian VXR 300
spectrometer operating at 299.9 and 75.42 MHz for the
observation of 1H and 13C, respectively. All spectra
were recorded at 25 ꢁC in either D2O or CDCl3 as lock
OBn
OCH2
1
2, 6
3, 5
4
74.45
136.90
127.82
128.52
128.09
—
—
—
1
solvent. H chemical shifts in D2O were measured rela-
tive to sodium 4,4-dimethyl-4-silapentane-1-sulfonate (d
0.0 ppm) as external reference. 1H and 13C chemical
aNo assignments were made for inositol ring.

M. A. Jardine et al. / Bioorg. Med. Chem. 10 (2002) 875–881
879
Table 2. 13C NMR data of compounds 9–12 (d, ppm) at 25 ꢁC in CDCl3
10
9
11
12
Dinitrohenyl
1
2
3
4
a
a
147.45
131.32
124.16
136.96
130.01
114.17
146.88
131.74
124.51
136.82
130.01
112.95
148.18
131.46
123.79
137.36
129.58
115.23
123.42
a
5
130.04
115.96
6
Glucosamine
1
2
3
4
92.77
54.52
71.96
96.92
54.89
71.17
67.36
69.45
61.71
103.36
57.44
72.37
67.96
72.30
61.61
99.26
57.63
72.24*
67.86*
72.07*
61.53*
71.02y
68.82
61.63
5
6
Inositol
10
69.02y
68.54{
69.17
71.99{
68.87{
69.93
68.77*
69.25*
73.24*
70.34*
69.79*
20
65.30{
30
70.02{
74.96{
40
69.64*72.35* 70.87
67.96y
69.496*8.98*68.96*66.35*
*y
50
70.90
70.83y
60
Acetyl
CH3
20.72, 20.57
20.51Â4
20.65Â2, 20.62
20.50Â2, 20.41
20.39, 20.09
20.96, 20.66
20.54, 20.50Â2
20.42, 20.34
20.10
20.76, 20.66
20.56, 20.49Â2
20.42, 20.38
19.67
20.44, 20.3
CO
170.58, 170.51
169.97, 169.74
169.55, 169.45
169.45, 169.06
170.63, 170.56
169.91, 169.76
169.69, 169.69
169.37, 168.66
170.71, 170.34
169.77, 169.71
169.44, 169.32
169.28, 168.91
aNot observed due to low concentration.
*,y,{Assignments are interchangeable in the same column.
Table 3. 13C NMR data for compounds 13–16 (d, ppm) at 25 ꢁC in
D2O
(chloroform–diethyl ether); [lit.22 mp 185–187 ꢁC); [a]D
ꢀ5.8ꢁ (c 1.8, CHCl3) (lit.22 [a]D ꢀ11.2ꢁ (c 0.05, CHCl3)].
Mycothiol4
13
14
15
16
(+)-2,3,4,5,6-Penta-O-acetyl-L-myo-inositol (L-7). Repe-
tition of the above procedure, using (L-6) afforded the
title product (L-7) (87%); mp 185–187 ꢁC (chloroform–
diethyl ether); (lit.22 mp 185–187 ꢁC); [a]D +8.5ꢁ (c 1.6,
CHCl3) [lit.22 [a]D +11.2ꢁ (c 0.1, CHCl3)].
Glucosamine
1
2
3
4
99.85
54.73
71.46
70.85
73.29
61.33
98.57
57.49
107.31
59.53
103.11
57.83
98.52
58.22
73.70*72.26*74.46*74.67
72.36*73.48*73.77*78.96*
70.87*74.33*72.50*72.34*
5
6
63.12*63.28
63.28
63.03
2,3,4,5,6-Penta-O-acetyl-1-O-(3,4,6-tri-O-acetyl-2-deoxy
-2-(2,4-dinitrophenylamino)-ꢀ-D-glucopyranosyl)-D-myo-
inositol (9)and the- ꢁ-D-glucopyranosyl derivative (10).
A mixture of (D-7) (1.0 g, 2.56 mmol), 2,6-di-tert-butyl-
pyridine (0.65 mL) and dichloromethane (15 mL) was
allowed to stir at 25 ꢁC for 1 h. Silver triflate (1.0 g,
3.89 mmol) and a solution of 3,4,6-tri-O-acetyl-2-deoxy-
2-(2,4-dinitrophenylamino)-a-d-glucopyranosyl bromide
(8)18 (1.95 g, 3.64 mmol) in dichloromethane (23 mL)
were then added. The mixture was allowed to stir at
20 ꢁC for 30 h, followed by neutralisation with triethyl-
amine and then filtration. The filtrate was concentrated
under reduced pressure to give a crude product mixture
(3.65 g). Elution of the mixture of products from a silica
gel (500 g) column (5 cm diameter) (diethyl ether–
chloroform 2:98) gave (9) (1.06 g, 49%) as a yellow
amorphous solid; (Rf 0.25 diethyl ether–chloroform
1:9); [a]D +11ꢁ (c 1.3, CHCl3). Found: MNH+4 ,
861.2524, C34H41O33N3 requires MNH4, 861.2525, fol-
lowed by (10) (626 mg, 29%) as a yellow amorphous
solid; (Rf 0.10, diethyl ether–chloroform 1:9); [a]D ꢀ34ꢁ
Inositol
10
20
30
40
50
60
79.86
72.46
71.81
72.83
74.99
72.86
78.13*83.72*82.03*79.55
76.88*78.55*76.83*71.79
76.40*78.42*75.59 *73.52
74.89*76.75*75.40*74.77
74.81*74.83*74.77*76.78
73.70*74.36*74.63*73.22
*Assignments are interchangeable in the same column.
afforded product (L-5) and subsequently, the title com-
pound (L-6) (95%); mp 185–186 ꢁC (ethyl acetate–hex-
ane); [a]D ꢀ30.1ꢁ (Æ0.4ꢁ) (c 0.39, CHCl3).
(ꢀ)-2,3,4,5,6-Penta-O-acetyl-D-myo-inositol (D-7). To a
solution of (D-6) (1.7 g, 3.54 mmol) in ethyl acetate
(78 mL) were added 10% Pd on charcoal (1.2 g). The
suspension was shaken under an atmosphere of hydro-
gen at 20 psi for 48 h. The catalyst was then removed by
filtration and the filtrate evaporated under reduced
pressure, to give (D-7) (1.2 g, 87%); mp 185–186 ꢁC

880
M. A. Jardine et al. / Bioorg. Med. Chem. 10 (2002) 875–881
Table 4. 1H Chemical shifts (d, ppm), multiplicities and H–1H cou-
pling constants (J, Hz) for compounds 6 and 7 at 25 ꢁC in CDCl3
(c 1.1, CHCl3). Found: MNH+4 , 861.2550, C34H41O33N3
requires MNH4, 861.2525.
1
D-6 or L-6
D-7 or L-7
2,3,4,5,6-Penta-O-acetyl-1-O-(3,4,6-tri-O-acetyl-2-deoxy
-2-(2,4-dinitrophenylamino)-ꢀ-D-glucopyranosyl)-L-myo-
inositol (11)and the- ꢁ-D-glucopyranosyl derivative (12).
Repetition of the above procedure, using (L7) afforded
the title products (11) (45%) as a yellow amorphous
solid; (Rf 0.21, diethyl ether–chloroform 1:9); [a]D +11ꢁ
(c 1.0, CHCl3). Found: MNH+4 , 861.2537, C34H41O33N3
requires MNH4, 861.2525, followed by (12) (25%) as a
yellow amorphous solid; (Rf 0.25, diethyl ether–chloro-
form 1:9); [a]D ꢀ60ꢁ (c 2.1, CHCl3). Found: MNH+4 ,
861.2547, C34H41O33N3 requires MNH4, 861.2525.
Inositol
10
3.608 (dd, 10.1/2.8)
5.757 (t, 2.8)
4.938 (dd, 10.6/2.8)
5.480 (dd, 10.6/9.7)
5.079 (t, 9.8)
3.901 (ddd, 10.0/7.0/3.0)
5.594 (t, 2.9)
4.978 (dd, 10.6/2.8)
5.457 (dd, 10.6/9.6)
5.148 (t, 9.8)
20
30
40
50
60
5.424 (t, 10.0)
5.310 (t, 10.0)
OBn
OCHA
OCHB
Phenyl
4.664 (d, 12.1)
4.399 (d, 12.1)
7.2–7.4 (m, 5H)
—
—
—
OH
CH3
—
2.199, 2.015
2.001, 1.998
1.989
2.569 (d, 7.0)
2.213, 2.088
2.023, 2.012
1.997
1D-1-O-(2-Amino-2-deoxy-ꢀ-D-glucopyranosyl)-myo-ino-
sitol (13). A mixture of (9) (580 mg, 0.69 mmol), metha-
Table 5. 1H Chemical shifts (d, ppm), multiplicities and 1H–1H coupling constants (J, Hz) for compounds 9–12 at 25 ꢁC in CDCl3
9
11
10
12
Inositol
10
20
30
40
50
60
4.015 (dd, 10.5/2.5)
5.611 (t, 2.6)
4.974 (dd, 10.4/2.7)
5.532 (dd, 10.4/9.8)
5.167 (t, 9.8)
4.157 (dd, 9.9/2.9)
5.660 (t, 2.9)
5.031 (dd, 10.6/2.9)
5.523 (dd, 10.6/9.8)
5.077 (t, 9.9)
3.941 (dd, 10.1/3.0)
5.706 (t, 2.8)
4.963 (dd, 10.6/2.7)
5.362 (dd, 10.6/9.7)
5.037 (t, 9.8)
4.042 (d, 10.1/2.8)
5.514 (t, 2.8)
4.894 (dd, 10.6/2.8)
5.353 (dd, 10.5/9.5)
5.157 (t, 9.7)
5.652 (dd, 10.5/9.8)
5.536 (t, 9.9)
5.186 (t, 10.0)
5.274 (t, 10.0)
Glucosamine
1
5.224 (d, 3.5)
5.169 (d, 3.9)
4.665 (d, 8.1)
4.675 (d, 8.0)
2
3
4
5
6A
6B
4.142 (ddd, 9.7/9.6/3.5)
5.243 (t, 9.6)
5.151 (t, 9.7)
4.099 (ddd, 9.9/4.5/2.5)
4.297 (dd, 12.5/4.5)
4.237 (dd, 12.5/2.5)
4.086 (ddd, 10.0/10.1/3.9)
5.293 (t, 9.9)
5.146 (t, 9.8)
4.209 (ddd, 9.8/4.7/1.9)
4.348 (dd, 12.1/4.7)
4.143 (dd, 12.1/1.9)
3.884 (ddd, 10.2/9.0/8.1)
5.278 (dd, 10.2/9.2)
5.147 (t, 9.6)
3.793 (dt, 9.9/3.7,3.7)
4.241 (d, 3.7)
4.241 (d, 3.7)
3.761 (ddd, 10.1/9.1/8.0)
5.133 (dd, 10.1,9.2)
5.327 (dd, 10.3,9.3)
3.813 (ddd, 10.0/4.2/2.3)
4.412 (dd, 12.5/4.2)
4.142 (dd, 12.5/2.3)
2,4-Dinitrophenyl
3
5
6
9.106 (d, 2.7)
8.265 (dd, 9.6/2.6)
7.097 (d, 9.6)
9.157 (d, 2.6)
8.251 (dd, 9.5/2.6)
7.010 (d, 9.5)
9.113 (d, 2.6)
8.194 (dd, 9.6/2.6)
7.092 (d, 9.6)
9.076 (d, 2.7)
8.263 (dd, 9.6/2.7)
7.155 (d, 9.6)
NH
8.571 (d, 9.7)
8.635 (d, 10.0)
8.496 (d, 9.0)
8.341 (d, 9.1)
CH3
2.181, 2.139
2.120, 2.052
2.025, 2.015
1.973, 1.798
2.396, 2.154
2.069, 2.038
2.007, 1.953
1.786, 1.549
2.207, 2.152
2.051, 1.992
1.975, 1.919
1.919, 1.756
2.110, 2.105
2.049, 2.014
1.973, 1.935
1.908, 1.445
Table 6. 1H Chemical shifts (d, ppm), multiplicities and 1H–1H coupling constants (J, Hz) for compounds 13–16 at 25 ꢁC in D2O
13
15
14
16
Inositol
10
3.584 (dd, 10.1/2.8)
4.236 (t,2.8)
3.491 (dd, 10.0/2.8)
3.643 (dd, 9.9/9.2)
3.296 (t, 9.3)
3.586 (dd, 9.9/2.8)
4.188 (t, 2.8)
3.515 (dd, 10.0/2.8)
3.614 (dd, 9.9/9.2)
3.286 (t, 9.3)
3.636 (dd, 10.0/2.8)
4.281 (t, 2.8)
3.515 (dd, 10.0/2.8)
3.608 (dd, 10.0/9.2)
3.278 (t, 9.2)
3.862 (dd, 10.1/2.6)
4.236 (t, 2.6)
3.495 (dd, 10.0,2.6)
3.640 (dd, 10.0/9.3)
3.291 (t, 9.3)
20
30
40
50
60
Glucosamine
3.707 (dd, 10.0/9.2)
3.755 (dd, 10.0/9.4)
3.743 (dd, 10.0/9.2)
3.734 (dd, 10.1/9.3)
1
2
3
4
5
6A
6B
5.031 (d, 3.60)
5.168 (d, 3.8)
4.551 (d, 8.3)
2.678 (t, 8.2)
3.31–3.42
3.31–3.42
3.31–3.42
4.872 (d, 8.3)
3.132 (dd, 10.6/8.3)
3.68a
2.755 (dd, 10.2/3.6)
3.637 (dd, 10.0/9.3)
3.414 (dd, 10.0/9.1)
3.963 (ddd, 10.0/4.7/2.6)
3.813 (dd, 12.3/2.6)
3.752 (dd, 12.3/4.7)
2.854 (dd, 10.3/3.8)
3.674 (dd, 10.2/9.0)
3.389 (t, 9.4)
3.832 (ddd, 9.4/6.1/2.1)
3.850 (dd, 12.7/2.1)
3.738 (dd, 12.7/6.1)
3.47a,b
3.48a,b
3.901 (dd, 12.4/1.9)
3.74a
3.880 (dd, 12.3/2.0)
3.723 (dd, 12.3/5.2)
aValue obtained from the COSY spectrum.
bValue obtained from the HETCOR spectrum.

M. A. Jardine et al. / Bioorg. Med. Chem. 10 (2002) 875–881
2. Fahey, R. C. Adv. Expt. Med. Biol. 1977, 86A, 1.
881
nol (73 mL), acetone (45 mL), water (27 mL), and
Amberlite IRA400 (OH) resin (90 mL) was allowed to
stir at 25 ꢁC for 3 days. After filtration of the mixture,
the filtrate was concentrated under reduced pressure,
followed by lyophilisation to give the product (13)
(200 mg, 85%) as a white solid; [a]D +109ꢁ (c 1.0, H2O).
Found: MH+, 342.1409, C12H23O10N requires MH,
342.1400.
3. Fahey, R. C.; Sundquist, A. R. Adv. Enzymol. 1991, 64, 1.
4. Spies, H. S. C.; Steenkamp, D. J. Eur. J. Biochem. 1994,
224, 203.
5. (a) 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. (b) Newton,
G. L.; Arnold, K.; Price, M. S.; Sherril, C.; Delcardayre, S. B.;
Aharonowitz, Y.; Cohen, G.; Davies, J.; Fahey, R. C. J. Bac-
teriol. 1995, 178, 1990.
1D-1-O-(2-Amino-2-deoxy-ꢁ-D-glucopyranosyl)-myo-ino-
sitol (14). Repetition of the above procedure, using (10)
afforded product (14) (62%); as a white solid; [a]D
+0.17ꢁ (c 1.2, H2O). Found: MH+, 342.1410,
C12H23O10N requires MH, 342.1400.
6. Sakuda, S.; Zhou, Z.-Y.; Amada, Y. Biosci. Biotech. Bio-
chem. 1994, 58, 1347.
7. Misset-Smiths, M.; Van Ophem, P. W.; Sakuda, S.; Duine,
J. A. FEBS Lett. 1997, 409, 221.
8. Storz, G.; Tartaglia, L. A. J. Nutr. 1992, 122, 627.
10. Deretic, V.; Song, J.; Pagan-Ramos, E. Trends Microbiol.
1997, 5, 367.
11. Newton, G. L.; Unson, M. D.; Anderberg, S. J.; Aguilera,
J. A.; Oh, N. N.; delCardayre, S. B.; Av-Gay, Y.; Faheym,
R. C. Biochem. Biophys. Res. Commun. 1999, 255, 239.
12. Bornemann, C.; Jardine, M. A.; Spies, H. S. C.; Steen-
kamp, D. J. Biochem. J. 1997, 325, 623.
1L-1-O-(2-Amino-2-deoxy-ꢀ-D-glucopyranosyl)-myo-ino-
sitol (15). Repetition of the above procedure, using (11)
afforded the title product (15) (88%); as a white solid;
[a]D +73ꢁ (c 1.1, H2O). Found: MH+, 342.1385,
C12H23O10N requires MH, 342.1400.
13. Newton, G. L.; Av-Gay, Y.; Fahey, R. C. J. Bacteriol.
2000, 182, 6958.
14. Patel, M. P.; Blanchard, J. S. J. Am. Chem. Soc. 1998, 51,
11538.
1D-1-O-(2-Amino-2-deoxy-ꢁ-D-glucopyranosyl)-myo-ino-
sitol (16). Repetition of the above procedure, using (12)
afforded the title product (16) (90%); as a white solid;
[a]D ꢀ27ꢁ (c 0.45, H2O). Found: MH+, 342.1411,
C12H23O10N requires MH, 342.1400. Enzymatic forma-
tion of mycothiol, 1D-1-O-(2-[N-acetyl-l-cysteinyl]-
amido-2-deoxy-a-d-glucopyranosyl)-myo-inositol, has
been fully described in a previous publication.12
15. Cole, S. T.; Brosch, R.; Parkhill, J.; Garnier, T.; Churcher,
C.; Harris, D.; Gordon, S. V.; Eiglmeier, K.; Gas, S.; Barry,
C. E.; Tekaia, F.; Badcock, K.; Basham, D.; Brown, D.; Chil-
lingworth, T.; Connor, R.; Davies, R.; Devlin, K.; Feltwell, T.;
Gentles, S.; Hamlin, N.; Holroyd, S.; Hornsby, T.; Jagels, K.;
Krogh, A.; McLean, J.; Moule, S.; Murphy, L.; Oliver, K.;
Osborne, J.; Quail, M. A.; Rajandream, M.-A.; Rogers, J.;
Rutter, S.; Seeger, K.; Skelton, J.; Squares, R.; Squares, S.;
Sulston, J. E.; Taylor, K.; Whitehead, S.; Barrell, B. G. Nature
1998, 393, 537.
Acknowledgements
16. Garegg, P.; Iverson, T.; Johansson, R.; Lindberg, B. Car-
bohydr. Res. 1984, 130, 322.
17. Vacca, J. R.; deSolmes, S. J.; Huff, H. R.; Billington,
D. C.; Baker, R.; Kulagowski, J. J.; Mawer, I. M. Tetrahedron
1989, 45, 5679.
18. Lloyd, P. F.; Stacey, M. Tetrahedron 1960, 9, 116.
19. Patt, S. L.; Shoolery, J. N. J. Magn. Reson. 1982, 46, 535.
20. Bax, A.; Freeman, R.; Morris, G. A. J. Magn. Reson.
1981, 42, 164.
This study was supported by grants from the University
of Cape Town, the South African Medical Research
Council and the the GlaxoWellcome ActionTB initia-
tive. M. Anwar Jardine was the recipient of a Mellon
postdoctoral fellowship.
References and Notes
21. Wilde, J. A.; Bolton, P. H. J. Magn. Reson. 1984, 59, 343.
22. Shvets, V. I.; Klyashchitskii, B. A.; Stepanov, A. E.;
Evstigneeva, R. P. Tetrahedron 1973, 29, 331.
1. Fahey, R. C.; Brown, W. C.; Adams, W. B.; Worsham,
M. B. J. Bacteriol. 1978, 133, 1126.