A family of mycothiol analogues

Article abstract of DOI:10.1081/CAR-200059965

A thioglycoside aminotriol scaffold has been elaborated by acylation, reductive alkylation, sulfonation, phosphorylation, and other procedures to produce a library of 40 functionalized thioglycosides that superficially resemble the enzyme-binding portions

Full text of DOI:10.1081/CAR-200059965

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A Family of Mycothiol Analogues
Spencer Knapp a , Benjamin Amorelli a , Etzer Darout a , Christian C.
Ventocilla a , Lawrence M. Goldman a , Richard A. Huhn a & Ellen C.
Minnihan a
a Department of Chemistry & Chemical Biology , Rutgers—The State
University of New Jersey , Piscataway, New Jersey
Published online: 16 Aug 2006.
To cite this article: Spencer Knapp , Benjamin Amorelli , Etzer Darout , Christian C. Ventocilla ,
Lawrence M. Goldman , Richard A. Huhn & Ellen C. Minnihan (2005) A Family of Mycothiol Analogues,
Journal of Carbohydrate Chemistry, 24:2, 103-130, DOI: 10.1081/CAR-200059965
To link to this article: http://dx.doi.org/10.1081/CAR-200059965
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Journal of Carbohydrate Chemistry, 24:103–130, 2005
Copyright # Taylor & Francis, Inc.
ISSN: 0732-8303 print
DOI: 10.1081/CAR-200059965
A Family of Mycothiol
Analogues
Spencer Knapp, Benjamin Amorelli, Etzer Darout,
Christian C. Ventocilla, Lawrence M. Goldman,
Richard A. Huhn, and Ellen C. Minnihan
Department of Chemistry & Chemical Biology, Rutgers—The State University of New
Jersey, Piscataway, New Jersey
A thioglycoside aminotriol scaffold has been elaborated by acylation, reductive alky-
lation, sulfonation, phosphorylation, and other procedures to produce a library of 40
functionalized thioglycosides that superficially resemble the enzyme-binding portions
of the Mycobacterium tuberculosis detoxifier mycothiol and its metabolic congeners.
To the extent that these analogues mimic the transition states derived from substrates
of the mycothiol-associated enzymes, they might prove useful as inhibitors and, ulti-
mately, as drug leads.
Keywords D-glucosaminide, Metalloprotein, Ligase, Glycosyltransferase, Acetyl-
transferase, Deacetylase, Reductase, Amidase, Mycothiol, Thioglycoside
aminotriol scaffold
INTRODUCTION
Most actinomycetes, including Mycobacterium tuberculosis, biosynthesize and
maintain high levels of mycothiol (MSH, 1)[1] as chemical protection against
oxidative stress[2–6] and electrophilic agents.[7–11] The unusual three-
component structure of 1 arises from a biosynthetic sequence commencing
formally with myo-inositol and N-acetylglucosamine precursors (Sch. 1).[12]
A
glycosyl transferase,[13] designated MshA, couples two components, not yet
identified, to give GlcNAc-Ins (3), which in turn is deacetylated by a second
enzyme (MshB)[14] to the aminoglycoside GlcN-Ins (4). An ATP-dependent
ligase (MshC)[6,10,15] joins 4 with cysteine, and the resulting amide 5 is
acetylated in an acetyl-CoA mediated process catalyzed by an acetyltransferase
(MshD).[17,18]
Received January 7, 2005; accepted January 12, 2005.
Address correspondence to Spencer Knapp, Department of Chemistry & Chemical
Biology, Rutgers—The State University of New Jersey, 610 Taylor Road, Piscataway,
New Jersey 08854-8087. E-mail: knapp@rutchem.rutgers.edu
103

S. Knapp et al.
104
Scheme 1: Biosynthesis of mycothiol.
Several MSH-processing enzyme systems have also been studied (Sch. 2).
Oxidative dimerization of 1 gives the mycothiol disulfide 6, and enzyme
mediated or spontaneous coupling of 1 with alkylating or otherwise electrophi-
lic species (represented by “Eþ”) gives rise to the S-substituted MSH deriva-
tives 7. These metabolites are respectively processed by mycothiol disulfide
reductase, which converts 6 back to 1, and mycothiol conjugate amidase,
which cleaves the S-conjugated N-acetylcysteine unit 8 from 7 for exportation
from the cell. The formaldehyde adduct of MSH (9) is oxidized to the S-formyl
derivative 10 by a mycothiol-dependent formaldehyde dehydrogenase.[7,19]
Drug-resistant tuberculosis infection is rampant globally: an estimated 16
million people were ill with the disease in 2002, 1.8 million died, and the
numbers are increasing.[20] The mycothiol-associated pathways offer an
opportunity for counteracting resistance to current antitubercular drugs.
MSH-deficient mutants of M. smegmatis survive and grow under laboratory
conditions, suggesting that MSH may not be critical for protection against
endogenous oxidizing or electrophilic agents in some mycobacteria. However,
direct correlation was found between MSH depletion and enhanced sensitivity
to exogenous toxins and antibiotics, including erythromycin, azithromycin,

A Family of Mycothiol Analogues
105
Scheme 2: Mycothiol-processing enzymes.
vancomycin, penicillin G, rifamycin, and rifampin.[21–24] Developing inhibitors
of the nonmammalian MSH biosynthetic or processing enzymes (Schemes 1
and 2) may therefore constitute an important approach toward improving
tuberculosis treatments.[25]
In order to establish the substrate requirements for the mycothiol S-
conjugate amidase (Mca, Sch. 2), we earlier prepared and evaluated the
mycothiol S-cyclohexyl thioglycoside analogue 14 (Sch. 3).[26] The fact that
Scheme 3: An aminotriol scaffold for construction of MSH analogues.

S. Knapp et al.
106
the bimane derivatives of MSH and 14 were cleaved by Mca at comparable
rates (14200 vs. 7500 nmol min21 mg-protein21, respectively) indicates that
the inositol ring and linking oxygen atom of MSH contribute little to substrate
binding, and thus might be dispensed with in the design of inhibitors of Mca.[26]
Likewise, the inositol ring of MSH was also shown to be noncritical for
substrate binding by mycothiol disulfide reductase (Sch. 2).[2] As most of the
enzymes in Schemes 1 and 2 involve attaching, cleaving, or maintaining an
acyl group at the nitrogen of a 2-amino-2-deoxy-a-D-glucopyranoside, the thio-
glycoside aminotriol 13 seemed to be a promising starting point for the prep-
aration of inhibitors. Thioglycosides are generally more resistant to
enzymatic degradation than are O-glycosides,[27–30] and are often, as is the
case with 13, easier to prepare in anomerically pure form.[31,32] Furthermore,
by attaching N-substituents to 13, we have the opportunity to mimic structural
and charge features of possible tetrahedral intermediates such as 15 or their
associated transition states. Evidence has been presented that both MshB
and Mca are zinc-containing metalloproteins that might catalyze amidolysis
through such intermediates.[33–35]
CONVERSION OF THE SCAFFOLD TO MSH ANALOGUES
The scaffold aminotriol 13 was efficiently prepared in gram quantities by our
previously published route,[26] and coupling reactions of 13 with a variety of
carboxylic acids and other commercially available or readily synthesized
partners were carried out. In this way, a family of 40 mycothiol analogues
(“MA”) featuring a 2-amino-2-deoxy-1-thio-a-D-glucopyranoside core was
generated, as shown in Charts 1 and 2 (structures MA-1 through MA-40).
The details of the preparation of the analogues are given in the experimental
1
section, and their H and 13C NMR spectra are listed in Tables 1 and 2. All
compounds were stable to storage in the freezer for several months.
The coupling agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(EDCI) was used in DMF solution to join 13 (¼MA-1) with carboxylic acids
in 13 cases (MA-3, MA-4, MA-5, MA-6, MA-10, MA-11, MA-12, MA-13, MA-
14, MA-37, MA-38, MA-39, and MA-40). The acids are mostly commercially
available, but for a few analogues the acids were made by literature procedures
(MA-11, MA-12, and MA-38) or by additions to (E)-3-iodoacrylic acid (MA-13
and MA-14). The EDCI coupling of unprotected oxime carboxylic acids with
13 failed, but the coupling was successful with a DCC/HOBT procedure to
give MA-34, MA-35, and MA-36. Reductive alkylation and direct alkylation
were used for MA-18 and MA-19, respectively. Sulfonylation of 13 with the
appropriate sulfonyl chloride led to MA-21 and MA-22. Similarly, acylation
of 13 gave MA-20 and MA-32, and acetimidation led to MA-24. Phosphoryl-
ation gave MA-25, and phosphonation produced MA-26. Condensation of 13
with various isocyanates and isothiocyanates gave MA-27, MA-28, MA-29,

A Family of Mycothiol Analogues
107
Chart 1: Mycothiol analogues prepared from the aminotriol scaffold.
MA-30, and MA-31. Ester hydrolysis of 12 gave the acetamidotriol MA-2. The
remaining analogues were made from other family members by acylation
(MA-9), reduction (MA-15, MA-16, and MA-17), hydrolysis (MA-7), oximation
(MA-33), thionation (MA-23), and displacement (MA-8) reactions, respectively.
CHARACTERIZATION OF THE MSH ANALOGUES
The mycothiol analogues MA-1 through MA-40 were characterized by their
1H and 13C NMR spectra (Tables 1 and 2), by their ESI-mass spectra (see
experimental section), and by spectral comparisons within the group and
with similar compounds. For example, all members showed characteristic

S. Knapp et al.
108
Chart 2: Mycothiol analogues subject to cyclization.
NMR signals for the cyclohexyl 2-amino-2-deoxy-1-thio-a-D-glucopyranoside
core, with expected differences attributable to the N-substituent.[31] The
oximes MA-33, MA-34, MA-35, and MA-36 were formed as single imino stereo-
isomers that we assign as E based on analogy to similar compounds.[36]
The a-ketoamides MA-37, MA-38, MA-39, and MA-40 showed a varying
tendency to cyclize with O-3’ to give hemiacetals (Chart 2).[37] The benzoylfor-
mamide MA-39 is mostly (ꢀ80%) in the keto form MA-39a, as indicated by the
C55O resonance at 190.7 ppm. Its hemiacetal exists as an approximately equal
mixture of presumed anomers MA-39b and MA-39c, ꢀ10% each, according to
integration of the respective anomeric protons. No evidence for –OCH3 or –
OCD3 mixed acetals[37] was seen by ESI-MS. The pyruvamide MA-37 exists
as a mixture of keto and two presumed hemiacetal forms, MA-37a, MA-37b,
and MA-37c, respectively, in a ratio of approximately 40:30:30, based on inte-
gration of the respective anomeric protons at 5.49, 5.54, and 5.58 ppm. In
contrast, the 3-(S-benzylthio)-2-oxopropionamide MA-38 exists entirely in
the cyclized form MA-38b (no keto carbon). The stereochemistry at the
acetal carbon C-2’’ is tentatively assigned as (S), with axial hydroxyl, based
on the anomeric effect. The 3-phenyl-2-oxopropionamide MA-40 also cyclizes
completely to the hemiacetal MA-40b (no keto carbon, anomeric proton at
5.82 ppm, acetal carbon at 96.9 ppm). The stereochemistry is again assumed
to be mostly (R) at C-2’’ (with axial hydroxyl), although a trace amount of
another isomer with anomeric proton at 5.84 ppm is present. Unlike MA-38,
however, MA-40 is accompanied by a single product of dehydration, alkene
MA-40c, to varying extent, commonly 50% or more. The alkene proton at
6.60 ppm[38] and the alkene carbon at 113.9 ppm are diagnostic. Stereo-
chemistry of MA-40c is assigned as (Z) based on the expected lower steric inter-
actions between the phenyl ring and the amide carbonyl oxygen. Dehydration
of MA-40b might be favored relative to MA-38b because the C55C forms in
conjugation with phenyl in the former case. In contrast to the ketoamides,

Table 1: 1H NMR data for mycothiol analogues.a
Cpd
H-10
H-20
H-50
H-60a
H-60b
H-30
H-40
MA-2
MA-3
MA-4
MA-5
5.53, d; 5.2
5.57, d; 5.3
5.49, d; 5.4
5.48, d; 5.1
4.01, dd; 5.6,
11.2
4.10, dd; 5.2,
11.0
4.05, dd; 5.1,
10.5
4.07, dd; 5.4,
11.1
3.97, ddd; 2.0,
5.2, 9.6
3.99, ddd; 2.5,
5.0, 9.7
3.98, ddd; 2.7,
5.4, 9.9
3.97, ddd; 2.1,
5.1, 9.6
3.80, dd; 2.4,
12.0
3.82, dd; 2.5,
12.0
3.80, dd; 2.4,
12.3
3.80, dd; 2.7,
12.0
3.72, dd; 5.2,
11.6
3.73, dd; 5.1,
12.1
3.73, dd; 5.4,
12.3
3.72, dd; 5.1,
12.0
3.54, dd; 8.8,
10.8
3.60, dd; 8.6,
11.0
3.53, dd; 8.7,
10.8
3.55, dd; 9.0,
11.1
3.35, dd; 8.8,
9.6
3.38, app t; 9.4
3.36, app t; 9.6
3.36, dd; 9.0,
9.6
7.92, d; 7.2
(NH)
MA-6
5.52, d; 5.2
5.50, d; 5.4
5.51, d; 5.4
5.49, d; 5.4
5.59, d; 5.4
5.54, d; 5.2
5.59, d; 5.2
5.70, d; 5.5
5.53, d; 5.7
5.54, d; 5.4
4.04, dd; 5.2,
10.8
4.08, dd; 5.1,
10.5
4.07, dd; 5.4,
11.1
4.06, dd; 5.7,
11.1
4.27, dd; 5.4,
10.5
4.05, dd; 5.2,
11.2
4.25, dd; 4.8,
10.8
4.07, dd; 5.4,
11.1
3.97, ddd; 2.4,
5.2, 9.6
4.00–3.95, m
3.80, dd; 2.4,
12.0
3.81, dd; 2.4,
12.0
3.81, dd; 2.7,
12.0
3.80, dd; 2.7,
12.3
3.84, dd; 2.7,
12.0
3.80, dd; 2.8,
12.4
3.83, dd; 2.4,
12.0
3.79, dd; 2.4,
12.0
3.73, dd; 5.2,
12.0
3.73, dd; 5.4,
12.3
3.73, dd; 4.8,
12.0
3.72, dd; 5.4,
12.3
3.77, dd; 4.8,
11.7
3.72, dd; 5.2,
12.0
3.76, dd; 5.2,
12.0
3.74, dd; 4.5,
12.0
3.55, dd; 8.8,
10.8
3.53, dd; 8.7,
10.8
3.55, dd; 8.7,
11.1
3.54, dd; 8.7,
11.1
3.65, dd; 8.7,
10.8
3.57, dd; 8.4,
10.8
3.62, dd; 8.8,
10.8
3.54, dd; 8.4,
10.8
3.37, dd; 8.8,
9.6
3.38, app t; 9.9
MA-7
MA-8
3.99, ddd; 2.4,
4.8, 9.9
3.98, ddd; 2.4,
5.1, 9.9
4.03, ddd; 2.7,
5.1, 9.6
3.98, ddd; 2.4,
5.6, 10.0
4.01, ddd; 2.8,
5.2, 10.0
3.96, ddd; 3.0,
5.7, 9.9
3.97, ddd; 2.1,
5.4, 9.3
3.99, ddd; 2.4,
4.8, 9.3
3.40–3.34, m
3.36, app t; 9.9
3.45, app t; 9.6
MA-9
MA-10
MA-11
MA-12
MA-13
MA-14
MA-15
3.36, dd; 8.8,
9.6
3.43, app t; 9.6
3.40, app t; 9.6
4.01, dd; 5.4,
11.1
2.87, dd; 5.1,
10.2
3.80, dd; 2.7,
12.0
3.82, dd; 2.4,
11.7
3.72, dd; 5.4,
12.0
3.74, dd; 5.4,
12.0
3.54, dd; 8.4,
10.5
3.45, app t;
8.7
3.38, dd; 9.0,
9.6
under MeOH
(continued)

Table 1: Continued
Cpd
H-10
H-20
H-50
H-60a
H-60b
H-30
H-40
MA-16
MA-17
MA-18
MA-19
MA-20
MA-21
MA-22
MA-23
MA-24
MA-25
MA-26
MA-27
MA-28
MA-29
MA-30
MA-31
5.54, d; 4.8
5.53, d; 5.1
5.52, d; 4.8
5.52, d; 5.1
5.54, d; 5.6
5.36, d; 5.1
5.42, d; 4.5
5.94, d; 5.4
5.53, d; 5.4
5.34, d; 4.5
5.44, d; 5.2
5.79, d; 5.4
5.51, d; 4.8
5.73, d; 4.8
5.76, d; 5.1
5.49, d; 5.1
3.10–2.99, m
4.00–3.91, m
3.79, dd; 2.7,
12.3
3.80, dd; 2.4,
11.7
3.79, dd; 2.4,
11.6
3.93–3.86, m
3.73, dd; 5.4,
12.3
3.72, dd; 4.8,
11.7
3.73, dd; 5.2,
11.6
3.86–3.73, m
3.42–3.30, m
3.75–3.57, m
3.42–3.30, m
2.86, dd; 5.1,
10.2
2.95, dd; 5.2,
10.4
3.97, ddd; 2.4,
5.4, 9.3
3.96, ddd; 2.8,
5.2, 9.6
3.41, dd; 9.0,
9.9
3.33, app t; 9.6
3.46, dd; 8.8,
10.0
3.86–3.73, m
3.01–2.91, m
4.04–3.96, m
3.45, dd; 8.1,
9.9
3.39, dd; 8.8,
9.6
3.37, dd; 8.1,
9.6
under MeOH
4.06, dd; 5.2,
10.8
3.51, dd; 5.4,
10.5
3.98, ddd; 2.4,
5.2, 10.0
3.96, ddd; 2.7,
4.8, 9.9
3.96, ddd; 2.4,
4.8, 9.6
3.99, ddd; 2.4,
4.8, 9.9
3.99, br dt;
3.6, 9.6
3.96, ddd; 2.4,
4.8, 9.3
4.00, ddd; 2.4,
4.8, 10.0
3.95, ddd; 2.4,
4.8, 9.6
3.97, ddd; 2.4,
5.2, 9.6
3.96, ddd; 2.4,
4.8, 9.6
3.95, ddd; 2.4,
4.8, 9.6
3.95, ddd, 2.7,
5.1, 9.3
3.81, dd; 2.4,
12.0
3.79, dd; 2.4,
11.7
3.77, dd; 2.4,
11.7
3.81, dd; 3.0,
12.3
3.74, dd; 5.2,
12.0
3.72, dd; 5.1,
12.3
3.70, dd; 5.1,
12.0
3.83–3.71, m
3.62, dd; 8.8,
10.8
3.44, dd; 8.1,
10.5
3.54–3.42, m
3.54–3.42, m
4.59, dd; 5.4,
11.1
3.84, dd; 5.1,
10.5
3.83–3.71, m
3.39, dd; 8.7,
9.9
3.40, dd; 8.7,
9.6
3.79, d; 3.6
3.79, d; 3.6
3.68, dd; 8.4,
10.2
3.45–3.30, m
3.45–3.30, m
3.78, dd; 2.7,
12.0
3.81, dd; 2.4,
12.4
3.81, dd; 2.4,
12.0
3.80, dd; 2.4,
11.7
3.80, dd; 2.4,
11.6
3.81, dd; 2.7,
12.0
3.80, dd; 2.4,
12.0
3.71, dd; 5.1,
12.0
3.77–3.67, m
3.45–3.30, m
3.77–3.70, m
3.42–3.35, m
3.38, app t; 9.6
3.39, app t; 9.9
4.57, dd; 4.8,
10.5
3.92, dd; 5.2,
10.4
4.57–4.48, br
m
4.59–4.50, m
3.73, dd; 5.4,
12.0
3.72, dd; 5.1,
11.7
3.73, dd; 5.2,
12.0
3.73, dd; 5.4,
12.3
3.51, dd; 8.7,
10.8
3.43, app t;
8.8, 10.4
3.59–3.49, br
m
3.51, br t, 9.0,
10.5
3.37, app t; 8.8,
9.6
3.39, app t;
10.0
3.38, app t; 9.9
3.89, dd; 5.1,
10.2
3.72, dd; 5.1,
12.0
3.45–3.27, m
3.45–3.27, m

MA-32
MA-33
5.48, d; 5.4
5.70, d; 5.4
5.58, d, 5.4
5.47, d; 5.6
3.79–3.72, m
3.95, ddd; 2.4,
5.1, 9.9
4.03, ddd; 2.7,
5.1, 9.9
3.79, dd; 2.7,
12.0
3.83, dd; 2.4,
12.0
3.71, dd; 5.1,
12.0
3.75, dd; 5.1,
12.0
3.46, app t;
8.7
3.59, dd; 8.7,
10.8
3.34, app t; 9.6
4.26, dd; 5.7,
11.4
4.14, dd; 5.1,
10.8
4.07, dd; 5.2,
10.8
7.47, d; 8.4
(NH)
4.06, ddd; 5.6,
8.0, 10.8
7.45, d; 8.0
(NH)
4.06, dd; 5.1,
10.8
3.42, dd; 9.0,
9.9
MA-34
MA-35
MA-36
3.94, ddd; 2.4,
5.2, 9.6
3.79, dd; 2.4,
12.0
3.72, dd; 5.2,
12.0
3.49, dd; 8.8,
10.4
3.37, app t; 9.6
3.36, app t; 9.6
3.36, app t; 9.6
5.46, d; 5.2
5.47, d; 5.1
3.93, ddd; 2.4,
5.2, 9.6
3.81–3.69, m
3.81–3.69, m
3.48, dd; 8.8,
10.8
3.93, ddd; 2.4,
4.8, 9.6
3.79, dd; 2.4,
12.0
3.72, dd; 5.1,
12.0
3.49, dd; 9.0,
10.5
7.47, d; 8.4
(NH)
MA-37a
MA-37b
5.49, d; 5.1
5.54, d; 5.4
4.05–3.95, m
4.05–3.95, m
3.81, dd; 2.4,
12.3
3.74, dd; 4.8,
12.3
3.73, dd; 5.1,
12.0
3.58, app t;
9.0
3.52, dd; 8.4,
11.1
3.39, app t; 9.9
3.38, app t; 9.6
MA-37c
MA-38b
5.58, d; 5.1
5.86, d; 5.6
3.88–3.76, m
4.10–4.02, m
3.68, dd; 2.4,
14.0
3.88–3.76, m
4.41, dd; 9.2,
11.6
4.10–4.02, m
MA-39
5.69, d; 5.1
4.19, dd; 5.4,
11.1
4.02, ddd; 2.4,
4.8, 9.6
3.83, dd; 2.7,
12.3
3.76, dd; 5.1,
12.3
3.63, dd; 8.7,
11.7
3.42, dd; 9.3,
9.9
MA-39a
MA-39b
MA-40b
5.45, d; 5.1
5.37, d; 5.1
5.82, d; 5.6
3.49–3.42, m
4.08–3.97, m
4.08–3.97, m
3.88–3.78, m
3.90–3.77, m
3.88–3.78, m
4.28, dd; 9.2,
11.6
4.42, app t;
9.0
3.90–3.77, m
MA-40c
5.70, d; 5.4
3.58, dd; 5.1,
9.9
4.05, dt; 3.9,
9.9
3.72, app t; 9.3
(continued)

Table 1: Continued
Cpd
H-1
H-2,3,4,5,6
H-100
H-200
H-300
H-400
Ar-H0s
MA-2
MA-3
2.86–2.78, m
2.90–2.76, m
2.01–1.26, m
2.04–1.20, m
1.97, s
6.35, dd; 9.0,
17.0
trans: 6.23,
dd; 2.8, 17.0
cis: 5.68, dd;
2.8, 9.0
MA-4
MA-5
MA-6
2.89–2.78, m
2.84–2.79, m
2.88–2.80, m
2.04–1.29, m
2.05–1.29, m
2.20–1.25, m
3.92, 3.85,
ABq; 17.1
4.61, 4.55,
ABq; 15.0
4.11, 4.10,
ABq; 13.6
4.00, s
3.40–3.34, m
3.98, 3.92,
ABq, 17.7
2.01, s
2.14, s
MA-7
MA-8
MA-9
2.88–2.79, m
2.84–2.79, m
2.84–2.78, m
2.01–1.25, m
2.15–1.29, m
2.01–1.26, m
4.13, s
MA-10
2.88–2.80, m
2.00–1.22, m
8.65, br d; 4.8
8.11, d; 7.8
7.97, td; 1.5, 7.5
7.57, ddd; 1.5,
5.1, 7.8
MA-11
MA-12
MA-13
MA-14
MA-15
2.83–2.79, m
2.84–2.81, m
2.81–2.72, m
2.86–2.70, m
2.90–2.76, m
1.99–1.24, m
2.10–1.25, m
2.00–1.25, m
1.82–1.29, m
2.14–1.30, m
7.08, d; 14.8
7.72, d; 14.8
9.03, d; 2.0
7.38–7.22, m
7.32–7.21, m
7.54, d; 15.0
2.97, d; 6.9
1.15, t; 6.9
6.01, d; 15.0
4.15, t; 6.9
4.05, s
3.83, s
2.90–2.76, m
2.61, dq; 7.2,
11.1
MA-16
2.86–2.82, m
2.13–1.29, m
2.86–2.82, m
3.10–2.99, m
3.10–2.99, m
1.32, t; 7.2
1.92, s
(2OCOCH3)
2.81–2.76, m

MA-17
MA-18
MA-19
2.92–2.83, m
2.93–2.87, m
3.01–2.91, m
2.09–1.23, m
2.05–1.30, m
2.05–1.26, m
2.92–2.83, m
2.70, ddd; 4.5,
7.2, 11.7
2.74, ddd; 6.4,
8.0, 14.4
2.93–2.87, m
3.67, dd; 3.9,
11.4
3.75–3.57, m
1.83–1.75, m
4.31–4.21, m
2.49, t; 7.2
3.73, s
7.34–7.20, m
4.04–3.96, m
2.37, s
(-OCNCH3)
3.56, dd; 5.4,
11.4
MA-20
MA-21
MA-22
2.88–2.80, m
2.91–2.83, m
2.80–2.72, m
2.20–1.26, m
2.10–1.27, m
2.01–1.20, m
4.34, q; 6.8
1.36, t; 7.2
3.03, s
7.10, d; 15.3
7.45, d; 15.9
7.60–7.57, m
7.43–7.41, m
MA-23
MA-24
2.86–2.77, m
2.94–2.84, m
1.99–1.25, m
2.04–1.29, m
2.48, s
2.26, s
1.91, s
(2OCOCH3)
7.43–7.31, m
3.19, q; 7.2
(NCH2CH3)3
1.31, t; 6.8
MA-25
MA-26
2.90–2.84, m
2.94–2.85, m
1.97–1.15, m
2.08–1.29, m
5.18–5.03, m
3.30–3.10, m
1.77–1.56, m
1.77–1.56, m
0.93, t; 7.2
(NCH2CH3)3
7.34–7.24, m
MA-27
MA-28
2.86–2.79, m
2.86–2.79, m
2.15–1.22, m
2.01–1.25, m
4.66, d; 15.0
d; under HOD
4.38, 4.27,
ABq; 15.0
2.97, s
3.83–3.63, m
7.30–7.20, m
7.32–7.17, m
MA-29
MA-30
2.86–2.80, m
2.93–2.79, m
2.01–1.27, m
2.03–1.28, m
2.91, dd; 2.1,
6.9
2.83, dd; 6.9,
10.5
MA-31
MA-32
MA-33
2.86–2.79, m
2.88–2.78, m
2.96–2.86, m
2.20–1.22, m
2.04–1.22, m
2.09–1.29, m
3.45-3.27, m
4.09, q; 6.9
2.77, t; 7.2
1.24, t; 6.9
7.30–7.16, m
7.76–7.72, m
7.51–7.46, m
(continued)

Table 1: Continued
Cpd
H-1
H-2,3,4,5,6
H-100
H-200
H-300
H-400
Ar-H0s
7.42–7.32, m
7.27, d; 7.6
7.21, t; 7.2
7.14, t; 6.8
7.09, d; 8.4
6.64, d; 8.4
7.37, d; 2.1
7.09, dd; 2.1,
8.4
MA-34
2.80–2.72, m
1.96–1.21, m
3.96, 3.88,
ABq; 13.2
MA-35
MA-36
2.78–2.65, m
2.79–2.50, m
1.96–1.20, m
1.97–1.20, m
3.87, 3.76,
ABq; 13.2
3.86, 3.74,
ABq; 13.2
6.76, d; 8.1
MA-
2.84–2.80, m
2.01–1.28, m
2.40, s
37a,b,c
1.47, s
MA-38b
3.05–2.96, m
2.94–2.86, m
2.78–2.64, m
2.07–1.26, m
2.05–1.26, m
3.88–3.76, m
3.88–3.76, m
3.10, d; 13.6
7.46, d; 7.2
7.32–7.24, m
8.16–8.13, m
MA-
39a,b,c
7.71–7.64, m
7.53, t; 7.8
7.34–7.31, m
7.33–7.27, m
7.57, d; 7.2
7.36, t; 7.5
MA-40b
MA-40c
2.96–2.87, m
2.96–2.87, m
2.14–1.09, m
2.14–1.26, m
3.90–3.77, m
6.60, s
7.22, t; 7.2
aSpectra were taken at 400 or 300 MHz (1H) and 100 or 75 MHz (13C) in CD3OD solutions. Chemical shifts, reported in ppm downfield from TMS, are followed
by multiplicities and then coupling constants, in hertz. Assignments are tentative and are based on related compounds and on first-order coupling
constants.

Table 2: 13C NMR for mycothiol analogues.a
Cpd
C-10
C-30
C-40
C-50
C-60
C-20
C-1
C-2
C-3
C-4
C-100
C-200
22.55
C-300
C-400
Ar-C’s
MA-2
MA-3
MA-4
MA-5
MA-6
MA-7
MA-8
MA-9
MA-10
84.01 74.36 72.79 72.62 62.60 55.92 44.71 35.30, 27.09, 26.83 173.58
34.96 26.90
87.09 77.45 75.77 75.64 65.61 59.03 47.86 38.31, 30.06, 29.81 171.29
134.94
43.77
63.29
43.15
62.47
44.62
43.08
130.01
37.93 29.89
84.63 74.75 73.34 72.77 62.84 55.98 45.35 35.66, 27.33, 27.15 172.02
172.02 22.69
179.47 20.48
35.22 27.15
84.32 74.57 72.89 72.45 62.54 55.56 45.16 35.38, 27.02, 26.86 170.25
34.94 26.86
84.10 74.59 72.96 72.44 62.53 56.09 45.15 35.35, 27.03, 26.85 169.32
34.96 26.85
84.53 74.71 73.42 72.27 62.54 55.11 45.27 35.39, 27.04, 26.86 175.23
34.94 26.86
84.06 74.49 72.96 72.74 62.61 55.11 44.97 35.53, 27.24, 27.04 176.47
35.09 27.11
84.28 74.47 73.01 72.54 62.58 55.78 45.05 35.39, 27.08, 26.90 171.12
34.95 26.90
84.73 74.88 73.63 72.25 62.59 55.78 45.40 35.35, 26.99, 26.81 166.53
34.92 26.81
174.39 43.55
150.54, 149.80,
138.88, 127.98,
123.13
MA-11
MA-12
MA-13
MA-14
MA-15
MA-16
83.97 74.46 72.52 72.52 62.52 55.95 44.98 35.30, 27.07, 26.93 166.82
140.01
94.70
34.93 26.93
84.83 74.99 73.61 72.40 62.72 55.90 45.53 35.50, 27.16, 26.97 166.97
35.07 26.97
84.07 73.82 72.52 72.02 62.22 55.42 44.74 34.98, 26.74, 26.52 166.90
34.54 26.52
83.99 74.35 72.77 72.60 62.59 55.91 45.01 35.30, 27.08, 26.90 173.57
34.96 26.90
84.37 75.06 74.31 72.61 63.56 62.88 44.14 35.66, 27.39, 27.17 42.58
35.17 27.23
183.05, 155.68,
125.36
137.20, 129.61,
129.43, 128.26
139.58, 130.18,
129.59, 128.13
142.97
27.63
15.44
43.87
117.53 37.14
57.77
36.20
85.41 75.28 74.45 72.57 63.38 62.79 44.78 35.81, 27.39, 27.18 under
43.65
11.89
179.67
35.34 27.27
MeOH
(2OCOCH3)
23.98
(2OCOCH3)
(continued)

Table 2: Continued
Ar-C’s and
others
Cpd
C-10
C-30
C-40
C-50
C-60
C-20
C-1
C-2
C-3
C-4
C-100
C-200
62.83
C-300
C-400
MA-17
MA-18
MA-19
MA-20
MA-21
MA-22
MA-23
MA-24
84.57 75.13 74.40 72.57 63.59 62.14 44.41 35.70, 27.39, 27.20 under
35.19 27.28
MeoH
83.54 74.23 74.09 72.19 62.72 62.42 44.62 35.47, 27.04, 26.89 46.61
29.16
70.60
161.26
29.61
63.65
64.05
36.78
139.96, 129.99,
129.45, 127.94
170.14 12.50
(2 OCNCH3)
34.85 26.89
83.89 74.56 72.77 62.21 61.03 58.59 46.10 35.57, 27.11, 26.90 50.70
35.00 27.06
83.88 74.67 72.83 72.18 62.47 56.28 45.28 35.34, 26.99, 26.83 159.20
14.25
34.91 26.83
87.06 74.63 73.69 72.83 62.65 59.57 45.34 35.56, 27.22, 27.06 41.89
34.97 27.06
84.21 72.36 71.06 70.49 60.47 57.32 43.12 33.45, 25.08, 24.84 132.64
126.89
33.28
33.32
138.41, 129.53,
128.08, 127.36
32.78 24.86
81.78 74.71 72.74 72.74 62.68 61.58 45.12 35.39, 27.21, 26.94 203.04
35.25 27.04
81.83 74.73 72.79 72.79 62.72 61.62 45.17 35.42, 27.24, 26.96 161.49
35.27 27.06
179.67
(2OCOCH3)
23.98
(2OCOCH3)
140.53, 129.51,
129.31, 128.90,
128.81
MA-25
MA-26
87.07 74.31 74.31 72.32 62.50 57.81 45.09 35.38, 26.97, 26.76 69.49, d;
34.75 26.85
4.5
69.26,
d; 4.5
85.72 74.58 72.34 72.18 62.31 53.57 45.37 35.44, 27.04, 26.83 27.63, d; 25.42,
21.48
14.25
47.33
34.88 26.91
114.6
app t;
10.7
[N(CH2CH3)]2
9.68
[N(CH2CH3)]2
140.18, 129.62,
128.74, 128.38
141.21, 129.42,
128.16, 127.95
MA-27
MA-28
84.78 74.81 73.95 72.68 62.72 59.86 45.44 35.53, 27.21, 27.03 167.44
under
MeOH
44.59
35.26 27.03
85.59 74.50 73.84 72.56 62.62 56.17 45.03 35.41, 27.07, 26.89 160.62
35.02 26.89

MA-29
MA-30
MA-31
MA-32
MA-33
MA-34
MA-35
MA-36
84.66 74.63 73.60 72.53 62.59 59.71 45.25 35.40, 27.07, 26.89
35.05 26.89
84.67 74.64 73.80 72.61 62.62 59.57 45.28 35.40, 27.06, 26.89 167.32
46.65
36.26
37.46
15.05
140.54, 129.88,
129.50, 127.32
140.79, 129.80,
129.46, 127.24
35.09 26.89
85.66 74.50 73.90 72.65 62.68 56.11 45.03 35.43, 27.07, 26.91 160.66
42.59
35.00 26.91
84.77 74.35 73.04 72.35 62.56 57.15 44.82 36.36, 27.11, 26.94 156.32
61.95
34.96 26.94
84.39 74.54 72.68 72.62 62.62 56.05 45.23 35.56, 26.96, 26.87 167.19
154.09
152.76
153.28
152.48
133.49, 130.63,
129.44, 127.64
138.12, 130.11,
129.27, 127.20
156.79, 131.15,
128.77, 116.01
153.54, 134.32,
130.35, 130.30,
116.79, 110.32
34.85 26.96
84.63 74.83 73.40 72.20 62.57 55.70 45.38 35.32, 26.99, 26.82 165.67
29.79
28.87
28.55
34.93 26.82
84.60 74.82 73.37 72.18 62.56 55.69 45.39 35.31, 26.99, 26.77 165.79
34.92 26.82
84.40 74.73 73.24 72.07 62.49 55.67 45.31 35.27, 27.05, 26.84 165.27
34.91 26.84
MA-
37a,b,c
84.31, 74.71, 73.18 72.32, 62.55 55.83, 45.34, 35.40, 27.01, 26.84 171.49
186.08
26.04,
25.74,
24.52
84.22 74.63
72.15
55.67, 45.19, 35.05, 26.84
55.60 45.07 34.91,
34.90
MA-38b
84.40 81.41 74.55 68.67 61.90 60.88 45.17 35.40, 26.87, 26.70 159.64
35.29 26.87
83.96, 74.57, 72.87, 72.49, 62.55 55.85, 45.15, 35.41, 27.00, 26.83 167.01
98.80
34.65
32.19
139.22, 130.65,
129.35, 128.12
135.59, 134.54,
131.44, 129.73,
129.52, 129.51,
128.96, 128.89,
127.7, 127.61
MA-
39a,b,c
190.65
84.39, 74.73, 73.14, 72.33
84.26 74.66 73.13
55.64 45.33 34.98, 26.88,
34.75 26.91
MA-
40b,c
84.38, 81.64, 74.51, 68.58, 61.96, 60.75, 45.00, 35.39, 27.16, 26.74, 165.31,
82.79 81.06 73.98 68.52 61.87 54.55 43.78 35.19, 27.05, 26.05 163.09
96.86,
156.98
41.15,
113.87
136.72, 136.65,
130.52 129.78,
129.68, 129.58
128.30, 127.98
34.88, 26.94,
34.75 26.89
aAssignments of the carbons are tentative and based on analogous compounds. Data for carbons C-20, C-30, C-40, C-50, and C-60 are presented in
arbitrary order; the assignments may be interchanged.

S. Knapp et al.
118
the hydroximinoamides MA-33, MA-34, MA-35, and MA-36 do not cyclize
onto O-3’.
FUTURE BIOLOGICAL EVALUATION
Although only 40 analogues are described here, it is clear from the straightfor-
ward nature of the various coupling reactions that the same procedures (alky-
lation, amide coupling, condensation, etc.) could be used to generate many new
members if enzymatic or biological evaluation indicates the need. Such studies
are presently underway and will be reported as results warrant. At this date 27
of the mycothiol analogues have been screened for inhibition against M. tuber-
culosis by the Tuberculosis Antimicrobial Acquisition & Coordinating Facility
of the Southern Research Institute, and four of them show weak activity as
evaluated in an Alamar blue assay (Table 3).
EXPERIMENTAL SECTION
General
Melting points (mp) were determined with a Electrothermal mp apparatus
and are uncorrected. TLC was performed on Merck glass silica gel 60 plates;
column chromatography employed 230–400 mesh flash silica. NMR spectra
were recorded with a Varian 300- or 400-MHz spectrometer on CD3OD sol-
utions. Mass spectra were obtained with a Finnigan LCQDUO LC/MS/MS
spectrometer.
Procedure A—Typical Amide Formation
A solution of aminotriol[26] 13 ( ¼ MA-1, 0.10 mmol, azeotropically dried
with 3 ꢀ 2 mL of toluene) and the appropriate carboxylic acid (0.20 mmol)
in 1 mL of dimethylformamide was cooled to 08C and then treated with
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 0.21 mmol). The
Table 3: Inhibitory activity of mycothiol analogues against
M. tuberculosis.a
Compound
Inhibition (%)
MA-6
51
27
4
MA-18
MA-23
MA-28
12
aCompounds MA-3–MA-13, MA-15–MA-25, MA-37, and
MA-39 were evaluated against M. tuberculosis strain H37RV at a
concentration of 6.25 mg/mL using an Alamar blue assay.

A Family of Mycothiol Analogues
119
reaction mixture was allowed to warm to 238C over a 3-hr period, quenched
with
3 drops of water, concentrated, and then purified by column
chromatography.
Procedure B—Coupling Unprotected Hydroxyimino Acids
A solution of aminotriol 13 ( ¼ MA-1, 0.10 mmol, azeotropically dried with
3 ꢀ 2 mL of toluene), N-hydroxybenzotriazole (0.10 mmol), and hydroxyimino
acid (0.10 mmol) in 1 mL of dimethylformamide was cooled to 08C and then
treated with dicyclohexylcarbodiimide (DCC, 0.15 mmol). The mixture was
allowed to warm to 238C, and then was stirred for an additional 16 hr. The
white precipitate (dicyclohexylurea) was removed by filtration, the super-
natant was concentrated, and then the crude product was purified by column
chromatography.
Cyclohexyl 2-Acetamido-2-deoxy-1-thio-a-D-glucopyranoside (MA-2)
De-O-acetylation of 12 (783 mg, 1.75 mmol) with methanolic sodium
methoxide as for MA-7 gave the acetamidotriol MA-2: 515 mg, 92%, colorless
oil; Rf 0.40 (9 : 1 dichloromethane/methanol); ESI-MS m/z 342 MNaþ.
Cyclohexyl 2-Acrylamido-2-deoxy-1-thio-a-D-glucopyranoside (MA-3)
Commercial acrylic acid was coupled with MA-1 by procedure A: 23 mg,
72%, colorless oil; Rf 0.54 (3 : 3 : 14 methanol/ethyl acetate/dichloromethane);
ESI-MS m/z 354 MNaþ.
Cyclohexyl 2-(2-N-Acetylglycinamido)-2-deoxy-1-thio-a-D-glucopyranoside
(MA-4)
Commercial N-acetylglycine was coupled with MA-1 by procedure
A: 65 mg, 86%, colorless oil; Rf 0.30 (3 : 3 : 14 methanol/ethyl acetate/dichloro-
methane); ESI-MS m/z 399 MNaþ.
Cyclohexyl 2-(2-Acetoxyacetamido)-2-deoxy-1-thio-a-D-glucopyranoside
(MA-5)
Commercial acetoxyacetic acid was coupled with MA-1 by procedure
A: 33 mg, 81%, colorless oil; Rf 0.23 (1 : 9 methanol/dichloromethane);
ESI-MS m/z 400 MNaþ.
Cyclohexyl 2-(2-Chloroacetamido)-2-deoxy-1-thio-a-D-glucopyranoside (MA-6)
Commercial chloroacetic acid was coupled with MA-1 by procedure
A: 21 mg, 72%, white solid, mp 183–1858C; Rf 0.33 (1 : 9 methanol/dichloro-
methane); ESI-MS m/z 376 MNaþ.

S. Knapp et al.
120
Cyclohexyl 2-Deoxy-2-(2-hydroxyacetamido)-1-thio-a-D-glucopyranoside
(MA-7)
A solution of the acetoxyglycinamide MA-5 (27 mg, 0.072 mmol) in 0.5 mL
of methanol was treated at 08C with methanolic sodium methoxide (2 drops,
25% w/w solution in MeOH), stirred at 238C for 30 min, and then concentrated.
Purification by column chromatography with 1 : 9 methanol/dichloromethane
as the eluant afforded 22 mg (92%) of MA-7 as a colorless oil: Rf 0.85 (3 : 7
methanol/dichloromethane); ESI-MS m/z 358 MNaþ.
Cyclohexyl 2-Deoxy-2-glycinamido-1-thio-a-D-glucopyranoside (MA-8)
Aqueous ammonia (approximately 0.75 mL) was added dropwise over a
period of 6 hr to a solution of the chloroacetamide MA-6 (58 mg, 0.150 mmol)
in 1.5 mL of methanol, by which time the reaction was complete according to
TLC analysis. The product was purified by column chromatography with 1 : 4
methanol/dichloromethane as the eluant to afford 52 mg (95%) of MA-8 as a
colorless oil: Rf 0.15 (3 : 17 methanol/dichloromethane); ESI-MS m/z 335 MHþ.
Cyclohexyl 2-[2-N-(2-Chloroacetamido)glycinamido]-2-deoxy-1-thio-a-D-
glucopyranoside (MA-9)
MA-8 was coupled with chloroacetic acid by procedure A: 20 mg, 72%, color-
less oil; Rf 0.15 (1 : 4 methanol/dichloromethane); ESI-MS m/z 433 MNaþ.
Cyclohexyl 2-Deoxy-2-(2-picolinamido)-1-thio-a-D-glucopyranoside (MA-10)
Commercial 2-picolinic acid was coupled with MA-1 by procedure A: 28 mg,
82%, white solid, mp 179–181 8C; Rf 0.60 (1 : 19 methanol/dichloromethane);
ESI-MS m/z 405 MNaþ.
Cyclohexyl 2-Deoxy-2-(3-E-iodoacrylamido)-1-thio-a-D-glucopyranoside
(MA-11)
(E)-3-Iodoacrylic acid was prepared by the literature method[39] and then
was coupled with MA-1 by procedure A: 29 mg, 76%, white solid, mp
212–2148C; Rf 0.40 (3 : 17 methanol/dichloromethane); ESI-MS m/z 480 MNaþ.
Cyclohexyl 2-Deoxy-2-(4-thiazolamido)-1-thio-a-D-glucopyranoside (MA-12)
4-Thiazolecarboxylic acid was prepared by the literature method[40] and
then was coupled with MA-1 by procedure A: 25 mg, 83%, colorless oil; Rf
0.60 (3 : 17 methanol/dichloromethane); ESI-MS m/z 411 MNaþ.
3-(S-Benzylthio)acrylic acid and 3,3-Bis(S-benzylthio)propionic acid
A solution of 3-Iodoacrylic acid (500 mg, 2.53 mmol), and N, N-diisopropyl-
ethylamine (Hu¨nig’s base, 1.32 mL, 7.59 mmol) in 1.0 mL of THF was treated
with benzyl mercaptan (0.33 mL, 3.04 mmol). A white solid precipitated

A Family of Mycothiol Analogues
121
after 1.5 hr. The reaction mixture was stirred for an additional 1.5 hr and then
diluted with 100 mL of ethyl ether. The resulting solution was washed with
saturated aqueous sodium bicarbonate (3 ꢀ 50 mL), and the combined
organic layer was dried over sodium sulfate, filtered, and then concentrated
1
to a residue containing both acid products (1 : 1 mixture respectively by H
NMR analysis). The products were purified by chromatography with 1 : 9
ethyl acetate/dichloromethane as the eluant to provide 245 mg (80%) of 3-(S-
benzylthio)acrylic acid and 322 mg (80%) of 3,3-bis(S-benzylthio)propionic
acid as colorless oils: Rf 0.38 and 0.45, respectively (3 : 7 ethyl acetate/dichloro-
1
methane); H NMR for 3-(S-benzylthio)acrylic acid (300 MHz, CD3OD) 7.73
1
(d, J ¼ 15.0, 1H), 7.41–7.25 (m, 5 H), 5.81 (d, J ¼ 15.0, 1H), 4.11 (s, 2 H); H
NMR for 3,3-bis(S-benzylthio)propionic acid (300 MHz, CD3OD) 3.98 (t,
J ¼ 7.5, 1H), 3.84 (d, J ¼ 13.2, 2H), 3.78 (d, J ¼ 13.2, 2H), 2.72 (d, J ¼ 7.2, 2 H).
Cyclohexyl 2-[(3-E-(S-benzylthio)acrylamido]-2-deoxy-1-thio-a-D-glucopyrano-
side (MA-13)
3-(S-Benzylthio)acrylic acid (synthesized by the procedure above) was
coupled with MA-1 by procedure A: 16.4 mg, 64%, colorless oil; Rf 0.30 (1 : 4
methanol/dichloromethane); ESI-MS m/z 454 MHþ.
Cyclohexyl 2-[3,3-Bis(S-benzylthio)propionamido]-2-deoxy-1-thio-a-D-glucopy-
ranoside (MA-14)
3,3-Bis(S-benzylthio)propionic acid (synthesized by the procedure above)
was coupled with MA-1 by procedure A: 13.5 mg, 65%, colorless oil; Rf 0.15
(1 : 4 methanol/dichloromethane); ESI-MS m/z 474 (MNa-BnSH)þ.
Cyclohexyl 2-Deoxy-2-ethylamino-1-thio-a-D-glucopyranoside MA-15)
A solution of MA-2 (46 mg, 0.151 mmol) and lithium aluminum hydride
(40 mg, 1.05 mmol) in 2.0 mL of dry THF was heated at reflux for 2 d. The
reaction was concentrated to half volume, cooled to 08C, and then quenched
by dropwise addition of 1 mL of aqueous sodium sulfate. Methanol (2.0 mL)
was added and the solution was stirred for 6 hr, filtered, and then concentrated.
Chromatography with 3 : 3 : 7 methanol/ethyl acetate/dichloromethane as the
eluant afforded 27 mg (61%) of MA-15 as a colorless oil: Rf 0.35 (5 : 1 : 14
methanol/ethyl acetate/dichloromethane); ESI-MS m/z 306 MHþ.
Cyclohexyl 2-Deoxy-2-(2-ethylamino)ethylamino-1-thio-a-D-glucopyranoside,
bis(acidic acid) salt (MA-16)
Reduction of MA-4 (38 mg, 0.085 mmol) as described for MA-15 (procedure
directly above) followed by chromatography with 1 : 2 : 5 acetic acid/methanol/
dichloromethane as the eluant afforded 24 mg (63%) of the salt MA-16 as a
colorless oil: Rf 0.18 (3 : 2 methanol/dichloromethane); ESI-MS m/z 349 MHþ.

S. Knapp et al.
122
Cyclohexyl 2-Deoxy-2-(2-hydroxyethylamino)-1-thio-a-D-glucopyranoside
(MA-17)
Reduction of MA-5 (31 mg, 0.082 mmol) as described for MA-15 followed by
chromatography with 3 : 17 methanol/dichloromethane as the eluant afforded
15 mg (55%) of MA-17 as a colorless oil: Rf 0.20 (3 : 17 methanol/dichloro-
methane); ESI-MS m/z 322 MHþ.
Cyclohexyl 2-[3-(S-benzylthio)propylamino]-2-deoxy-1-thio-a-D-glucopyrano-
side (MA-18)
3-(S-Benzylthio)propionaldehyde[41] (46 mg, 0.260 mmol) and MA-1 (51 mg,
0.185 mmol) in dichloromethane (1.0 mL) and DMF (0.10 mL) was treated with
sodium triacetoxyborohydride (55 mg, 0.260 mmol). The solution became
cloudy within 15 min. The reaction mixture was stirred for 1.5 hr and then con-
centrated. Chromatography with 1 : 19 methanol/dichloromethane as the
eluant provided 64 mg (78%) of MA-18 as a colorless oil: Rf 0.44 (3 : 17
methanol/dichloromethane); ESI-MS m/z 442 MHþ.
Cyclohexyl 2-Deoxy-2-[(4-R-2-methyl-2-oxazolin-4-yl)methylamino]-1-thio-a-D-
glucopyranoside acetic acid salt (MA-19)
(R)-4-Hydroxymethyl-2-methyl-2-oxazoline was prepared by the literature
method,[42] and then converted to its O-methanesulfonyl derivative as follows.
Methanesulfonyl chloride (357 mL, 1.05 equiv) was added to a solution of (R)-
4-hydroxymethyl-2-methyl-2-oxazoline (504.8 mg, 4.39 mmol) in 8 mL of dichlo-
romethane at 08C, followed by triethylamine (673 mL, 1.1 equiv.). The solution
was allowed to slowly come to room temperature over a 2-hr period, and then
was concentrated under vacuum. The unstable crude mesylate was used for
the subsequent alkylation reaction without further purification. A solution of
crude (4-R-2-methyl-2-oxazolin-4-yl)methyl methanesulfonate (100 mg,
0.518 mmol), MA-1 (46 mg, 0.166 mmol), and potassium carbonate (30 mg,
0.217 mmol) in 2.0 mL of DMF was heated at 708C for 12 hr. The reaction
mixture was concentrated and then chromatographed with 1:1 methanol/
dichloromethane as the eluant to give 56 mg (78%) of MA-19 as a colorless
oil: Rf 0.10 (1 : 4 methanol/dichloromethane); ESI-MS m/z 375 MHþ.
Cyclohexyl 2-Deoxy-2-(2-ethoxy-2-oxoacetamido)-1-thio-a-D-glucopyranoside
(MA-20)
Commercial ethyl chlorooxoacetate (14 mL, 0.121 mmol) was added to a
solution of MA-1 (28 mg, 0.101 mmol) and Hu¨nig’s base (21 mL, 0.117 mmol)
in 1.0 mL of DMF at 08C. The reaction mixture was stirred at 238C for 12 hr
and then concentrated. Chromatography with 4 : 1 ethyl acetate/dichloro-
methane as the eluant provided 30 mg (79%) of 35 as a colorless white solid:

A Family of Mycothiol Analogues
123
mp 164–1658C; Rf 0.50 (1 : 9 methanol/dichloromethane); ESI-MS m/z
401 MNaþ.
Cyclohexyl 2-Deoxy-2-methanesulfonamido-1-thio-a-D-glucopyranoside
(MA-21)
Methanesulfonyl chloride (12 mL, 0.155 mmol) was added to a solution of
MA-1 (40 mg, 0.144 mmol) in 2.0 mL of dioxane at 08C. Triethylamine (22 mL,
0.157 mmol) was then added, and the solution was stirred for 2 hr at 238C. Con-
centration and then chromatography with 1 : 19 methanol/dichloromethane as
the eluant gave 39 mg (77%) of the sulfonamide MA-21 as a colorless oil: Rf 0.20
(1 : 9 methanol/dichloromethane); ESI-MS m/z 378 MNaþ.
Cyclohexyl 2-Deoxy-2-(2-phenylethenylsulfonamido)-1-thio-a-D-glucopyrano-
side (MA-22)
Commercial (E)-2-styrenesulfonyl chloride (19 mg, 0.095 mmol) was added
to a solution of MA-1 (22 mg, 0.079 mmol) and Hu¨nig’s base (17 mL, 0.095 mmol)
in 1.0 mL of 1 : 1 DMF/THF at 08C, whereupon the mixture turned from clear to
yellow. The reaction mixture was stirred at 238C for 4 hr, and then concen-
trated. Chromatography with 13 : 7 ethyl acetate/dichloromethane as the
eluant provided 28 mg (86%) of MA-22 as a colorless oil: Rf 0.83 (9 : 41
methanol/dichloromethane); ESI-MS m/z 466 MNaþ.
Cyclohexyl 2-Amino-2-N-thioacetamido-2-deoxy-1-thio-a-D-glucopyranoside
(MA-23)
Lawesson’s reagent (52 mg, 0.130 mmol) was added to a solution of 12
(68 mg, 0.153 mmol) in 2 mL of THF, and the suspension was stirred at
238C for 12 hr under an argon atmosphere. The reaction mixture was cooled
to 238C and then chromatographed directly with 3 : 7 ethyl acetate/dichloro-
methane as the eluant to afford 60 mg (100%) of the thioamide triacetate as a
yellow oil. This product was dissolved in 2.0 mL of methanol, and then was
treated with sodium methoxide (4 drops, 25% w/w solution in MeOH) under
an argon atmosphere. After 30 min the solution was concentrated and then
chromatographed with 1 : 19 methanol/dichloromethane as eluant to afford
44 mg (98%) of MA-23 as a light yellow oil: Rf 0.50 (1 : 9 methanol/dichloro-
methane); ESI-MS m/z 358 MNaþ.
Cyclohexyl 2-Acetamidino-2-deoxy-1-thio-a-D-glucopyranoside acetic acid salt
(MA-24)
A solution of MA-1 (30 mg, 0.108 mmol), ethylacetimidate hydrochloride
(14 mg, 0.113 mmol), and triethylamine (16 mL, 0.113 mmol) in 1.5 mL of 1 : 1
dioxane/methanol was stirred at 238C for 6 hr. The reaction mixture was con-
centrated and then chromatographed with 3 : 7 : 90 methanol/acetic
acid/dichloromethane as the eluant to give 31 mg (75%) of the acetate salt

S. Knapp et al.
124
MA-24 as a colorless oil: Rf 0.10 (1 : 4 methanol/dichloromethane); ESI-MS m/z
319 MHþ.
Cyclohexyl 2-Deoxy-2-(O,O0-dibenzylphosphoramido)-1-thio-a-D-glucopyrano-
side (MA-25)
Tetrabenzylpyrophosphate[43] (77 mg, 0.143 mmol) was added to a solution
of MA-1 (33 mg, 0.119 mmol) and Hu¨nig’s base (93 mL, 0.179 mmol) in 1.0 mL of
1 : 1 DMF/THF at 08C. The mixture was allowed to stir at 238C for 6 hr. Concen-
tration and chromatography with 17 : 3 ethyl acetate/dichloromethane as the
eluant provided 38 mg (60%) of MA-25 as a colorless oil: Rf 0.38 (1 : 9
methanol/dichloromethane); ESI-MS m/z 560 MNaþ.
Cyclohexyl 2-(1-butanephosphonamido)-2-deoxy-1-thio-a-D-glucopyranoside
triethylamine salt (MA-26)
Commercial n-butylphosphonic dichloride (45 mL, 0.320 mmol) was added
to a solution of MA-1 (59 mg, 0.213 mmol) and Hu¨nig’s base (78 mL,
0.447 mmol) in 1.0 mL of DMF at 08C. The mixture was allowed to stir at
238C for 4 hr. Concentration and then chromatography with 13 : 7 ethyl
acetate/dichloromethane as the eluant provided 29 mg (27%) of the triethyl-
amine salt MA-26 as a colorless oil: Rf 0.20 (3 : 2 : 15 methanol/triethyl-
amine/dichloromethane); NI-ESI-MS m/z 498 MH2.
Cyclohexyl 2-[N(3)-Benzylthioureido]-2-deoxy-1-thio-a-D-glucopyranoside
(MA-27)
Commercial benzylisothiocyanate (29 mL, 0.217 mmol) was added to a
solution of MA-1 (50 mg, 0.181 mmol) in 1.5 mL of 1 : 1 DMF/THF at 08C.
The reaction mixture was stirred at 238C for 2 hr, concentrated, and then chro-
matographed with 1 : 19 methanol/dichloromethane as the eluant to afford
65 mg (85%) of MA-27 as a white solid: mp 185–1878C; Rf 0.30 (1 : 9
methanol/dichloromethane); ESI-MS m/z 449 MNaþ.
Cyclohexyl 2-[N(3)-Benzylureido]-2-deoxy-1-thio-a-D-glucopyranoside
(MA-28)
Commercial benzylisocyanate (27 mL, 0.220 mmol) was added to a solution
of MA-1 (50 mg, 0.183 mmol) in 1.5 mL of 1 : 1 DMF/THF at 08C. The reaction
mixture was stirred at 238C for 2 hr, concentrated, and then chromatographed
with 1 : 9 methanol/dichloromethane as the eluant to afford 59 mg (79%) of
MA-28 as a white solid: mp 167–169.58C; Rf 0.20 (1 : 9 methanol/dichloro-
methane); ESI-MS m/z 433 MNaþ.

A Family of Mycothiol Analogues
125
Cyclohexyl 2-Deoxy-2-[N(3)-methylthioureido]-1-thio-a-D-glucopyranoside
(MA-29)
Commercial methylisothiocyanate (13 mL, 0.192 mmol) was added to a
solution of MA-1 (44 mg, 0.159 mmol) in 1.5 mL of 1 : 1 DMF/THF at 08C.
The reaction mixture was stirred at 238C for 2 hr, concentrated, and then chro-
matographed with 9 : 1 ethyl acetate/dichloromethane as the eluant to afford
41 mg (73%) of MA-29 as a colorless oil: Rf 0.26 (ethyl acetate); ESI-MS m/z
373 MNaþ.
Cyclohexyl 2-Deoxy-2-[N(3)-2-phenylethylthioureido]-1-thio-a-D-glucopyrano-
side (MA-30)
Commercial phenethylisothiocyanate (27 mL, 0.178 mmol) was added to a
solution of MA-1 (41 mg, 0.149 mmol) in 1.5 mL of 1 : 1 DMF/THF at 08C.
The reaction mixture was stirred at 238C for 2 hr, concentrated, and then
chromatographed with 7 : 3 ethyl acetate/dichloromethane as the eluant to
afford 46 mg (73%) of MA-30 as a colorless oil: Rf 0.70 (3 : 17 methanol/dichloro-
methane); ESI-MS m/z 425 MHþ.
Cyclohexyl 2-Deoxy-2-[N(3)-2-phenylethylureido]-1-thio-a-D-glucopyranoside
(MA-31)
Commercial phenethylisocyanate (24 mL, 0.176 mmol) was added to a
solution of MA-1 (41 mg, 0.149 mmol) in 1.5 mL of 1 : 1 DMF/THF at 08C.
The reaction mixture was stirred at 238C for 2 hr, concentrated, and then
chromatographed with ethyl acetate as the eluant to afford 59 mg (91%) of
MA-31 as a colorless oil: Rf 0.71 (3 : 17 methanol/dichloromethane); ESI-MS
m/z 447 MHþ.
Cyclohexyl 2-Deoxy-2-(ethoxyformamido)-1-thio-a-D-glucopyranoside (MA-32)
Ethyl chloroformate (20 mL, 0.212 mmol) was added to a solution of MA-1
(56 mg, 0.202 mmol) and Hu¨nig’s base (39 mL, 0.216 mmol) in 1.5 mL of 1 : 1
dioxane/DMF at 08C. The reaction mixture was stirred at 238C for 6 hr, concen-
trated, and then chromatographed with 4 : 1 ethyl acetate/dichloromethane as
the eluant to provide 54 mg (74%) of MA-32 as a white solid: mp 144–1468C; Rf
0.42 (ethyl acetate); ESI-MS m/z 372 MNaþ.
Cyclohexyl 2-Deoxy-2(2-hydroxyimino-2-phenylacetamido)-1-thio-a-D-gluco-
pyranoside (MA-33)
Hydroxylamine hydrochloride (36 mg, 0.059 mmol) was added to a solution
of MA-39 (20 mg, 0.049 mmol) in 0.75 mL of methanol at 08C. The solution was
allowed to warm to 238C over a 4-hr period, and then was concentrated.
Chromatography with ethyl acetate as the eluant provided 16 mg (76%) of

S. Knapp et al.
126
MA-33 as a colorless oil: Rf 0.22 (1 : 9 methanol/dichloromethane); ESI-MS m/z
447 MNaþ.
2-Hydroxyimino-3-phenylpropionic Acid
Phenylpyruvic acid (300 mg, 1.83 mmol) and sodium hydroxide (73 mg,
1.83 mmol) were dissolved in 4.6 mL of water at 08C. In a separate flask, hydro-
xylamine hydrochloride (127 mg, 1.83 mmol) and sodium bicarbonate (153 mg,
1.83 mmol) were dissolved in 3.7 mL of water at 08C. The hydroxylamine
solution was then added to the phenylpyruvate solution, and the resulting
solution was allowed to warm to 238C and was stirred for 6 h. During this
period the pH was maintained at 5–6 by dropwise addition of concentrated
aqueous hydrochloric acid. The reaction mixture was acidified to pH 2, extracted
with ethyl acetate (3 ꢀ 50 mL). The organic extract was dried over magnesium
sulfate, filtered, and then concentrated to afford 294 mg (90%) of 2-hydroxy-
imino-3-phenylpropionic acid as a white solid: 164–1658C (dec.); 1H NMR
(200 MHz, DMSO-d6) 7.31–7.17 (m, 5 H), 3.81 (s, 2 H), 3.32 (br s, 1 H); 13C
NMR (50 MHz, DMSO-d6) 165.2, 150.2, 136.8, 128.6, 128.4, 126.2, 29.9; NI-
ESI-MS m/z 178 MH2.
Cyclohexyl 2-Deoxy-2(2-hydroxyimino-3-phenylpropionamido)-1-thio-a-D-
glucopyranoside (MA-34)
2-Hydroxyimino-3-phenylpropionic acid (prepared by the procedure above)
was coupled with MA-1 by procedure B: 50 mg, 79%, white solid, mp 144–
1468C; Rf 0.33 (1 : 9 methanol/dichloromethane); ESI-MS m/z 461 MNaþ.
2-Hydroxyimino-3-(4-hydroxyphenyl)propionic Acid
Commercial (4-hydroxyphenyl)pyruvic acid (670 mg, 3.74 mmol) was con-
verted to its oxime as for 2-hydroxyimino-3-phenylpropionic acid to afford
725 mg (94%) of 2-hydroxyimino-3-(4-hydroxyphenyl)propionic acid as an off-
1
white solid: 170–1728C (dec.); H NMR (400 MHz, CD3OD) 7.10 (d, J ¼ 8.4,
2 H), 6.68 (d, J ¼ 8.4, 2 H), 3.82 (s, 2H); 13C NMR (50 MHz, CD3OD) 167.0,
156.8, 152.7, 131.0, 128.6, 116.1, 30.0; NI-ESI-MS m/z 194 MH2.
Cyclohexyl 2-Deoxy-2[2-hydroxyimino-3-(4-hydroxyphenyl)propionamido]-1-
thio-a-D-glucopyranoside (MA-35)
2-Hydroxyimino-3-(4-hydroxyphenyl)propionic acid (prepared by the pro-
cedure above) was coupled with MA-1 by procedure B: 56 mg, 76%, white
solid, mp 214–2168C; Rf 0.30 (3 : 17 methanol/dichloromethane); ESI-MS
m/z 477 MNaþ.
2-Hydroxyimino-3-(3-bromo-4-hydroxyphenyl)propionic Acid
(3-Bromo-4-hydroxyphenyl)pyruvic acid[44] (300 mg, 1.16 mmol) was con-
verted to its oxime by using the procedure described above for

A Family of Mycothiol Analogues
127
2-hydroxyimino-3-phenylpropionic acid, affording 265 mg (84%) of 2-hydroxy-
imino-3-(3-bromo-4-hydroxyphenyl)propionic acid as a white solid: 147–
1488C (dec.); 1H NMR (400 MHz, CD3OD) 7.37 (d, J ¼ 2.0, 1 H), 7.08 (dd,
J ¼ 2.0, 8.0, 1 H), 6.78 (d, J ¼ 8.0, 1 H), 3.80 (s, 2H); 13C NMR (100 MHz,
CD3OD) 166.7, 153.8, 152.0, 134.4, 130.4, 130.3, 117.0, 110.5, 29.7; NI-ESI-
MS m/z 273 and 275 MH2.
Cyclohexyl 2-Deoxy-2[2-hydroxyimino-3-(3-bromo-4-hydroxyphenyl)
propionamido]-1-thio-a-D-glucopyranoside (MA-36)
2-Hydroxyimino-3-(3-bromo-4-hydroxyphenyl)propionic acid (prepared by
the procedure above) was coupled with MA-1 by procedure B: 96 mg, 86%,
white solid, mp 218–2208C; Rf 0.42 (3 : 17 methanol/dichloromethane); ESI-
MS m/z 555 and 557 MNaþ.
Cyclohexyl 2-Deoxy-2-(2-oxopropionamido)-1-thio-a-D-glucopyranoside
(MA-37a, MA-37b/c)
Commercial pyruvic acid was coupled with MA-1 by procedure A: 41 mg,
82%, 2 : 3 mixture respectively by 1H NMR analysis, colorless oil; Rf 0.30
(1 : 9 methanol/dichloromethane); ESI-MS m/z 370 MNaþ.
Cyclohexyl 2-Deoxy-2-[3-(S-benzylthio)-2-oxopropionamido]-1-thio-a-D-gluco-
pyranoside (MA-38b)
3-(S-Benzylthio)pyruvic acid was prepared by the literature method[45] and
then was coupled with MA-1 by procedure A: 48 mg, 62%, completely cyclized
1
as one isomer by H NMR analysis, colorless oil; Rf 0.35 (1 : 4 ethyl acetate/
dichloromethane); ESI-MS m/z 474 (MNa-H2O)þ.
Cyclohexyl 2-Deoxy-2(2-oxo-2-phenylacetamido)-1-thio-a-D-glucopyranoside
(MA-39a, MA-39b, MA-39c)
Commercial benzoylformic acid was coupled with MA-1 by procedure A:
46 mg, 77%, 8 : 1 : 1 respectively by 1H NMR analysis, colorless oil; Rf 0.64
(3 : 17 methanol/dichloromethane); ESI-MS m/z 432 MNaþ.
Cyclohexyl 2-Deoxy-2(2-oxo-3-phenylpropionamido)-1-thio-a-D-glucopyrano-
side (MA-40b, MA-40c)
Commercial phenylpyruvic acid was coupled with MA-1 by procedure
1
A: 22 mg, 84%, 1 : 1 respectively by H NMR analysis, colorless oil; Rf 0.58
(4 : 1 ethyl acetate/dichloromethane); ESI-MS m/z 428 MNaþ.
ACKNOWLEDGMENT
We are grateful to NIH (AI055760) for financial support and UNCF-Merck for a
graduate fellowship to ED. CCV, LMG, and RAH are Rutgers University

S. Knapp et al.
128
Undergraduate Fellows and Rutgers College Honors Program awardees. We
thank David S. Myers and Silvia Gonzalez for their contributions early in
this project.
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