Synthesis and evaluation of an N-acetylglucosamine biosynthesis inhibitor

Article abstract of DOI:10.1016/j.carres.2011.07.004

The structural rationale, synthesis and evaluation of an inhibitor designed to block glucosamine synthesis by competitively inhibiting the action of glutamine: fructose-6-phosphate amidotransferase and subsequently reducing the transformation of any gluco

Full text of DOI:10.1016/j.carres.2011.07.004

Carbohydrate Research 346 (2011) 2294–2299
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Synthesis and evaluation of an N-acetylglucosamine biosynthesis inhibitor
Juliana L. Sacoman a, Rawle I. Hollingsworth b,
⇑
a Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA
b Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The structural rationale, synthesis and evaluation of an inhibitor designed to block glucosamine synthesis
by competitively inhibiting the action of glutamine: fructose-6-phosphate amidotransferase and
subsequently reducing the transformation of any glucosamine-6-phosphate formed to UDP-N-acetylglu-
Received 11 April 2011
Received in revised form 28 June 2011
Accepted 6 July 2011
cosamine are described. The inhibitor 2-acetamido-2,6-dideoxy-6-sulfo-D-glucose (D-glucosamine-6-
Available online 18 July 2011
sulfonate) is an analog of glucosamine-6-phosphate in which the phosphate group in the latter is
replaced with a sulfonic acid group. The inhibitor is designed to function by three different modes which
together reduce UDP-N-acetylglucosamine synthesis. This reduction was confirmed by evaluating the
effect of the inhibitor on bacterial cell-wall synthesis and by demonstrating that it inhibits acetylation
of glucosamine-6-phosphate competitively and by acting as
a surrogate substrate. Inhibition of
glucosamine production or suitably activated glucosamine in bacteria leads to disruption of the
peptidoglycan structure, which results in softening, bulging, deformation, fragility and lysis of the cells.
These modifications were documented by scanning electron microscopy for bacteria treated with the
inhibitor. They were observed for inhibitor concentrations in the 20 mg/mL range for Escherichia coli
and Bacillus subtilis and the 5 mg/mL range for Rhizobium trifolii.
Ó 2011 Elsevier Ltd. All rights reserved.
Amino sugars such as
D
-glucosamine (2-amino-2-deoxy-
D
-glu-
It is of universal importance to prokaryotes and eukaryotes.
Because of its wide occurrence in bacteria and fungi, the produc-
tion of inhibitors for this enzyme is an appealing strategy in the
development of new antibiotics and antifungals.
cose), galactosamine and mannosamine (the galacto- and manno-
isomers, respectively) are characterized by a nitrogen atom being
attached directly to the carbon chain of a carbohydrate molecule.
These are all hexose sugars and are members of the hexosamine
Hexosamines in mammals have great clinical significance.
These compounds are now known to be involved in the establish-
ment and development of many diseases, such as type II diabetes,
Alzheimer’s disease, rheumatoid arthritis and cancer.10–14
Although the molecular basis of these diseases is well appreci-
ated, the development of cures is still a significant challenge. A
limited availability of therapies with a low percentage of cures
is currently available. The development of new drugs that func-
tion by interfering with the hexosamine pathway is an active
strategy. The main enzyme of the hexosamine biosynthetic path-
way (HBP) responsible for glucosamine biosynthesis is called
glutamine fructose-6-phosphate amidotransferase (GFAT), and it
has two structural domains. One of these is for binding glutamine
(N-terminal or glutaminase domain) and another for binding
fructose-6-phosphate (C-terminal or isomerase domain). There is
also a hinge region that allows the interaction of the two sub-
strates. In prokaryotes, GFAT consists of four polypeptide chains,
and it seems to be active as a homodimer,15,16 while in eukary-
otes it is thought to be active as an heterodimer. This enzyme
catalyzes the transfer of the amino group from glutamine to
fructose-6-phosphate producing glutamic acid and glucosamine-
6-phosphate. This enzyme is a thiol protease, and the proposed
mechanism of action is through the formation of an acyl
group. D-Glucosamine is the most common amino sugar, and it is
an integral part of all living systems. It is now known that amino
sugars, make up the most abundant form of organic matter in
the oceans.1 Many bacteria can utilize glucosamine as the only
source of carbon and are able to transform it to fructose-6-phos-
phate and initiate the glycolytic pathway.2 Amino sugars and their
derivatives are present in many glycolipids and most glycopro-
teins, showing the crucial importance of hexosamine biosynthesis
for cell survival.3–8 They are also major components of bacterial
and fungal cell walls.9
The conversion of glucose to glucosamine is a critical biochem-
ical pathway. Fructose-6-phosphate is the species that primes
glucosamine biosynthesis. The main limiting step of this pathway
is catalyzed by the enzyme glucosamine synthase, also known as
glutamine: fructose-6-phosphate amidotransferase or GFAT (EC
2.6.1.16) in eukaryotes. This enzyme catalyzes the conversion of
fructose-6-phosphate in the presence of glutamine into glucosa-
mine-6-phosphate.
⇑
Corresponding author. Tel.: +1 517 4321113; fax: +1 517 353 9334.
E-mail addresses: sacomanj@msu.edu (J.L. Sacoman), holling7@msu.edu (R.I.
Hollingsworth).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.07.004

J. L. Sacoman, R. I. Hollingsworth / Carbohydrate Research 346 (2011) 2294–2299
2295
Fructose-6-phosphate
GFAT
OPO3H
OH
OPO3H
OH
AGM1
AGX1
UTP
GAT
HO
HO
O
HO
HO
O
HO
HO
O
HO
HO
O
OH
UDP
OH
OPO3H
NH2
NH
NH
O
NH
O
O
Glucosamine-6-phosphate
UDP-N-acetylglucosamine
GFAT Glutamine:fructose-6-phosphate amidotransferase
GAT Acetyl-CoA:D-glucosamine-6-phosphate N-acetyltransferase
AGM1 Phospho-N-acetylglucosamine mutase
AGX1 UDP-GlcNAc Pyrophosphorylase
Scheme 1. Biosynthetic steps in the conversion of fructose-6-phosphate to UDP-N-acetylglucosamine.
OH OH
Br
BnOH / H+
Ph3P / Py / CCl4
Na2SO3
HO
HO
O
HO
HO
O
HO
HO
O
OH
NHAc
NH
NH
OBn
OBn
O
O
2
3
4
SO3H
O
SO3H
O
SO3H
O
H2 Pd/C
NH2NH2
HO
HO
HO
HO
HO
HO
OH
H2N
NH2
NH
OBn
OBn
O
5
1
6
Scheme 2. Synthetic route to 2-amino-2,6-dideoxy-6-O-sulfo-D-glucose.
intermediate between the cysteine residue (Cys1) and the
glutamine in the glutaminase domain, releasing ammonia; the
intermediate is hydrolyzed forming glutamic acid and regenerat-
ing the free enzyme. In the isomerase domain, the carbonyl of
fructose-6-phosphate reacts with a lysine residue (Lys603) to
form a Schiff base. This is attacked by ammonia produced in
the glutaminase domain, liberating the free lysine and forming
a 2-imine derivative. Through the action of a glutamic acid resi-
due (Glu488), the substrate is then isomerized to glucosamine-
6-phosphate, the product of the overall reaction.15–17
A structural analog of glucosamine-6-phosphate in which the
phosphate group is replaced with a similar charged group presents
many modes of inhibiting UDP-N-acetylglucosamine synthesis. It
would bind to GFAT inhibiting formation of the glucosamine-6-
phosphate. It would also inhibit GAT by again competing with gluco-
samine-6-phosphate binding. If the similarity is close it would be
converted to an acyl derivative that could bind to AGM1 but not lead
to the formation of N-acetylglucosamine-1-phosphate. Such an
inhibitor could function by three effectively different modes. To this
end, 2-amino-2,6-dideoxy-6-sulfo-
D
-glucose (D-glucosamine-6-sul-
Glucosamine is incorporated into other biomolecules through its
activated form, UDP-N-acetylglucosamine. This is formed by the
successive action of three enzymes on glucosamine-6-phosphate
fonate, compound 1), an analog of
D
-glucosamine-6-phosphate in
which the phosphate group is replaced with a sulfonate group, was
synthesized and evaluated for its ability to inhibit glucosamine syn-
thesis.
(Scheme 1). The first is acetyl-CoA:
D-glucosamine-6-phosphate
N-acetyltransferase (GAT) which converts the amino group to an
acetamido group using acetyl-CoA as the acyl donor. The second
step is the transfer of the phosphate group from the 6-position to
the 1-position by phospho-N-acetylglucosamine mutase (AGM1)
to form N-acetylglucosamine-1-phosphate. The third and last step
is by the action of the enzyme UDP-GlcNAc pyrophosphorylase
(AGX1), which catalyzes the process in which uridine triphosphate
(UTP) reacts with N-acetylglucosamine-1-phosphate to form
UDP-GlcNAc and pyrophosphate.
SO3H
HO
HO
O
OH
NH2
1
The synthetic method used in the synthesis of 1 is illustrated in
Scheme 2.

2296
J. L. Sacoman, R. I. Hollingsworth / Carbohydrate Research 346 (2011) 2294–2299
0.56 mg/mL; Escherichia coli DH5a, 4.72 mg/mL. Complete inhibi-
tion of cell growth was observed for R. trifolii, B. subtilis and
E. coli by the inhibitor at concentrations 5, 20, and 20 mg/mL,
respectively.
In order to evaluate whether the bacterial growth inhibition ob-
served was connected to the disruption of cell-wall synthesis, the
ultra structures of the cell walls were assessed by scanning elec-
tron microscopy. Glucosamine is the primary building block of
peptidoglycan in the cell wall. This is a complex macromolecule
that gives bacteria cells their strength and rigidity and that results
in the characteristic cigar shapes of many bacteria. Inhibition of
peptidoglycan synthesis results in a loss of shape in cells with frag-
ile walls that might be enlarged or those with spherical or irregular
shapes.18,19 Cells treated with glucosamine-6-sulfonate show a
high frequency of lysis and many have bloated irregular shapes
(Fig. 1) when compared to control cells (Fig. 2). More examples
of these morphological changes can be found in Supplementary
data. These results are consistent with the inhibition of peptidogly-
can synthesis. This is accord with the expectation that enough glu-
cosamine would not be available for this because of the several
modes by which glucosamine-6-sulfonate would inhibit N-acetyl-
glucosamine synthesis.
Figure 1. Representative Scanning Electron Microscopy image of the bacterial
strains. R. trifolli: 1.5 mg/mL of inhibitor.
As mentioned earlier the glucosamine-6-phosphate analog can
potentially bind to several enzymes on the hexosamine pathway.
It could inhibit GFAT as a competitive inhibitor and also act as a
competitive and allosteric inhibitor of GAT. It is also possible for
1 to act as a substrate for GAT thus diverting acyl equivalents away
from the synthesis of N-acetylglucosamine. The sulfonated N-
acetylglucosamine thus formed cannot be converted to UDP-N-
acetylglucosamine. To evaluate the latter possibilities, GAT activity
was measured in an assay in which the transfer of acetyl groups
from acetyl-CoA to glucosamine-6-phosphate was evaluated by
measuring coenzyme A formation during the process using 5,50-
dithio-bis(2-nitrobenzoic acid). This forms the highly colored
5-mercapto-2-nitro-benzoic acid on reaction with coenzyme A by
disulfide exchange. This colored substance can be readily moni-
tored at 412 nm. An extract of lysed yeast cells was used as the
source of the enzyme. The results are presented in Figure 3. Com-
pound 1 inhibits the acetylation of glucosamine-6-phosphate by
about 60% at two and four times the substrate concentration and
almost completely at eight times the substrate concentration.
The very small difference observed between the two lower inhibi-
tor concentrations hints that there is another aspect to the interac-
tions besides a simple competition. This was made clear when the
natural substrate was removed and compound 1 was used as a
substrate. It was a very effective acceptor for acetyl groups from
acetyl-CoA under catalysis by GAT at a level of 40% of the native
substrate. The GAT enzyme also presents an allosteric site for
Figure 2. Representative Scanning Electron Microscopy image of the R. trifolli:
control cells.
The D-glucosamine-6-sulfonate concentration that resulted in a
50% reduction in growth of the bacterial cells (IC50) was obtained
by optical density measurement at 600 nm. The results are as fol-
lows: Bacillus subtilis PY74, 2.88 mg/mL; Rhizobium trifolii ANU843,
Figure 3. Glucosamine-6-phosphate acetyltransferase (GAT) activity in presence of 1 mM of glucosamine-6-phosphate and 8, 4, 2 and 1 mM of D-glucosamine-6-sulfonate
inhibitor. Controls: glucosamine-6-phosphate (Glcn-6-P): 1 mM; inhibitor: 1 mM.

J. L. Sacoman, R. I. Hollingsworth / Carbohydrate Research 346 (2011) 2294–2299
2297
binding N-acetylglucosamine-6-phosphate, which is its natural
inhibitor.20,21 This could explain the complete inhibition by 1 at
very high concentrations. The glucosamine-6-sulfonate is not capa-
ble of being transformed to UDP-N-acetylglucosamine-6-phos-
phate because the sulfonate group cannot be removed by the
same mechanism that removes the phosphate group.
The glucosamine-6-phosphate analog was designed to act as a
competitive inhibitor for the isomerase active site and for the en-
zymes GAT and AGM1, leading to a decrease in the production of
N-acetylglucosamine-6-phosphate within the cell. The fragility of
cell walls resulting in cell deformation, and (at higher concentra-
tions) cell lysis, was expected and detected in all strains incubated
with the inhibitor (Fig. 1 and Figs. S1–S7 in the Supplementary
data). Gram-negative strains should be even more susceptible to
the inhibitory effects of these inhibitors since they also depend
on N-acetylglucosamine production for the synthesis of lipid A, a
main constituent of the lipopolysaccharide (LPS). Lipopolysaccha-
rides form regular crystalline arrays in the outer membrane.22
These features make bacteria cells good systems for the evaluation
of the efficacy of these inhibitors. In summary, 2-amino-2,6-dide-
oxy-6-sulfo-D-glucose-inhibited bacterial cell wall biosynthesis is
consistent with expectations. This compound should be a valuable
tool in elucidating the contribution of amino sugars to biochemi-
cal processes and in developing antimicrobial and therapeutic
agents.
Figure 4. View of the active site of the isomerase domain of E. coli in presence of the
fructose-6-phosphate substrate (in red).
Figure 5. (A) 1H NMR spectrum of the pure
a
anomeric form of compound 1 obtained directly after heating in acid and cooling. (B) 1H NMR spectrum after allowing the
product to mutarotate to a 2:1 mixture of
showing a strong molecular ion.
a
and b anomers. (C) 13C NMR spectrum of the mixture of anomers. (D) Negative-ion electrospray-ionization mass spectrum of 1

2298
J. L. Sacoman, R. I. Hollingsworth / Carbohydrate Research 346 (2011) 2294–2299
70% EtOH in water as the eluent. Evaluation of the product by 1H
1. Experimental
1.1. Structural comparison of
NMR spectroscopy (D2O) at this stage revealed the expected loss
of the signal at ꢂ2.1 ppm for the N-acetyl group as well as the sig-
nals between 7.3 and 7.5 ppm for the benzyl group and the appear-
ance of multiplets at 2.9–3.1 ppm characteristic of the protons on
the sulfonated C6 position. The product existed almost exclusively
D
-glucosamine-6-sulfonate (1)
and D-glucosamine-6-phosphate
D
-Glucosamine-6-phosphate contains
a
phosphate group
(—O—PO32ꢀ) attached to the carbon 6 of the sugar structure. The
glucosamine-6-phosphate analog contains a sulfonic acid group
as the
tated to a 2:1 mixture of
while. IR [CaF2 film, cmꢀ1] 3408, 2911, 2592, 1873, 1734 1H
NMR: 5.29, d, J = 3 Hz, H1 ; 4.84, d, J = 8 Hz, H1b; 4.12, t, J = 8 Hz,
H3 ; 3.76, t, J = 8 Hz, H4 ; 3.71, t, J = 8 Hz, H3b; 3.58, t, J = 8 Hz,
H3b; 3.2–3.5, m, H5 and H6
and b; 2.90–3.01, m, H60a and b.
a
anomer when heated in acid and cooled, but this mutaro-
a
and b anomers after standing for a
(—SO32ꢀ) replacing this —O—PO3 group, with the sulfur atom
2ꢀ
a
being directly attached to the C6 of the aminosugar. In its mono-
ionized form the phosphate group is the same in charge and similar
in shape and size to the sulfonic acid group but with an extra atom
connecting the carbon. The analog should easily fit the active site.
The structure of the binding site of glucosamine-6-phosphate
synthase of E. coli (PDB ID: 2vf523) is shown in Figure 4. The active
site of the isomerase domain contains a P-loop, which is composed
by residues 347–352. This loop is responsible for stabilizing the
phosphate group in the correct orientation to allow the transfer
of ammonia to the C2 of fructose phosphate. The P-loop residues
interact with the oxygen atoms of the phosphate group through
hydrogen bonds between the hydroxyl groups of the Ser347,
Ser349 and Thr352 and the amino group of the main chain of
Ser349 and Gln348.16 These interactions do not require the pres-
ence of any metals or other components that would make these
strong interactions; therefore, the replacement of the phosphate
group by a sulfonate group should give a molecular species that
binds comparably well compared to the phosphorylated species.
a
a
a
13C NMR (mixture of anomers): 89.2, 93.0, 72.4, 72.0, 70.0, 69.7,
68.2, 68.1, 56.8, 54.4, 52.2. HRMS: Calcd for C6H12NO7S, m/z
242.0334; found m/z 242.0345. A composite presentation of the
spectral analyses of 1 is shown in Figure 5.
1.2. Evaluation of glucosamine-6-sulfonate activity in bacterial
systems
1.2.1. Activity assay
D-Glucosamine-6-sulfonate was evaluated for its ability to
inhibit cell wall formation in three bacteria strains (E. coli DH5
a
,
R. trifolii ANU843 and B. subtilis PY74). The first two strains are
Gram negative and the last one is Gram positive. This allowed
the efficacy of the compound as an N-acetyl-D-glucosamine syn-
thesis inhibitor over a broad spectrum of bacteria to be evaluated.
The strains cultured in liquid media were treated in the log phase
with the concentrations of the analog varying from 0 to 20 mg/mL.
Their growth rates were evaluated after 6 h, 18 h and 24 h of incu-
bation by monitoring the optical density at 600 nm. The results are
expressed as the concentration that inhibits 50% of the bacteria
growth (IC50) in mg/mL after 24 h of treatment.
1.1.1. Benzyl 2-acetamido-6-bromo-2,6-dideoxy-
glucopyranoside
a-D-
2-Acetamido-2-deoxy-
ture of the - and b-benzyl glycosides as described by Kushida and
Ichiro.24 This consisted of heating 10 g of 2-acetamido-2-deoxy-
D-glucopyranose was converted to a mix-
a
D
-glucopyranose in 200 g of dry benzyl alcohol in the presence of
2 g of Amberlite IR120 (H+) at 60 °C for 3 h. The resin was filtered
1.2.2. Scanning electron microscopy
off and the excess benzyl alcohol was removed under reduced pres-
The bacteria after 24 h of treatment with the inhibitor were shad-
owed with gold and their morphology was visualized by scanning
electron microscopy (JEOL 6300F with field emission (Oxford EDS).
sure at 60 °C. One gram of the 5:1
formed was converted to a corresponding mixture of benzyl 2-acet-
amido-6-bromo-2,6-dideoxy- -glucopyranosides without further
a:b mixture of benzyl glycosides so
D
purification using triphenylphosphine and pyridine as described
by Galemmo and Horton.25 The product was purified by chromatog-
raphy on silica using 2:1 acetone–dichloromethane. Yield 0.42 g
1.2.3. Glucosamine-6-phosphate acetyl transferase activity
assay
The assay to verify if the analog has inhibitory activity over that
of the GAT enzyme we performed the assay as previously de-
scribed.26 Yeast cells were lysed in 0.6 M sorbitol, 0.02 M HEPES–
KOH buffer and used as source of enzyme. Tris–HCl 50 mM, pH
7.4, 1 mM EDTA, 1 mM glucosamine-6-phosphate, 0.5 mM acetyl-
(40%) of the pure
a
anomer: mp 183–184 (1H and 13C NMR data
match that reported).24
1.1.2. 2-Amino-2,6-dideoxy-6-sulfo-
D-glucopyranose
CoA, 0.5 mM of 5,50-dithio-bis(2-dinitrobenzoic acid), 75
yeast lysate and 20, 10, 5, 2.5 mg/mL of inhibitor were mixed up
to 250 L per well to observe if it has any inhibitory activity over
l
L of
Benzyl
2-acetamido-6-bromo-2,6-dideoxy-a-D-glucopyrano-
side (0.4 g) was dissolved in 5 mL of water. Sodium sulfite (0.5 g)
was added and the mixture was heated at 80 °C for 3 h. The solu-
tion was cooled and poured down a column (20 g) of strong base
anion-exchange resin (Dowex Monosphere 550A, OH form). The re-
sin bed was washed with water (200 mL) and then with 5% NaCl
(100 mL). The NaCl wash was concentrated almost to dryness
and then treated with 200 mL MeOH at room temperature. The
mixture was stirred for 10 min and filtered. The filtrate was con-
centrated to a syrup that was dissolved in 0.5 mL of hydrazine
and heated at 90 °C to remove the acetyl group. Excess hydrazine
was removed on a rotary evaporator at 60 °C under high vacuum,
followed by successive evaporation of several 10 mL volumes of
water at the same temperature. The resulting solid was dissolved
in 1 mL of water and a few drops of HCl added to adjust the pH
to between 5 and 6. MeOH (10 mL) was then added followed by
0.4 g of 10% palladium-on-carbon. The mixture was hydrogeno-
lyzed at 30 psi pressure for 5 h to remove the benzyl group yielding
the desired product (110 mg) after filtration and evaporation of
solvent. This was purified on an XAD-7 column (10 ꢁ 1 cm) using
l
the GAT enzyme. The possibility of the inhibitor acting as a sub-
strate of GAT was also investigated by evaluating the result with-
out glucosamine-6-phosphate and with the same concentration
of inhibitor as was used for glucosamine-6-phosphate.
Supplementary data
Supplementary data (scanning electron micrographs) associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.carres.2011.07.004.
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