Rational design of reversible inhibitors for trehalose 6-phosphate phosphatases

Article abstract of DOI:10.1016/j.ejmech.2017.02.001

In some organisms, environmental stress triggers trehalose biosynthesis that is catalyzed collectively by trehalose 6-phosphate synthase, and trehalose 6-phosphate phosphatase (T6PP). T6PP catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to trehalose and inorganic phosphate and is a promising target for the development of antibacterial, antifungal and antihelminthic therapeutics. Herein, we report the design, synthesis and evaluation of a library of aryl D-glucopyranoside 6-sulfates to serve as prototypes for small molecule T6PP inhibitors. Steady-state kinetic techniques were used to measure inhibition constants (K_i) of a panel of structurally diverse T6PP orthologs derived from the pathogens Brugia malayi, Ascaris suum, Mycobacterium tuberculosis, Shigella boydii and Salmonella typhimurium. The binding affinities of the most active inhibitor of these T6PP orthologs, 4-n-octylphenyl α-D-glucopyranoside 6-sulfate (9a), were found to be in the low micromolar range. The K_iof 9a with the B.?malayi T6PP ortholog is 5.3?±?0.6?μM, 70-fold smaller than the substrate Michaelis constant. The binding specificity of 9a was demonstrated using several representative sugar phosphate phosphatases from the HAD enzyme superfamily, the T6PP protein fold family of origin. Lastly, correlations drawn between T6PP active site structure, inhibitor structure and inhibitor binding affinity suggest that the aryl D-glucopyranoside 6-sulfate prototypes will find future applications as a platform for development of tailored second-generation T6PP inhibitors.

Full text of DOI:10.1016/j.ejmech.2017.02.001

European Journal of Medicinal Chemistry 128 (2017) 274e286
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Rational design of reversible inhibitors for trehalose 6-phosphate
phosphatases
Chunliang Liu, Debra Dunaway-Mariano*, Patrick S. Mariano**
Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, NM 87131, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In some organisms, environmental stress triggers trehalose biosynthesis that is catalyzed collectively by
trehalose 6-phosphate synthase, and trehalose 6-phosphate phosphatase (T6PP). T6PP catalyzes the
hydrolysis of trehalose 6-phosphate (T6P) to trehalose and inorganic phosphate and is a promising target
for the development of antibacterial, antifungal and antihelminthic therapeutics. Herein, we report the
Received 3 January 2017
Received in revised form
1 February 2017
Accepted 3 February 2017
Available online 4 February 2017
design, synthesis and evaluation of a library of aryl D-glucopyranoside 6-sulfates to serve as prototypes
for small molecule T6PP inhibitors. Steady-state kinetic techniques were used to measure inhibition
constants (Ki) of a panel of structurally diverse T6PP orthologs derived from the pathogens Brugia malayi,
Ascaris suum, Mycobacterium tuberculosis, Shigella boydii and Salmonella typhimurium. The binding af-
Keywords:
HAD phosphatase
Trehalose 6-sulfate
Trehalose 6-phosphate phosphatase
T6PP inhibitor
finities of the most active inhibitor of these T6PP orthologs, 4-n-octylphenyl
sulfate (9a), were found to be in the low micromolar range. The Ki of 9a with the B. malayi T6PP
ortholog is 5.3 0.6 M, 70-fold smaller than the substrate Michaelis constant. The binding specificity of
a-D-glucopyranoside 6-
m
Antihelminthic
9a was demonstrated using several representative sugar phosphate phosphatases from the HAD enzyme
superfamily, the T6PP protein fold family of origin. Lastly, correlations drawn between T6PP active site
structure, inhibitor structure and inhibitor binding affinity suggest that the aryl D-glucopyranoside 6-
sulfate prototypes will find future applications as a platform for development of tailored second-
generation T6PP inhibitors.
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
Aspergillus fumigatus, Candida albicans, Caenorhabditis elegans and
Brugia malayi, demonstrated the sensitivity of microbial and
The (a-1,
a
0-1)-glucose dimer trehalose is produced by special-
nematodal human pathogens to the disruption of the trehalose
pathway [9e16]. T6PP, the subject of our work, is required both for
trehalose production and for the prevention of T6P accumulation
and associated toxicity [11,15]. Despite the considerable discussion
given to T6PP as a platform for the development of antibacterial,
antifungal and antihelminthic therapeutics [9,11,12,15,16], T6PP
inhibitors have not yet been reported. In this article, we report the
synthesis and evaluation of a library of first generation T6PP in-
hibitors designed using a modular approach.
ized bacteria, fungi, plants and nematodes in response to envi-
ronmental stress [1e6]. Trehalose biosynthesis is catalyzed,
collectively, by trehalose 6-phosphate synthase (T6PS), which cat-
alyzes the condensation of glucose 6-phosphate and uridine 50-
diphosphoglucose (UDP-glucose) to trehalose 6-phosphate (T6P),
and trehalose 6-phosphate phosphatase (T6PP), which catalyzes
the hydrolysis of T6P to trehalose and inorganic phosphate (see
Fig. 1) [7,8]. Gene silencing experiments, carried out with Myco-
bacterium tuberculosis, Cryptococcus neoformans and C. gattii,
2. Results and discussion
* Corresponding author. Department of Chemistry and Chemical Biology, Uni-
versity of New Mexico, MSC03 2060, 300 Terrace St. NE, Albuquerque, NM 87131-
0001, USA.
2.1. Inhibitor design
** Corresponding author. Department of Chemistry and Chemical Biology, Uni-
versity of New Mexico, MSC03 2060, 300 Terrace St. NE, Albuquerque, NM 87131-
0001, USA.
E-mail addresses: dd39@unm.edu (D. Dunaway-Mariano), mariano@unm.edu
(P.S. Mariano).
The T6PP inhibitor design was guided by the X-ray crystal
structure of the T6PP ortholog from the archaeon Thermoplasma
acidophilum (Ta-T6PP) [17]. The Ta-T6PP backbone fold conforms to
that of the type C2 Haloalkanoic Acid Dehalogenase (HAD)
http://dx.doi.org/10.1016/j.ejmech.2017.02.001
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

C. Liu et al. / European Journal of Medicinal Chemistry 128 (2017) 274e286
275
Fig. 1. An illustration of the trehalose 6-phosphate phosphatase-catalyzed hydrolysis of trehalose 6-phosphate to trehalose and inorganic phosphate and the sequential modifi-
cations made to the substrate structure in forming the first generation inhibitor 4-n-octylphenyl -D-glucopyranoside-6-sulfate.
a
superfamily phosphatase [18]. As with other phosphatases of this
structural class, T6PP is comprised of a Rossmann-fold catalytic
domain and the inserted a/b-fold cap domain. Ensuing structure
determinations, first on the T6PP ortholog from the pathogenic
nematode B. malayi (Bm-T6PP) [19], and most recently on the T6PP
orthologs from fungal pathogens C. albicans, C. neoformans and
A. fumigatus [20] and the bacterial pathogen M. tuberculosis [21],
have illuminated the structural determinants of substrate
recognition.
consistent with the large steady-state Km values (200e1600 mM)
observed for T6PP orthologs [19e21,24e28], and confirmed by
using substrate-fragment analysis [28]. The analysis showed that
phosphate, glucose 6-phosphate and trehalose do not generate
sufficient binding energy to overcome the entropy penalty intrinsic
to protein-ligand association. Thus, stable complexes are not
formed [28].
With this information in hand and the original apo-TaT6PP
structure to guide inhibitor design, we pursued the divide-and-
conquer strategy depicted in Fig. 1. The plan was to retailor the
substrate by first replacing the labile phosphate group with a stable
mimetic, and then substitute the carbohydrate portion with a
structural module tailored to complement the substrate binding
site. Because the T6P phosphate group binds to a polar pocket,
where it favorably interacts with the Mg2þ cofactor and several
stringently conserved electropositive amino acid residues (Fig. 2B),
we limited our search to tetrahedral oxyanions. This led to the ul-
timate discovery that the sulfate group is best suited for this pur-
pose [28]. Trehalose 6-sulfate (T6S) thus became the platform for
tailoring the trehalose unit for enhanced binding affinity.
Inspection of the T6PP structure given in Fig. 2A, reveals that the
two substrate glucosyl units are bound in starkly different envi-
ronments. Whereas the hydroxyl groups of inner glucosyl moiety
(the one that is phosphorylated) collectively form multiple
hydrogen bonds with the charged side chains of a cap domain Glu-
Lys-Glu triad, the outer glucosyl unit (the one that is not phos-
phorylated) is bound through interactions with the main chain
amide groups of an encompassing loop from the catalytic domain.
For the purpose of inhibitor design, the inner glucosyl unit was left
intact and used for tethering a structural motif designed to extract
binding energy through optimized interaction with the active site.
Because the “cap-closing” interactions between the T6P inner
glucosyl hydroxyl groups are retained, the T6PP inhibitor will
A comparative analysis of the T6PP X-ray structures identifies
the active site formed at the cap domain-catalytic domain interface
and provides insight into the domain dynamics associated with
catalytic cycling. To illustrate, the superposition of the structures of
the T6P complex of the T6PP-D24 N (site-directed mutant) from
C. neoformans (colored cyan) and the (unbound) apo-TPP from
A. fumigatus (green) is shown in Fig. 2A. From the outset, we had
assumed that T6PP, like other HAD phosphatases possessing a
mobile cap domain, would undergo changes in domain-domain
association as required to open the catalytic site for substrate
binding, close it for catalysis, and open it for product release.
Indeed, the structures of the fungal T6PP orthologs define two cap
domain-catalytic domain orientations, one in which the active site
is open to enable ligand exchange (hereafter referred to as the
“open conformer”) and the other in which the active site is closed
for catalysis (the “closed conformer”).
Although the suggested T6PP substrate induced-fit mechanism
(depicted by the cartoon in Fig. 2B) confers substrate specificity, it
also subtracts from the intrinsic binding energy, hence binding
affinity [22]. Furthermore, the carbohydrate portion of the sub-
strate is an unlikely source of binding energy because its parti-
tioning between solvent and the T6PP binding site relies primarily
on the entropic advantage gained from intramolecular multi-site
binding created by the active site [23]. Weak T6P binding is

276
C. Liu et al. / European Journal of Medicinal Chemistry 128 (2017) 274e286
Fig. 2. (A). Left: Superposition of the structures of the T6P complex of the D24 N mutant T6PP from C. neoformans (cyan, PDB: 5DX9) and the apo-T6PP from A. fumigatus (green,
PDB:5DXL). Right: View of the bound T6P ligand and the binding interacts that occur with the phosphate group and with the Glu-Lys-Glu triad of the cap domain. (B). Cartoon
depiction of the trehalose 6-phosphate phosphatase mechanism of substrate binding by induced cap domain-catalytic domain association stabilized by the noncovalent bonding
interactions with the bridging substrate T6P. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
complement the space-restricted, desolvated-binding site of the
closed conformer. Given that we were initially unaware of the key
role played by the inner-glucosyl unit in cap domain binding, the
choice to use it in the template was fortuitous.
simple modular design.
2.2. Inhibitor library synthesis and evaluation
In the design of T6PP inhibitors, we selected an aryl group to
replace the outer glucosyl unit in view of the fact that it carries both
binding site compatibility and synthetic versatility. Specifically, as a
The library of aryl D-glucopyranoside 6-sulfate derivatives (and
related structures), and the strategies and protocols used in their
synthesis are given in Fig. 3 and the Experimental section. The re-
sults from testing the derivatives for T6PP inhibition are given in
Tables 1 and 2.
Our objective in preparing and evaluating the synthetic library
was to identify a tight binding inhibitor and to correlate the binding
affinity with specific features of the inhibitor structure. To begin,
we selected the phenyl derivatives to test the aryl D-glucopyrano-
side 6-sulfate platform with the T6PP ortholog in hand, Bm-T6PP.
The selection of Bm-T6PP to serve in the initial exploratory study
protein ligand, the p-electron cloud of the aromatic ring engages in
electrostatic binding interactions with nearby polar residues [29],
and its departure from solvent gives rise to the desired “hydro-
phobic effect” [30] associated with tight ligand binding. Just as
important, the aromatic ring also serves as an ideal scaffold to
introduce strategically placed substituents. In the sections that
follow, we describe the synthesis and evaluation of the library of
aryl
D-glucopyranoside 6-sulfate derivatives created using this

C. Liu et al. / European Journal of Medicinal Chemistry 128 (2017) 274e286
277
Fig. 3. The library of compounds synthesized for testing as inhibitors of T6PP.
inhibitors with Ki ¼ 320 30
m
M and 800 100
m
M, respectively.
Table 1
Steady-state inhibition constants determined at pH 7.5 and 25 ꢁC for the targeted
inhibitors of T6PP-catalyzed hydrolysis of trehalose 6-phosphate (T6P). See Fig. 3 to
find the chemicals structures of the inhibitors and the Experimental section for
details.
We imagined that the phenyl D-glucopyranoside 6-sulfate ligand
would bind to the active site oriented in a manner analogous to that
of bound substrate, with the glucosyl 6-sulfate substituting for the
glucosyl 6-phosphate and the phenyl ring substituting for the outer
Inhibitor
Bm-T6PP
Ki ( M)
As-T6PP
Ki ( M)
Sb-T6PP
St-T6PP
Mt-T6PP
Ki ( M)
glucosyl unit. To check this assumption, we prepared phenyl b-D-
m
m
Ki (
m
M)
Ki (
mM)
m
glucopyranoside 6-phosphate (7) and tested its substrate activity
by measuring the rate of T6PP-catalyzed phosphate release.
Whereas the glucose 6-phosphate itself shows no substrate activity
(<0.0001 sꢀ1) [28], the corresponding O-phenyl derivative un-
dergoes catalyzed hydrolysis at the rate ca. 0.01 sꢀ1 (Table S1,
Supplementary Information). This activity, although no greater
than 1% that of T6P hydrolysis, suggests that 7 binds, albeit weakly,
to the active site in a “T6P-like” orientation. Furthermore, one
9a
9b
8p
8o
5.3 0.6
80 10
60 10
~200
80 10
350 30
180 20
~200
21
78
13
3
9
2
56
6
~100
>400
>300
>400
280 30
120 20
>400
140 20
T6P Km
(m
M) 360 60a 230 70b 690 70b 310 40b 500 100b
a
Reported in Farelli et al. [19].
Reported in Liu et al. [28].
b
might speculate that the phenyl
anomer, compared to the (accessible)
likely display more activity because the T6P C1 configuration is
To enhance the binding affinity of the corresponding phenyl
glucopyranoside 6-sulfate inhibitor, a nonpolar group was added to
the phenyl ring. The anticipated desolvation of this group into a
D
-glucopyranoside 6-phosphate
a
-
was also fortuitous because it turned out to be the most highly
inhibited among the five T6PP orthologs probed.
b
-anomer tested, would
a
.
The phenyl
a
- (5c) and
b- (8a)
D-glucopyranoside 6-sulfate
D
-
anomers prepared and tested for inhibition of T6P hydrolysis
catalyzed by Bm-T6PP, proved to be reversible competitive

278
C. Liu et al. / European Journal of Medicinal Chemistry 128 (2017) 274e286
Table 2
T6PP) and Salmonella typhimurium (St-T6PP). This effort provided a
more comprehensive picture of T6PP inhibition. Unfortunately, the
group did not include the recently reported fungal T6PP orthologs
[20]. The Ki values measured for the p-octylphenyl (9a) and p-
hexylphenyl (9b) glucopyranoside 6-sulfate inhibitors with the
panel of five T6PP orthologs are listed along with the T6P Km values
in Table 1. It is evident that the pattern of inhibitor recognition is
somewhat different for each enzyme, underscoring the importance
of using the panel of enzymes to evaluate inhibitor-binding
strength and ultimately to identify the structural determinants of
tight binding.
VI/V values (fraction of enzyme that is not bound with the inhibitor) measured at pH
7.5 and 25 ꢁC for the library of inhibitors shown in Fig. 3. The initial velocity V of
T6PP-catalyzed hydrolysis of T6P at half saturation ([T6PP] ¼ Km) is compared to the
initial velocity (VI) measured in the presence of 1 mM inhibitor. See the Experi-
mental section for details.
VI/V
Compound
Bm-T6PP
Mt-T6PP
Sb-T6PP
As-T6PP
St-T6PP
T6S
Trehalose
1
2a
2b
2c
2d
3
4
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
7
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8p
0.13
0.98
0.96
1.12
1
0.99
0.85
0.59
1.05
0.93
0.8
0.42
0.53
0.67
0.68
0.07
0.72
0.85
0.98
0.79
0.52
0.58
0.55
0.74
0.64
0.63
0.87
0.79
1.05
1
0.43
0.83
0.88
0.81
0.88
1.04
0.95
0.96
1.17
0.97
0.91
1.17
1.01
1.16
1.11
0.83
0.83
0.85
0.98
0.94
0.94
0.58
1.09
0.8
0.22
1.01
0.86
0.9
0.92
0.92
1
0.21
0.97
0.97
1
0.01
0.88
1
0.87
0.77
1.09
0.9
Through screening T6PP inhibition by members of the assem-
0.97
0.81
0.96
0.98
0.96
0.94
1.07
0.71
0.96
1.04
0.99
0.86
0.9
0.91
0.76
0.98
0.92
0.9
0.94
0.88
0.83
0.74
0.9
bled library of aryl
D-glucopyranoside 6-sulfate (and related)
compounds (Fig. 3), we were able to scrutinize the design strategy
used in constructing the lead inhibitor 9a. Each inhibitor was tested
(at a concentration of 1 mM) for inhibition of the T6PP orthologs by
separately measuring the initial velocities of T6PP-catalyzed hy-
drolysis at half saturation ([T6P] ¼ Km) in the presence (VI) and
absence (V) of the inhibitor. The VI/V values given in Table 2 reflect
the fraction of enzyme that is not bound with the inhibitor. Under
these conditions VI/V ¼ 0.5 would correspond to a Ki ¼ 2Km or
0.4e1.4 mM for this group of T6PP orthologs. Therefore, inhibition
by substances possessing Ki values as high as 2 mM are detected.
For calibration purposes, the T6PP inhibitors trehalose (Ki > 10 mM)
0.8
0.85
1
0.85
0.75
1
0.76
1
0.84
0.95
0.74
0.79
0.85
0.9
0.94
0.76
0.84
1
0.92
0.77
1.03
0.88
0.96
0.81
1.06
0.85
0.46
0.67
0.04
0.84
1.04
0.64
0.95
0.99
0.97
0.47
0.54
0.8
0.97
0.77
0.81
0.85
0.96
0.89
0.68
0.94
0.85
0.85
0.95
0.87
0.73
0.77
0.79
0.01
and T6S (Ki ¼ 50
T6PP), 180 M (St-T6PP) and 330
included in the screen.
Firstly, we prepared and tested the
-1-N-phenylsulfamide (4) derivatives of
m
M (As-T6PP), 80
m
M (Bm-T6PP), 130
mM (Mt-
m
mM (Sb-T6PP) [19,28]) were
b
-1-O-benzylsulfate (3) and
-glucose to determine
1.27
1
b
D
whether an alternate strategy, wherein a benzylsulfate (or sulfa-
mide) group replaces the glucose 6-phosphate moiety, might also
be used for inhibitor design. However, neither 3 nor 4 displayed
inhibition with the T6PP orthologs (an exception being the weak
Bm-T6PP inhibition observed with 3) (Table 2). An inspection of the
recent X-ray structures of T6PP bound with T6P [20,21] provides
insight into why the glucose-sulfate is better suited for the glucose-
phosphate binding site than is benzylsulfate. Specifically, the C2, C3
and C4 OH functions of the glucose 6-sulfate moiety in T6S are
expected to form hydrogen bonds with the Glu-Lys-Glu triad of the
cap domain like those observed for C2, C3 and C4 OH functions in
the inner glucosyl moiety of the substrate T6P (see Fig. 2).
Secondly, we determined if the O-phenyl group at C1 of the
glucose 6-sulfate template is the best option. Thus, the 1-O-methyl
(5b), O-(n-octyl) (6b), O-benzyl (6a), O-naphthyl (6c) O-biphenyl
(8b) and O-phenyl-triazole (2d and 6d) derivatives were prepared
and screened as were the 1-N-phenyl (2b) and N-phenyl-
thiocarbamoyl (2c) derivatives (Table 2). The benzyl derivative (6a
0.94
1.17
0.67
0.87
0.89
0.69
0.85
0.42
0.99
0.78
0.81
0.9
0.63
0.76
0.02
0.89
0.69
0.68
0.07
suitable sub-compartment of the active site, should in principle,
augment inhibitor binding via the “hydrophobic effect”. n-Hexyl
and n-octyl substituents were selected because each provides a
sufficient number of methylene units for desolvation. In addition,
the conformational flexibility of the long aliphatic chain facilitates
binding to a compatible surface in the active site. The Ki values
determined for the a- and b-anomers of the p-hexylphenyl sub-
strates, 9b and 8o, and those of the p-octylphenyl substrates, 9a and
8p, are given in Table 1. A comparison of Ki values reveals that the
aliphatic group enhances binding, that the octyl group is superior to
the hexyl group, and that inhibitors having the
their anomeric centers display the tightest binding. The Ki of 9a,
determined with the B. malayi T6PP ortholog to be 5.3 0.6 M, is
70-fold smaller than the substrate Michaelis constant. The feasi-
bility of module-based T6PP inhibitor design was thus demon-
strated. However, two questions remained, which are (i) do T6PP
orthologs respond similarly to the inhibitors and (ii) how should
the phenyl group be tailored for inhibitor optimization?
Ki ¼ 160 20
m
M) proved to be a more potent inhibitor than the
phenyl analog (5c Ki ¼ 320 30
mM) towards Bm-T6PP, and but to
a-configuration at
have comparable potency to that of the phenyl derivative towards
the other T6PP orthologs. The O-methyl adduct 5b showed no in-
hibition, and inhibition by the O-(n-octyl) 6b derivative was weak
or absent. Substances containing the larger aryl groups, O-naphthyl
(6c), O-biphenyl (8b) and O-phenyl-triazole (6d) do not display
significant inhibition, with the exception of the modest inhibition
observed for 6c and 8b toward Bm-T6PP.
m
Next, we compared the effectiveness of the O-benzyl group at
enhancing inhibitor binding affinity when it is located at the glu-
cosyl C2 (5d), C3 (5e) or C4 (5f) position. The relatively strong in-
hibition conveyed by the 1-O-benzyl group (6a) towards the Bm-
T6PP is attenuated in these analogs and the modest inhibition
conveyed by the 1-O-benzyl group towards St-T6PP and Sb-T6PP is
lost. These findings demonstrate that attachment of an O-benzyl or
O-phenyl group to C1 of the glucosyl moiety is optimal.
Because the T6PP orthologs targeted for the development of
therapeutics derive from evolutionarily distant organisms (bacte-
ria, fungi and nematodes) and thus have structures that have
diverged a great deal, they are likely to present different and un-
predictable profiles for inhibitor recognition. Therefore, we
expanded our analysis of the structural determinants of 9a and Bm-
T6PP association by adding T6PP orthologs from Ascaris suum (As-
T6PP), Mycobacterium tuberculosis (Mt-T6PP), Shigella boydii (Sb-
Finally, a brief survey of the impact of polar ring substituents on

C. Liu et al. / European Journal of Medicinal Chemistry 128 (2017) 274e286
279
the O-phenyl group at C1 was carried out. In one group of sub-
4. Experimental
stances prepared for this purpose, the ortho, meta and para phenyl
ring positions contain a formyl (8h, 8i, 8j) and a hydroxymethyl
group (8k, 8l, 8m). In another group, nitro, amino and N-acetyl
moieties are present at the para phenyl ring position (8e, 8f and 8g).
Also, the impact of multi-functionalization was tested using sub-
4.1. General
The structures of the inhibitors selected for this study are given
in Fig. 3. Among members of this family, phenyl -glucopyrano-
side (1) and phenyl -glucopyranoside (2a) and octyl -gluco-
a-D
stances containing
a
combination of 2,3-dimethoxy-4-formyl
b-
D
b-D
groups (8c) or two fluoride substituents (8d). The results show
that the formyl substituent has no impact, the hydroxymethyl
substituent increases inhibition but only to a small degree, and the
amino, nitro, N-acetyl and fluoro substituents have only a slight or
no impact on inhibition. Furthermore, the multiple ring sub-
stituents in 8c do not result in increased binding affinity.
pyranoside are commercially available substances and glucose
derivatives 2b [34], 2c [35], 2d [36], 4 [37], glucose 6-sulfate 5a [38],
10 [39], along with trehalose 6-phosphatate (T6P) [40] and treha-
lose 6-sulfate (T6S) [19,28] were synthesized by using known
procedures. The routes utilized for preparation of the other mem-
bers of the T6PP inhibitor library are displayed in Schemes 1e9 and
described in the Experimental section. Either commercially avail-
able or previously synthesized aryl b- and a-D-glucopyranosides
2.3. Inhibition specificity
were employed as starting materials in most of these sequences
(Schemes 2e4). In certain cases, the starting materials were pre-
viously unknown substances. Accordingly, 3-sulfatomethylphenyl
glucoside 11 was prepared from the known aldehyde 10 [39]
(Scheme 1). In addition, aryl glucopyranosides 12e14 and 16
The specificity of T6PP inhibitor 9a was explored using other
HAD phosphatases which, unlike T6PP, are promiscuous in their
activity with phosphorylated carbohydrate metabolites and, as a
result, are predisposed to off-target inhibition. The phosphatases
were selected include (i) E. coli Ybiv [31], which hydrolyzes a wide
variety of phosphates, including glucose 6-, imido-di-, fructose 1-,
ribose 5-, acetyl-, glycerol 1- and glycerol 2-phosphate, (ii) Bacter-
oides thetaiotaomicron BT1713 [32], a phosphatase that hydrolyzes
(Scheme 6) were prepared from 2,3,4,6-tetra-O-acetyl-a-D-gluco-
pyranosyl bromide, and 17e20 (Scheme 9) were prepared from
1,2,3,4,6-penta-O-benzoyl-D-glucopyranose by reaction with the
appropriate phenol followed by chromatography on silica gel to
separate the
6-phosphate (7) was produced starting with commercially avail-
able phenyl -glucopyranoside (2a) (Scheme 5). All synthetic
b and a anomers. Finally, phenyl b-D-glucopyranoside
in addition to its physiological 2-keto-3-deoxy-D-glycero-D-galacto
9-phosphonononic acid (KDN-9-P), 2-keto-3-deoxy-8-phospho-d-
manno-octulosonic acid (KDO-8-P), N-acetylneuraminate 9-
phosphate (Neu5Ac-9-P), pNPP, phosphoenol-pyruvate, and
glucose 6-phosphate, tyrosine-phosphate and gluconate 6-
b-D
substances were shown to be >95% pure by using NMR spectros-
copy and HPLC analysis.
Except where specified, all solvents and reagents were pur-
chased and used without further purification. Analytical thin-layer
chromatography (TLC) was performed on silica gel plates contain-
ing F254 fluorescence indicator and column chromatography was
performed using the indicated eluants on silica gel (230e400
mesh). 1H (300 and 500 MHz), 13C NMR (75 MHz and 125 MHz), and
31P (121.5 MHz) NMR spectra were recorded on Bruker Avance 500
and Bruker Avance III 300 spectrometers. 1H and 13C NMR chemical
phosphate and (iii) Pseudomonas putida KT2440 GmhB (
-manno-heptose-1,7-bisphosphate 7-phosphatase) [33], which
hydrolyzes a variety of hexose and heptose bisphosphate metabo-
lites in addition to its physiological substrate -glycero- -manno-
heptose-1,7-bisphosphate. As the data in Table S2 (Supplementary
Information) show, when used at a concentration of 200 M in the
presence of the respective substrates at limiting concentrations
(equal to 1/3Km), 9a does not inhibit any of the three HAD phos-
phatases tested. We infer from this result that 9a is not a promis-
cuous inhibitor towards HAD phosphatases, including ones that like
T6PP bind and hydrolyze phosphorylated carbohydrate metabo-
lites, but that unlike T6PP, have lax substrate specificities.
D-glycero-
b-D
D
b-D
m
shifts are reported in parts per million (d) relative to tetrame-
thylsilane (TMS; 0.00 ppm) or solvent peaks as the internal refer-
ence, multiplicities are given as s ¼ singlet, d ¼ doublet, t ¼ triplet,
q ¼ quartet, m ¼ multiplet, and coupling constant (J) are in Hz; 31
P
NMR chemical shifts are reported in ppm relative to 50% aq H3PO4
as a standard.
3. Conclusion
Taken together, our findings validate the aryl
D
-glucopyranoside
4.1.1. 3-Sulfatomethylphenyl
b-D-glucopyranoside (3)
6-sulfate template as a promising platform to use in combination
with T6PP X-ray structures for the construction of potent second-
generation inhibitors. We have identified the phenyl group and
the benzyl group as superior in the role of the aryl group. A brief
survey of phenyl ring substituents showed that small polar ones
have little to no impact on binding affinity whereas the long and
flexible nonpolar substituents enhance binding. Future inhibitor
designs will focus on optimizing binding affinity for target ortho-
logs and on creating active site-directed irreversible inhibitors.
A solution of 3-formylphenyl 2,3,4,6-tetra-O-acetyl-b-D-gluco-
pyranoside (10) (0.976 g, 2.2 mmol) in 50 mL of a 1:1 mixture of
THF/EtOH containing NaBH4 (42 mg, 1.1 mmol) was stirred at r.t.
until complete disappearance of starting material occurred (TLC).
The mixture was poured into ice-water, the pH was adjusted to 7.0
and extracted with chloroform. The extracts were dried and
concentrated in vacuo, giving a residue that was dissolved in
anhydrous DMF (4 mL). To this solution at ꢀ20 ꢁC was added a
solution of SO3.pyridine (0.7 g, 4.4 mmol) in 2 mL anhydrous DMF.
Scheme 1. Synthesis of 3.

280
C. Liu et al. / European Journal of Medicinal Chemistry 128 (2017) 274e286
The resulting solution was stirred at r.t. for 12 h and concentrated in
vacuo, giving a residue that was subjected to silica gel chromato-
graph (10:1 to 5:1 DCM/MeOH) to give 3-sulfatomethylphenyl
2,3,4,6-tetra-O-acetyl-
NMR (300 MHz, CDCl3)
(m, 2H), 6.82e6.79 (m,1H), 5.22e5.00 (m, 4H), 4.91 (s, 2H), 4.18 (dd,
J1 ¼12.6 Hz, J2 ¼ 5.4 Hz, 1H), 4.07e4.02 (m, 1H), 3.85e3.79 (m, 1H),
1.95 (s, 3H), 1.93 (s, 3H), 1.92 (s, 3H), 1.91 (s, 3H). 13C NMR (75 MHz,
b
-
D
-glucopyranoside (11) (0.857 g, 73%). 1H
(ppm) 7.14 (t, J ¼ 8.1 Hz, 1H), 6.98e6.96
d
CDCl3)
d (ppm) 170.4, 169.9, 169.3, 169.2, 156.6, 138.5, 129.3, 122.7,
116.4, 116.0, 98.6, 72.5, 71.7, 70.9, 68.9, 68.0, 61.7, 20.5, 20.5, 20.4.
Following addition of sodium (38 mg, 1.64 mmol) to 70 mL of
anhydrous methanol, a solution of 11 (768 mg, 1.6 mmol) in
anhydrous methanol (4.5 mL) was added dropwise. After stirring at
r.t. for 2 h, the mixture was neutralized with solid carbon dioxide,
and concentrated in vacuo, giving a residue that was subjected to
silica gel chromatography to give 0.485 g (78%) of 3.1H NMR
Scheme 2. Synthesis of 5b, 5c, 6a and 6d.
(300 MHz, CD3OD)
d
(ppm) 7.30 (t, J ¼ 8.1 Hz,1H), 7.21e7.20 (m,1H),
7.10e7.06 (m, 2H), 5.01 (s, 2H), 4.95 (d, J ¼ 6.9 Hz, 1H), 3.93 (dd,
J1 ¼ 12.0 Hz, J2 ¼ 1.2 Hz, 1H), 3.74 (dd, J1 ¼ 12.0 Hz, J2 ¼ 4.5 Hz, 1H),
3.50e3.47 (m, 4H). 13C NMR (75 MHz, CD3OD)
d (ppm) 159.1, 139.4,
130.4,122.8,117.3,102.3, 78.0, 77.9, 74.9, 71.3, 70.4, 62.4. HRMS (ESI)
m/z calcd. for C13H18O10S [M-H]ꢀ 365.0542, found 365.0535.
Scheme 3. Synthesis of 5d, 5e and 5f.
4.1.2. Methyl
a-D-glucopyranoside-6-sulfate (5b)
To a solution of methyl
a-D-glucopyranoside (0.58 g, 3.0 mmol)
in anhydrous DMF (9 mL) at ꢀ20 ꢁC, was added a solution of
SO3.pyridine (0.50 g, 3.15 mmol) in 1 mL anhydrous DMF, The
resulting solution was stirred at r.t. for 12 h and concentrated in
vacuo, giving a residue that was subjected to silica gel chromato-
graph to give 0.435 g (50%) of 5b. 1H NMR (300 MHz, CD3OD)
d
(ppm) 4.69 (d, J ¼ 3.9 Hz, 1H), 4.29 (dd, J1 ¼ 10.8 Hz, J2 ¼ 1.8 Hz,
1H), 4.16 (dd, J1 ¼ 10.8 Hz, J2 ¼ 5.7 Hz, 1H), 3.77e3.71 (m, 1H), 3.64
(t, J ¼ 9.2 Hz,1H), 3.45e3.41 (m,1H), 3.43 (s, 3H), 3.56e3.33 (m,1H).
13C NMR (75 MHz, CD3OD)
d (ppm) 101.2, 74.9, 73.4, 71.6, 71.53,
68.3, 55.6. HRMS (ESI) m/z calcd. for C7H14O9S [M-H]ꢀ 273.0208,
found 273.0278.
Scheme 4. Synthesis of 6b, 6c, 8a, 8b, 8e, 8h and 8j.
4.1.3. Phenyl
This substance was prepared starting with phenyl
pyranoside (1) using the same procedure employed for the prep-
aration of 5b. Yield 42%. 1H NMR (500 MHz, CD3OD)
(ppm)
a-D-glucopyranoside-6-sulfate (5c)
a-D-gluco-
d
7.30e7.27 (m, 2H), 7.16e7.15 (m, 2H), 7.01 (t, J ¼ 7.5 Hz, 1H), 5.45 (d,
J ¼ 3.5 Hz, 1H), 4.22 (dd, J1 ¼10.0 Hz, J2 ¼ 4.0 Hz, 1H), 4.17e4.15 (m,
1H), 3.88 (t, J ¼ 9.5 Hz,1H), 3.77e3.70 (m, 1H), 3.59 (dd, J1 ¼10.0 Hz,
J2 ¼ 4.0 Hz, 1H), 3.52 (t, J1 ¼ 10.0 Hz, 1H). 13C NMR (125 MHz,
CD3OD) d (ppm) 158.6, 130.5, 123.5, 118.3, 99.4, 74.6, 73.2, 72.5, 71.1,
67.8. HRMS (ESI) m/z calcd. for C12H16O9S [M-H]ꢀ 335.0407, found
Scheme 5. Synthesis of 7.
335.0438.
Scheme 6. Synthesis of 8c, 8d, 8i and 8n.

C. Liu et al. / European Journal of Medicinal Chemistry 128 (2017) 274e286
281
Scheme 7. Synthesis of 8g.
HRMS (ESI) m/z calcd. for C7H14O9S [M-H]ꢀ 363.0750, found
363.0739.
4.1.6. Methyl 4-O-benzyl-
This substance was prepared starting with methyl 4-O-benzyl-
-glucopyranoside [43] using the same procedure employed for
the preparation of 5b. Yield 51%. 1H NMR (500 MHz, CD3OD)
(ppm) 7.44e7.43 (m, 2H), 7.33e7.30 (m, 2H), 7.27e7.24 (m, 1H),
a-D-glucopyranoside-6-sulfate (5f)
a-D
d
4.93e4.91 (m, 1H), 4.75 (m, 1H), 4.67 (d, J ¼ 3.5 Hz, 1H), 4.25 (s, 2H),
3.81e3.75 (m, 1H), 3.45e3.43 (m, 2H), 3.40 (s, 3H). 13C NMR
Scheme 8. Synthesis of 8k, 8l and 8m.
(75 MHz, CD3OD)
d (ppm) 139.9, 129.4, 129.2, 128.6, 101.2, 79.3,
75.9, 75.5, 73.6, 70.6, 67.8, 55.7. HRMS (ESI) m/z calcd. for C14H20O9S
[M-H]ꢀ 363.0750, found 363.0740.
4.1.4. Methyl 2-O-benzyl-
a-D-glucopyranoside-6-sulfate (5d)
This substance was prepared starting with methyl 2-O-benzyl-
a
-
D
-glucopyranoside [41] using the same procedure employed for
4.1.7. Benzyl
This substance was prepared from benzyl
[44] using the same procedure described for the preparation of 5b.
Yield 47%. 1H NMR (300 MHz, CD3OD)
(ppm) 7.45e7.43 (m, 2H),
a-D-glucopyranoside-6-sulfate (6a)
the preparation of 5b. Yield 43%. 1H NMR (300 MHz, CD3OD)
a-D-glucopyranoside
d
(ppm) 7.46e7.41 (m, 2H), 7.40e7.32 (m, 3H), 4.80 (d, J ¼ 12.0 Hz,
1H), 4.68 (d, J ¼ 12.0 Hz, 1H), 4.66 (d, J ¼ 3.9 Hz, 1H), 4.29 (dd,
J1 ¼ 7.8 Hz, J2 ¼ 2.1 Hz, 1H), 4.17 (dd, J1 ¼ 10.8 Hz, J2 ¼ 5.4 Hz, 1H),
3.79 (t, J ¼ 9.3 Hz,1H), 3.75e3.70 (m,1H), 3.39e3.37 (m,1H), 3.37 (s,
d
7.34e7.30 (m, 3H), 4.88 (d, J ¼ 3.6 Hz, 1H), 4.77 (d, J ¼ 11.7 Hz, 1H),
4.56 (d, J ¼ 11.7 Hz, 1H), 4.26 (dd, J1 ¼ 10.8 Hz, J2 ¼ 3.8 Hz, 1H), 4.15
(dd, J1 ¼ 10.8 Hz, J2 ¼ 5.4 Hz, 1H), 3.83e3.80 (m, 1H), 3.72 (t,
J ¼ 9.3 Hz, 1H), 3.46e3.37 (m, 2H). 13C NMR (75 MHz, CD3OD)
3H), 3.36e3.34 (m, 1H). 13C NMR (75 MHz, CD3OD)
d (ppm) 139.8,
129.4, 129.2, 128.8, 99.2, 80.9, 74.1, 74.0, 71.6, 71.4, 68.2, 55.5. HRMS
(ESI) m/z calcd. for C14H20O9S [M-H]ꢀ 363.0750, found 363.0738.
d
(ppm) 138.7, 129.3, 129.2, 128.7, 99.1, 74.7, 73.2, 71.8, 71.4, 70.3,
62.1. HRMS (ESI) m/z calcd. for C13H18O9S [M-H]ꢀ 349.0593, found
349.0585.
4.1.5. Methyl 3-O-benzyl-
This substance was prepared starting with methyl 3-O-benzyl-
-glucopyranoside [42] using the same procedure employed for
the preparation of 5b. Yield 50%. 1H NMR (300 MHz, CD3OD)
(ppm) 7.44e7.41 (m, 2H), 7.33e7.16 (m, 3H), 4.86 (s, 2H), 4.65 (d,
a-D-glucopyranoside-6-sulfate (5e)
a
-D
4.1.8. n-Octyl
This substance was prepared starting with n-octyl
pyranoside using the same procedure described for the preparation
of 5b. Yield 56%. 1H NMR (300 MHz, CD3OD)
(ppm) 4.33e4.23 (m,
2H), 4.13 (dd, J1 ¼ 11.1 Hz, J2 ¼ 5.6 Hz, 1H), 3.90e3.83 (m, 1H),
3.56e3.44 (m, 2H), 3.38e3.33 (m, 2H), 3.18 (t, J ¼ 7.2 Hz, 1H),
1.64e1.56 (m, 2H), 1.38e1.26 (m, 10H), 0.90 (t, J ¼ 6.6 Hz, 3H). 13C
b-D-glucopyranoside-6-sulfate (6b)
b-D-gluco-
d
J ¼ 3.6 Hz, 1H), 4.28 (dd, J1 ¼ 10.8 Hz, J2 ¼ 1.8 Hz, 1H), 4.16 (dd,
d
J1 ¼10.8 Hz, J2 ¼ 5.4 Hz, 1H), 3.78e3.72 (m, 1H), 3.62e3.60 (m, 1H),
3.55e3.45 (m, 2H), 3.41 (s, 3H). 13C NMR (75 MHz, CD3OD)
d (ppm)
140.5, 129.1, 129.0, 128.4, 101.4, 83.5, 76.2, 73.5, 71.7, 71.4, 68.2, 55.6.
Scheme 9. Synthesis of 8o, 8p, 9a and 9b.

282
C. Liu et al. / European Journal of Medicinal Chemistry 128 (2017) 274e286
NMR (75 MHz, CD3OD)
d
(ppm) 104.3, 77.7, 75.9, 75.0, 71.4, 71.0,
4.1.14. 2,6-Dimethoxy-4-formylphenyl
sulfate (8c)
To a solution of 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bro-
b-D-glucopyranoside-6-
68.2, 33.0, 30.8, 30.6, 30.4, 27.1, 23.7, 14.4. HRMS (ESI) m/z calcd. for
C
14H28O9S [M-H]ꢀ 371.1376, found 371.1373.
mide (0.824 g, 2 mmol) in CH2Cl2 (8 mL), containing tetrabuty-
lammonium bromide (0.76 g, 2 mmol) and syringaldehyde (1.09 g,
6 mmol) was added 8 mL of 1 M NaOH. The resulting mixture was
stirred vigorously at 35 ꢁC for 6 h. The mixture was diluted with
EtOAc and the organic phase was separated, washed three times
with a 1 M NaOH aqueous solution, water and brine, dried and
concentrated in vacuo. The resulting residue was dissolved in 20 mL
of a 1:1 mixture of THF/MeOH containing anhydrous K2CO3 (0.5 g).
The resulting mixture was stirred for 20 h at r.t. and concentrated in
vacuo, giving a residue that was subjected to silica gel chroma-
4.1.9. 1-Naphthyl
b-D-glucopyranoside-6-sulfate (6c)
This substance was prepared starting with 1-naphthyl
b-D-
glucopyranoside (purchased from Carbosynth Limited) using the
same procedure described for the preparation of 5b. Yield 45%. 1H
NMR (500 MHz, CD3OD)
d (ppm) 8.38e8.36 (m, 1H), 7.79e7.77 (m,
1H), 7.50e7.44 (m, 3H), 7.40e7.37 (m, 1H), 7.24e7.22 (m, 1H), 5.10
(d, J ¼ 7.5 Hz, 1H), 4.39 (m, 1H), 4.21 (dd, J1 ¼ 11.0 Hz, J2 ¼ 5.5 Hz,
1H), 3.77e3.67 (m, 2H), 3.59e3.50 (m, 2H). 13C NMR (125 MHz,
CD3OD)
d (ppm) 154.4, 135.8, 128.4, 127.3, 127.1, 127.0, 126.3, 123.2,
tography to give 2,6-dimethoxy-4-formylphenyl
side (12) [46] (364 mg, 53%). 1H NMR (300 MHz, CD3OD/D2O (v/v, 1/
2))
(ppm) 9.91 (s, 1H), 7.39 (s, 2H), 5.24 (d, J ¼ 7.2 Hz, 1H), 4.02 (s,
6H), 3.87e3.78 (m, 2H), 3.65e3.58 (m, 3H), 3.42e3.39 (m, 1H). 13C
NMR (75 MHz, CD3OD/D2O (v/v, 1/2)) (ppm) 195.0, 154.2, 140.6,
b-D-glucopyrano-
123.0, 110.6, 102.6, 77.7, 75.9, 75.0, 71.2, 68.1. HRMS (ESI) m/z calcd.
for C16H18O9S [M-H]ꢀ 385.0593, found 385.0589.
d
4.1.10. (4-Phenyl-1H-1,2,3-triazol-1-yl) b-D-glucopyranoside-6-
sulfate (6d)
d
133.6, 108.4, 103.8, 77.7, 77.0, 74.9, 70.4, 61.6, 57.2.
This substance was prepared starting with 2d using the same
The target 8c was prepared from 12 using the same procedure as
procedure described for the preparation of 5b. Yield 55%. 1H NMR
described for the preparation of 5b. Yield 58%. 1H NMR (300 MHz,
(300 MHz, D2O)
d (ppm) 8.35 (s, 1H), 7.68e7.65 (m, 2H), 7.46e7.42
D2O)
4.30e4.18 (m, 2H), 3.82 (s, 6H), 3.64e3.60 (m, 4H). 13C NMR
(75 MHz, D2O) (ppm) 194.3, 152.6, 138.8, 132.1, 107.1, 102.7, 75.4,
d
(ppm) 9.44 (s, 1H), 6.90 (s, 2H), 5.07 (d, J ¼ 5.7 Hz, 1H),
(m, 3H), 5.74 (d, J ¼ 9.0 Hz, 1H), 4.47e4.43 (m, 1H), 4.37e4.35 (m,
1H), 4.05e4.02 (m, 2H), 3.78e3.74 (m, 2H), 13C NMR (75 MHz, D2O)
d
d
(ppm) 147.5, 129.0, 128.9, 125.6, 121.0, 87.4, 76.7, 75.7, 75.3, 72.2,
74.1, 73.5, 69.1, 66.8, 56.1. HRMS (ESI) m/z calcd. for C15H20O12S [M-
68.8. HRMS (ESI) m/z calcd. for C14H17N3O8S [M-H]ꢀ 386.0664,
H]ꢀ 423.0597, found 423.0599.
found 386.0666.
4.1.15. 3,4-Difuorophenyl
3,4-Difuorophenyl
the same procedure as that for 2,6-dimethoxy-4-formylphenyl
glucopyranoside (12). Yield 45%. 1H NMR (300 MHz, D2O)
(ppm)
b
-D-glucopyranoside-6-sulfate (8d)
4.1.11. Phenyl
b
-D-glucopyranoside-6-phosphate (7)
-glucopyranoside (2a) (1.08 g,
b-D-glucopyranoside (13) was prepared using
To a solution of phenyl
b-D
b-D-
4.0 mmol) in 5.08 mL of a 2.5:1:0.1 mixture of acetonitrile/pyridine/
water at 0 ꢁC was added POCl3 (1.6 mL, 8 mmol). The resulting
solution was stirred at 0 ꢁC for 2 h and poured onto 20 g of ice. After
the ice melted, the pH of the mixture was adjusted to 7.0 by slowly
adding 1 M aqueous NaOH. The resulting solution was concentrated
in vacuo, giving a residue that was subjected to silica gel chro-
matograph (ethyl acetate/isopropyl alcohol/H2O/NH3$H2O ¼ 5/5/2/
d
7.14e7.04 (m, 1H), 7.01e6.94 (m, 1H), 6.85e6.81 (m, 1H), 4.77 (d,
J ¼ 5.1 Hz, 1H), 3.83 (dd, J1 ¼ 12.0 Hz, J2 ¼ 2.0 Hz, 1H), 3.63 (dd,
J1 ¼ 12.0 Hz, J2 ¼ 5.4 Hz, 1H), 3.45e3.31 (m, 4H). 13C NMR (75 MHz,
CD3OD)
d
(ppm) 155.4 (d, JC,F ¼ 6.6 Hz), 150.90 (dd, JC,F1 ¼ 319.1 Hz,
JC,F2 ¼ 13.7 Hz), 147.8 (dd, JC,F1 ¼ 313.8 Hz, JC,F2 ¼ 14.0 Hz), 118.2 (d,
JC,F ¼ 18.7 Hz), 113.9, 107.6 (d, JC,F ¼ 20.3 Hz), 102.8, 78.1, 77.8, 74.1,
71.2, 62.4.
0.2, v/v/v/v) to give 7 (0.38 g, 29%). 1H NMR (300 MHz, D2O)
d (ppm)
7.42e7.37 (m, 2H), 7.16e7.12 (m, 3H), 5.13 (d, J ¼ 7.2 Hz, 1H),
The target 8d was prepared from 13 using the same procedure
4.08e4.06 (m, 2H), 3.68e3.59 (m, 4H). 13C NMR (75 MHz, D2O)
as that for 5b. Yield 33%. 1H NMR (300 MHz, CD3OD)
d (ppm)
d
(ppm) 156.5, 129.9, 123.3, 116.5, 100.2, 75.3, 75.1, 73.0, 68.7,
62.8.31P NMR (121.5 MHz, D2O)
(ppm) 3.32. HRMS (ESI) m/z calcd.
for C12H17O9P [M-H]ꢀ 335.0532, found 335.0529.
7.10e7.01 (m, 1H), 6.94e6.92 (m, 1H), 6.86e6.79 (m, 1H), 4.45 (d,
J ¼ 10.8 Hz, 1H), 4.29e0.20 (m, 1H), 4.03e3.82 (m, 2H), 3.65 (t,
J ¼ 9.0 Hz, 1H), 3.53e3.38 (m, 2H). 13C NMR (75 MHz, CD3OD)
d
d
(ppm) 155.2 (d, JC,F ¼ 4.7 Hz), 150.9 (dd, JC,F1 ¼ 298.4 Hz,
4.1.12. Phenyl b-D-glucopyranoside-6-sulfate (8a)
JC,F2 ¼ 4.7 Hz), 147.74 (dd, JC,F1 ¼ 312.2 Hz, JC,F2 ¼ 12.8 Hz), 118.3 (d,
JC,F ¼ 18.8 Hz), 113.9, 107.8 (d, JC,F ¼ 19.2 Hz), 102.4, 75.8, 75.5, 73.5,
70.0, 68.0. HRMS (ESI) m/z calcd. for C12H14F2O9S [M-H]ꢀ 371.0248,
found 371.0239.
This substance was prepared using starting with 2a using the
same procedure described for the preparation of 5b. Yield 49%. 1H
NMR (300 MHz, CD3OD)
d (ppm) 7.32e7.27 (m, 2H), 7.13e7.10 (m,
2H), 7.04e6.99 (m,1H), 4.90 (d, J ¼ 7.5 Hz,1H), 4.39 (dd, J1 ¼10.8 Hz,
J2 ¼ 1.4 Hz, 1H), 4.19 (dd, J1 ¼ 10.8 Hz, J2 ¼ 5.7 Hz, 1H), 3.69 (t,
J ¼ 7.4 Hz, 1H), 3.51e3.48 (m, 3H). 13C NMR (75 MHz, CD3OD)
4.1.16. 4-Nitrophenyl
This substance was prepared using the same procedure as that
for 5b starting with 4-nitrophenyl -glucopyranoside [47]. Yield
31%. 1H NMR (300 MHz, CD3OD)
b-D-glucopyranoside-6-sulfate (8e)
d
(ppm) 159.1, 130.4, 123.4, 117.8, 102.3, 77.6, 75.9, 74.8, 71.2, 68.1.
b-D
HRMS (ESI) m/z calcd. for C12H16O9S [M-H]ꢀ 335.0437, found
335.0429.
d
(ppm) 8.19 (d, J ¼ 9.3 Hz, 2H),
7.24 (d, J ¼ 9.3 Hz, 2H), 5.08 (d, J ¼ 7.2 Hz, 1H), 3.40 (dd, J1 ¼11.1 Hz,
J2 ¼ 1.8 Hz, 1H), 4.17 (dd, J1 ¼11.1 Hz, J2 ¼ 6.0 Hz, 1H), 3.85e3.80 (m,
4.1.13. 4-Biphenylyl
This substance was prepared starting with 4,4’-biphenyl
glucopyranoside [45] using the same procedure described for the
preparation of 5b. Yield 49%. 1H NMR (500 MHz, CD3OD)
(ppm)
b
-D-glucopyranoside-6-sulfate (8b)
1H), 3.57e3.47 (m, 3H). 13C NMR (75 MHz, CD3OD)
d (ppm) 163.6,
b
-D
-
143.7, 126.6, 117.7, 101.3, 77.3, 75.9, 74.5, 71.0, 68.0. HRMS (ESI) m/z
calcd. for C12H15NO11S [M-H]ꢀ 380.0288, found 380.0284.
d
7.54e7.51 (m, 4H), 7.38e7.34 (m, 2H), 7.26e7.23 (m, 1H), 7.16e7.15
(m, 2H), 4.90 (d, J ¼ 7.0 Hz, 1H), 4.37 (m, 1H), 4.14 (dd, J1 ¼ 11.0 Hz,
J2 ¼ 6.0 Hz, 1H), 3.68 (t, J ¼ 7.3 Hz, 1H), 3.50e3.40 (m, 3H). 13C NMR
4.1.17. 4-Aminophenyl b-D-glucopyranoside-6-sulfate (8f) [48]
To a solution of 8e (120 mg, 0.3 mmol) in DI water (6 mL), was
added Pd(OH)2/C (32 mg, 0.06 mmol). The resulting mixture was
stirred under a H2 atmosphere for 2 h. The mixture was filtered
through Celite pad and the filtrate was concentrated in vacuo,
(125 MHz, CD3OD)
d (ppm) 158.6, 142.0, 136.7, 129.8, 129.0, 127.6,
118.2, 102.4, 77.7, 76.1, 74.9, 71.3, 68.2. HRMS (ESI) m/z calcd. for
C
18H20O9S [M-H]ꢀ 411.0705, found 411.0746.
giving 8f (105 mg, 100%). 1H NMR (500 MHz, D2O)
d (ppm)

C. Liu et al. / European Journal of Medicinal Chemistry 128 (2017) 274e286
283
7.06e7.03 (m, 2H), 6.91e6.88 (m, 2H), 4.98 (d, J ¼ 8.0 Hz, 1H),
4.38e4.35 (m, 1H), 4.29e4.26 (m, 1H), 3.82e3.80 (m, 1H),
concentrated in vacuo giving a residue that was subjected to silica
gel chromatography, giving 8k (61 mg, 64%). 1H NMR (500 MHz,
3.63e3.55 (m, 3H). 13C NMR (125 MHz, D2O)
d
(ppm) 143.9,
D2O) d (ppm) 7.38e7.35 (m, 2H), 7.22e7.21 (m, 1H), 7.15e7.12 (m,
132.4111.3, 111.0, 94.2, 68.3, 66.6, 65.8, 62.0, 59.7. HRMS (ESI) m/z
1H), 5.10 (d, J ¼ 7.5 Hz, 1H), 4.73e4.65 (m, 2H), 4.35e4.32 (m, 1H),
calcd. for C12H17NO9S [M-H]ꢀ 350.0546, found 350.0536.
4.23e4.20 (m, 1H), 3.83e3.82 (m, 1H), 3.65e3.55 (m, 3H). 13C NMR
(125 MHz, D2O)
d (ppm) 122.6, 122.4, 122.2, 116.3, 108.3, 93.5, 68.3,
4.1.18. 4-Acetamidophenyl
b
-D-glucopyranoside-6-sulfate (8g) [48]
66.7, 65.7, 61.9, 59.7, 52.1. HRMS (ESI) m/z calcd. for C13H18O10S [M-
A mixture of 8f (95 mg, 0.27 mmol) in 4.5 mL H2O containing
H]ꢀ 365.0548, found 365.0540.
K2CO3 (112 mg, 0.81 mmol) was stirred at 0 ꢁC before adding Ac2O
(77
m
L, 0.81 mmol) dropwise. Following stirring at for 3 h at 0 ꢁC,
4.1.23. 3-Hydroxymethylphenyl
This substance was prepared from 3-formylphenyl
pyranoside-6-sulfonate (8i) using the same procedure as that for
8k. Yield 80%. 1H NMR (500 MHz, D2O)
(ppm) 7.37e7.36 (m, 1H),
b-D-glucopyranoside-6-sulfate (8l)
the mixture was concentrated in vacuo, giving a residue that was
b-D
-gluco-
subjected to silica gel chromatography, to give 8g (65 mg, 62%). 1H
NMR (300 MHz, D2O)
d
(ppm) 7.34 (d, J ¼ 8.7 Hz, 2H), 7.13 (d,
d
J ¼ 8.7 Hz, 2H), 5.08 (d, J ¼ 7.2 Hz, 1H), 4.38e4.35 (m, 1H), 4.23 (dd,
7.10e7.07 (m, 3H), 5.10 (d, J ¼ 4.5 Hz, 1H), 4.61e4.59 (m, 2H),
J1 ¼11.4 Hz, J2 ¼ 5.4, 1H), 3.87e3.83 (m,1H), 3.62e3.56 (m, 3H), 2.13
4.36e4.34 (m, 1H), 4.20 (dd, J1 ¼11.0 Hz, J2 ¼ 5.0 Hz, 1H), 3.86e3.85
(s, 3H). 13C NMR (75 MHz, D2O)
d
(ppm) 172.9, 154.1, 132.0, 123.9,
(m, 1H), 3.61e3.56 (m, 3H). 13C NMR (125 MHz, D2O)
d (ppm) 126.2,
117.1, 100.5, 75.3, 73.9, 72.8, 69.1, 66.8, 22.6. HRMS (ESI) m/z calcd.
123.0, 119.5, 114.9, 108.6, 108.1, 93.1, 68.2, 66.7, 65.7, 62.0, 59.7, 56.4.
HRMS (ESI) m/z calcd. for C13H18O10S [M-H]ꢀ 365.0548, found
365.0538.
for C14H19NO10S [M-H]ꢀ 392.0651, found 392.0645.
4.1.19. 2-Formylphenyl
This substance was prepared using the same procedure as that
for 5b starting with 2-formylphenyl -glucopyranoside [49].
Yield 39%. 1H NMR (500 MHz, D2O)
(ppm) 10.03 (s, 1H), 7.72e7.64
b-D-glucopyranoside-6-sulfate (8h)
4.1.24. 4-Hydroxymethylphenyl b-D-glucopyranoside-6-sulfate
(8m)
b-
D
d
This substance was prepared from 4-formylphenyl
pyranoside-6-sulfonate (8j) using the same procedure as that for
8k. Yield 58%. 1H NMR (500 MHz, D2O)
(ppm) 7.35e7.33 (m, 2H),
b-D-gluco-
(m, 2H), 7.27e7.17 (m, 2H), 5.20 (d, J ¼ 7.5 Hz, 1H), 4.38e4.35 (m,
1H), 4.26e4.23 (m, 1H), 3.87e3.86 (m, 1H), 3.69e3.58 (m, 3H). 13C
d
NMR (125 MHz, D2O)
108.7, 92.8, 68.3, 66.9, 65.6, 61.9, 59.7. HRMS (ESI) m/z calcd. for
13H16O10S [M-H]ꢀ 363.0386, found 363.0379.
d
(ppm) 186.1, 151.9, 130.2, 121.4, 117.6, 116.1,
7.12e7.10 (m, 2H), 5.09 (d, J ¼ 4.5 Hz, 1H), 4.56e4.55 (m, 2H),
4.34e4.32 (m, 1H), 4.22e4.20 (m, 1H), 3.84e3.83 (m, 1H),
C
3.58e3.54 (m, 3H). 13C NMR (125 MHz, D2O)
d (ppm) 149.0, 127.8,
122.1, 109.5, 93.1, 68.2, 66.7, 65.7, 61.9, 59.7, 56.2. HRMS (ESI) m/z
4.1.20. 3-Formylphenyl -D-glucopyranoside-6-sulfate (8i)
b
calcd. for C13H18O10S [M-H]ꢀ 365.0548, found 365.0538.
3-Formylphenyl b-D-glucopyranoside (14) was prepared from 3-
hydroxybenzaldehyde and 2,3,4,6-tetra-O-acetylglucosyl bromide
using the same procedure as that for 12. Yield 29%. 1H NMR
4.1.25. 4-n-Butylphenyl
4-n-Butylphenyl
4-n-butylphenol using the same procedure as that for 12. Yield 30%
in two steps. 1H NMR (300 MHz, CD3OD)
(ppm) 7.18e7.07 (m, 4H),
b
-D-glucopyranoside-6-sulfate (8n)
b-D-glucopyranoside (16) was prepared from
(300 MHz, CD3OD)
J ¼ 6.3 Hz, 1H), 3.88e3.84 (m, 1H), 3.67 (dd, J1 ¼12.0 Hz, J2 ¼ 5.1 Hz,
1H), 3.48e3.39 (m, 4H). 13C NMR (75 MHz, CD3OD)
(ppm) 193.9,
d (ppm) 9.88 (s, 1H), 7.54e7.34 (m, 4H), 4.96 (d,
d
d
4.98 (d, J ¼ 7.2 Hz, 1H), 3.98e3.94 (m, 1H), 3.80 (dd, J1 ¼ 11.1 Hz,
J2 ¼ 4.5 Hz, 1H), 3.61e3.53 (m, 4H), 2.59 (t, J ¼ 7.7 Hz, 2H), 1.62e1.57
(m, 2H), 1.40e1.33 (m, 2H), 0.96 (t, J ¼ 7.4 Hz, 3H). 13C NMR
159.5, 139.2, 131.3, 125.0, 124.1, 117.7, 102.0, 78.1, 77.8, 74.7, 71.2,
62.3.
The target 8i was prepared from 14 substance was prepared
(75 MHz, CD3OD) d (ppm) 156.6, 138.2, 130.3, 117.6, 102.2, 77.5, 77.3,
using the same procedure as that for 5b. Yield 39%. 1H NMR
74.5, 70.9, 62.1, 35.5, 34.8, 23.0, 14.2.
(300 MHz, D2O)
d
(ppm) 9.83 (s, 1H), 7.63e7.49 (m, 3H), 7.41e7.37
The target 8n was prepared from 16 using the same procedure
(m, 1H), 5.14 (d, J ¼ 6.6 Hz, 1H), 4.33e4.29 (m, 1H), 4.15 (dd,
as that for 5b. Yield 51%. 1H NMR (300 MHz, D2O/CD3OD (v/v, 1/1))
d
J1 ¼11.4 Hz, J2 ¼ 5.7 Hz, 1H), 3.87e3.82 (m, 1H), 3.58e3.50 (m, 3H).
(ppm) 7.21e7.06 (m, 4H), 4.99 (d, J ¼ 6.9 Hz, 1H), 4.39e4.35 (m,
13C NMR (125 MHz, D2O)
d
(ppm) 188.6, 149.8, 130.1, 123.6, 118.0,
1H), 4.24 (dd, J1 ¼ 11.1 Hz, J2 ¼ 5.1 Hz, 1H), 3.78e3.77 (m, 1H),
3.59e3.56 (m, 3H), 2.59 (t, J ¼ 7.7 Hz, 2H), 1.63e1.53 (m, 2H),
1.40e1.30 (m, 2H), 0.93 (t, J ¼ 7.4 Hz, 3H). 13C NMR (75 MHz, D2O/
116.4, 109.6, 92.9, 68.2, 66.8, 65.6, 61.9, 59.7. HRMS (ESI) m/z calcd.
for C13H16O10S [M-H]ꢀ 363.0386, found 363.0384.
CD3OD (v/v, 1/1))
d (ppm) 156.1, 138.8, 130.4, 117.6, 102.1, 76.7, 75.1,
4.1.21. 4-Formylphenyl
b
-D-glucopyranoside-6-sulfate (8j)
-glucopyranoside (15) was
74.1, 70.4, 67.8, 35.2, 34.4, 22.7, 14.2. HRMS (ESI) m/z calcd. for
C
The precursor 4-Formylphenyl
b
-D
16H24O9S [M-H]ꢀ 391.1063, found 391.1075.
synthesized by using a previously described procedure [50]. The
target 8j was prepared starting with 15 using the same procedure
4.1.26. 4-n-Hexylphenyl
hexylphenyl -D-glucopyranoside (18) [51]
To a solution of penta-O-benzoyl- -glucose (2.8 g, 4 mmol) and
b-D-glucopyranoside (17) and 4-n-
as that for 5b. Yield 59%. 1H NMR (300 MHz, D2O)
d
(ppm) 9.81 (s,
a
1H), 7.93 (d, J ¼ 8.7 Hz, 2H), 7.27 (d, J ¼ 8.7 Hz, 2H), 5.30 (d,
J ¼ 6.9 Hz, 1H), 4.46e4.43 (m, 1H), 4.31 (dd, J1 ¼11.4 Hz, J2 ¼ 5.1 Hz,
1H), 4.01e3.97 (m, 1H), 3.73e3.64 (m, 3H). 13C NMR (75 MHz, D2O)
D
4-n-hexylphenol (1.42 g, 8 mmol) in dichloromethane (28 mL)
under argon at 0 ꢁC was added BF3$Et2O (1.67 mL, 13.2 mmol). The
mixture was stirred at 50 ꢁC for 48 h, diluted with water (30 mL)
and stirred for 15 min. The mixture was diluted with dichloro-
methane (60 mL), washed with water, brine, and concentrated in
vacuo, giving a residue that was dissolved in THF/MeOH (100 mL/
20 mL) and cooled to 0 ꢁC. A 3 N solution of aq NaOH (9.52 mL) was
added and the mixture was stirred for 9 h at room temperature,
diluted with water (40 mL), neutralized with AG 50W-X8 resin
(Bio-Rad), filtered, and concentrated in vacuo, giving a residue that
was subjected to silica gel chromatography, giving 4-n-hexylphenyl
d
(ppm) 188.5, 155.3, 126.1, 124.3, 110.0, 92.8, 68.8, 67.5, 66.2, 62.6,
60.3. HRMS (ESI) m/z calcd. for C13H16O10S [M-H]ꢀ 363.0386, found
363.0381.
4.1.22. 2-Hydroxymethylphenyl
b-D-glucopyranoside-6-sulfate
(8k)
To
a solution of 2-formylphenyl b-D-glucopyranoside-6-
sulfonate (8h) (98 mg, 0.26 mmol) in MeOH (2 mL) at 0 ꢁC, was
added NaBH4 (40 mg, 1.03 mmol). The mixture was warmed to
room temperature and stirred for 1 h, then diluted with water, and
b-D-glucopyranoside (17) (237 mg, 17%) and 4-n-hexylphenyl a-D-

284
C. Liu et al. / European Journal of Medicinal Chemistry 128 (2017) 274e286
glucopyranoside (18) (639 mg, 47%).
29.4, 22.6, 13.9. HRMS (ESI) m/z calcd. for C20H32O9S [M-H]ꢀ
447.1689, found 447.1693.
17: 1H NMR (300 MHz, CDCl3/CD3OD (v/v, 1/1))
d (ppm)
7.59e7.45 (m, 4H), 5.39 (d, J ¼ 6.9 Hz, 1H), 4.39e4.26 (m, 2H),
4.06e4.04 (m, 3H), 3.93e3.92 (m, 1H), 3.04 (t, J ¼ 7.7 Hz, 2H),
2.07e2.05 (m, 2H), 1.84e1.79 (m, 6H), 1.38 (t, J ¼ 6.6 Hz, 3H). 13C
4.1.31. 4-n-Octylphenyl
This substance was prepared from 4-n-octylphenyl
pyranoside (20) using the same procedure as that for 5b. Yield 68%.
1H NMR (300 MHz, D2O)
(ppm) 6.96e6.83 (m, 4H), 5.38 (d,
a-D-glucopyranoside-6-sulfate (9a)
a-D-gluco-
NMR (75 MHz, CDCl3/CD3OD (v/v, 1/1))
d (ppm) 155.1, 137.0, 129.0,
116.3, 100.8, 76.1, 75.9, 73.1, 69.4, 61.0, 34.8, 31.4, 31.4, 28.6, 22.3,
13.6.
d
J ¼ 3.6 Hz, 1H), 4.28e4.19 (m, 1H), 3.96e3.90 (m, 2H), 3.76e3.59 (m,
18: 1H NMR (300 MHz, CDCl3/CD3OD (v/v, 1/1))
d (ppm)
3H), 2.3 (t, J ¼ 7.7 Hz, 2H), 1.43e1.42 (m, 2H), 1.24e1.23 (m, 10H),
0.87 (t, J ¼ 6.6 Hz, 3H). 13C NMR (75 MHz, D2O)
d (ppm) 154.8, 136.7,
7.36e7.29 (m, 4H), 5.75 (d, J ¼ 3.6 Hz, 1H), 4.19 (t, J ¼ 9.3 Hz, 1H),
4.10e3.98 (m, 3H), 3.93e3.82 (m, 2H), 2.81 (t, J ¼ 7.7 Hz, 2H),
1.86e1.84 (m, 2H), 1.58e1.57 (m, 6H), 1.56 (t, J ¼ 6.6 Hz, 3H). 13C
129.1, 117.0, 97.8, 72.8, 71.2, 70.5, 68.7, 66.2, 35.0, 31.9, 31.6, 29.6,
29.5, 22.7, 13.9. HRMS (ESI) m/z calcd. for C20H32O9S [M-H]ꢀ
447.1689, found 447.1695.
NMR (75 MHz, CDCl3/CD3OD (v/v, 1/1))
d (ppm) 154.7, 136.8, 129.0,
116.6, 97.7, 73.4, 72.2, 71.6, 69.3, 60.7, 34.8, 31.4, 31.3, 28.6, 22.3,13.6.
4.2. T6PP inhibition measurement using high throughput screening
4.1.27. 4-n-Hexylphenyl
This substance was prepared from 4-n-hexylphenyl
pyranoside (17) using the same procedure as that for 5b. Yield 46%.
1H NMR (300 MHz, D2O/CD3OD (v/v, 1/1))
(ppm) 7.19e7.09 (m,
b-D-glucopyranoside-6-sulfate (8o)
b-D-gluco-
In order to determine the level of inhibition of T6PP, initial ve-
locities of hydrolysis of T6P in its presence (VI) and in its absence (V)
of 1 mM inhibitor were measured and translated into inhibition
activities expressed as the VI/V ratio. T6PP proteins were prepared
by using methods reported previously [19,28]. For the initial ve-
d
4H), 4.99 (d, J ¼ 6.6 Hz,1H), 4.43e4.40 (m,1H), 4.29 (dd, J1 ¼11.1 Hz,
J2 ¼ 5.4 Hz, 1H), 3.80e3.76 (m, 1H), 3.65e3.57 (m, 3H), 2.60 (t,
J ¼ 7.5 Hz, 2H), 1.65e1.60 (m, 2H), 1.38e1.35 (m, 6H), 0.95 (t,
locities determinations (V), 50
well plate was prepared to contain T6P (for Mt-T6PP: 400
T6PP: 200 M; Sb-T6PP: 690 M; St-T6PP: 310 M and As-T6PP:
230 M), buffer (for Mt-T6PP: 25 mM Hepes pH 7.5, 50 mM NaCl,
mL of a solution in each well of a 96-
J ¼ 6.6 Hz, 3H). 13C NMR (75 MHz, CDCl3/CD3OD (v/v, 1/1))
d (ppm)
mM; Bm-
156.6, 138.3, 130.2, 117.7, 102.4, 77.0, 75.4, 74.4, 70.7, 67.9, 35.8, 32.6,
32.5, 29.6, 23.4, 14.4. HRMS (ESI) m/z calcd. for C18H28O9S [M-H]ꢀ
419.1376, found 419.1388.
m
m
m
m
5 mM MgCl2, and 5% glycerol; for Sb-T6PP and St-T6PP: 50 mM
Hepes pH 7.5, 25 mM NaCl, 2 mM MgCl2, and 5% glycerol; for As-
T6PP: 50 mM Tris pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT
and 5% glycerol; for Bm-T6PP: 25 mM Tris pH 7.5, 25 mM NaCl,
2 mM MgCl2, 1 mM DTT) and 5e25 nM of freshly purified T6PP, the
4.1.28. 4-n-Hexylphenyl
This substance was prepared from 4-n-hexylphenyl
pyranoside (18) using the same procedure as that for 5b. Yield 55%.
1H NMR (300 MHz, CD3OD)
(ppm) 7.12e7.05 (m, 4H), 5.44 (d,
a-D-glucopyranoside-6-sulfate (9b)
a-D-gluco-
d
plate was incubated at room temperature for 5 min and a 5
aliquot of the mixture was removed from each and added to a well
of another 96 well plate containing 45 L of the corresponding
buffer to generate a 50 L solution. To each well of the new plate
was added 100 L BioMol Green (Enzo Life Sciences) and the plate
mL
J ¼ 3.6 Hz,1H), 4.29e4.17 (m, 2H), 3.94e3.88 (m, 2H), 3.65e3.52 (m,
2H), 2.56 (t, J ¼ 7.7 Hz, 2H), 1.61e1.54 (m, 2H), 1.33e1.31 (m, 6H),
m
0.91 (t, J ¼ 6.6 Hz, 3H). 13C NMR (75 MHz, CD3OD)
d (ppm) 156.5,
m
138.0, 130.2, 118.2, 99.5, 74.5, 73.1, 72.3, 71.1, 67.8, 36.0, 32.8, 29.9,
23.2, 14.4. HRMS (ESI) m/z calcd. for C18H28O9S [M-H]ꢀ 419.1376,
found 419.1375.
m
was incubated for 30 min. The absorbance of each well at 625 nm
was measured using a SpectraMax i3 Multi-Mode Microplate
Detection Platform. For determining initial velocities in the pres-
ence of inhibitor (VI), solutions containing T6P, buffer, T6PP, all at
the same concentrations as above, and 1 mM inhibitor were incu-
bated in the wells of a 96-well plate and analyzed in the same
manner as described above. The initial velocity data (triplicate in all
cases) were used to calculate the VI/V values displayed in Table 2.
4.1.29. 4-n-Octylphenyl
phenyl -D-glucopyranoside (20) [51]
These substances were prepared using the same procedure as
those for 4-n-hexylphenyl -glucopyranoside (17) and 4-n-hex-
ylphenyl -glucopyranoside (18).
19: Yield 25%. 1H NMR (300 MHz, CD3OD)
b-D-glucopyranoside (19) and 4-n-octyl
a
b-D
a-D
d
(ppm) 7.03e6.92 (m,
4H), 4.80 (d, J ¼ 6.6 Hz,1H), 3.84e3.80 (m,1H), 3.64 (dd, J1 ¼11.4 Hz,
J2 ¼ 3.6 Hz,1H), 3.40e3.35 (m, 4H), 2.47 (t, J ¼ 7.5 Hz, 2H),1.54e1.48
(m, 2H), 1.24e1.23 (m, 10H), 0.83 (t, J ¼ 6.3 Hz, 3H). 13C NMR
4.3. Enzyme inhibition studies
Initial velocities (V0) for T6PP catalyzed hydrolysis of T6P in
presence of different concentrations of inhibitor (0, Ki and 2xKi)
were determined at 25 ꢁC using an EnzCheck Phosphate Assay Kit
(Invitrogen). The assay solutions contained 1 mM MgCl2, 0.1 mM
sodium azide, 1.0 unit/mL purine nucleoside phosphorylase, and
0.2 mM MESG in 50 mM Tris-HCl (pH 7.5). Absorbance changes
(75 MHz, CD3OD) d (ppm) 157.2, 137.9, 130.2, 117.7, 102.5, 78.0, 77.9,
74.9, 71.3, 62.5, 36.1, 33.0, 32.9, 30.6, 30.4, 30.2, 23.7, 14.4.
20: Yield 44%. 1H NMR (300 MHz, CD3OD)
d
(ppm) 7.10e7.09 (m,
4H), 5.46 (d, J ¼ 3.6 Hz, 1H), 3.90 (t, J ¼ 9.3 Hz, 1H), 3.77e3.71 (m,
3H), 3.59 (dd, J1 ¼ 9.6 Hz, J2 ¼ 3.6 Hz, 1H), 3.47 (t, J ¼ 9.2 Hz, 1H),
2.57 (t, J ¼ 7.7 Hz, 2H),1.62e1.58 (m, 2H),1.33e1.32 (m,10H), 0.92 (t,
were monitored at 360 nm (
D
ε ¼ 9.8 mMꢀ1cmꢀ1). The steady-state
J ¼ 6.8 Hz, 3H). 13C NMR (75 MHz, CD3OD)
d (ppm) 156.7, 138.0,
130.2, 118.2, 99.6, 74.9, 74.2, 73.3, 71.5, 62.3, 36.1, 33.0, 32.9, 30.6,
30.4, 30.3, 23.7, 14.4.
kinetic constants for T6PP competitive inhibition constants, Ki,
were determined for inhibitors by fitting initial velocity data,
measured as a function of T6P (0.5 Km to 5Km) and inhibitor (0, Ki,
2Ki) concentration, to the following equation:
4.1.30. 4-n-Octylphenyl
This substance was prepared from 4-n-octylphenyl
pyranoside (19) using the same procedure as that for 5b. Yield 42%.
1H NMR (300 MHz, D2O)
(ppm) 6.97e6.90 (m, 4H), 4.72 (d,
b-D-glucopyranoside-6-sulfate (8p)
b-D-gluco-
V0 ¼ Vmax½Sꢂ=fKmð1 þ ½Iꢂ=KiÞ þ ½Sꢂg
d
where V0 is the initial velocity, Vmax the maximum velocity, [S] the
substrate concentration and Km the Michaelis-Menten constant
calculated for trehalose 6-phosphate, [I] is the inhibitor concen-
tration and Ki the inhibition constant, calculated using SigmaPlot
Enzyme Kinetics Module.
J ¼ 6.3 Hz, 1H), 4.30e4.27 (m, 1H), 4.17e4.13 (m, 1H), 3.61e3.43 (m,
4H), 2.37 (t, J ¼ 7.1 Hz, 2H), 1.44e1.43 (m, 2H), 1.24e1.23 (m, 10H),
0.88 (t, J ¼ 6.2 Hz, 3H). 13C NMR (75 MHz, D2O)
d (ppm) 155.2, 137.3,
129.3, 117.3, 101.7, 75.2, 73.8, 72.8, 68.7, 66.3, 35.0, 31.9, 31.5, 29.5,

C. Liu et al. / European Journal of Medicinal Chemistry 128 (2017) 274e286
285
substrate recognition and catalysis, Proc.Natl. Acad. Sci. U. S. A. 113 (2016)
7148e7153.
Acknowledgements
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This study is supported by NIH grant R21 AI103484. We are
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.02.001.
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