Synthesis of substrate analogues as potential inhibitors for Mycobacterium tuberculosis enzyme MshC

Article abstract of DOI:10.1016/j.carres.2017.10.014

Mycothiol cysteine ligase (MshC) is a key enzyme in the mycothiol (MSH) biosynthesis and a promising target for developing new anti-mycobacterial compounds. Herein, we report on the synthesis of substrate analogues, as potential inhibitors, for the MshC e

Full text of DOI:10.1016/j.carres.2017.10.014

Carbohydrate Research 453-454 (2017) 10e18
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Carbohydrate Research
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Synthesis of substrate analogues as potential inhibitors for
Mycobacterium tuberculosis enzyme MshC
Krishnakant Patel, Fengling Song, Peter R. Andreana*
Department of Chemistry and Biochemistry and School of Green Chemistry and Engineering, The University of Toledo, 2801 W. Bancroft Street, Toledo,
Ohio 43606, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Mycothiol cysteine ligase (MshC) is a key enzyme in the mycothiol (MSH) biosynthesis and a promising
target for developing new anti-mycobacterial compounds. Herein, we report on the synthesis of sub-
strate analogues, as potential inhibitors, for the MshC enzyme. The target molecules were synthesized
employing a Schmidt glycosylation strategy using an enantiomerically pure inositol acceptor and 2-
deoxy trichloroacetimidate glycosyl donors with glycosylation yields greater than 70% and overall
yields >5%. The inositol acceptor was obtained via chiral resolution of ( )-myo-inositol.
© 2017 Elsevier Ltd. All rights reserved.
Received 1 September 2017
Received in revised form
20 October 2017
Accepted 21 October 2017
Keywords:
Chiral resolution
Glucosamine inositol
Mycothiol
MshC
Substrate analogues
Tuberculosis
1. Introduction
are required to avert further complications from TB.
Low molecular weight thiols [3] such as coenzymeA (CoA),
Tuberculosis (TB), an airborne infectious disease caused by ba-
cillus Mycobacterium tuberculosis, is an epidemic disease and the
second largest cause of human death from an infection only after the
human immunodeficiency virus (HIV). As per the Global Tuberculosis
Report 2015 [1] an estimated 10 million new cases of TB were re-
ported, leading to some 1.5 million deaths worldwide, where
approximately 30% of those individuals were also infected with HIV.
To treat TB, isoniazid (isonicotinylhydrazide [INH]), rifampicin
(rifampin), ethambutol (EMB, E), amikacin are commonly used,
however, with the emergence of multidrug-resistance (MDR) and
extensively drug-resistant (XDR) strains, the remedy for tuberculosis
has become increasingly more difficult due to compromised im-
munity and compatibility issues, such as drug-drug interactions,
drug toxicities, high pill burden and the immune reconstitution
inflammatory syndrome (IRIS) amongst combination drug therapies
[2]. The demand for new drug targets is critical due to MDR and XDR
TB strains and therefore targeting important cellular processes, such
as cell wall synthesis, DNA and RNA replication, detoxification
process (removal of antibiotics which are toxic to bacterial cell) etc.,
glutathione (GSH), bacillithiol (BSH) and mycothiol (MSH) help in
regulating redox conditions in variety of eukaryotes, gram-positive
and gram-negative bacteria. Among these, mycothiol (MSH, 7) is a
major, low-molecular weight thiol found in most actinomycetes.
MSH is the functional equivalent of GSH, which facilitates the sur-
vival of the M. tuberculosis in an oxygen-rich environment by pro-
tecting it from oxidative stress [4,5] and antibiotics [6]. MSH
biosynthesis is a complex process involving 5 different enzymes as
shown in Fig. 1 [7e9]. In short, conjugation of UDP-GlcNAc (1) with
Ins-3-P (2) is catalyzed by the enzyme MshA [10] to form GlcNAc-
Ins-3-P (3), which upon dephosphorylation by MshA2 forms
GlcNAc-Ins (4). Deacetylation of the GlcNAc-Ins by MshB leads to
the formation of glucosamine inositol (GlcN-Ins, 5). The ATP-
dependent ligation of cysteine to GlcN-Ins is catalyzed by MshC
[11] to form Cys-GlcN-Ins (6). Finally, N-acetylation of cysteine is
carried out by the enzyme MshD to form AcCys-GlcN-Ins (MSH, 7).
Enzyme MSTconjugates MSH with the drug molecules to form drug
conjugates (MSH-R, 8) which are processed by enzyme Mca to
eliminate toxins such as alkylating agents, free radicals generated
by the biological processes of the cell and administered antibiotics
[12]. In the process of toxin elimination (8 /Cys-R/ elimination),
GlcN-Ins is regenerated and incorporated back into the MSH
* Corresponding author.
E-mail address: peter.andreana@utoledo.edu (P.R. Andreana).
https://doi.org/10.1016/j.carres.2017.10.014
0008-6215/© 2017 Elsevier Ltd. All rights reserved.

K. Patel et al. / Carbohydrate Research 453-454 (2017) 10e18
11
Pi
Eliminated
from the
bacterial cell
UDP GlcNAc
(1)
GlcNAc-Ins-3-P
(3)
GlcNAc-Ins
(4)
+
MshA2
MshA
Ins P
(2)
MshB
AcOH
Cys-R
MSH-R
(8)
OH
O
GlcN-Ins
ATP
(5)
HO
HN
HO
HO
Mca
MshD
X
+
+
Cys
AMP
MshC
MST
HO
PPi
AcHN
HS
RX
CH2OH
OH
O
HO
Cys-GlcN-Ins
(6)
AcCys-GlcN-Ins
MSH
O
,
(
7)
MSH,
(
7)
AcCoA CoA
Fig. 1. Mycothiol biosynthesis and metabolic pathway.
biosynthesis cycle (Fig. 1). Cysteine is found to be prone to auto-
oxidation. Because of this, high levels of intracellular cysteine are
toxic to the cell. MSH acts a cysteine reservoir [13] in order to keep
levels in check and important to note is that MSH oxidizes slower
than cysteine [14].
stage, the reacting substrates and coenzymes are held in place by
the amino acid residues W227, H55, T46, and D251. Gene knockout
studies [16,19] have revealed that MshC plays a pivotal role in MSH
biosynthesis in comparison to other enzymes in the mechanistic
sequence. Functions of the enzymes in the depicted sequence can
be performed by other similar enzymes in the bacterial cell, how-
ever and most importantly, the function of MshC is irreplaceable.
Therefore, understanding the enzyme structure is critical to
developing potential inhibitors. Along those lines, the first partial
crystal structure of MshC was solved by Blanchard et al., in 2008,
leading to a breakthrough in the organization of the active site [20].
However, in their attempt to obtain the crystal structure, the
enzyme was subjected to trypsin digestion which lead to some of
the amino acid residues being proteolyzed and hence only a partial
crystal structure was obtained. New molecules, such as substrate
analogues, are required to obtain a full-length crystal structure and
identify the missing amino acids residues in the sequence for a
better understanding of enzyme function.
Amongst all the enzymatic processes depicted in Fig. 1, the role
of the essential enzyme MshC, elucidated by Bornemann and co-
workers in 1997, remains to be a critical drug target [15,16]. Based
on sequence identification, it has been concluded that MshC shares
a close relation to class I cysteinyl-tRNA-synthetases and the
mechanism of MshC was proposed as Bi-Uni-Uni-Bi ping pong
using steady state kinetics [17] and positional isotope exchange
studies [18]. As shown in Fig. 2, in the first phase of the enzymatic
reaction, the nucleophilic attack of carboxylate of cysteine onto the
phosphate of ATP leads to the formation of the cysteineadenylate
intermediate releasing inorganic pyrophosphate (PPi). This cys-
teineadenylate intermediate is attacked by the amine of GlcN-Ins to
form Cys-GlcN-Ins and in the process releases AMP, which com-
pletes the second phase of the enzymatic reaction. During this
As the mechanism indicates, the enzymatic reaction involves
H
H55
N
OH
HO
GlcN-Ins
HO
HO
NH2
N
Mg2+
O
H
O
N
N
HO
H
O
N
O
O
CH2OH
OH
N
O
NH2
N
O
P
H
O
P
O
P
O
H
P
O
N
HO
N
N
O
O
P
T46
O
O
B
H2N
O
O
O
ATP
O
O
O
Mg2+
O
O
NH2
O
N
HS
O
O
H
HO
Zn2+
P
S
O
O
O
O
OH
HO
H
O
PPi
Cysteineadenylate
Cysteine
N
D251
O
W227
OH
NH2
N
HO
HO
HO
N
N
O
HO
H2N
O
P
BH
O
N
O
O
H
N
O
CH2OH
OH
O
HS
OH
HO
HO
Cys-GlcN-Ins
O
AMP
Fig. 2. Proposed enzyme mechanism of MshC.

12
K. Patel et al. / Carbohydrate Research 453-454 (2017) 10e18
two different substrates, which gives an extensive range of op-
portunities to design substrate analogues as potential inhibitors.
Screening of chemical libraries led to the identification of some
molecules exhibiting notable MshC inhibitory activity, however the
antibacterial activities were considered to be caused by their
interaction with other biological targets and not specifically against
MshC [19,21]. 50-O-[N-(L-cysteinyl)sulfamoyl]adenosine (CSA, 11)
(Fig. 2), an analogue for the cysteineadenylate intermediate of the
enzymatic reaction, was found to be an inhibitor of MshC. It was
further determined to be a competitive inhibitor when compared to
ATP (inhibition constant ~306 nM) and a non-competitive inhibitor
when compared to cysteine [17]. Other molecules that showed
inhibition were NTF1836 (12) [22] and dequalinium chloride (13)
[23], but they proved to be harmful to mammalian cells and hence
were not pursued further. With all considered to date, there has not
been a considerable effort towards designing substrate analogues
as inhibitors. Therefore, based on this information, we elected to
synthesize substrate-based analogues 2-deoxy-azido (GlcN3-Ins, 9)
and 2-deoxy-fluoro sugars (GlcF-Ins, 10) (Fig. 3). We rationalized
our choice of design based on the crystal structure obtained by
Blanchard et al. [20]. As per the crystal structure and the modeled
proteolyzed loop [20], it is evident that the glucosamine ring of
GlcN-Ins is positioned to interact with W227 and D86 as to allow
nucleophilic attack of the amine on the electrophilic center of the
cysteineadenylate intermediate (Fig. 2). Fluorine, being an elec-
tronegative atom, and azide being a bipolar and linear functional
group, can both form strong hydrogen bonding interactions with
amino acid residues [24e26]. Substituting the amine 2-deoxy po-
sition of GlcN-Ins with fluorine and azide is proposed to result in
eliminating nucleophilic attack and secondly form tight in-
teractions with histidine (H55) and aspartic acid (D99) residues in
the MshC pocket. These interactions may, in turn, allow the enzyme
to crystallize and assist in attaining the full-length crystal structure
[25]. Fluorine based glycosides have previously been used as in-
hibitors and transition state analogues for Mycobacterium tuber-
material myo-inositol, which was resolved into a pure, single dia-
stereomer before conjugating it with 2-deoxy glycosyl donors via
Schmidt glycosylation, yielding enzyme substrate GlcN-Ins and
proposed inhibitors GlcF-Ins and GlcN3-Ins.
2. Results and discussion
Our strategy to synthesize the desired molecules includes the
use of trichloroacetimidate (TCA) donor and an orthogonally pro-
tected acceptor to assist in a [1 þ 1] Schmidt glycosylation [30].
Since both the donor molecules were 2-deoxy-2-substituted
sugars, we expected to obtain a a-selective glycosylation product
owing to the anomeric effect in the absence of neighboring group
participation (NGP). The target molecules were a set of pseudo-
disaccharides, i.e., a combination of sugar and a polyhydroxy-
cyclohexane motif [31e34]. Achieving the target molecules was
challenging accrediting to the resolution and diastereomeric
intermediates.
2.1. Synthesis of glycosyl acceptor (18)
The synthesis of myo-inositol acceptor commenced by following
an established protocol by Schmidt et al. [35] with required mod-
ifications to facilitate our coupling strategy. ( )-Myo-inositol (14)
was protected using 1,1-dimethoxy cyclohexane under acidic con-
ditions to yield a mixture of di-O-cyclohexylidene derivatives
(Scheme 1). The undesired racemates were separated from the
mixture by crystallization removing the unwanted, differently
protected derivatives of myo-inositol and then this was followed by
column chromatography to afford the desired molecule 15. Under
refluxing conditions in toluene, 15 was then treated with tributyltin
oxide followed by addition of (1R)-(-)-menthyl chloroformate in
the presence of NaHCO3, resulting in the regioselective protected
product 16. Compound 16 was then subjected to benzylation under
unique silver oxide conditions. Since, under basic conditions, the
carbonate moieties are prone to migration to the adjacent available
hydroxyl groups, in order to prevent this, silver oxide was used
instead of sodium hydride [35,36]. Use of freshly prepared silver
oxide [37] is highly important in order to obtain complete con-
version of the starting material. Separation of the desired diaste-
reomer from the reaction mixture was achieved by precipitation
using 100% petroleum ether to afford protected D-myo-inositol 17
culosis GlmM and GlmU and Agrobacterium faecalis b-glucosidase
[24,27,28]. Azide based molecules have been widely used in
various commercial drugs such as Retrovir® and Azidocillin [29].
Having the target molecule design in mind, we began the syn-
theses and as expected it proved difficult due to one half of the
pseudo-disaccharide being an enantio-pure polyhydroxy-cyclo-
hexane moiety. The reaction sequence began with racemic starting
OH
HO
OH
O
OH
O
HO
N3
HO
F
HO
HO
HO
HO
HO
HO
O
HO
HO
HO
O
O
O
CH2OH
OH
CH2OH
OH
CH2OH
OH
H2N
HO
HO
HO
GlcN3-Ins 9
5
GlcN-Ins
GlcF-Ins
10
O
H2N
S
H
N
O
N
N
HS
Cl
Cl
S
O
CH3
N
NH2
O
O
N
N
O
N
O
8
N
O
N
NH
H2N
NH2
HO
Cl
OH
5'-O-(N-(L-Cysteinyl)-Sulfamoyl)
chloride
NTF1836
12
Dequalinium
13
Adenosine
(CSA)
11
Fig. 3. Substrate for MshC (5) and analogues (9, 10). Known inhibitors for MshC (11-13) [17,22,23].

K. Patel et al. / Carbohydrate Research 453-454 (2017) 10e18
13
O
OH
O
O
OH
OH
HO
HO
OH
OH
a
b
O
O
O
O
O
O
O
OH
14
HO
O
15
16
O
O
O
O
c
d
O
O
O
O
16
O
O
OH
BnO
BnO
O
17
18
(D-)
Scheme 1. Reagents and conditions: a) 1,1- dimethoxy cyclohexane, N,N-Dimethylformamide(DMF), p-TsOH, 95-100 ꢁC, 3 h, (23%); b) (i) (Bu3Sn)2O, toluene(Tol), reflux, 3 h (ii)
NaHCO3, (1R)-(-)-menthyl chloroformate, Tol, 0 ꢁC-RT, 24 h, (73%); c) (i) Ag2O, BnBr, MS-4 Å, anhydrous dichloromethane (DCM), reflux, 6 h, (ii) recrystallization in petroleum ether
(45%); d) K2CO3, isopropanol:methanol (IPA:MeOH, 1:1), reflux, 6 h, (97%).
(Scheme 1). Enantiopurity confirmation of 17 was achieved
characterized and confirmed using 1H-NMR. This compound was
then subjected to conditions of bromination generating a a-
employing optical rotation where obtained
a
values matched quite
nicely with those reported in the literature (obs. ½a D22
ꢀ
¼ -22.2ꢁ,
glycosyl bromide donor. Surprisingly, we observed the molecule to
be quite unstable so we elected to pursue the trichloroacetimidate
donor. Therefore, compound 21 was subjected to conditions for
anomeric deacetylation using ammonium acetate [39] in DMF to
afford hemiacetal 22. Under anhydrous conditions and in the
presence of K2CO3, compound 22 was then treated with tri-
chloroacetonitrile to yield the TCA donor (23) in 95% yield [40,41].
Glycosylation was conducted using standard glycosylation pro-
c ¼ 0.5, CHCl3; lit. ½a D22
ꢀ
¼ -25.0ꢁ, c ¼ 0.5, CHCl3). Treatment of the 6-
O-benzyl derivative 17 with K2CO3 to remove the auxillary (1R)-
(-)-menthyl in isopropanol:methanol (IPA:MeOH, 1:1) furnished
desired acceptor 18.
2.2. Synthesis of GlcF-Ins (10)
With our acceptor in hand, the synthesis of the desired glycosyl
donor was initiated using commercially available 3,4,6-tri-O-
acetyl-D-glucal (19) (Scheme 2). Compound 19 was subjected to
fluorination using Selectfluor® in N,N-dimethylformamide:water
(DMF:water ¼ 1:1) solvent mixture affording an inseparable
mixture of 2-fluoro axial/equatorial epimers. Difficulties in sepa-
ration were overcome by acetylating the anomeric hydroxyl group
upon which molecules 20 and 21 [38] were readily separated
via silica gel chromatography. The required epimer 21 was fully
cedures which selectively afforded the
a-selective product 24 in
78% yield (a:b
¼ 99:1). Initially we observed low yields on the
glycosylation step, however, after careful investigation we realized
that the cyclohexane ketal protecting groups on the acceptor, which
are sensitive to acidic conditions, were being cleaved by trime-
thylsilyltriflate (TMSOTf), which was used as the promoter in the
Schmidt glycosylation reaction. In order to prevent the ketal pro-
tecting groups from leaving, the reaction temperature was main-
tained at -20 ꢁC, lower than the usual temperature for these types
OAc
OAc
OAc
O
R
c
a
O
O
AcO
AcO
AcO
AcO
AcO
AcO
O
OR"
F
R'
CCl3
NH
19
23
20 R= F R'=
R"=Ac
,
H,
21 R=
22 R=
R'= F R"=Ac
,
H,
H,
b
R'= F
R"=OH
,
OH
O
HO
F
O
O
HO
HO
d
f
O
O
O
HO
23
18
O
CH2OH
OH
RO
O
CH2OAc
OAc
F
HO
10
=
99:1
AcO
α:β
24 R=
OBn
e
25 R=OH
Scheme 2. Reagents and conditions: a) (i) Selectfluor®, DMF:Water, RT, 12 h, (ii) Ac2O, Pyridine, DMAP, RT, 12 h, (20 ¼ 33%, 21 ¼ 58%); b) Ammonium acetate, DMF, RT, 12 h, (96%); c)
Trichloroacetonitrile, K2CO3, anhydrous DCM, 0 ꢁC, 16 h, (93%); d) TMSOTf, anhydrous DCM, MS-4 Å, -20 ꢁC, 1 h, (78%); e) 10% w/w Pd-C, H2, MeOH, RT, 5 h, (98%); f) (i) NaOMe/
MeOH, RT, 1 h, (ii) DOWEX 50WX8-100 ion exchange (Hþ) resin, MeOH, 35 ꢁC, 1 h, (98%).

14
K. Patel et al. / Carbohydrate Research 453-454 (2017) 10e18
of glycosylation reactions and hence we experienced a longer re-
action duration. Furthermore, another reason for our initial lower
yielding glycosylation step was as a direct result of protection of the
acceptor by the trimethylsilyl group from the TMSOTf activator,
which was readily confirmed by both 1H-NMR (Compound 18a in
SI) and MS analysis. The silyl protected acceptor was recovered and
recycled for use in a simultaneous batch of glycosylation reactions
after the removal of the TMS protecting group using tetrabuty-
lammoniumfluoride (TBAF, 1.0 M in THF). Compound 24 was then
subjected to palladium catalyzed hydrogenation using 10% w/w Pd-
C at 60 psi to remove the benzyl protecting group and furnish 25
target molecule (Scheme 3). Therefore, in order to obtain GlcN-Ins
(5), compound 30 was subjected to palladium catalyzed hydroge-
nation using 10% w/w Pd-C at 60 psi to remove the benzyl pro-
ꢀ
tecting group, followed by Zemplen deacetylation conditions with
DOWEX 50WX8ꢂ100 ion exchange (Hþ) resin under heat ulti-
mately giving rise to GlcN-Ins (5) in 90% overall yield. In order to
obtain GlcN3-Ins (9), first the benzyl group was removed selectively
via oxidative debenzylation [46] as to avoid unwanted azido group
reduction. This was accomplished using NaBrO3/Na2S2O4 in a
biphasic solvent system. Without purification, the mixture was
ꢀ
then subjected to global deprotection with Zemplen deacetylation
[42] (Scheme 2). Compound 25 was further subjected to Zemplen
conditions and finally heating at 45 ꢁC in the presence of DOWEX
50WX8ꢂ100 ion exchange (Hþ) resin to remove the cyclo-
hexylidene groups respectively to give rise to GlcN3-Ins (9).
The desired target molecules described herein, were achieved
by overcoming difficulties in separating diastereomeric mixtures of
important intermediates. Contrary to utilizing enzymes [47] for
resolution, a chiral auxiliary was used to assist in the separation of
diastereomers. The glycosylation reaction was optimized to prevent
ꢀ
deacetylation conditions [43] followed by gentleþheating in the
presence of DOWEX 50WX8ꢂ100 ion exchange (H ) resin at 45 ꢁC
[44] to assist in a one-pot removal of acetyl and cyclohexylidene
protecting groups respectively with a 90% yield over two steps
affording 10.
2.3. Synthesis of GlcN-Ins (5) and GlcN3eIns (9)
loss of yield and a-selective product formation was confirmed by
After obtaining GlcF-Ins (10), our focus turned towards syn-
thesizing the azido analogue as well as the natural substrate for
MshC. We commenced by performing a diazo transfer reaction on
glucosamine hydrochloride (26) using Stick's diazo transfer reagent
[45] (prepared in-house) followed by peracetylation to afford
compound 27 in 87% yield over two steps (Scheme 3). Compound
27 was then subjected to anomeric deacetylation using ammo-
nium acetate in DMF to afford hemiacetal 28. Reaction with tri-
chloroacetonitrile, under anhydrous conditions, afforded TCA
donor (29) in 90% yield. Following the general Schmidt glycosyla-
tion procedure, acceptor 18 and donor 29 were coupled to furnish
1H-NMR analysis. The H-1 proton of the glycosylation products 24
and 30 showed coupling constant values of 4.14 and 2.88 Hz
respectively confirming the desired
a-configuration at the
anomeric position. 19F-NMR was also recorded for all steps in the
synthetic route for GlcF-Ins (10) in order to confirm the presence of
fluorine. GlcF-Ins was obtained from 24 using palladium catalyzed
hydrogenation followed by a one-pot acetyl and ketal group
deprotection. GlcN3-Ins (9) was obtained from 30 following an
oxidative debenzylation procedure to avoid the reduction of azide
and then a one-pot global deprotection. We were also able to
synthesize GlcN-Ins (5), the naturally occurring MshC substrate, by
subjecting 30 to palladium catalyzed hydrogenation and one-pot
global deprotection. As previously mentioned, the fluorine and
azide moieties can interact with amino acid residues such as
aspartic acid and histidine in the enzyme pocket to induce
conformational changes in the enzyme for inhibition purposes as
well as aide in obtaining the full crystal of MshC enzyme. The idea
of including the azide moiety as a substrate-based analogue can
compound 30 in 81% yield with high
a
-selectivity (
a:b
¼ 99:1)
(Scheme 3). The glycosylation reaction presented problems such as
low yield due to removal of the ketal groups on acceptor and pro-
tection of acceptor by TMSOTf. These issues were also observed in
the case of GlcF-Ins and were overcome by carefully monitoring
reaction temperature and duration. Compound 30 was then sub-
jected to different deprotection strategies depending on the desired
OAc
OAc
O
OH
a
c
O
O
AcO
AcO
AcO
HO
HO
HCl
AcO
.H N
O
N3
CCl3
NH
OR
OH
N3
2
26
29
27 R=Ac
28 R=OH
b
OH
HO
O
O
e
HO
HO
d
O
O
+
29
18
N
O
O
HO
O
CH2OH
OH
H2N
BnO
O
CH2OAc
N3
AcO
99:1
HO
OAc
30
5
O
S
N.
HCl
α
:
=
N3
β
OH
O
O
HO
N3
HO
HO
Stick's Diazo-
f
transfer
reagent
HO
O
CH2OH
OH
9
HO
Scheme 3. Reagents and conditions: a) (i) Stick's diazo transfer reagent, K2CO3, CuSO4.5H2O, MeOH, RT, 3 h, (ii) Ac2O, Pyridine, DMAP, RT, 12 h (87%); b) Ammonium acetate, DMF,
RT, 12 h, (98%); c) Trichloroacetonitrile, K2CO3, anhydrous DCM, 0 ꢁC to RT, 16 h, (90%); d) TMSOTf, anhydrous DCM, MS-4 Å, -20 ꢁC, 1 h, (81%); e) (i) 10% w/w Pd-C, H2, MeOH, RT, 5 h;
(ii) NaOMe/MeOH, RT, 1 h, (iii) DOWEX 50WX8-100 ion exchange (Hþ) resin, MeOH, 35 ꢁC, 1 h, (92%); f) (i) NaBrO3, Na2S2O4, EtOAc:H2O, RT, 6 h, (ii) NaOMe/MeOH, RT, 1 h, (iii)
DOWEX 50WX8-100 ion exchange (Hþ) resin, MeOH, 35 ꢁC, 1 h, (90%).

K. Patel et al. / Carbohydrate Research 453-454 (2017) 10e18
15
also be supported by commercial azide-based drug molecules like
using flash column chromatography to furnish highly pure
lective products.
a
-se-
Retrovir® and Azidocillin.
3. Conclusion
4.3. Synthesis
We have synthesized substrate analogues for the Mycobacterium
tuberculosis enzyme MshC. The synthesis was accomplished
employing chiral resolution of the acceptor, stereoselective glyco-
sylation and modified purification procedures leading to overall
good yields. Access to the naturally occurring substrate GlcN-Ins is
important for probing enzyme function as well as for developing
inhibitors. We anticipate that these molecules will be useful in
further MshC-based studies leading to the development active
molecules against the enzyme and the Tuberculosis disease in a
broader spectrum.
4.3.1. 2,3; 4,5-Di-O-cyclohexylidene-D/L-myo-inositol (15) [35]
To a solution of ( )-myo-inositol (14) (50 g, 277 mmol) in N,N-
dimethylformamide (200 mL), 1,1 dimethoxycyclohexane (108 mL,
721 mmol) and p-toluenesulfonic acid (1.36g, 7.2 mmol) were
added and the mixture was heated to 95-100 ꢁC for 4 h. After the
noted duration, the mixture was cooled to room temperature and
diluted with ethyl acetate (450 mL). It was washed with saturated
sodium bicarbonate (150 mL) and with water (2 ꢃ 100 mL). The
organic layer was separated and removed in vacuo to give a
yellowish syrup. The syrup was dissolved in minimal amount of
acetone followed by addition of hexanes until solids began to
separate (acetone:hexanes, 1:10). This was left overnight depos-
iting the crystals. The crystals were filtered and washed with
hexanes. The remaining mother liquor was purified using silica gel
column chromatography with DCM:acetone (24:1 to 9:1) as the
eluent to furnish the desired product 15 as a white solid (21.71 g,
63.85 mmol, 23%).
4. Experimental
4.1. General methods
All chemicals and solvents were of commercial grade and were
used without further purification unless otherwise stated. Chem-
icals were purchased from Alfa Aesar, Acros Organic, Fisher Scien-
tific, Oakwood Chemicals, Sigma Aldrich and TCI Chemicals.
Solvents were purchased from EMD, Fisher Scientific and Sigma
Aldrich. Molecular sieves (MS-4 Å) were activated by heating at
150 ꢁC overnight under a vacuum in a high temperature vacuum
oven equipped with an inert gas line. All reactions performed were
monitored using thin layer chromatography over silica gel-coated
TLC plates purchased from Silicycle. TLC plates were visualized
under UV light or by charring (5% H2SO4-MeOH and Anisladehyde
solutions). Flash column chromatography was performed using
200-400 mesh silica gel, Siliaflash®P60, purchased from Silicycle.
Size exclusion column was performed using Bio-Rad Bio-Gel® P-2
gel. Optical rotations were measured with Autopol-IV digital
polarimeter; concentrations are expressed as g/100 mL. 1H, 13C-APT,
NMR spectra were recorded on an Avance 600 MHz NMR spec-
trometer with CDCl3, MeOD, D2O as solvents. 19F-NMR was recor-
ded on a GEM 200 MHz spectrometer with CDCl3 and MeOD as the
4.3.2. 2,3; 4,5-Di-O-cyclohexylidene- (1R)-menthyloxycarbonyl-D/
L-myo-inositol (16) [35]
A mixture of 15 (4.36 g, 12.8 mmol) and dibutyltinoxide (3.5 g,
14.08 mmol) in toluene (100 mL) was refluxed employing a Dean-
Stark apparatus for 4 h, after observing disappearance of starting
material on TLC, the volume of toluene was reduced to 30 mL. The
reaction mixture was then cooled to 0 ꢁC followed by addition of
dry sodium bicarbonate (0.43 g, 1.28 mmol). (1R)-(-)-menthyl
chloroformate (3.6 g, 16.64 mmol) was added dropwise. After
stirring for 24 h at room temperature the reaction mixture was
filtered, concentrated in vacuo and purified using silica gel column
chromatography with hexanes:ethyl acetate (24:1 to 5:1) as the
eluent to furnish the desired product 16 as a white crystalline solid
(4.88 g, 9.35 mmol, 73% yield).
4.3.3. 6-O-benzyl-2,3; 4,5-Di-O-cyclohexylidene- (1R)-
solvents. The residual CHCl3 was referenced to
77.27 ppm in proton and carbon spectra respectively. The residual
MeOH was references to 3.31 and 49.00 ppm for proton and
carbon spectra respectively. The residual HDO was referenced to
4.9 with spectra taken in D2O and CFCl3 was used as internal
standard for 19F-NMR. Chemical shifts are reported in
ppm. Data
d
7.27 and
menthyloxycarbonyl-D-myo-inositol (17) [35]
d
To a solution of 16 (3.2 g, 6.13 mmol) in anhydrous dichloro-
methane, freshly prepared Ag2O (3.5 g, 15.3 mmol), molecular
sieves (MS 4 Å, 0.5 g) and benzylbromide (1.7 mL, 14.7 mmol) were
added and the reaction mixture was heated to 35 ꢁC for 8 h. After
allotted time, the reaction mixture was cooled to room temperature
and filtered through Celite®-545 and the organic layer was evap-
orated in vacuo to produce a thick syrup. The syrup was purified
using silica gel column chromatography to remove excess benzyl
bromide to yield a crude diastereomeric-mixture, which was then
dissolved in petroleum ether ultimately giving crystals. The crystals
were filtered and washed with petroleum ether to give the desired
product 17 (1.07 g, 1.7 mmol, 45%).
d
d
d
d
for 1H NMR are reported as follows: chemical shift, integration,
multiplicity (s ¼ singlet, d ¼ doublet, dd ¼ doublet of doublet,
dt ¼ doublet of triplet, ddd ¼ doublet of doublet of doublet,
t ¼ triplet, m ¼ multiplet) and coupling constants in Hertz (Hz).
Low resolution mass spectra were obtained using electrospray
ionization (ESI) technique. Yields refer to chromatographically and
spectroscopically pure material unless otherwise noted.
4.2. General glycosylation procedure
4.3.4. 6-O-benzyl-2,3; 4,5-Di-O-cyclohexylidene-D-myo-inositol
(18)
To a solution of acceptor (1 mmol) and trichloroacetimidate
donor (1.1 mmol) in anhydrous DCM (30 mL), preactivated molec-
ular sieves (MS-4 Å, 0.25 g) were added and stirred at room tem-
perature for 25 min under argon and then the mixture was cooled
to -20 ꢁC. After 5 min, a catalytic amount of the activator, trime-
thylsilyltriflate (TMSOTf), was added and the reaction was stirred
for 1 h until completion as was determined by TLC. Upon comple-
tion, the reaction was quenched with triethylamine and then
passed through a pad of Celite®-545. The organic layer was
concentrated under vacuo and the crude mixture was purified
To a solution of 17 (1.07 g, 1.7 mmol) in isopropanol:methanol
(IPA:MeOH, 1:1) was added K2CO3 (0.35 g, 2.55 mmol) and the
reaction mixture was allowed to stir vigorously for 5 h at 60 ꢁC. The
reaction was monitored by TLC. After noted spot-to-spot comple-
tion, the reaction mixture was cooled to room temperature and
filtered through Celite®-545. The solvents were removed in vacuo
and the mixture was purified employing silica gel column chro-
matography using hexanes:ethyl acetate (12:1 to 6:1) as the eluent
to yield the product 18 as colorless syrup (0.730 g, 1.69 mmol, 97%).
½
a 2D2
ꢀ
¼ þ4.5ꢁ (c ¼ 0.5, CHCl3). 1H-NMR (600 MHz, CDCl3)

16
K. Patel et al. / Carbohydrate Research 453-454 (2017) 10e18
d
¼ 1.65 (m, 20 H, 20-Hcyclohex), 3.53 (1H, dd, J ¼ 9.27, 7.05 Hz, 4-H),
trichloroacetimidate donor 23 (0.408 g, 0.904 mmol) were dis-
solved in anhydrous dichloromethane and the reaction was con-
ducted as per the general glycosylation procedure noted earlier. The
crude mixture was purified using flash column chromatography
using hexanes:ethyl acetate (9:1 to 3:1) as the eluent to furnish
3.73 (1H, dd, J ¼ 3.12, 9.90 Hz, 6-H), 3.87 (1H, dd, J ¼ 5.79, 6.93 Hz,
3-H), 4.08 (1H, t, J ¼ 9.60 Hz, 5-H), 4.22 (1H, t, J ¼ 5.58 Hz, 2-H), 4.64
(1H, dd, J ¼ 3.09, 5.49 Hz, 1-H), 4.83 (1H, dd, J ¼ 11.70, 158.81 Hz,
CH2Ph), 7.30e7.41 ((m, 5H, Ar-H).). 13C-NMR (150 MHz, CDCl3)
d
¼ 23.66, 24.03, 24.06, 24.15, 25.21, 25.28, 35.12, 36.12, 36.93, 37.47,
pure
a-24 as a colorless syrup (0.422 g, 0.587 mmol, 78%).
ꢀ
71.58, 71.97, 72.61, 73.41, 75.34, 75.58, 77.43, 80.51, 81.43, 112.74,
112.79, 128.05, 128.38, 128.68, 138.39. ESI-LRMS [(M þ Na)þ] calcd
for C25H34NaO6 is 543.23, found 543.67.
½
a 2D2
¼ þ82.5ꢁ, (c ¼ 1.0, CHCl3). 1H-NMR (600 MHz, CDCl3)
d
¼ 1.68 (m, 20 H, 20-Hcyclohex), 2.02 (s, 3H, CH3), 2.03 (s, 3H, CH3),
2.08 (s, 3H, CH3), 3.71 (d, J ¼ 2.82 Hz, 1H, 2-H), 3.71 (d, J ¼ 1.56 Hz,
1H, 40-H), 3.78 (dd, J ¼ 2.04, 12.06 Hz, 1H, 6b-H), 3.96 (dd, J ¼ 3.78,
12.60 Hz, 1 H, 6a-H), 3.99 (dd, J ¼ 4.92, 6.84 Hz, 1H, 50-H), 4.11 (t,
J ¼ 9.69 Hz, 1H, 30-H), 4.27 (qd, J ¼ 1.89, 10.41 Hz, 1H, 5-H), 4.35 (t,
J ¼ 5.28 Hz, 1 H, 60-H), 4.48 (ddd, J ¼ 3.84, 9.59, 49.23 Hz, 1H, 20-H),
4.63 (dd, J ¼ 3.03, 5.67 Hz, 1H, 10-H), 4.82 (d, Jgem ¼ 11.46, 140.51 Hz,
2H, CH2Ph), 4.99 (t, J ¼ 9.93 Hz, 1H, 4-H), 5.49 (t, J ¼ 9.54, 12.00 Hz,
1H, 3-H), 5.51 (d, J ¼ 41.4 Hz, 1H, 1-H), 7.29e7.41 (m, 5H, Ar-H). 13C-
4.3.5. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-fluoro-a-D-
mannopyranose (20) and 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-fluoro-
D-glucopyranose (21) [38]
To a solution of 3,4,6-tri-O-acetyl-D-glucal (19) (5 g, 18.3 mmol)
in N,N-dimethylformamide:water (1:1, 100 mL) was added Select-
fluor® (12.9 g, 36.6 mmol) and the reaction was stirred at room
temperature for 12 h until noted TLC completion. The reaction
mixture was diluted with ethyl acetate and then washed thrice with
water. The organic layer was separated, dried and concentrated
NMR (150 MHz, CDCl3)
d
¼ 20.64, 20.73, 20.82, 23.48, 23.83, 23.89,
23.96, 24.94, 25.05, 34.79, 35.88, 36.72, 37.08, 61.27, 67.17, 67.68,
67.72, 70.65, 70.78, 71.94, 72.45, 75.15, 75.33, 79.43, 80.42, 81.37,
86.52, 87.81, 94.28, 94.42, 127.84, 128.44, 137.95, 169.61, 170.12,
under reduced pressure to give an inseparable mixture of a/b 2-
fluoro epimers. The crude product was then dissolved in pyridine
(30 mL) followed by addition of acetic anhydride (1.72 mL,
18.3 mmol) and a catalytic amount of 4-dimethylaminopyridine
(DMAP). The reaction mixture was allowed to stir for 8 h. Pyri-
dine was removed in vacuo and the remaining syrup was diluted
with ethyl acetate and washed with 1N HCl (3 ꢃ 50 mL) followed by
saturated sodium bicarbonate (2 ꢃ 50 mL) and a solution of brine
(2 ꢃ 25 mL). The organic layer was dried over sodium sulfate and
the ethyl acetate evaporated in vacuo to afford a peracetylated
mixture of 20 and 21 in a 1:2 ratio. The mixture was separated by
column chromatography using hexanes:ethyl acetate (9:1 to 3.5:1)
as the eluent to furnish the desired product 21 as colorless syrup
(3.7 g, 10.5 mmol, 58%) and product 20 as a colorless syrup (2.1 g,
5.96 mmol, 33%).
170.59.19F-NMR (200 MHz, CDCl3)
d
¼ -201.51 (1F, dd, J ¼ 12.19,
49.11 Hz). ESI-LRMS [(M þ Na)þ] calcd for C37H49FNaO13 is 743.31,
found 743.56.
4.3.9. O-(3,4,6-Tri-O-acetyl-2-deoxy-2-fluoro-a-D-
glucopyranosyl)-(1 / 1)-2,3:4,5-di-0-cyclohexylidene-D-myo-
inositol (25)
Compound 24 (0.200 g, 0.277 mmol) was dissolved in methanol
in a Parr hydrogenation flask and a catalytic amount of 10% w/w Pd-
C was added. The mixture was stirred under hydrogen at 40 psi on
the Parr apparatus until completion of reaction was noted using
TLC. The mixture was then filtered through Celite®-545 and the
solution was concentrated in vacuo. The residue was purified using
silica gel column chromatography employing hexanes:ethyl acetate
(3:2) as the eluent to furnish 25 as an off-white solid (0.175 g,
0.277 mmol, 99%).
4.3.6. 3,4,6-Tri-O-acetyl-2-deoxy-2-fluoro-D-glucopyranose (22)
[41]
½
a 2D2
ꢀ
¼ þ50.3ꢁ (c ¼ 1.5, CHCl3). 1H-NMR (600 MHz, CDCl3)
Compound 21 (0.510 g, 1.45 mmol) was dissolved in N,N-
dimethylformamide (5 mL) followed by the addition of ammonium
acetate (0.224 g, 2.91 mmol). The reaction mixture was allowed to
stir at room temperature for 12 h. The reaction mixture was then
diluted with cold water and extracted with ethyl acetate. The
organic layer was dried over sodium sulfate and concentrated in
vacuo to yield a thick syrup which was subjected to purification
conditions using silica gel column chromatography employing
hexanes:ethyl acetate (3:2) as the eluent to afford 22 as a colorless
syrup (0.440 g, 1.42 mmol, 98%).
d
¼ 1.68 (m, 20H, 20-Hcyclohex), 2.05 (s, 3H, CH3), 2.09 (s, 3H, CH3),
2.10 (s, 3H, CH3), 3.71 (dd, J ¼ 2.94, 9.90 Hz, 1H, 20-H), 3.87 (m, 2 H,
40,60-H), 4.00 (t,1H, 30-H), 4.10 (dd, J ¼ 2.16,12.36 Hz,1H, 6b-H), 4.25
(dd, J ¼ 4.92, 12.46 Hz, 1 H, 6a-H), 4.36 (d, J ¼ 8.94 Hz, 1H, 5-H), 4.37
(d, J ¼ 2.58 Hz,1H, 50-H), 4.53 (ddd, J ¼ 3.93, 9.60, 49.14 Hz,1H, 2-H),
4.64 (dd, J ¼ 3.03, 5.49 Hz, 1H, 10-H), 5.00 (t, J ¼ 9.90 Hz, 1H, 4-H),
5.49 (d, J ¼ 3.84 Hz, 1H. 1-H), 5.54 (dt, J ¼ 9.59, 11.85 Hz, 1H, 3-H).
13C-NMR (150 MHz CDCl3)
d
¼ 20.86, 20.97, 20.99, 23.67, 23.91,
23.93, 24.10, 25.13, 25.17, 29.58, 29.81, 29.92, 35.08, 36.19, 36.72,
37.42, 62.13, 67.69, 68.35, 68.40, 70.66, 70.79, 72.31, 73.78, 74.96,
75.17, 80.63, 85.03, 86.61, 87.90, 95.34, 95.48, 113.14, 113.21, 169.93,
4.3.7. 3,4,6-Tri-O-acetyl-2-deoxy-2-fluoro-D-glucopyranosyl
Trichloroacetimidate (23) [41]
170.35, 170.92.19F-NMR (200 MHz, CDCl3)
d
¼ -202.34 (1F, dd,
J ¼ 12.64, 49.37 Hz). ESI-LRMS [(M þ Na)þ] calcd for C30H43FNaO13
To a solution of compound 22 (0.300g, 0.97 mmol) in anhydrous
dichloromethane, 10 mL of aqueous K2CO3 (0.06g, 0.48 mmol) was
added. The reaction mixture was maintained at 0 ꢁC until the
addition of trichloroacetonitrile (0.07 mL, 0.71 mmol) was
completed using a drop-wise technique. The reaction was stirred at
room temperature for 16 h. The reaction was then filtered through
Celite®-545 and the organic layer was removed in vacuo. The res-
idue was purified using silica gel column chromatography
employing hexanes:ethyl acetate (3:1) as the eluent to furnish 23 as
a colorless syrup (0.408 g, 0.905 mmol, 93%).
is 653.26, found 653.78.
4.3.10. O-(2-deoxy-2-fluoro-
myo-inositol (GlcF-Ins) (10)
a-D-glucopyranosyl)-)-(1 / 1)-D-
Compound 25 (0.175 g, 0.277 mmol) was dissolved in methanol
and sodium metal was added until the pH read ~10. The reaction
mixture was stirred for 1 h until completion as noted by TLC. Once
the reaction was complete, DOWEX 50WX8-100 ion exchange (Hþ)
resin was added until the pH was acidic (pH~1). The reaction
mixture was then heated to 45 ꢁC for another 45 min to remove the
cyclohexylidene protecting groups and the reaction was monitored
via TLC until completion. The reaction was filtered through a pad of
Celite®-545 and concentrated in vacuo to give a precipitate which
was purified using Bio-Rad Bio-Gel® P-2 gel size exclusion chro-
matography to furnish product 10 as yellowish syrup (0.95 g,
4.3.8. 6-O-benzyl-O-(3,4,6-Tri-O-acetyl-2-deoxy-2-fluoro-a-D-
glucopyranosyl)-(1 / 1)-2,3:4,5-di-0-cyclohexylidene-D-myo-
inositol (24)
The acceptor 18 (323.7 mg, 0.753 mmol) and the

K. Patel et al. / Carbohydrate Research 453-454 (2017) 10e18
17
0.277 mmol, 99%).
dichloromethane, 10 mL of aqueous K2CO3 (0.155 g, 1.125 mmol)
was added. The reaction mixture was maintained at 0 ꢁC until the
addition of trichloroacetonitrile (0.165 mL, 1.65 mmol) was
completed. The reaction was stirred at room temperature for 16 h.
The reaction was filtered through Celite®-545 and the organic layer
was removed in vacuo. The residue was purified using silica gel
column chromatography employing hexanes:ethyl acetate (3:1) as
the eluent to furnish 29 as light-yellow colored syrup (0.780 g,
1.64 mmol, 90%).
½
a 2D2
ꢀ
¼ þ36.5ꢁ, (c ¼ 1.0, MeOH). 1H-NMR (600 MHz, CD3OD)
d
¼ 3.22 (t, J ¼ 9.27 Hz,1H, 60-H), 3.32 (d, J ¼ 11.28 Hz,1H, 20-H), 3.34
(t, J ¼ 9.45 Hz, 1H,40-H), 3.53 (dd, J ¼ 2.79 Hz, 9.63 Hz, 1H, 4-H), 3.60
(t, J ¼ 9.54 Hz, 1H, 50-H), 3.64 (dd, J ¼ 5.01,11.79 Hz, 1H, 6b-H), 3.74
(t, J ¼ 9.21 Hz, 1H, 5-H), 3.78 (d, J ¼ 2.34 Hz, 1H, 6a-H), 3.88 (t,
J ¼ 5.61Hz, 1H, 30-H), 3.89 (dd, J ¼ 9.54, 21.85 Hz, 1H, 3-H), 4.03
(ddd, J ¼ 2.32, 4.93,10.09 Hz, 1H, 10-H), 4.20 (ddd, J ¼ 4.02, 9.51,
49.97 Hz, 1 H, 2-H), 5.46 (d, J ¼ 4.02 Hz, 1H, 1-H). 13C-NMR
(150 MHz, CD3OD)
d
¼ 61.66, 70.66, 70.72, 72.46, 72.58, 72.66,
73.19, 73.92, 73.95, 74.57, 81.19, 90.82, 92.07, 97.56, 97.70.19F-NMR
4.3.15. 6-O-benzyl-O-(3,4,6-Tri-O-acetyl-2-deoxy-2-azido-a-D-
(200 MHz, CD3OD)
d
¼ -201.14 (1F, dd, J ¼ 13.10, 49.36 Hz). ESI-
glucopyranosyl)-(1 / 1)-2,3:4,5-di-0-cyclohexylidene-D-myo-
inositol (30)
LRMS [(M þ Na)þ] calcd for C12H21FNaO10 is 367.10, found 367.57.
The acceptor 18 (0.500 g, 1.162 mmol) and trichloroacetimidate
donor 29 (0.605 g, 1.278 mmol) were dissolved in anhydrous
dichloromethane (30 mL) and then the reaction was conducted as
per the general glycosylation procedure explained earlier in the
text. The crude was purified via flash column chromatography us-
ing hexanes:ethyl acetate (9:1 to 3:1) as the eluent to furnish pure
4.3.11. Stick's diazo transfer reagent [45]
To an ice cold suspension of sodium azide (NaN3, 10 g,
153.8 mmol) in acetonitrile (175 mL), sulfuryl chloride (12.4 mL,
153.8 mmol) was added and the reaction mixture was stirred
overnight at room temperature followed by addition of imidazole
(19.89 g, 292.2 mmol) in portions under ice-cold conditions. The
resulting mixture was stirred for 4 h at room temperature. The
mixture was diluted with ethyl acetate (350 mL), washed with
a-selective products 30 as off-white solid (0.696 g, 0.936 mmol,
81%).
½
a 2D2
ꢀ
¼ þ86.5ꢁ, (c ¼ 1.0, CHCl3). 1H-NMR (600 MHz, CDCl3)
water (2
ꢃ
200 mL) and saturated sodium bicarbonate
d
¼ 1.3e1.8 (m, 20 H, 20-Hcyclohex), 2.02 (s, 3H, CH3), 2.03 (s, 3H,
(2 ꢃ 200 mL). The organic layer was then dried over Na2SO4. To the
organic layer, a dropwise solution of HCl in ethanol was added
under ice cold conditions and then the mixture was filtered and
washed with ethyl acetate to give a solid of Stick's salt.
CH3), 2.10 (s, 3H, CH3), 3.36 (dd, J ¼ 3.54, 10.56 Hz, 1H, 2-H), 3.67
(dd, J ¼ 7.02, 9.30 Hz, 1H, 40H), 3.71 (dd, J ¼ 3.00, 9.96 Hz, 1H, 20-H),
3.75 (dd, J ¼ 1.86, 12.60 Hz, 1H, 6b-H), 3.94 (dd, J ¼ 4.53, 13.47 Hz,
1H, 6a-H), 3.97 (dd, J ¼ 5.16, 7.02 Hz, 1H, 50-H), 4.10 (t, J ¼ 9.66 Hz,
1H, 30-H), 4.26 (qd, J ¼ 1.81, 10.17 Hz, 1H, 5-H), 4.36 (t, J ¼ 5.31 Hz,
1H, 60-H), 4.64 (dd, J ¼ 3.09, 5.55 Hz,1H,10-H), 4.81 (dd, Jgem ¼ 11.46,
143.51 Hz, 2H,CH2Ph), 5.01 (t, J ¼ 9.81 Hz, 1H, 4-H), 5.43 (d,
J ¼ 2.88 Hz, 1H, 1-H), 5.43 (t, J ¼ 9.87 Hz, 1H, 3-H), 7.33e7.44 (m, 5H,
4.3.12. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-azido-D-glucopyranose
(27) [45]
To a mixture of D-glucosamine HCl 26 (9.69 g, 44.81 mmol),
K2CO3 (16.75 g, 121.19 mmol), CuSO4 H2O (0.112 g, 0.448 mmol) in
methanol (400 mL) and Stick's diazo transfer reagent (imidazole-1-
sulfonyl azide hydrochloride), (11.3 g, 53.9 mmol) was added at 0 ꢁC
and stirred at room temperature for 3 h until completion as noted
by TLC. The reaction mixture was evaporated and then co-
evaporated with toluene to remove any moisture. The resulting
mass (8.5 g, 414 mmol) was dried on a vacuum pump and then
dissolved in 200 mL of pyridine followed by the addition of acetic
anhydride (42.44 mL, 448 mmol) and a catalytic amount of DMAP
at 0 ꢁC. The reaction mixture was stirred for 12 h at room tem-
perature. It was then concentrated and diluted with ethyl acetate
and washed with 1N HCl (4 ꢃ 75 mL) followed by saturated sodium
bicarbonate (2 ꢃ 75 mL) and brine solution (2 ꢃ 25 mL). The
organic layer was dried over sodium sulfate and evaporated in
vacuo to give a crude mass of the product which was purified using
silica gel chromatography employing hexanes:ethyl acetate (3:1) as
the eluent to yield the desired product 27 as yellowish solid (13.5 g,
36.19 mmol, 87%).
Ar-H). 13C-NMR (150 MHz CDCl3)
d
¼ 20.86, 20.93, 20.97, 23.67,
24.01, 24.09, 24.16, 25.13, 25.24, 29.92, 35.02, 36.08, 36.90, 37.32,
61.08, 61.53, 67.69, 68.34, 70.74, 72.17, 75.32, 75.54, 79.49, 80.54,
81.35, 96.28, 112.94, 113.12, 128.07, 128.66, 128.69, 138.08, 169.86,
170.26, 170.80. ESI-LRMS [(M þ Na)þ] calcd for C37H49FNaO13 is
743.31, found 743.56.
4.3.16. O-(a-D-glucosaminepyranosyl)-(1 / 1)-D-myo-inositol
(GlcN-Ins) (5)
To a solution of compound 30 (0.200 g, 0.269 mmol) in meth-
anol (4 mL) was added a catalytic amount of 10% w/w Pd-C. The
mixture was stirred under hydrogen at 40 psi until completion of
the reaction was noted by TLC. The mixture was then filtered
through a pad of Celite®-545 and the organic solvent was removed
in vacuo. The crude product was then dissolved in methanol and
sodium metal was added until the pH reached ~10. The reaction
mixture was stirred for 1 h until noted completion of deacetylation.
Once the reaction was complete, DOWEX 50WX8-100 ion exchange
(Hþ) resin was added until the pH read acidic (~1) and the reaction
mixture was then heated to 45 ꢁC for another 45 min until
completion to remove the cyclohexylidene protecting groups. The
reaction mixture was then filtered through Celite®-545 and
concentrated in vacuo to give a precipitate which was purified using
Bio-Rad Bio-Gel® P-2 gel size exclusion chromatography to furnish
product 5 as an off-white solid (0.085 g, 0.249 mmol, 92%).
4.3.13. 3,4,6-Tri-O-acetyl-2-deoxy-2-azido-D-glucopyranose (28)
[40]
Compound 27 (0.70 g, 1.87 mmol) was dissolved in N,N-dime-
thylformamide followed by the addition of ammonium acetate
(0.206 g, 2.68 mmol). The reaction mixture was stirred at room
temperature for 12 h. The mixture was diluted with cold water and
then extracted with ethyl acetate. The organic layer was dried over
sodium sulfate and concentrated in vacuo to yield a syrup which
was then purified using silica gel chromatography employing
hexanes:ethyl acetate (3:2) as the eluent to afford 28 as colorless
syrup (0.608 g, 1.83 mmol, 98%).
½
a 2D2
ꢀ
¼ þ41.1ꢁ (c ¼ 1.15, H2O). 1H-NMR (600 MHz, D2O)
¼ 3.29
d
(t, J ¼ 9.36 Hz, 1H, 50-H), 3.36 (dd, J ¼ 3.54, 10.68 Hz, 1H, 2-H), 3.49
(t, J ¼ 9.39 Hz, 1H, 4-H), 3.53 (dd, J ¼ 2.46, 9.90 Hz, 1H, 30-H), 3.61 (t,
J ¼ 9.63 Hz, 1H, 40-H), 3.69 (dd, J ¼ 2.70, 10.08 Hz, 1H, 10-H), 3.77 (m,
J ¼ 4.83 Hz, 2H, 6b, 60-H), 3.85 (m, 2H, 5,6a-H), 3.93 (t, J ¼ 8.76 Hz,
1H, 3-H), 4.20 (t, J ¼ 2.37 Hz, 1H, 20-H), 5.42 (d, J ¼ 3.54 Hz, 1H, 1-H).
4.3.14. 3,4,6-Tri-O-acetyl-2-deoxy-2-azido-D-glucopyranosyl
trichloroacetimidate (29) [40]
13C-NMR (150 MHz D2O)
d
¼ 54.17, 60.18, 69.37, 69.46, 70.93, 71.67,
71.86, 71.94, 72.70, 74.11, 78.92, 97.18. ESI-LRMS [(M þ Na)þ] calcd
To a solution of compound 28 (0.608 g, 1.83 mmol) in anhydrous
for C12H23NNaO10 is 364.12, found 364.42.

18
K. Patel et al. / Carbohydrate Research 453-454 (2017) 10e18
[7] G.L. Newton, R.C. Fahey, Archives Microbiol. 178 (2002) 388e394.
4.3.17. O-(2-deoxy-2-azid-
inositol (GlcN3-Ins) (9)
a-D-glucopyranosyl)-(1 / 1)-D-myo-
[8] F. Fan, M.W. Vetting, P.A. Frantom, J.S. Blanchard, Curr. Opin. Chem. Biol. 13
(2009) 451e459.
To a solution of 30 (0.100 g, 0.134 mmol) in ethyl acetate (5 mL)
was added 3 mL of aqueous sodium bromate (NaBrO3, 0.904 g,
0.603 mmol). A solution of sodium dithionite (Na2S2O4, 85%,
0.932 g, 0.536 mmol) in water (6 mL) was added over 20 min and
the reaction was vigorously stirred for 6 h at room temperature. The
mixture was then diluted with ethyl acetate, quenched with 10%
sodium thiosulphite (1 mL) and finally washed with water. The
organic layer was separated and dried over sodium sulfate and
concentrated in vacuo. The crude mixture was then dissolved in
methanol and sodium metal was added until the pH read ~10. The
reaction mixture was stirred for 1 h until completion. Once the
reaction was complete, DOWEX 50WX8-100 ion exchange (Hþ)
resin was added to until the pH read acidic (~1). The reaction
mixture was then heated to 45 ꢁC for another 45 min until
completion to remove the cyclohexylidene protecting groups.
Finally, the mixture was filtered through Celite®-545 and concen-
trated in vacuo to give a precipitate which was purified using Bio-
Rad Bio-Gel® P-2 gel size exclusion chromatography to furnish
product 9 as a colorless syrup (0.044 g, 0.119 mmol, 90%).
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reviews, MMBR 72 (2008) 471e494.
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140e158.
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M. Trujillo, Antioxid. Redox. Signaling (2017).
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6736e6740.
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11421e11429.
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4843e4850.
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Agents Chemother. 46 (2002) 3348e3355.
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13326e13335.
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Screen. 14 (2009) 643e652.
½
a 2D2
ꢀ
¼ þ46.5ꢁ (c ¼ 0.35, H2O). 1H-NMR (600 MHz, D2O)
¼ 3.26
d
(t, J ¼ 9.45 Hz, 1H, 40-H), 3.36 (dd, J ¼ 3.72, 10.56 Hz, 1H, 2-H), 3.45
(t, J ¼ 9.39 Hz,1H, 4-H), 3.50 (dd, J ¼ 2.79, 9.99 Hz,1H, 60-H), 3.60 (d,
J ¼ 19.09 Hz, 1H, 20-H), 3.61 (d, J ¼ 10.14 Hz, 1H, 50-H), 3.74 (dd,
J ¼ 5.28, 11.88 Hz, 1H, 6b-H), 3.78 (t, J ¼ 9.75 Hz, 1H, 30-H), 3.82 (dd,
J ¼ 2.07, 4.47 Hz, 1H, 5-H), 3.83 (dd, J ¼ 2.13, 29.14 Hz, 1H, 6a-H),
3.92 (dd, J ¼ 9.12, 10.50 Hz, 1H, 3-H), 4.15 (t, J ¼ 2.82 Hz, 1H, 10-H),
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J. Fluor. Chem. 129 (2008) 731e742.
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Biomol. Chem. 13 (2015) 7542e7550.
€
[26] M. Braitsch, H. Kahlig, G. Kontaxis, M. Fischer, T. Kawada, R. Konrat,
W. Schmid, Beilstein J. Org. Chem. 8 (2012) 448e455.
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119 (1997) 5792e5797.
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91 (1981) 313e318.
5.29 (d, J ¼ 3.72 Hz, 1H, 1-H). 13C-NMR (150 MHz D2O)
d
¼ 60.38,
63.08, 69.78, 70.75, 70.98, 71.68, 71.98, 72.39, 74.27, 79.00, 99.49.
ESI-LRMS [(M þ Na)þ] calcd for C12H21N3NaO10 is 390.11, found
390.84.
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1996 (1996) 673e677.
Acknowledgement
^
The authors acknowledge The University of Toledo for financial
support of this research.
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[36] M. Dvorakova, M. Pribylova, R. Pohl, M.E. Migaud, T. Vanek, Tetrahedron 68
(2012) 6701e6711.
Appendix A. Supplementary data
[37] D.Y. Curtin, R.J. Harder, J. Am. Chem. Soc. 82 (1960) 2357e2368.
[38] M.D. Burkart, Z. Zhang, S.-C. Hung, C.-H. Wong, J. Am. Chem. Soc. 119 (1997)
11743e11746.
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.carres.2017.10.014.
[39] C. Srinivas, H. Blake, W. Qian, ChemInform 37 (2006).
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