Design, synthesis and in vitro evaluation on glucosamine-6P synthase of aromatic analogs of 2-aminohexitols-6P

Article abstract of DOI:10.1590/S0103-50532010000400014

The aminosugars are very important structural components of bacterial and fungi cell walls. Glucosamine-6-phosphate synthase (GlmS), which catalyses the first step of the aminosugar biosynthetic pathway i.e. the formation of D-glucosamine-6-phosphate from

Full text of DOI:10.1590/S0103-50532010000400014

J. Braz. Chem. Soc., Vol. 21, No. 4, 680-685, 2010.
Printed in Brazil - ©2010 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Design, Synthesis and In Vitro Evaluation on Glucosamine-6P Synthase
of Aromatic Analogs of 2-Aminohexitols-6P
Danielle F. Dias,a Céline Roux,b Philippe Durand,b Bogdan Iorga,b
Marie A. Badet-Denisot,b Bernard Badetb and Ricardo J. Alves*,a
aFaculdade de Farmácia, Universidade Federal de Minas Gerais,
31970-201 Belo Horizonte-MG, Brazil
bInstitut de Chimie des Substances Naturelles-CNRS, 1 Avenue de la Terrasse,
91198 Gif-sur-Yvette, France
A glicosamina-6-fosfato sintase (GlmS) catalisa a formação de glicosamina-6-fosfato a partir
de frutose-6-fosfato. Esta é a primeira etapa da via biossintética de aminoaçúcares, componentes
estruturais importantes da parede celular de fungos e bactérias. Este trabalho descreve o
planejamento, a síntese e a avaliação da atividade inibitória da GlmS de dois novos compostos
aromáticos enantioméricos, análogos de 2-amino-2-desoxi-D-glucitol-6-fosfato (ADGP) e de seu
epímero 2-amino-2-desoxi-D-manitol-6-fosfato (ADMP), dois importantes inibidores da GlmS.
Os análogos aromáticos apresentaram atividade inibitória modesta, na faixa de mmol L-1.
The aminosugars are very important structural components of bacterial and fungi cell walls.
Glucosamine-6-phosphate synthase (GlmS), which catalyses the first step of the aminosugar
biosynthetic pathway i.e. the formation of D-glucosamine-6-phosphate from D-fructose-6-
phosphate, is therefore an interesting target in the fight against microorganisms. In this work is
described the synthesis of aromatic analogs of 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP)
and its epimer 2-amino-2-deoxy-D-manitol-6-phosphate (ADMP), two important inhibitors of
GlmS. The aromatic analogs displayed modest inhibitory activity against GlmS, with IC50 in the
mmol L-1 range.
Keywords: glucosamine synthase, inhibitors, aminohexitols derivatives
Introduction
GlmS has two catalytic domains, namely, a glutaminase
and an isomerase domains. The glutaminase domain is
responsible for the hydrolysis of glutamine and generation
of ammonia and glutamate. The ammonia released travels
across a hydrophobic tunnel to reach the isomerase domain,
where it is transferred to enzyme-bound Fru-6P.6,7
Several inhibitors of both domains have been
reported.8-11 Inhibitors of the glutaminase domain are
L-glutamine mimics whereas inhibitors of the isomerase
domain have been trying to mimic the putative transition
state intermediates.4 Therefore 2-amino-2-deoxy-D-
glucitol-6-phosphate (ADGP) and 2-amino-2-deoxy-D-
manitol-6-phosphate (ADMP) are, along with D-arabinose-
oxime-6-phosphate, the most potent inhibitors described
thus far.9,10 Nevertheless, these compounds possess weak
in vitro antifungal activity which was attributed to low
cell penetration due to the high hydrophilic character
of these compounds.12 Lipophilic derivatives of ADGP
Systemic fungi infections remain a problem in
modern chemotherapy. Current therapeutic arsenal does
not fulfill all medical needs. The increasing number
of immunocompromised patients, due to anticancer
chemotherapy, organ transplantation and AIDS, along
with drug resistance, makes things more complicated.1,2
Thus the search for new antifungal agents is mandatory.
Glucosamine-6-phosphate synthase (GlmS) catalyses
the transformation of D-fructose-6-phosphate (Fru-6P)
into D-glucosamine-6-phosphate (GlcN-6P). This is, in
turn, converted into UDP-GlcNAc, the nucleotide form
of N-acetylglucosamine that is the building block for the
biosynthesis of chitin.3,4 The enzyme has been validated as a
new target for the development of new antifungal agents.4,5
*e-mail: ricardodylan@farmacia.ufmg.br

Vol. 21, No. 4, 2010
Dias et al.
681
were prepared and some of them did display enhanced in
vitro activity,11 reinforcing the importance of hydrophilic/
lipophilic balance for good activity.
Results and Discussion
Initially, the commercially available protected serine 3
was reacted with LiBH4 in THF/CH3OH at -10 oC to give
the alcohol 4.14 Reaction of 4 with 3-benzoyloxyphenol
under Mitsunobu conditions15 at 80 °C afforded the
corresponding ether 5. Removal of the benzoyl protecting
group was achieved upon treatment with CH3ONa in
CH3OH at 0 °C to afford 6 in high yield. Phosphorylation
with dibenzylphosphite (HPO(BnO)2), in the presence of
NaOH and CBr4,16 gave the aromatic phosphate 7 in 90%
yield. Hydrogenolysis of the benzyl phosphate (H2, Pd-
C, Parr hydrogenator, 40 psi) and subsequent removal of
N-Boc and dimethyl acetal groups with TFA17 followed by
preparative HPLC purification gave the target compounds
1S or 2R, in 19% and 30% overall yield, respectively.
Inhibitory assay against GlmS from E. coli was performed
according to published procedures.18 Results are given
in Table 1.
The replacement of carbohydrate moieties of bioactive
compounds by an aromatic ring has been a useful strategy
for the obtention of active analogs.13 Besides increasing
the lipophilic character, the ring introduces conformational
restriction, which might eventually enhances activity by
selecting the bioactive conformation. The 6-phosphate and
2-amino groups of ADGP have been suggested as crucial
for its enzyme inhibitory potency.11 Substitution of the C3-
C6 backbone chain by a meta phenyloxy ring linking the
two groups was envisaged as one possibility for aromatic
introduction. This was supported by molecular modeling
(docking) which points out a similar distance between C2
and O6 atoms in the two molecules (5.3 Å for ADGP vs
5.7 Å for 2), and the good fitting for some of the aromatic
molecule conformers with the crystallographic structure
of ADGP in the active site of GlmS (E. coli) (Figure 1).
With this in mind we planned the synthesis of
compounds 1 and 2, according to the retrosynthetic analysis
shown in Figure 2.
As shown in Table 1, compounds 1 and 2 were poor
inhibitors of GlmS, displaying IC50 in the millimolar
range.Although the replacement of the hydroxyl groups in
ADGP and ADMP by an aromatic ring can provide higher
lipophilicity to the resulting analogs, as predict by ACD
The synthesis of 1 and 2 were then carried out according
to the synthetic route shown in Figure 3.
Figure 1. Comparison between crystal structure of ADGP (Stick model, colored by heteroatom) and docked conformers of 2 (orange, green and magenta)
in the active site of Glms (E.coli). Potential hydrogen bonds are represented by Blue lines. Conserved crystallographic water molecules are shown as grey
spheres.

682
Design, Synthesis and In Vitro Evaluation on Glucosamine-6P Synthase
J. Braz. Chem. Soc.
OH
OH
R
HO
HO
O
P
OH
R1
O
OH
O
ADGP: R= H; R1= NH2
ADMP: R= NH2; R1= H
HO
O
BnO
P
BzO
O
BnO
O
O
O
N
O
O
P
Boc
N
N
HO
HO
Boc
Boc
O
O
OH
1: R= NH2; R1= H
2: R= H; R1= NH2
R
R1
O
P
BnO
BnO
HO
O
O
O
O
BzO
O
N
O
N
Boc
N
Boc
Boc
Figure 2. Retrosynthetic analysis leading to compounds 1 and 2, aromatic analogs of ADGP and ADMP, respectively.
BocN
BocN
BocN
BocN
O
O
O
HO
HO
O
O
O
BzO
O
a
c
b
4 R (71%)
S (90%)
3 R
S
5 R (67%)
S (76%)
6 R (95%)
S (85%)
O
d
NH2
BocN
O
HO
HO
O
P
O
OH
e
BnO
BnO
O
O
P
2 R (68%)
1 S (48%)
7 R (89%)
S (76%)
O
O
Figure 3. Synthetic route to compounds 1 and 2. Reagents and conditions (a) LiBH4, THF, CH3OH, -10 °C; (b) 3-benzoyloxyphenol, DEAD, PPh3, toluene,
80 °C; (c) CH3ONa/ CH3OH, 0 °C; (d) CBr4, TEBA, 30% NaOH, CH2Cl2, HPO(OBn)2; (e) 1- H2, Pd-C, THF; 2- TFA, CH3OH.
Table 1. Inhibition of GlmS by ADGP and compounds 1-2
Experimental
Compounds
IC50 / (mmol L-1)a
0.294 ( 0.07)
3.156 ( 1.0)
General procedures
ADGP
1
2
The reactions were monitored by thin-layer
chromatography (TLC) performed on silica gel 60 F254
(Merck®). Melting points were determined with a
Microquímica MQAPF 301 apparatus and are uncorrected.
Flash chromatography was performed on silica gel
230-600 mesh (Merck®). NMR spectrum was recorded on a
BrukerAC300orAC500spectrometeratambienttemperature
or under heating at 363K. Chemical shifts (d) were reported
inppmandstandardizedtotheresidualundeuteratedsolvents
as the reference peaks and coupling constants (J) in Hz. The
products 1S and 2R were purified on Atlantis® Prep dC18
OBD preparative column (19 mm × 150 mm, 5 µm) using an
HPLC apparatus (Waters) equipped with a photodiode array
detector. Gradient elution was employed using solvent A
(10mmolL-1NH4OAc),solventB(CH3CN);gradient:0-7min
100% A; 7-9 min linear change to A-B (40:60); 9-30 min,
isocratic elution with 60% B (column rinsing); 30-50 min
100% A (column equilibration). The flow rate was kept at
3.220 ( 1.7)
aValues are means of three experiments, standard deviation is given in
parentheses.
software calculations of Clog D pH 7 (-6.5 for ADGP
and ADMP vs -4.6 for 1 and 2), it is deleterious for their
interaction with the enzyme active site.
Conclusions
In conclusion, we reported the design, synthesis
and evaluation of two chiral aromatic analogs of the
GlmS inhibitors ADGP and ADMP. Although the target
compounds displayed weak inhibitory activity, they
represent a new class of Glms inhibitors whose activity can
be improved by further modification. This is in progress
and will be reported in due time.

Vol. 21, No. 4, 2010
Dias et al.
683
17mLmin-1 andthesampleinjectionvolumewas0.5mL.The
alcohol 4 was synthesized following standard procedure.14,19
DMSO-d6, 363K) d 1.45 (s, 9H, CH3-tBu); 1.47 (s, 3H,
CH3-iPr); 1.53 (s, 3H, CH3-iPr); 3.87 (t, 1H, 2J 9.4Hz, CH2);
3.93 (d, 1H, 2J 8.5Hz, CH2); 4.00 – 4.02 (m, 1H, CH2); 4.05
(t, 1H, 2J 9.4Hz, CH2); 4.13 (br, s, 1H, CH); 6.38 – 6.42 (m,
3H, CH-aromatics); 7.04 (t, 1H, 2J 8 Hz, mCH-aromatic);
8.98 (br, s, 1H, OH). 13C NMR (125 MHz, DMSO-d6,
363 K) d 23.37 (CH3-iPr); 26.17 (CH3-iPr); 27.59 (CH3-tBu);
55.30 (CH); 64.51 (CH2); 66.52 (CH2); 79.02 (C-tBu); 92.76
(C-iPr); 102.08 (CH-aromatic); 105.04 (CH-aromatic);
108.04 (CH-aromatic); 129.16 (CH-aromatic); 150.86
(C=O NBoc); 158.16 (C-ipso phenolic); 159.22 (C-ipso).
Synthesis of (R)-tert-butyl 4-((3-(benzoyloxy)phenoxy)
methyl)-2,2-dimethyl-oxazolidine-3-carboxylate 5R
Diethyl azodicarboxylate (0.20 mL, 1.20 mmol)
was added to a mixture of 3-benzoyloxyphenol (0.25 g,
1.20 mmol), alcohol 4R (0.20 g, 1.00 mmol) and PPh3
(0.31 g, 1.20 mmol) in toluene (8 mL) at room temperature
under an argon atmosphere and the reaction mixture was
stirred for 40 h at 80 °C. The solution was cooled to room
temperature and was washed with 1 mol L-1 NaOH solution
(20 mL) and then H2O (2 × 20 mL). The organic phase was
dried over MgSO4, filtered and evaporated under reduced
pressure. The residue was purified by flash chromatography
on silica gel (n-hexane/EtOAc 9:1) to give the desired ether
Synthesis of (S)-tert-butyl 4-((3-hydroxyphenoxy)methyl)-
2,2-dimethyloxazolidine-3-carboxylate 6S
The S enantiomer was prepared following the same
procedure described above to prepare 6R (0.18 g,
0.42 mmol) to give pure phenol 6S (0.12 g, 85%). mp
110.7-111.6 °C. [α]D20 36.0 (c 1.02, CHCl3). HRMS (ESI+)
calculated for C17H25NO5Na (M+Na), 346.1631. Found:
346.1636. 1H NMR and 13C NMR spectra, as for 6R.
20
5R (0.23 g, 67%). [α]D -49.0 (c 1.0, CHCl3). HRMS
(ESI+) calculated for C24H29NO6Na (M+Na), 450.1893.
Found: 450.1898. 1H NMR (500 MHz, DMSO-d6, 363 K)
d 1.44 (s, 9H, tBu); 1.48 and 1.54 (2s, 6H, iPr); 3.96-4.18
(m, 5H); 6.88-6.94 (m, 3H, CH-aromatics); 7.36 (t, 1H, 2J
7.75 Hz, CH-aromatic); 7.60 (t, 2H, 2J 7 Hz; mCH-benzoyl
group); 7.73 (t, 1H, 2J 7.5 Hz, pCH-benzoyl group); 8.13 (d,
2H, 2J 7.5 Hz, oCH-benzoyl group). 13C NMR (125 MHz,
DMSO-d6, 363K) δ 23.32 (CH3-iPr); 26.17 (CH3-iPr); 27.56
(CH3-tBu); 55.15 (CH); 64.47 (CH2); 67.0 (CH2); 79.09
(C-tBu); 92.81 (C-iPr); 108.15 (C-aromatic); 112.09 (CH-
aromatic); 113.74 (CH-aromatic); 128.24 (mCH-benzoyl
group); 128.80 (C-benzoyl group); 129.10 (oCH-benzoyl
group); 129.37 (mCH-aromatic); 133.19 (pCH-benzoyl
group); 150.86 (C=O NBoc); 151.32 (C-ipso); 158.89
(C-ipso); 163.83 (C=O benzoyl group).
Synthesis of (R)-tert-butyl 4-((3-(bis(benzyloxy)
phosphoryloxy)phenoxy)methyl)-2,2-dimethyloxazolidine-
3-carboxylate 7R
A solution of dibenzylphosphite (0.12 g, 0.46 mmol)
and phenol 6R (0.10 g, 0.31 mmol) in CH2Cl2 (2 mL) was
added to an ice-cold two-phase system consisting of CBr4
(0.053 g, 0.16 mmol), 30% (m/v) aqueous NaOH (100 µL),
H20 (1 mL) and CH2Cl2 (1 mL). The system was stirring for
30 min at room temperature. CH2Cl2 (10 mL) was added
and the organic layer was separated, washed with H2O
(3 × 5 mL), dried over MgSO4 and filtrated. Concentration
followed by flash chromatography on silica gel of the crude
material afforded the product 7R (0.17 g, 89% yield) as
Synthesis of (S)-tert-butyl 4-((3-(benzoyloxy)phenoxy)
methyl)-2,2-dimethyl-oxazolidine-3-carboxylate 5S
The S enantiomer was prepared following the same
procedure described above to prepare 5R (0.21 g, 0.9 mmol)
to give 5S (0.29 g, 76%). [α]D20 49.2 (c 1.05, CHCl3). HRMS
(ESI+) calculated for C24H29NO6Na (M+Na), 450.1893.
Found: 450.1892. 1H NMR and 13C NMR spectra, as for 5R.
20
colorless oil. [α]D -37.0 (c 1.03, CHCl3). HRMS (ESI+)
calculated for C31H38NO8PNa (M+Na), 606.2233. Found:
606.2230. 1H NMR (500 MHz, DMSO-d6, 363 K) d 1.44
(br, s, 9H, CH3-tBu); 1.47 (s, 3H, CH3-iPr); 1.53 (s, 3H,
CH3-iPr); 3.90-4.14 (m, 5H); 5.15-5.17 (m, 4H, CH2-benzyl
group); 6.77-6.64 (m, 3H, CH-aromatics); 7.26 (t, 1H, 2J
8.5 Hz; CH-aromatic); 7.36 (br, s, 10H, CH-benzyl group).
13C NMR (75 MHz, CDCl3) d 23.30; 24.50; 27.00; 27.80
(CH3-iPr); 28.60; 28.70 (CH3-tBu); 56.00; 56.20 (CH); 65.40;
65.6 (CH2); 66.50; 67.10 (CH2); 70.10; 70.20 (CH2-benzyl
group); 80.50; 80.80 (C-tBu); 93.80; 94.30 (C-iPr); 107.20;
107.30 (CH-aromatic); 111.40; 111.7 (CH-aromatic); 112.7;
112.80 (CH-aromatic); 128.20; 128.80 (CH-aromatic benzyl
group); 130.30 (CH-aromatic); 135.60; 135.70 (C-ipso
benzyl group); 151.60; 151.90 (C-aromatic and C=O NBoc),
159.70(C-ipso). 31PNMR(121.5MHz, CDCl3, external85%
H3PO4) d -6.38 and -6.43.
Synthesis of (R)-tert-butyl 4-((3-hydroxyphenoxy)methyl)-
2,2-dimethyloxazolidine-3-carboxylate 6R
To a solution of 5R (0.19 g, 0.44 mmol) in dry CH3OH
(3 mL), was added CH3ONa (24 mg, 0.44 mmol) at 0 °C.
After 30 min. the solvent was removed under reduced
pressure. The residue was purified by flash chromatography
on silica gel (n-hexane/ AcOEt 8:2) to give pure 6R as a
20
white solid, (0.14 g; 95%). mp 110.6-111.6 °C. [α]D -34.5
(c 1.0, CHCl3). HRMS (ESI+) calculated for C17H25NO5Na
(M+Na), 346.1631. Found: 346.1634. 1H NMR (500 MHz,

684
Design, Synthesis and In Vitro Evaluation on Glucosamine-6P Synthase
J. Braz. Chem. Soc.
Synthesis of (S)-tert-butyl 4-((3-(bis(benzyloxy)
phosphoryloxy)phenoxy)methyl)-2,2-dimethyloxazolidine-
3-carboxylate 7S
assayed using the modified microplate Morgan-Elson
method.18 The assay mixture contained, in a final volume
of 100 µL for each well of the microplate, 100 mmol L-1
phosphate buffer, pH 7.2, 50 mmol L-1 KCl, 1 mmol L-1
EDTA, 0.4 mmol L-1 D-Fru-6P (a concentration close to
the Km value was chosen), and 4 mmol L-1 Gln (saturating
concentration). Inhibitors were used at the following
concentrations: ADGP at 0-0.5 mmol L-1, 1 or 2 at
0-7 mmol L-1. Following preincubation at 37 °C for 10 min,
the enzymatic reaction was initiated by the addition of 0.06
unit of GlmS. Precisely 30 min later (initial rate conditions),
the reaction was stopped by the addition of 10 µL of a
1.5% (v/v) acetic anhydride solution in acetone (prepared
daily) followed by 50 µL of a 0.2 mol L-1 borate solution.
After being heated to 80 °C for 30 min, the microplate
(containing the various reaction mixtures) was centrifuged
before addition of 130 µL of Ehrlich reagent (10% (m/v)
solution of p-dimethyl-aminobenzaldehyde in acetic acid/
concentrated HCl mixture (87/13; v/v) to each well. Then,
the amount of GlcN-6P formed was measured from the
absorbance of the Ehrlich adduct at 585 nm (e value was
determined daily from a GlcN-6P standard solution). The
initial rate of formation of Glc-N6P as a function of the
inhibitor concentration allowed the determination of the
IC50 value (inhibition that gives an initial rate equal to 50%
of the rate in the absence of inhibitor).
The S enantiomer was prepared following the same
procedure described above to prepare 7R (0.12 g,
20
0.37 mmol) to give 7S (0.15g, 76% yield). [α]D
37.4 (c 1.01, CHCl3). HRMS (ESI+) calculated for
C31H38NO8PNa (M+Na), 606.2233. Found: 606.2228.
1H NMR and 13C NMR spectra, as for 7R.
Synthesis of 3-(2-(S)-amino-3-hydroxypropoxy)phenyl
dihydrogenphosphate 1
A solution of dibenzylphosphate 7R (0.16 g, 0.27 mmol)
inTHF(2mL)washydrogenolyzedunderhydrogenpressure
(40 psi) in presence of 10% activated Pd-C (40 mg) at room
temperature for 1h. The reaction mixture was filtered and
the THF evaporated under vacuum. TFA (1 mL) was then
added to a solution of the residue in CH3OH (2 mL). The
reaction mixture was stirred at room temperature for 3h.TFA
was eliminated by co-distillation with CH2Cl2 and CH3OH.
The product 1S was purified by preparative RP-HPLC and
obtained as an amorphous white solid (35 mg, 48% yield).
mp 168.0-168.7 °C. [α]D20 7.2 (c 0.5, H2O). HRMS (ESI+)
calculated for C9H14NO6PNa (M+Na), 286.0456. Found:
286.0455. 1H NMR (500 MHz, D2O) d 3.79-3.84 (m, 1H,
CH); 3.92 (dd, 1H, Jgem 12.3 Hz, 2J 6.6 Hz, CH2); 3.99 (dd,
1H, Jgem 12.2 Hz, 2J 4.7 Hz, CH2); 4.26 (dd, 1H, Jgem 10.5 HZ,
2J 6.6 Hz, CH2); 4.36 (dd, 1H, Jgem 10.5 Hz, 2J 3.8 Hz, CH2);
6.77 (dd, 1H, 2J 8.2 Hz, 3J 2.2 Hz, CH-aromatic); 6.89 (d,
Molecular modeling
2
1H, J 8.3 Hz, CH-aromatic); 6.95 (s, 1H, CH-aromatic);
The tridimensional structures of compounds 1 and 2
were constructed using Corina v3.44. Docking studies
were performed on the dimer structure of GlmS (PDB code
2J6H)21 with Gold 4.0,22 using the default parameters and
the GoldScore scoring function. A number of five water
molecules (W166, W251, W254, W256, W257), which are
conserved in all GlmS structures, were considered in the
docking procedure. Ligand 2-deoxy-2-amino-D-glucitol-6-
phosphate (ADGP) from the structure 1MOS 23 was used
as reference in the docking analysis. Raytraced image was
produced with UCSF Chimera24 and POV-Ray.25
7.32 (t, 1H, 2J 8.1 Hz, CH-aromatic). 13C NMR (125 MHz,
D2O) d 52.18 (CH); 58.74 (CH2); 65.01 (CH2); 107.15
3
(d, J 4.6 Hz, CH-aromatic); 108.81 (CH-aromatic);
113.96 (d, 3J 4.6 Hz, CH-aromatic); 129.96 (CH-aromatic);
2
31
154.87 (d, J 6.4 Hz, C-ipso); 158.31 (C-ipso). P NMR
(202.5 MHz, D2O, external 85% H3PO4) d -0.273.
Synthesis of 3-(2-(R)-amino-3-hydroxypropoxy)phenyl
dihydrogenphosphate 2
The R enantiomer was prepared following the same
procedure using 7S (0.13g, 0.22 mmol) to give the desired
phosphate 2R (39 mg; 68% yield). mp 163.9-165.0 °C.
Acknowledgments
20
[α]D -7.5 (c 0.5, H2O). HRMS (ESI+) calculated for
C9H14NO6PNa (M+Na), 286.0456. Found: 286.0451.
The authors thank the CNPq (Conselho Nacional
de Desenvolvimento Científico e Tecnológico - Brasil),
CAPES (Coordenação de Aperfeiçoamento Pessoal de
Nível Superior - Brasil), FAPEMIG (Fundação deAmparo
à Pesquisa do Estado de Minas Gerais - Brasil), CNRS
(Centre National de la Recherche Scientifique - France)
and ICSN (Insitut de Chimie des Substances Naturelles)
for grant and fellowships.
1H NMR and 13C NMR spectra, as for 1.
Determination of enzyme activity
GlmS was purified to homogeneity using a protocol
adapted from the procedure first reported by Badet and
co-workers.20 GlmS activity was spectrophotometrically

Vol. 21, No. 4, 2010
Dias et al.
685
References
13. Raiber, E-A.; Wilkinson, J.A.; Manetti, F.; Bota, M.; Deakin,
J.; Gallagher, J.; Lyon, M.; Ducki, S.W.; Bioorg. Med. Chem.
Lett. 2007, 17, 6321. Finley, J.B.; Atigadda, V.R.; Duarte, F.;
Zhao, J.J.; Brouillette, W.J.; Air, G.M.; Luo, M.; J. Mol. Biol.
1999, 293, 1107.
1. Ascioglu, S.; Rex, J. H.; Pauw, B.; Bennett, J. E.; Bille, J.;
Crokaert, F.; Denning, D. W.; Donnelly, J. P.; Edwards, J. E.;
Erjavec, Z.; Fiere, D.; Lortholary, O.; Maertens, J.; Meis, J. F.;
Patterson, T. F.; Ritter, J.; Selleslag, D.; Shah, P. M.; Stevens,
D. A.; Walsh, T. J.; Clinical Infectious Diseases 2002, 34, 7.
2. Barnes, R. A.; J. Antimicrob. Chemother. 2008, 61, i3.
3. Durand, P; Golinelli-Pimpaneau, B.; Mouilleron, S.; Badet, B.;
Badet-Denisot, M.-A.; Arch. Biochem. Biophys. 2008, 474, 302.
4. Milewski, S.; Biochim. Biophys. Acta 2002, 1597, 173.
5. Borowski, E.; Il Farmaco 2000, 55, 206.
14. Meffre, P.; Durand, P.; Branquet, E.; Le Goffic, F.; Synth.
Commun. 1994, 24, 2147; Garner, P.; Park, J. M.; J. Org. Chem.
1987, 52, 2361.
15. Pavé, G.; Usse-Versluyus, S.; Viaud-Massuard, M. C.;
Guillaumet, G.; Org. Lett. 2003, 5, 4253.
16. Zwierzak, A.; Synthesis 1975, 507.
17. Kobayashi, H.; Ohashi, J.; Fujita, T.; Iwashita, T.; Nakao, Y.;
Matsunaga, S.; Nobuhiro, F.; J. Org. Chem. 2007, 72, 1218;
Meffre, P.; Gauzy, L.; Durand, P.; Branquet, E.; Le Goffic, F.;
Tetrahedron 1996, 52, 11215.
6. Mouilleron, S.; Badet-Denisot, M.-A.; Golinelli-Pimpaneau,
B.; J. Biol. Chem. 2006, 281, 4404.
7. Floquet, N.; Mouilleron, S.; Daher, R.; Maigret, B.; Badet, B.;
Badet-Denisot, M.-A.; FEBS Lett. 2007, 581, 2981.
18. Ghosh, S.; Roseman, S.; Methods Enzymol. 1962, 5, 414;
Burghardt, C. K.; Kochan, J. P.; Eur. Pat. Appl. EP1431396,
2004.
8. Le Camus, C.; Chassagne, A.; Badet-Denisot, M.-A.; Badet,
B.; Tetrahedron Lett. 1998, 39, 287; Le Camus, C.; Badet-
Denisot, M.-A.; Badet, B.; Tetrahedron Lett. 1998, 39, 2571;
Chittur, S. V.; Griffith, R. K.; Bioorg. Med. Chem. Lett. 2002,
12, 2639; Zgódka, D.; Jedrzejczak, R.; Milewski, S.; Borowski,
E.; Bioorg. Med. Chem. 2001, 9, 931.
19. Dondoni, A.; Perrone, D.; Synthesis 1997, 527;Williams, L.;
Zhang, Z.; Shao, F.; Carroll, P. J.; Joullié, M. M.; Tetrahedron
1996, 52, 11673.
20. Badet, B.;Vermoote, P.; Haumont, P. V.; Lederer, F.; Le Goffic,
F.; Biochemistry 1987, 26, 1940.
9. Badet-Denisot, M.-A.; Leriche, C.; Masserie, F.; Badet, B.;
Bioorg. Med. Chem Lett. 1995, 5, 815.
21. Mouilleron, S.; Badet-Denisot, M.-A.; Golinelli-Pimpaneau,
B.; J. Biol. Chem. 2006, 281, 4404.
10. Milewski, S.; Janiak,A. M.;Wojciechowski, M.; Arch. Biochem.
Biophys. 2006, 450, 39.
22. Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.;
Taylor, R. D.; Proteins 2003, 52, 609.
11. Janiak, A. M.; Hoffmann, M.; Milewska, M. J.; Milewski, S.;
Bioorg. Med. Chem. 2003, 11, 1653; Melcer, A. Lacka, I.;
Gabriel, I.; Wojciechowski, M.; Liberek, B.; Wisniewski, A.;
Milewski , S.; Bioorg. Med. Chem. Lett. 2007, 17, 6602.
12. Janiak, A. M.; Cybulska, B.; Szlinder-Richert, J.; Borowski,
E.; Milewski, S.; Acta Biochim. Pol. 2002, 49, 77.
23. Teplyakov, A.; Obmolova, G.; Badet-Denisot, M.-A.; BAdet,
B.; Protein Sci. 1999, 8, 596.
24. Pettersen, E. F.; Goddard, T. D.; Huang,C. C.; Couch,G. S.;
Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E.; J. Comput. Chem.
2004, 25, 1605.
25. http://www.povray.org, accessed in January 2009.
Received: July 1, 2009
Web Release Date: December 21, 2009