Synthesis and evaluation of non-hydrolyzable d-mannose 6-phosphate surrogates reveal 6-deoxy-6-dicarboxymethyl-d-mannose as a new strong inhibitor of phosphomannose isomerases

Article abstract of DOI:10.1016/j.bmc.2009.09.005

Non-hydrolyzable d-mannose 6-phosphate analogues in which the phosphate group was replaced by a phosphonomethyl, a dicarboxymethyl, or a carboxymethyl group were synthesized and kinetically evaluated as substrate analogues acting as potential inhibitors o

Full text of DOI:10.1016/j.bmc.2009.09.005

Bioorganic & Medicinal Chemistry 17 (2009) 7100–7107
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of non-hydrolyzable D-mannose 6-phosphate
surrogates reveal 6-deoxy-6-dicarboxymethyl-D-mannose as a new strong
inhibitor of phosphomannose isomerases
Johanna Foret a,b, Benoit de Courcy c,d,e, Nohad Gresh e, Jean-Philip Piquemal c,d, Laurent Salmon a,b,
*
a Univ Paris-Sud, Equipe de Chimie Bioorganique et Bioinorganique, ICMMO, UMR8182, Bâtiment 420, 15 rue Georges Clemenceau, Orsay F-91405, France
b CNRS, Equipe de Chimie Bioorganique et Bioinorganique, ICMMO, UMR8182, Bâtiment 420, 15 rue Georges Clemenceau, Orsay F-91405, France
c UPMC Univ Paris 06, UMR 7616, Laboratoire de Chimie Théorique, Case Courrier 137, 4 Place Jussieu, F-75252 Paris Cedex 05, France
d CNRS, UMR 7616, Laboratoire de Chimie Théorique, Case Courrier 137, 4 Place Jussieu, F-75252 Paris Cedex 05, France
e Univ Paris Descartes, Laboratoire de Pharmacochimie Moléculaire et Cellulaire, U648 INSERM, UFR Biomédicale des Saints-Pères, 45 rue des Saint-Pères, F-75006 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2009
Revised 28 August 2009
Accepted 3 September 2009
Available online 6 September 2009
Non-hydrolyzable D-mannose 6-phosphate analogues in which the phosphate group was replaced by a
phosphonomethyl, a dicarboxymethyl, or a carboxymethyl group were synthesized and kinetically eval-
uated as substrate analogues acting as potential inhibitors of type I phosphomannose isomerases (PMIs)
from Saccharomyces cerevisiae and Escherichia coli. While 6-deoxy-6-phosphonomethyl-
deoxy-6-carboxymethyl- -mannose did not inhibit the enzymes significantly, 6-deoxy-6-dicarboxym-
ethyl- -mannose appeared as a new strong competitive inhibitor of both S. cerevisiae and E. coli PMIs with
D-mannose and 6-
D
D
Keywords:
Phosphomannose isomerase inhibitors
Sugar phosphates
Malonates
Phosphonates
Km/Ki ratios of 28 and 8, respectively. We thus report the first malonate-based inhibitor of an aldose–
ketose isomerase to date. Phosphonomethyl mimics of the 1,2-cis-enediolate high-energy intermediate
postulated for the isomerization reaction catalyzed by PMIs were also synthesized but behave as poor
inhibitors of PMIs. A polarizable molecular mechanics (SIBFA) study was performed on the complexes
of D-mannose 6-phosphate and two of its analogues with PMI from Candida albicans, an enzyme involved
Polarizable molecular mechanics
Zinc metalloenzymes
in yeast infection homologous to S. cerevisiae and E. coli PMIs. It shows that effective binding to the cat-
alytic site occurs with retention of the Zn(II)-bound water molecule. Thus the binding of the hydroxyl
group on C1 of the ligand to Zn(II) should be water-mediated. The kinetic study reported here also sug-
gests the dianionic character of the phosphate surrogate as a likely essential parameter for strong binding
of the inhibitor to the enzyme active site.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
nidulans.9 Therefore, PMI can be considered as a suitable target
for the development of antibacterial, antiparasitic, and antifungal
Phosphomannose isomerases (PMIs, E.C. 5.3.1.8) are metal-
dependent aldose–ketose isomerases involved in the reversible
agents. Indeed, in most human tissues, its inhibition should not im-
isomerization of
D-fructose 6-phosphate (F6P) to D-mannose 6-
phosphate (M6P) in prokaryotic and eukaryotic cells (Chart 1).1
This reaction is the first step of the mannose metabolism pathway
resulting in the generation of GDP-D-mannose, an important pre-
cursor of many mannosylated structures such as glycoproteins,
nucleotide sugars, glycolipids, fungi cell wall components, and bac-
terial exopolysaccharides.2 PMI was reported to be essential for the
survival or pathogenesis of bacteria from Mycobacterium smegma-
tis3 and Pseudomonas aeruginosa,4 and for the virulence of the
protozoan parasite Leishmania mexicana.5 PMI activity also ap-
peared essential in the case of yeasts like Saccharomyces cerevisiae,6
Candida albicans,7 Cryptococcus neoformans,8 and Aspergillus
Chart 1. Mannose 6-phosphate (M6P) to fructose 6-phosphate (F6P) reversible
isomerization reaction catalyzed by phosphomannose isomerases (PMIs) involving
the postulated 1,2-cis-enediol(ate) high-energy intermediate.
* Corresponding author. Tel.: +33 1 69 15 63 11; fax: +33 1 69 15 72 81.
E-mail address: laurent.salmon@u-psud.fr (L. Salmon).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.005

J. Foret et al. / Bioorg. Med. Chem. 17 (2009) 7100–7107
7101
pair human metabolism, because the bulk of the central metabo-
lism M6P that is utilized for glycoprotein synthesis is likely not de-
rived from F6P but originates from efficient uptake of -mannose
D
through a specific exogenous mannose transporter followed by
phosphorylation by hexokinase.10 In some tissues such as liver
and intestine where this pathway appears less efficient, a defi-
ciency in human PMI activity leads to the severe metabolic disor-
der with hepatic–intestinal presentation CDGS 1b (carbohydrate-
deficient glycoprotein syndrome type 1b),11,12 that is successfully
treated by oral
combining the enzyme inhibitor and
D
-mannose.13 Hence, a bitherapeutical approach
-mannose should alleviate
D
in such cases the side-effects on humans of PMI inhibition. In most
microorganisms, the efficiency of such M6P formation from exog-
enous D-mannose remains to be evaluated. In the pathogenic yeast
C. NEOFORMANS, this pathway of exogenous mannose uptake was re-
ported to be much less efficient than in humans, and PMI was val-
idated as an excellent therapeutic target.8 Finally, in the case of
local treatments such as against yeast infections (candidiasis)
and cutaneous leishmaniasis, PMI inhibition will most likely not af-
fect human metabolism.
Chart 2. Structure of the mannose 6-phosphate (M6P) analogues synthesized and
evaluated in this study: 6-deoxy-6-phosphonomethyl-
1), 6-deoxy-6-dicarboxymethyl- -mannopyranose (6DCM, 2), and 6-deoxy-6-car-
boxymethyl- -mannopyranose (6CM, 3).
D-mannopyranose (6PMM,
D
D
and 6-deoxy-6-carboxymethyl-D-mannose (6CM, 3) as depicted
in Chart 2. The methylated analogues of compounds 1–3 in which
the anomeric hydroxyl group is replaced by a methoxy group in the
The existence of PMI was first demonstrated by Slein.14 It was
then isolated by Gracy and Noltmann in brewer’s yeast and shown
to be a monomeric 45 kDa protein containing an essential zinc cat-
ion.15 S. cerevisiae, Escherichia coli, C. albicans, and human PMIs,
among others, belong to the type I class of three structurally unre-
lated families of proteins identified with PMI activity,16 except for a
very small conserved amino acid sequence motif, which makes up
part of the active site. Type I PMIs have been shown to have very
similar characteristics with a high level of sequence identity
(>40%), particularly in the region of the active site.16,17 Therefore,
conclusions that may be drawn from a type I PMI can most likely
be applied to other type I PMIs, including the prokaryotic and
eukaryotic PMIs studied in this paper. One of the sole high-resolu-
tion X-ray crystal structures of a PMI was reported by Cleasby et al.
a
configuration, respectively, methyl 6-deoxy-6-phosphonometh-
yl- -mannopyranoside, methyl 6-deoxy-6-dicarboxymethyl
-mannopyranoside, and methyl 6-deoxy-6-carboxymethyl-
-mannopyranoside, were previously synthesized and evaluated
a
-D
-a
-D
a-
D
for binding affinity to a cation-independent M6P receptor, namely
the M6P/insulin-like growth factor II, by the Berkowitz et al.29 and
Montero/Morère groups.43 We do not expect the above methylated
analogues to bind tightly to PMI, notably because the a-anomer of
M6P, in contrast to the b-anomer, has no activity as a substrate and
is, at best, a poor inhibitor of the enzyme.49 Because high-energy
intermediate (or transition state) analogues acting as inhibitors
are expected to bind more tightly to the enzyme active site, we also
report the synthesis and kinetic evaluation of phosphonomethyl
analogues of the reaction intermediate postulated for the isomeri-
zation reaction catalyzed by PMIs (Chart 1). Modeling of the com-
plexes of the M6P substrate and of two of its analogues, 6PMM (1)
and 6DCM (2), with the active site of C. albicans PMI was performed
by the SIBFA polarizable molecular mechanics procedure devel-
oped by some of us.50 It sheds light on the essential interactions
taking place between PMI and these ligands at both the Zn-binding
site and the cavity entrance which has two cationic residues,
Arg304 and Lys310.
for C. albicans.18 We previously reported 5-phospho-
D-arabi-
nonohydroxamic acid as the strongest known PMI inhibitor,19 as
well as the theoretical structure of the inhibitor bound to the zinc
cofactor at the active site of the enzyme from C. albicans.20 How-
ever, phosphorylated compounds are likely to be of limited thera-
peutical interest, not only because of their ionic character which do
not allow them to cross cells membranes, but also because of their
high sensitivity to hydrolysis by phosphatases.
For these reasons, numerous analogues of phosphorylated mol-
ecules containing a phosphate surrogate were reported in the liter-
ature as enzyme inhibitors, ligand of protein receptors, or
molecular tools for the study of nucleic acid metabolism, including
phosphorothioate,21,22 phosphonomethyl (or phosphonate),23–35
fluoroalkyl phosphonate,29,36 sulfonate,37 malonyl ether,29,38–42
malonate,29,39,40,43,44 fluoroalkyl malonates,45,46 and carboxy-
methyl (or carboxylate) analogues.43 Numerous phosphate surro-
gates were notably used in the mechanistic study of 3-
dehydroquinate synthase.47,48 In the case of ribose-5-phosphate
2. Results and discussion
2.1. Synthesis of enzyme inhibitors
The dipotassium salt of 6-deoxy-6-phosphonomethyl-
D-man-
nopyranose24 (6PMM, 1, Chart 2) was obtained from the commer-
cially available methyl
a-D-mannopyranoside in 30% yield (seven
steps) using reported procedures.23,24,51
Synthesis of the disodium salt of 6-deoxy-6-dicarboxymethyl-
isomerase, we reported 4-deoxy-4-phosphonomethyl-D-erythro-
D
-mannopyranose (6DCM, 2) was achieved in four steps from the
nate as the only known efficient hydrolytically stable competitive
inhibitor of an aldose–ketose isomerase to date.30 In the case of
phosphomannose isomerase, no such hydrolytically stable sub-
strate or reaction intermediate analogues were evaluated to date
regarding their possible enzyme inhibitory properties.
known benzyl 2,3,4-tri-O-benzyl-
a-D
-mannopyranoside 452 by
the route shown in Scheme 1. The synthetic strategy, similar to
that reported for the synthesis of the methyl mannopyranoside
analogue of 2,29,43 involves the displacement of sugar triflate by
dimethylmalonate as the key step to accessing the title compound.
Indeed, triflate compounds are known to be very versatile electro-
philes in making sugar derivatives with a whole range of function-
ality in place of phosphorous.53,54 A benzyl instead of a methyl
group was chosen as the protecting group of the hydroxyl function
on C1 in 4 in order to achieve its deprotection at room temperature
without hydrolysis and decarboxylation of the dimethylmalonate
function. Trifluoromethanesulfonic anhydride was added to the
In our general search for isosteric and isoelectronic non-hydro-
lyzable inhibitors of aldose–ketose isomerases of potential thera-
peutical interest, we report here the synthesis and kinetic
evaluation of three mimics of the PMI substrate M6P in which
the phosphate group has been replaced by a phosphonomethyl, a
dicarboxymethyl (or 2-malonyl), and a carboxymethyl group, to
give, respectively, 6-deoxy-6-phosphonomethyl-
(6PMM, 1), 6-deoxy-6-dicarboxymethyl- -mannose (6DCM, 2),
D
-mannose24
D

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J. Foret et al. / Bioorg. Med. Chem. 17 (2009) 7100–7107
tained in 50% yield from 1 (no yield nor data were reported in
the literature25 for its synthesis from 6-deoxy-6-phosphonometh-
yl-
-glucose58). Alternatively, evaporation under high vacuum
and lyophilization yielded the synthon 5-deoxy-5-(dihydro-
genophosphonomethyl)-
-arabinono-1,4-lactone 1155 in 50% yield
from 1 (lit.55 71% yield from 6-deoxy-6-phosphonomethyl-
-glu-
D
D
D
cose.58 1H, 13C, and 31P NMR spectral data matched that re-
ported55). Subsequent nucleophilic addition of aqueous
hydroxylamine or aqueous hydrazine followed by lyophilization,
gave, respectively, the dihydroxylammonium salt of 955 (yield
100%, lit.55 72% for the disodium salt of 9) or the dihydrazinium salt
of 10 (yield 100%).
We were also interested in the synthesis and kinetic evaluation
of the respective 2-malonyl analogues of the reaction intermediate
postulated for the isomerization reaction catalyzed by PMIs. How-
ever, oxidative cleavage of 2 according to the Spengler–Pfannenst-
iel conditions described above followed by cation exchange on a
DowexÒ resin (H+ form) did not yield the expected 5-deoxy-5-
Scheme 1. Synthesis of the M6P analogues 6-deoxy-6-dicarboxymethyl-D-manno-
pyranose (6DCM, 2) and 6-deoxy-6-carboxymethyl-D-mannopyranose (6CM, 3)
from benzyl 2,3,4-tri-O-benzyl- -mannopyranoside 4. Reagents and conditions:
a-D
(a) (CF3SO2)2O, (iPr)2EtN, CH2Cl2, ꢀ40 °C, 3 h; (b) H2C(COOMe)2, THF, NaH, reflux,
15 h, 64% (two steps); (c) H2 (15 bar), Pd(OH)2/C, MeOH, rt, 2 d, 94%; (d) 1—NaOH
1 M, rt, 15 h, 2—DowexÒ-50WX8 (H+), 3—DowexÒ-50WX8 (Na+), 99%; (e) 1—
DowexÒ-50WX8 (H+), H2O, reflux, 18 h, 2—DowexÒ-50WX8 (Na+), 100%.
primary alcohol 4 in the presence of diisopropylethylamine in
dichloromethane to form benzyl 2,3,4-tri-O-benzyl-6-O-trifluoro-
dicarboxymethyl-D-arabinonic acid in pure form. Furthermore,
methanesulfonyl-a-D-mannopyranoside 5. Following work-up,
subsequent lyophilization yielded several lactone derivatives as a
consequence of the three carboxylic acid groups present in the
molecule.
compound 5 was used immediately for the nucleophilic substitu-
tion with dimethylmalonic acid in the presence of sodium hydride
in THF to give benzyl 2,3,4-tri-O-benzyl-6-deoxy-6-dimethylmalo-
nate-
a-D-mannopyranoside 6 in 64% yield (two steps). Hydrogen-
2.2. Kinetic evaluation of enzyme inhibitors
olysis over Pd(OH)2/C afforded 6-deoxy-6-dimethylmalonate-
D
-
mannopyranose 7 in 94% yield, which, upon hydrolysis in 1 M so-
dium hydroxide aqueous solution, cation-exchange chromatogra-
phy on a DowexÒ resin column (H+ form, then Na+ form) and
lyophilization gave the malonate M6P analogue 2 in 99% yield.
Compound 2 also served as the starting compound for the syn-
Kinetic studies were performed on E. coli and S. cerevisiae PMIs
as model targets for the development of PMI-inhibiting antibacte-
rial and antifungal agents, respectively. Inhibition parameters of
M6P analogues 6PMM (1), 6DCM (2) and 6CM (3) for the M6P to
F6P isomerization reaction catalyzed by these two type I PMIs
(Table 1) were determined using the phosphoglucose isomerase/
thesis of the sodium salt of 6-deoxy-6-carboxymethyl-D-mannopy-
ranose (6CM, 3, Scheme 1). The carboxylate M6P analogue 3 was
obtained in one step in quantitative yield through simple decar-
boxylation of 2 in aqueous medium under reflux in the presence
of DowexÒ resin (H+ form).
D-glucose-6-phosphate dehydrogenase (PGI/G6PDH) coupled en-
zymes method (50 mM Hepes buffer, pH 7.1, 25 °C) in which PMI
activity was spectrophotometrically assayed at 340 nm (NADPH
formation) as previously described.19 Following isomerization of
As depicted in Scheme 2, 1 was also used as a starting
M6P to F6P by PMI, then to D-glucose 6-phosphate by PGI, D-gluc-
compound for the synthesis of 5-deoxy-5-phosphonomethyl-
arabinonate (5PMAA, 825), 5-deoxy-5-phosphonomethyl-
-arabi-
nonohydroxamic acid (5PMAH, 955), and 5-deoxy-5-phosphonom-
ethyl- -arabinonohydrazide (5PMAHz, 10). As reported for the
D-
onolactone 6-phosphate and NADPH were formed by G6PDH with
NADP+ as the cofactor. Both auxiliary enzymes were added in ex-
cess so that the rate-limiting reaction was the PMI-catalyzed isom-
erization of M6P to F6P.
Both phosphonomethyl (6PMM, 1) and carboxymethyl (6CM, 3)
substrate analogues gave high IC50 values, respectively, 4.7 and
4.6 mM against EcPMI, and 1.6 and 0.9 mM against ScPMI. Both
compounds therefore behave as quite weak inhibitors of the en-
zymes, thus indicating that the phosphonomethyl and carboxy-
methyl functions are not good phosphate surrogates. In the case
of the carboxymethyl analogue (6CM, 3), the monoanionic state
D
D
synthesis of 5-phospho-
D
-arabinonate from 6-phosphate
D-fruc-
tose56,57 or 6-phosphate
D
-glucose,57 oxidative cleavage of 1 by
molecular oxygen in 0.5 M sodium hydroxide aqueous solution
(Spengler–Pfannenstiel conditions) followed by cation exchange
on a DowexÒ resin (H+ form) yielded 5-deoxy-5-(dihydrogeno-
phosphono)-
change on a DowexÒ resin (Na+ form), the trisodium salt of 5-
deoxy-5-phosphonomethyl-
-arabinonate (5PMAA, 825) was ob-
D-arabinonic acid (not isolated). Following cation ex-
D
Scheme 2. Synthesis of 5-deoxy-5-phosphonomethyl-
D
-arabinonate (5PMAA, 8), 5-deoxy-5-phosphonomethyl-
D-arabinonohydroxamic acid (5PMAH, 9), and 5-deoxy-5-
phosphonomethyl-
D
-arabinonohydrazide (5PMAHz, 10) from the synthon 5-deoxy-5-(dihydrogenophosphono)-D-arabinono-1,4-lactone (5PMAL, 11). Reagents and
conditions: (a) 1—O2, NaOH 0.5 M, rt, 7 d, 2—concd HCl, 3—satd Ba(OH)2, 4—DowexÒ-50WX8 (H+); (b) DowexÒ-50WX8 (Na+), 50% (two steps); (c) lyophilization, 50%
(two steps); (d) NH2OH/H2O, rt, 20 min, 100%; (e) H2NNH2/H2O, rt, 20 min, 100%.

J. Foret et al. / Bioorg. Med. Chem. 17 (2009) 7100–7107
7103
confirm earlier findings on other targets,29,39,40,43,47 namely that
the hydrolase-resistant 2-malonyl function is an efficient mimic
of the phosphate group. Although the structure of a malonate as
a phosphate bioisoster is chemically different, molecular dynamic
simulations previously performed by one of us revealed that the
malonate group occupies only about 13% more volume than the
phosphate.39 The crucial point thus appears to be the dianionic ion-
ization state of the 2-malonyl function (ethyl malonic acid has
pKa1 = 3.0 and pKa2 = 5.8),60 as likely is the phosphate function at
pH 7.1 used for the kinetics.
Table 1
Inhibition parameters of M6P analogues 1–3 for the M6P to F6P isomerization
catalyzed by E. coli and yeast PMIsa
Inhibitor
Name
E. coli PMIb
IC50 (Ki) ( M)
S. cerevisiae PMIc
IC50 (Ki) ( M)
l
l
1
2
6PMM
6DCM
4700 800
199
1610 90
18.1 0.6
(10.5 0.5)
900 100
ndd
5
(115 5)
4600 400
>3000
370 20
>3000
3
8
9
10
6CM
5PMAA
5PMAH
5PMAHz
2200 200
ndd
Phosphonomethyl mimics of the 1,2-cis-enediolate high-energy
intermediate postulated for the isomerization reaction catalyzed
by PMIs, namely 5PMAA (8), 5PMAH(9), and 5PMAHz (10), were
also evaluated against PMIs from E. coli and S. cerevisiae (9 only)
using the same conditions as reported above (Table 1). With IC50
values above 3 mM against E. coli PMI, both 5PMAA (8) and
5PMAHz (10) behave as quite weak inhibitors. In the case of
5PMAA (8), the IC50 value obtained is sixfold higher than the value
of 0.5 mM we determined for its phosphate analogue, namely 5-
a
PMI activity assays were achieved using the PGI/G6PDH coupled enzymes
method (50 mM Hepes buffer, pH 7.1, 25 °C) as previously described.19
b
Km (M6P) = 910 80
Km (M6P) = 290 30
nd: not determined.
l
l
M (lit.68 1.21 mM).
c
M (lit.19 121 M; lit.17 650 M; lit.69 1350
l l lM).
d
of this phosphate surrogate would likely explain its low binding
affinity to the PMI active site. With a generally accepted pKa2 of
about 7.5–8 for phosphonate compounds as compared to 6.5 for
phosphate compounds,55,59 6PMM (1) could be monoprotonated
at the pH used for the kinetics (7.1), hence a lowered affinity to
the PMI active site.
In the opposite, the 2-malonyl substrate analogue (6DCM, 2)
displayed low IC50 values on EcPMI, and particularly on ScPMI,
respectively, 199 and 18.1 lM (Table 1). Double reciprocal plot of
the initial reaction velocity Vo versus M6P concentration at differ-
ent inhibitor concentrations revealed that 6DCM (2) is a competi-
tive inhibitor of the isomerization reaction catalyzed by EcPMI
and ScPMI as depicted in Figure 1A (for ScPMI). Secondary plot of
the Km/Vmax ratio obtained at different inhibitor concentrations
phospho-
value obtained for 5PMAHz (10) is much higher than the value of
D
-arabinonate, a bad PMI inhibitor.19 However, the IC50
2 lM obtained for its phosphate analogue, namely 5-phospho-D-
arabinonohydrazide.61 5PMAH (9) is also a quite weak inhibitor
of S. cerevisiae PMI with an IC50 value of 2.2 mM, but a reasonably
good inhibitor of E. coli PMI with an IC50 value of 0.37 mM. How-
ever, the loss of binding affinity is of several orders of magnitude
when compared to the Ki value of 0.084
lM obtained for its phos-
phate analogue 5-phospho-
D
-arabinonohydroxamic acid.19 Overall,
replacement of the phosphate group by a phosphonomethyl group
in known 1,2-cis-enediolate high-energy intermediate PMI ana-
logue inhibitors strongly impairs their binding affinity to the en-
zyme active site.
gave the respective Ki values of 115 and 10.5 lM (Fig. 1B), from
Interestingly, Frost and coworkers reported a study of 3-dehy-
droquinate (DHQ) synthase active site in which a large number
of substrate analogue carbocyclic inhibitors bearing a phosphate
mimic were evaluated.47 They clearly demonstrated that access
to a dianionic ionization state of the phosphate surrogate yielded
strong inhibitors, as in the case of their dianionic carbamalonate
for which an apparent pKa of 4.40 was measured, as compared to
the monoanionic carbacarboxylate or carbaacetate analogues
which appeared as much weaker inhibitors. These results are in
perfect agreement with the kinetic results we report here for
6DCM (2) as compared to 6CM (3). On the other hand, in contrast
to our results, their carbaphosphonate analogues were reported to
which the respective Km/Ki ratio values of 8 and 28 could be calcu-
lated. The Ki values appear to be about an order of magnitude in fa-
vor of the fungal enzyme as compared to the bacterial enzyme.
However the ratio of the respective Km/Ki values is only 3.5. It is
not significant in terms of specificity, which confirms that the
two type I PMIs have comparable active sites. Although these Ki
values are only in the micromolar range, 6DCM appears as the sec-
ond best inhibitor of a PMI reported to date, 5-phospho-D-arabi-
nonohydroxamic acid being in the submicromolar range.19
Furthermore, 6DCM represents the first 2-malonyl-based phos-
phate surrogate inhibitor of an aldose–ketose isomerase ever re-
ported in the literature. Our results regarding PMI inhibition
Figure 1. (A) Inhibition of S. cerevisiae PMI (50 mM Hepes buffer, 25 °C, pH 7.1) by 6-deoxy-6-dicarboxymethyl-
D-mannopyranose (6DCM, 2); double reciprocal plot of initial
reaction velocity versus M6P concentration obtained at various concentrations of inhibitor: d, no inhibitor; h, [I] = 8
l
M; j, [I] = 15 M; s, [I] = 40 M. Lines drawn were
l
l
obtained from a linear regression fit of the observed data using Michaelis–Menten equation for competitive inhibition. (B) Secondary plot of apparent Km/Vmax values (slopes
of the straight lines in the primary graph A) versus inhibitor concentration. The x intercept, which is equal to ꢀKi, was determined by linear regression.

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J. Foret et al. / Bioorg. Med. Chem. 17 (2009) 7100–7107
be strong inhibitors of DHQ synthase, implying that they might
bind as dianions rather than monoanions. This might originate
from the slightly higher pH of 7.7 used in their study as compared
to 7.1 for our kinetics on PMIs, or from possible modulations of the
pKa of phosphonate as a function of the enzyme active site.
from the present study is the need to retain the Zn-bound water
molecule in the optimized complex. Alternative energy-minimiza-
tions in its absence showed that none of the ligand hydroxyl
groups was unable to bind Zn(II) at distances closer than 2.5 Å,
while the C–O–Zn angle was non-optimal. This constitutes an
uncommon instance in which ligand binding to the catalytic Zn(II)
occurs through an interposed water molecule. Figure 2 clearly
shows that both the phosphate group of bM6P (Fig. 2A) and the
2-malonyl group of 6DCM (Fig. 2B) can fit snugly between with
Arg304, and Lys310, with additional stabilization involving
2.3. Polarizable molecular mechanics study of enzyme
substrate and inhibitors
Because we did not succeed into getting high-resolution crystal
structures of PMI complexes, the polarizable molecular mechanics
procedure SIBFA (Sum of Interactions Between Fragments
computed ab initio)50 was used to study the PMI-M6P and PMI-
inhibitor interactions. This procedure was previously applied suc-
cessfully to study several inhibitor–metalloprotein com-
plexes.20,62–67 We used the three-dimensional structure of the
type I C. albicans PMI18 as a starting point to generate a theoretical
model of the enzyme, and thereafter the different complexes of the
model with the substrate or the inhibitors as depicted in Figure 2.
Energy-minimized representations of the active site of C. albicans
Ser109. In fact, as previously observed with 5-phospho-D-arabi-
nonohydroxamic acid,20 this array of interactions propagates to
the entire recognition site. Thus Ser109 in turn behaves as an H-
bond acceptor to Gln111, which donates a second proton to the li-
gand ring oxygen, as well as acting as one of the Zn-ligands. Zn(II)
is coordinated to Glu138, which also accepts a proton from the hy-
droxyl group on C2 of the ligand, while as mentioned above its
water ligand donates a proton to the hydroxyl group on C1 of the
ligand.
The case of the phosphonomethyl substrate analogue 6PMM (1)
is interesting. In its dianionic form (Fig. 2C), the binding pattern of
6PMM (1) is quite similar to that observed for bM6P (and so for
6DCM) as shown by the superposition of the two complexes
PMI complexed with b-D-mannopyranose 6-phosphate (bM6P),
6DCM (2), dianionic 6PMM (1), and monoanionic 6PMM(H) (1)
are depicted, respectively, in Figure 2A–D. An important finding
Figure 2. SIBFA representations of the active site of C. albicans PMI complexed to: (A) b-
D
-mannopyranoside 6-phosphate (bM6P); (B) 6-deoxy-6-dicarboxymethyl-
D-
mannopyranose (6DCM, 2); (C) 6-deoxy-6-phosphonomethyl- -mannopyranose (6PMM, 1); (D) 6-deoxy-6-hydrogeno-phosphonomethyl-
D
D
-mannopyranose [6PMM(H)]. H-
bonds are depicted as green dashed lines and H-bonds distances are given in angstrom. Ligand interactions at the zinc binding site are depicted as pink dashed lines. Some
hydrogen atoms, active site residues, water molecules, and H-bonds were omitted for clarity of the figure. DS Visualizer 2.0 and Microsoft PowerPoint software’s were used to
prepare the figure.

J. Foret et al. / Bioorg. Med. Chem. 17 (2009) 7100–7107
7105
be known as hydrolase-resistant, they could be vectorized by
hydrophobic labile chains in order to increase their intracellular
uptake, and thereby their potential efficiency toward the targeted
enzyme. The demonstration in the present work that the malonate
derivative has a one order of magnitude better binding property
than the parent phosphate is the incentive for related comparisons
in the 5-phospho-D-arabinonohydroxamic acid series of deriva-
tives. Synthesis of the 2-malonyl analogue of the reaction interme-
diate postulated for the isomerization reaction catalyzed by PMIs,
namely
5-deoxy-5-(dicarboxymethyl)-D-arabinonohydroxamic
acid, will thus be undertaken as an extension of the present work.
4. Experimental
4.1. General materials and methods
Unless otherwise stated, all chemical reagents were of analyti-
cal grade, obtained from Acros, Alfa Aesar, or Aldrich, and used
without further purification. Solvents were obtained from SDS or
VWR-Prolabo. MeOH, CH2Cl2, and THF were dried by refluxing
with, respectively, Mg/I2, CaH2, and Na/benzophenone, then dis-
tilled and used immediately. Flash chromatography was performed
Figure 3. Superposition of the active sites of C. albicans PMI complexed to (i)
b- -mannopyranose 6-phosphate (bM6P) in green, (ii) 6-deoxy-6-phosphonometh-
yl- -mannopyranose (6PMM, 1) in purple, and (iii) 6-deoxy-6-hydrogenophosph-
onomethyl- -mannopyranose [6PMM(H)] in pink, obtained by structural alignment
(DS Visualizer 2.0). Hydrogen atoms, H-bonds, and some active site residues and
water molecules were omitted for clarity of the figure. DS Visualizer 2.0 and
Microsoft PowerPoint software’s were used to prepare the figure.
D
D
D
using silica gel (35–70 lm, E. Merck) under N2 pressure. Ion-ex-
change chromatography was performed on DowexÒ-50WX8 cat-
ion-exchange resin (H+ or Na+ form, 50–100 or 100–200 mesh,
Acros), eluting with purified water (Millipore, 18.2 MX). Unless
depicted in Figure 3. Replacement of the oxygen atom on carbon C-
6 of the substrate M6P by a methylene group does not impair bind-
ing of the inhibitor to the PMI active site. However, this observa-
tion does not agree with the kinetic results which indicate that
the phosphonomethyl analogue is not a PMI inhibitor. Represent-
ing the inhibitor in its likely monoanionic form, 6PMM(H), shows
that only one attractive interaction of the phosphonomethyl group
takes place with Arg304 (Fig. 2D). Superimposition with the two
previous PMI complexes (bM6P and dianionic 6PMM) reveals a dis-
tinct and most likely weaker binding pattern (Fig. 3).
In order to unravel the origin of the experimentally observed
preferential binding of 6DCM (2) than 6PMM (1) to PMI, energy
balances are underway. As in our previous studies,19,62–65 they take
into account the ligand–PMI intermolecular interaction and solva-
tion energy of the complex on the one hand, and the conforma-
tional energy cost and desolvation energies of the ligand and the
protein on the other hand. These results will be detailed and pub-
lished separately in due course. They indicate that a sole contin-
otherwise stated, all organic extracts were dried over Na2SO4 and
filtered. Concentration of solutions was performed under dimin-
ished pressure at temperature <30 °C using a rotary evaporator.
All air- and moisture-sensitive reactions were performed under
an atmosphere of argon. Analytical TLC was performed using Silica
Gel 60 F254 pre-coated aluminum plates (E. Merck). Spots were
visualized by treatment with 5% ethanolic H2SO4 followed by
heating and/or by absorbance of UV light at 254 nm. Optical rota-
tions were measured on a Jasco DIP-370 digital polarimeter at
589 nm. NMR spectra were recorded at 297 K in CDCl3, CD3OD,
and D2O with a Bruker DPX 250 (1H at 250.13 MHz, 13C at
62.90 MHz, and 31P at 101.26 MHz), DRX 300 (1H at 300.13 MHz,
13C at 75.47 MHz, and 31P at 121.50 MHz), or Avance 360 (1H at
360.13 MHz and 13C at 90.56 MHz) spectrometer using NMR Note-
book 2.0 WinNMR software. Chemical shifts are reported in ppm
(d) and coupling constants in Hz (Jij). 1H NMR spectra were refer-
enced to internal residual chloroform (d 7.26), CD2HOD (d 3.31),
and HOD (d 4.80) for solutions in CDCl3, CD3OD, and D2O, respec-
tively. For CH2 groups, H-50, H-60, and H-70 resonances appear arbi-
trarily at lower field than H-5, H-6, and H-7 resonances,
respectively. 13C NMR spectra were referenced to solvent for solu-
tions in CDCl3 (d 77.0) and CD3OD (d 49.1), and to dioxane (d 67.4)
for solutions in D2O. 31P NMR spectra were referenced externally to
85% aq H3PO4 (d 0.0). In most cases, COSY, HSQC, and/or DEPT135
NMR spectra were recorded for assigning resonances. Infrared
spectra were recorded with a FTIR Bruker IFS-66 spectrometer.
Low-resolution mass spectrometry (MS) and high-resolution mass
spectrometry (HRMS) analyses were performed by Imagif (ICSN-
CNRS, Gif-sur-Yvette) by electrospray with positive (ESI+) or nega-
tive (ESIꢀ) ionization mode.
uum representation of solvation cannot account for such
a
preferential binding. On the other hand, including a limited array
(nine) of highly polarized discrete water molecules in the binding
site can exert a decisive effect in favor of 6DCM. The so-augmented
interaction site is shown as Supplementary data for the 6DCM
complex. It is seen to extend to three anionic residues of PMI.
3. Conclusion
In conclusion, we report 6-deoxy-6-(dicarboxymethyl)-
D-man-
nopyranose, a non-hydrolyzable -mannose 6-phosphate analogue
D
in which the phosphate group was replaced by a dicarboxymethyl
(or 2-malonyl) group, as a strong inhibitor of PMIs from E. coli and
S. cerevisiae. This compound also represents the first malonate-
based inhibitor of an aldose–ketose isomerase reported to date.
By contrast, phosphonomethyl and carboxymethyl mimics of
M6P do not inhibit significantly the enzymes, most likely because
of their monoanionic state. SIBFA molecular modeling showed the
binding to the catalytic zone to occur with retention of the Zn-
bound water molecule, while both dianionic phosphate and malon-
ate can snugly bind to both Arg304 and Lys310 residues at the en-
trance of the binding site. Malonate-based inhibitors offer
promising therapeutic outcomes considering that, in addition to
4.2. Synthesis
4.2.1. 6-Deoxy-6-dicarboxymethyl-D-mannopyranose,
disodium salt (2)
Compound 7 (0.92 g, 3.12 mmol) was dissolved in 1 M aq NaOH
(22 mL) and vigorously stirred at rt for 15 h. Following concentra-
tion of the reaction mixture, the residue was successively eluted
with water on two DowexÒ-50WX8 ion-exchange columns (1-H+

7106
J. Foret et al. / Bioorg. Med. Chem. 17 (2009) 7100–7107
form, 2-Na+ form) and lyophilized to afford 2 (0.96 g, 99%) as a
under diminished pressure. The residue was purified by flash-chro-
matography (2:1 petroleum ether/ethyl acetate) to give 6 (4.65 g,
64% for two steps) as a pale yellow oil: Rf 0.31 (2:1 petroleum
ether/ethyl acetate); 1H NMR (300 MHz, D2O): d 7.44–7.38 (m,
20H, Ph), 5.06 (d, 1H, J 10.8 Hz, OCH2Ph), 4.97 (s, 1H, H-1), 4.86–
4.66 (m, 6H, OCH2Ph), 4.48 (d, 1H, J 11.9 Hz, OCH2Ph), 4.05 (m,
white powder: ½a D25
ꢁ
ꢀ159 (c 1, H2O); FTIR (KBr): mmax 3402, 2930,
1596, 1387, 1061 cmꢀ1
;
1H NMR (300 MHz, D2O): d 4.98 (s, 0.6H,
H-1a), 4.84 (s, 0.4H, H-1b), 3.79 (m, 1H, H-2), 3.67 (dd, 0.6H, J3–4
9.7, J3–2 3.0 Hz, H-3
a
), 3.63 (t, 0.6H, J5–6 9.4, J5–4 9.4 Hz, H-5 ),
a
3.47 (dd, 0.4H, J3–4 9.5, J3–2 2.8 Hz, H-3b), 3.37 (m, 2H, H-4 and
0
H-7), 3.10 (t, 0.4 H, J5–6 9.4, J5–4 9.4 Hz, H-5b), 2.28 (br d, 1H, J6–6
1H, H-3), 3.90 (m, 2H, H-2 and H-7), 3.82 (m, 2H, H-4 and H-5),
14.0 Hz, H-60), 1.80 (dd, J6–5 9.4, J6–6 14 Hz, 1H, H-6); 13C NMR
3.81 (s, 3H, CH3 ), 3.72 (s, 3H, CH3), 2.79–2.71 (ddd, 1H, J6 –6 14.2,
0
0
0
0
0
(75 MHz, D2O): d 177.9 and 177.6 (2COONa), 93.8 (C-1
(C-1b), 74.0 (C-5b), 72.8 (C-3b), 71.1 (C-4b), 70.5 (C-2b), 70.8,
a
), 93.7
J6 –5(7) 9.8, J6 –7(5) 4.7 Hz, H-60), 2.32–2.22 (ddd, 1H, J6–5(7) 10.2,
J6–7(5) 2.0 Hz, H-6); 13C NMR (75 MHz, CDCl3): d 169.6 and 169.4
(2COOMe), 138.2, 138.1, 138.0, and 137.0 (4Cq Ph), 128.2–127.4
(20 CH Ph), 96.7 (C-1), 79.9 (C-3), 78.5 (C-4), 75.1 (CH2Ph), 74.5
(C-2), 72.5 (CH2Ph), 72.1 (CH2Ph), 69.2 (C-5), 68.4 (CH2Ph), 52.3
(2CH3), 48.2 (C-7), 31.2 (C-6); ESI+MS: m/z 677.3 (100) [M+Na]+,
678.3 (43), 679.3 (11); HRESI+MS: calcd for C39H42O9Na:
677.2721 [M+Na]+, found m/z 677.2722.
70.6, 70.1, and 70.0 (C-2a, C-3a, C-4a, and C-5a), 52.6 (C-7b),
52.4 (C-7
a
), 31.6 C-6); ESI+MS: m/z 289.0 (100) [MꢀNa+2H]+,
311.0 (38) [M+H]+; HRESI+MS: calcd for C9H13O9Na2: 311.0355
[M+H]+, found m/z 311.0356.
4.2.2. 6-Deoxy-6-carboxymethyl-D-mannopyranose, sodium
salt (3)
Compound 2 (100 mg, 0.32 mmol) was dissolved in water
(5 mL). DowexÒ-50WX8 ion-exchange resin (H+ form, 2 g) was
added to the soln and the reaction mixture was stirred under reflux
for 18 h. The soln was then filtered and directly eluted on a Dow-
exÒ-50WX8 ion-exchange column (Na+ form). Following concen-
tration and lyophilization, the title compound (80 mg, 100%) was
4.2.4. 6-Deoxy-6-dimethylmalonate-D-mannopyranose (7)
Compound 6 (2.4 g, 3.7 mmol) dissolved in anhyd MeOH and
Pd(OH)2/C (420 mg) were stirred under H2 (15 bar) for 2 d. The
mixture was then filtered and concentrated to give the intermedi-
ate compound benzyl 6-deoxy-6-dimethylmalonate-D-mannopy-
ranoside. The latter dissolved in anhyd MeOH (50 mL) and
Pd(OH)2/C (420 mg) were stirred again under H2 (15 bar) for 3 d,
filtered and concentrated to give 7 (1.017 g, 94%) as a colorless
oil: Rf 0.47 (9:1 ethyl acetate/MeOH); 1H NMR (360 MHz, CD3OD):
obtained as a white solid: ½a D25
ꢁ
+19 (c 0.95, H2O); FTIR (KBr): mmax
3427, 2924, 1582, 1413, 1063 cmꢀ1
;
1H NMR (250 MHz, D2O): d
5.04 (s, 0.4H, H-1
(dd, 0.6H, J3–4 9.6, J3–2 2.3 Hz, H-3b), 3.65 (m, 0.6H, H-5b), 3.52
(dd, 0.4H, J3–4 9.6, J3–2 1.9 Hz, H-3 ), 3.41 (t, 0.6H, J4–3 9.6, J4–5
9.6 Hz, H-4b), 3.32 (t, 0.4H, J4–3 9.6, J4–5 9.6 Hz, H-4 ), 3.15 (t,
0.4H, J5–6 9.6, J5–4 9.6 Hz, H-5
), 2.33–2.11 (2H, m, H-7 and H-70),
2.03 (m, 1H, H-60), 1.59 (m, 1H, H-6); 13C NMR (62.5 MHz, D2O):
d 183.0 (COONa), 93.9 (C-1b), 93.7 (C-1 ), 75.2 (C-5 ), 72.9 (C-
), 70.6 (C-2b), 70.4 (C-3b), 70.3 (C-4),
a), 4.76 (s, 0.6H, H-1b), 3.84 (m, 1H, H-2), 3.72
d 5.03 (s, 0.7H, H-1a), 4.65 (s, 0.3H, H-1b), 3.83–3.65 (m, 2H, H-5
a
and H-7), 3.73 and 3.72 (2s, 6H, 2OCH3), 3.48–3.37 (m, 3H, H-2,
0
0
0
a
H-3, and H-4), 2.50 (ddd, 1H, J6 –6 14.2, J6 –5(7) 10.1, J6 –7(5) 5.2 Hz,
H-60), 1.98 (ddd, 1H, J6–5(7) 9.5, J6–7(5) 2.5 Hz, H-6); 13C NMR
(90.5 MHz, CD3OD): d 171.8, 171.7, 171.3, and 171.2 (4COOMe:
a
a
a
a
- and b-anomers), 95.4 and 95.3 (C-1
a
and C-1b), 74.8, 74.5,
3a
), 71.3 (C-5b), 71.1 (C-2
a
72.9, 72.6, 72.4, 71.9, 70.4 (C-2, C-3, C-4, and C-5: a- and b-ano-
33.4 (C-7, 27.4 C6); ESI+MS: m/z 229.1 (100) [MꢀOH+H]+, 245.1
(75) [M+H]+, 267.1 (6) [M+Na]+; HRESI+MS: calcd for C8H14O7Na:
245.0637 [M+H]+, found m/z 245.0641.
mers), 53.1 and 53.0 (2CH3), 49.3 (C-7), 32.0 (C-6); ESI+MS: m/z
316.9 (100) [M+Na]+, 317.9 (12); HRESI+MS: calcd for C11H18O9Na:
317.0843 [M+Na]+, found m/z 317.0836.
4.2.3. Benzyl 2,3,4-tri-O-benzyl-6-deoxy-6-dimethylmalonate-
4.2.5. Preparation of 5-deoxy-5-phosphonomethyl-
D-
a-
D-mannopyranoside (6)
arabinonate, trisodium salt (8)25
Benzyl 2,3,4-tri-O-benzyl-
a-
D
-mannopyranoside 452 (6 g, 11.1
The dipotassium salt of 6-deoxy-6-phosphonomethyl-D-man-
mmol) was dissolved in anhyd CH2Cl2 (50 mL) under argon. Diiso-
propylethylamine (4.9 mL, 28.3 mmol) was added and the soln was
stirred and cooled to ꢀ40 °C. Following dropwise addition of tri-
fluoromethanesulfonic anhydride (2.8 mL, 16.6 mmol), the reac-
tion mixture was stirred for 3 h. Thereafter, the soln was allowed
to warm to rt and ethyl acetate (50 mL) was added. The resulting
soln was washed with satd aq NH4Cl (50 mL), then neutralized
with satd aq NaHCO3 (50 mL). The organic phase was dried
and concentrated under diminished pressure to afford
nopyranose24 (460 mg, 1.38 mmol) was dissolved in 1 M NaOH
(6.8 mL, 3.4 mmol). The reaction mixture was vigorously stirred
at rt under O2 for 7 d, using a volumetric device which allowed
measurement of dioxygen consumption. The barium salt of 8 was
obtained using the reported procedure,56 dispersed in a few mL
of water, and eluted with water on a DowexÒ-50WX8 ion-ex-
change columns (H+ form). The aq solution was then directly
eluted on a DowexÒ-50WX8 ion-exchange columns (Na+ form),
concentrated under vacuum and lyophilized to afford the triso-
dium salt of 8 (215 mg, 50%) as a white solid: 1H NMR (300 MHz,
D2O): d 4.15 (s, 1H, H-2), 3.60 (d, 1H, J3–4 8.4 Hz, H-3), 3.53 (td,
benzyl 2,3,4-tri-O-benzyl-6-O-trifluoromethanesulfonyl-a-D-man-
nopyranoside 5 (7.8 g) as an intense red oil which was not further
purified and used immediately for the next reaction: Rf 0.71 (4:1
petroleum ether/ethyl acetate).
In a two-neck round bottom flask placed under argon and con-
taining anhyd THF (85 mL) was dissolved dimethyl malonate
(1.7 mL, 28.8 mmol). NaH (60% in oil, 574 mg, 14.4 mmol) was then
slowly added to the solution under vigorous stirring. The reaction
mixture was further stirred at rt for 1 h. A soln of the benzyl 2,3,4-
1H, J4–5 8.4, J4–5 2.4 Hz, H-4), 1.94–1.41 (m, 4H, H-50, H-5, H-60,
0
H-6); 13C NMR (75 MHz, D2O): d 179.7 (C-1), 74.8 (C-3), 71.6 (C-
2), 71.2 (d, J4-P 16.7 Hz, C-4), 27.2 (C-5), 23.9 (d, J6-P 134 Hz, C-6);
31P NMR (121.5 MHz, D2O): d 26.1; ESIꢀMS: m/z 264.9 (48)
[Mꢀ2Na+H]ꢀ, 246.9 (23) [Mꢀ2Na+HꢀH2O]ꢀ, 243.0 (88)
[Mꢀ3Na+2H]ꢀ, 224.9 (100) [Mꢀ3Na+2HꢀH2O]ꢀ; HRESIꢀMS: calcd
for C6H12O8P: 243.0270 [Mꢀ3Na+2H]ꢀ, found m/z 243.0278.
tri-O-benzyl-6-O-trifluoromethanesulfonyl-a-D-mannopyranoside
5 previously synthesized in anhyd THF (17 mL) was then added
dropwise to the sodium dimethyl malonate THF soln. The reaction
mixture was stirred under reflux for 15 h, then cooled to rt, and
neutralized by addition of MeOH (5 mL) and satd aq NH4Cl
(100 mL). The aq phase was extracted with diethyl ether
(3 ꢂ 100 mL). The combined organic phases were washed with
satd aq NaCl (100 mL), dried (Na2SO4), filtered, and concentrated
4.2.6. Preparation of 5-deoxy-5-phosphonomethyl-
D-
arabinonohydroxamic acid, dihydroxylammonium salt (9)55
6-Deoxy-6-dihydrogenophosphono-D-arabinono-1,4-lactone
1155 (110 mg, 0.486 mmol) was dissolved in a 50% aq NH2OH soln
(2.4 mL, 40 mmol). The reaction mixture was stirred at rt for
20 min, concentrated under high vacuum at rt, and lyophilized to
afford the title compound as a white solid (160 mg, 100%): ½a D25
ꢁ

J. Foret et al. / Bioorg. Med. Chem. 17 (2009) 7100–7107
7107
ꢀ21 (c 0.95, H2O); FTIR (KBr): mmax 3194, 1651, 1042; 1H NMR
(300 MHz, D2O): d 4.05 (s, 1H, H-2), 3.27 (d, 1H, J3–4 8.4 Hz, H-3),
15. Gracy, R.; Noltman, E. J. Biol. Chem. 1968, 243, 3161.
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3.22 (td, 1H, J4–5 8.4, J4–5 2.4 Hz, H-4), 1.36–1.04 (m, 4H, H-50,
0
H-5, H-60, H-6), not in good agreement with the literature55 for
the disodium salt; 13C NMR (75 MHz, D2O): d 171.3 (C-1), 73.6
(C-3), 70.4 (d, J4-P 14.8 Hz, C-4), 70.1 (C2), 27.0 (C-5), 23.6 (d, J6-P
133 Hz, C-6)), not in good agreement with the literature55 for the
disodium salt; 31P NMR (121.5 MHz, D2O): d 25.3, lit.55 26.18 and
26.16 (2s) for the disodium salt; ESIꢀMS: m/z 258.1 (100)
[Mꢀ2NH3OH+H]ꢀ, lit.55 258 [Mꢀ2Na+H]ꢀ; ESIꢀMS: m/z calcd for
C6H13NO8P: 258.0379 [Mꢀ2NH3OH+H]ꢀ, found 258.0383.
24. Belakhov, V.; Dovgolevsky, E.; Rabkin, E.; Shulami, S.; Shoham, Y.; Baasov, T.
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3943.
4.2.7. 5-Deoxy-5-phosphonomethyl-
dihydrazinium salt (10)
D
-arabinonohydrazide,
6-Deoxy-6-dihydrogenophosphono-
D-arabinono-1,4-lactone
1155 (70 mg, 0.309 mmol) was dissolved in a 64% aq hydrazine
soln. The reaction mixture was stirred at rt for 20 min, concen-
trated under high vacuum at rt, and lyophilized to afford the title
compound (100 mg, 100%) as a white solid: 1H NMR (360 MHz,
D2O): d 4.28 (s, 1H, H-2), 3.53 (d, 1H, J3–4 8.4 Hz, H-3), 3.46 (td,
1H, J4–5 8.4, J4–5 2.4 Hz, H-4), 1.82–1.19 (m, 4H, H-50, H-5, H-60,
0
H-6); 13C NMR (90.5 MHz, D2O): d 173.8 (C-1), 74.0 (C-3), 70.8 (d,
J4-P 14.8 Hz, C-4), 70.6 (C-2), 27.6 (C-5), 24.4 (d, J6-P 131 Hz, C-6);
31P NMR (101 MHz, D2O):
d
23.2; ESIꢀMS: m/z 257.0 (45)
[Mꢀ2NH2NH3+H]ꢀ, 224.9 (100) [Mꢀ2NH2NH3–NH2NH2+H]ꢀ;
HRESIꢀMS: calcd for C6H14N2O7P 257.0539 [Mꢀ2NH2NH3+H]ꢀ,
found 257.0552.
Acknowledgements
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The ab initio computations were performed using resources
from GENCI (CINES/IDRIS), Grant 2009-075009, and the Centre de
Ressources Informatiques de Haute Normandie (CRIHAN, Rouen,
France), Grant 1998053. We wish to thank Nicole Audiffren (CINES)
and Guy Moebs (CRIHAN) for their help. We also wish to thank the
Ligue Nationale contre le Cancer (Comité de Paris) for financial
support.
Supplementary data
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Supplementary data (structure of the active site of CaPMI
complexed to 6DCM including additional discrete water molecules
and 13C NMR spectra of new compounds) associated with this
article can be found, in the online version, at doi:10.1016/
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