Synthesis and biochemical evaluation of selective inhibitors of class II fructose bisphosphate aldolases: Towards new synthetic antibiotics

Article abstract of DOI:10.1002/chem.200800857

We report the synthesis and biochemical evaluation of selective inhibitors of class II (zinc-dependent) fructose bisphosphate aldolases. The most active compound is a simplified analogue of fructose bisphosphate, bearing a well-positioned metal chelating

Full text of DOI:10.1002/chem.200800857

FULL PAPER
DOI: 10.1002/chem.200800857
Synthesis and Biochemical Evaluation of Selective Inhibitors of Class II
Fructose Bisphosphate Aldolases: Towards New Synthetic Antibiotics
Matthieu Fonvielle,[a] Mathieu CoinÅon,[b] Racha Daher,[a] Nicolas Desbenoit,[a]
Katarzyna Kosieradzka,[a] Nathalie Barilone,[c] Brigitte Gicquel,[c] Jurgen Sygusch,[b]
Mary Jackson,[c] and Michel Therisod*[a]
Abstract: We report the synthesis and
biochemical evaluation of selective in-
hibitors of class II (zinc-dependent)
fructose bisphosphate aldolases. The
most active compound is a simplified
analogue of fructose bisphosphate,
bearing a well-positioned metal chelat-
ing group. It is a powerful and highly
selective competitive inhibitor of isolat-
ed class II aldolases. We report crystal-
lographic studies of this inhibitor
bound in the active site of the Helico-
bacter pylori enzyme. The compound
also shows activity against Mycobacte-
rium tuberculosis isolates.
Keywords: aldolases · antibiotics ·
bioorganic chemistry · enzyme in-
hibitors
Introduction
ulcers and the sole known microorganism directly involved
in the development of cancer.[1]
Bacterial infections, including nosocomial diseases contract-
ed in hospital from antibiotic-resistant strains of pathogenic
bacteria, are a public health problem of major concern. Tu-
berculosis (TB) is one of the most common infectious dis-
eases known to man. The increasing occurrence of multiple-
drug resistant TB, even in developed countries, is also
alarming because there are only a few effective drugs avail-
able, and infection with drug-resistant Mycobacterium tuber-
culosis (M. tb) could give rise to a potentially untreatable
form of disease. It must be noted that there have been no
new major anti-TB drugs introduced into widespread use
since rifampicin in the 1960s. Helicobacter pylori is another
very widespread bacterium, responsible for most gastric
There is clearly an urgent need for new drugs with bacter-
icidal mechanisms different from those of currently avail-
able agents.[2] This implies identification of new metabolic
targets, specific to microbes, as likely sites of action for ra-
tionally designed drugs. The search for new therapeutic tar-
gets resulted in our interest in ATP synthesis from glucose
in microbial pathogens, and more specifically in fructose-
1,6-bisphosphate aldolase (Fba; E.C. 4.1.2.13).
Fba is an enzyme involved in glycolysis, in which it cataly-
ses the retro-ketolic cleavage of fructose-1,6-bisphosphate
(FBP) to yield dihydroxyacetone phosphate (DHAP) and
glyceraldehyde-3-phosphate (G3P), and it also plays a role
in gluconeogenesis and the Calvin cycle, in which it catalyses
the condensation of DHAP with G3P.
Selective inhibition of microbial Fba would be expected
to disrupt glycolysis, thereby affecting a major metabolic
pathway and hindering survival and persistence of patho-
gens within their human host.[3] Although ubiquitous in
living organisms, Fbas can be divided into two classes that
differ in their structures and catalytic mechanisms. Class I
Fbas are found in mammals and higher plants and form
Schiff-base intermediates between the carbonyl substrates
(FBP or DHAP) and lysine residues in their active sites.
Class II Fbas, in contrast, require divalent metal ions (usu-
ally zinc or cobalt) to polarize the carbonyl groups of the
substrates (FBP or DHAP) and to stabilize the enediolate
intermediates during catalysis (Figure 1). They are found
mainly in micro-organisms such as bacteria, yeasts, micro-
[a] Dr. M. Fonvielle, R. Daher, N. Desbenoit, K. Kosieradzka,
Prof. M. Therisod
LCBB–ICMMO, UMR 8182, UniversitØ Paris-Sud
91405 Orsay (France)
Fax : (+33)1-6915-7281
E-mail: therisod@icmo.u-psud.fr
[b] M. CoinÅon, Prof. J. Sygusch
Biochimie, UniversitØ de MontrØal, CO 6128, Stn centre-ville
MontrØal, PQ H3C 3J7 (Canada)
[c] N. Barilone, Dr. B. Gicquel, Dr. M. Jackson
UnitØ de GØnØtique MycobactØrienne
Institut Pasteur, 75724 Paris (France)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200800857.
Chem. Eur. J. 2008, 14, 8521– 8529
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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ful inhibitor of several other enzymes. PGH has been shown
to act both as a stable analogue of the high-energy inter-
mediate of the reaction (Figure 2) and as a strong chelator
of Zn2+. The report of structural data on PGH-fuculose
phosphate aldolase complexes[15] was a major breakthrough
for understanding of the role of PGH as an inhibitor of met-
alloenzymes.
We first reasoned that through changing the hydroxamate
chelating group for other functions we might be able to
retain strong inhibition while at the same time increasing se-
lectivity and resistance to hydrolysis. This goal was only
partly achieved with PGHz (phosphoglycolohydrazide) and
PGA (phosphoglycoloamidoxime; Figure 2). The selectivi-
ties of both compounds for class II aldolases were increased
and they were much more stable in water, but their Ki
values were much higher than that of PGH (Table 1).[16]
Figure 1. Mechanisms of class I (e.g., human) and class II (e.g., bacterial)
FBP aldolases.
algae and protozoa (occasionally in combination with class I
enzymes).
Because of their occurrence in many pathogenic bacteria
(M. tb, Pseudomonas aeruginosa, Burkholderia cepacia,
H. pylori, Yersinia pestis, Clostridium difficile), yeasts (Can-
dida albicans) and parasites (Giardia lamblia) and their ab-
sence in animals, it has been suggested that class II Fbas
may serve as drug targets.[4] This assumption was further
supported by the inability to knock out the class II FBP al-
dolase genes of E. coli and Streptomyces.[5,6] Also, the condi-
tional loss of class II aldolase activity in temperature-sensi-
tive mutants of E. coli harbouring point mutations in the
class II FBP aldolase gene (fda) was found to result in loss
of viability attributable to inhibition of rRNA synthesis.[7,8]
M. tb, like E. coli, is distinctive in that it possesses both a
class I and a class II FBP aldolase.[9,10] A recombinant form
of the class II M. tb Fba protein was recently produced in
E. coli.[11] However, hypoxic conditions, generally believed
to be determinant in establishment of a latent infection by
M. tb, have been shown to induce both increased levels and
increased activity of the class II Fba.[9,12] The apparent es-
sentiality of class II Fbas in various micro-organisms and the
likely importance of these enzymes during latent infection
thus make Fbas an attractive and specific target for the de-
velopment of novel anti-TB drugs.
Table 1. Activities of DHAP analogues as selective inhibitors of class II
Fbas.
Inhibitor
Ki [mm] class I Fba
Ki [mm] class II Fba
Selectivity[b]
PGH[a]
PGHz
PGA
2
370
>1000
0.05
0.34
2.3
273
8900
3560
[a] Data from ref. [4]. [b] (KM/Kiclass II)/(KM/Kiclass I). KM class I (rabbit
muscle)=55 mm; KM class II (S. cerevisiae)=450 mm.
PGHz and PGA probably behaved as analogues of
DHAP rather than as analogues of a high-energy intermedi-
ate. PGH was unambiguously shown to be present in its s-Z
conformation within the active sites of several enzymes
(Fbas from E. coli and H. pylori,[17–19] fuculose bisphosphate
aldolase from E. coli,[15] methylglyoxal synthase[20] and triose
phosphate isomerase[21]). Similarly, PGHz in complexation
with H. pylori Fba chelates the Zn2+ ion in the s-Z confor-
mation (M. CoinÅon, J. Sygusch, unpublished results). Al-
though we have no structural data on the structure of PGA
in the active site of Fba yet, the Z configuration is very
likely.
Surprisingly, while dozens of class I Fba inhibitors have
been reported,[13] only one potent inhibitor of class II en-
zymes was known when we started this work. Phosphoglyco-
lohydroxamate (PGH; Figure 2) was first prepared inde-
pendently by two groups in 1973.[4,14] It is a very powerful
inhibitor of the E. coli class II Fba (Ki ꢀ10–50 nm). Howev-
er, PGH is poorly stable in water, releasing (toxic) hydroxyl-
amine through hydrolysis within a few hours. It has limited
selectivity for class II Fbas over class I Fbas, and is a power-
Results and Discussion
Chemistry: We therefore decided to design and synthesize
analogues of the substrate of the forward reaction: FBP.
Previous studies on class I Fbas had indicated that very
simple structures can be potent inhibitors, provided that two
phosphate groups, separated by an appropriate distance, are
present.[13] The selectivity for class II Fbas would be induced
by the presence, at the appropriate position, of a function
capable of chelating the divalent Zn2+ ion in the active site.
Taking into consideration the good inhibitory properties of
PGH and PGHz,[16] we chose an N-substituted hydroxamate
and an N-substituted hydrazide as basic structures. We fur-
ther decided to omit the two secondary hydroxyl groups
present in FBP, leading as a first step to the simplified ana-
logues 1 and 2 (Figure 3).
Figure 2. Substrate, reaction intermediate and inhibitors of class II Fbas.
8522
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Chem. Eur. J. 2008, 14, 8521– 8529

Bisphosphate Aldolase Inhibitors
FULL PAPER
Fba from H. pylori was a recombinant form produced in
E. coli. The activities of the M. bovis BCG (Pasteur strain;
an attenuated strain of M. bovis used for vaccination against
tuberculosis) and yeast enzymes were monitored in crude or
partially purified cell-free extracts obtained by breaking
fresh cells in a French press. The primary sequence of the
M. bovis BCG enzyme is 100% identical to that of the
pathogenic species: M. tuberculosis H37Rv. The compounds
behaved as purely competitive inhibitors of all tested en-
zymes. Results are shown in Table 2.
Figure 3. Substrate analogues designed as selective inhibitors of class II
Fbas (with labelling of phosphates 1and 6 on FBP).
Even if active in vitro, these bisphosphorylated com-
pounds would be very unlikely readily to undergo passive
diffusion across cellular membranes. The removal of one
phosphoryl group could, however, favour permeation. The
phosphoryl group at the 1-position of FBP has been report-
ed to be essential for recognition by Fbas, while the one at
the 6-position is not. Compound 3 can thus be considered a
simplified analogue of fructose-1-phosphate, which is a
rather weak inhibitor of class I Fbas, with Ki >2 mm.[13] Syn-
theses of these three products are shown in Scheme 1.
Table 2. In vitro inhibition of various class I and class II Fbas by FBP an-
alogues.
Inhibitor
Class I Fba
Class II Fba
Rabbit muscle
S. cerevisiae
H. pylori
M. bovis
Ki [mm] (selectivity)[a]
[b]
1
2
3
524
264
49 (90)
21(23)
3.1 (180)
0.025[b] (86500) 0.013 (18500) 0.013 (22200)
2500[b]
1.6 (12800)
4.7[b] (480)
0.17 (16000)
[a] (KM/Kiclass II)/(KM/Kiclass I). [b] Estimated from IC50 value. KM rabbit muscle
55 mm; KM S. cerevisiae =450 mm; KM H. pylori =50 mm; KM M. bovis =60 mm.
=
Biochemical studies: All three compounds were tested as in-
hibitors of class I (from rabbit muscle) and class II (from
Saccharomyces cerevisiae, H. pylori and Mycobacterium
bovis BCG) aldolases.
The hydrazide 1 acted as a modest inhibitor, both in
terms of Ki and in terms of selectivity. In contrast, the hy-
droxamate 2 was very active, with a Ki comparable to that
of PGH but a greatly increased selectivity for class II Fbas.
The important difference be-
tween compounds 1 and 2 can
be explained in part by the dif-
ferent affinities of a hydrazide
and a hydroxamate for Zn2+
.
1
Also, the H NMR spectrum of
1 as a cyclohexylammonium
salt indicates the presence of
three cyclohexylamines (see
Experimental Section), which
means that one acidity of a
phosphate is neutralized by
the NH2 of the neighbouring
hydrazide (pKa values of
normal hydrazides are usually
close to 7). Should this ampho-
teric form exist within the
active site of the enzyme, it
could lower the affinity of the
hydrazide function for the zinc
ion. As would be expected for
N-alkylated hydroxamic acids,
2 and 3 were also much more
stable in water than PGH (as
verified by NMR of the prod-
uct dissolved in D2O). The
higher Ki value of 3 confirmed
that a phosphoryl group at the
6-position is an important, al-
though non-essential, element
of class II enzyme inhibitors.
Scheme 1. Synthesis of hydrazide 1 and of hydroxamates 2 and 3. Reagents and conditions: i) a) NaH, BnBr
(0.2equiv), 75%; b) PCC/CH2Cl2, 47%; c) BocNHNH2/THF, 68%. ii) DIBAH/toluene, 62%. iii) a) ClCO-
CH2OAc/THF/pyridine, 76%; b) NEt3/MeOH/H2O 2:8:1, 100%. iv) a) ClPO
C, 96%; c) ClPO(OPh)2/pyridine, 81%. v) a) H2/PtO2, 83%; b) H2O/TFA 2:1, 100%. vi) a) BnONH2/EtOH
reflux, 63%; b) ClCOCH2OAc/MeOH/NEt3, 70%. vii) a) ClPO(OPh)2/pyridine, 81%; b) NEt3/MeOH/H2O
2:8:1, 100%; c) ClPO(OPh)2/pyridine, 52%. viii) a) H2/Pd-C, 98%; b) H2/PtO2, 98%. ix) a) NaH, BnBr
ACHTRE(UNG OPh)2/pyridine, 82%; b) H2/Pd-
ACHTREUNG
AHCTREUNG
ACHTREUNG
(0.2equiv), 67%; b) PCC/CH2Cl2, 88%. c) BnONH2, EtOH/pyridine 808C, 24 h. x) NaBH3CN, EtOH/AcOH
1:1, RT, 4 h, 61% from ix. xi) ClCOCH2OAc/THF/pyridine, 90%. xii) a) NEt3/MeOH/H2O 2:8:1, 68%; b)
PACHTREUNG(OBn)2NiPr/imidazole/triazole/AcCN, 24 h RT; c) tBuOOH, 77% from xi. xiii) H2/Pd-C, 100%.
Chem. Eur. J. 2008, 14, 8521– 8529
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M. Therisod et al.
Against the M. bovis BCG enzyme, 3 was “only” ten times
less active than 2, a particularly encouraging result for an
analogue of fructose-1-phosphate. We believe that com-
pound 3 might represent an interesting basis lead compound
for the further synthesis of improved monophosphorylated
compounds and lipophilic prodrugs.
Biological studies—growth inhibition of cultivated bacteria:
Minimal inhibitory concentration values of compound 2
against M. tb H37Rv were determined in 7H9-ADC broth at
378C by the colorimetric resazurin microtiter assay de-
scribed by Martin and collaborators.[22] The bacteria were
grown aerobically under conditions compatible with class II
Fba expression[9] and similar to those used for the prepara-
tion of the M. bovis BCG extracts (see above). Compound 2
inhibited the growth of M. tb H37Rv at a concentration of
1 mm. Although modest, this result was unexpected in view
of the high polarity of compound 2 at physiological pH. The
presence of the class I aldolase (not verified) might in part
have offset the effect of 2. We have no evidence yet that
growth inhibition of cultivated M. tuberculosis by 2 corre-
lates with inhibitory properties observed in vitro. If it is as-
sumed, however, that this product kills M. tb through the in-
hibition of Fba, this effect could also be accounted for by an
extra-cytosolic location of the enzyme.[12] We are now inves-
tigating the question of the permeation of compound 2 into
M. tb cells (active transport or passive diffusion) and wheth-
er the inhibition of mycobacterial growth results directly
from the effect of this compound on the M. tb class II Fba.
Experiments to measure the MIC of 2 against M. tb grown
under anaerobic conditions—under which substrate-level
phosphorylation-dependent (and thus Fba-dependent) syn-
thesis of ATP would be expected to prevail—are also under-
way.
Figure 4. Difference electron density (F0ÀFC) annealed omit map show-
ing fit of compound 2 bound in the active site of H. pylori Fba. Confor-
mational changes induced upon binding of 2 are also highlighted. Loops
and catalytic zinc ion that undergo positional changes are shown in red
for the native Fba structure and in yellow for the bound Fba structure.
P1and P6 phosphoryls are identified. Figure and superposition were pre-
pared with the program PyMOL.[23]
dues 210–214) adjusts by 1.7 Š, enabling Ser213 also to bind
the P1 phosphoryl group. Concomitantly with this move-
ment, the catalytic zinc ion is displaced by 3.7 Š from its
buried position in the native structure towards the active
À
site surface, chelating the C=O and N O oxygens of 2 while
maintaining its interaction with the chelating His residues of
the active site. Interactions between 2 and neighbouring res-
idues in the H. pylori Fba active site are summarized in
Figure 5.
The structural data obtained with 2 in H. pylori Fba indi-
cate, consistently with data previously reported for PGH
bound in E. coli Fba,[17,18] that the interaction with the hy-
droxamate-based inhibitor results in a loosely coordinated
trigonal bipyramid geometry around the catalytic Zn2+ ion
(Figure 6). The conformational change stabilized by attach-
ment of the P1 phosphoryl results in tighter oxyanion bind-
ing (seven interactions; Figure 5) in relation to the interac-
tions stabilizing attachment with the P6 phosphoryl (three
interactions; Figure 5) and corroborates P1 phosphoryl as es-
sential for active site binding. The hydroxamate moiety
atoms C2, O2, N3 and O3 in compound 2 are coplanar (di-
hedral angle <18) and in the s-Z conformation (Figure 6 A),
mimicking an enediolate transition-state analogue, which
would favour a potent Ki value. Because the catalytic Zn2+
ion is significantly out of the plane containing the putative
enediolate, however, optimal chelation is unlikely. The same
hydroxamate atoms in active site complexes formed by
PGH bound with E. coli[17,18] and H. pylori Fba,[19] although
not coplanar (dihedral angle ꢀ458), do chelate the catalytic
Zn2+ ion in an approximately s-Z conformation (Figure 6B).
Further synthetic exploration of simplified substrate ana-
logues using the PGH scaffold that optimizes Zn2+ ion che-
Crystallographic studies: To examine the high affinity and
selectivity of 2 for class II aldolases, crystallographic struc-
tures of Fba from H. pylori alone and bound with compound
2 were solved to 1.8 and 2.3 Š resolutions, respectively.
Crystals of H. pylori Fba used for the studies were grown by
vapour diffusion from a protein solution (10 mgmLÀ1) dilut-
ed with an equal volume of precipitant buffer [PEG 1000
(12%), PEG 8000 (12%), calcium acetate (0.2m) and Tris/
HOAc (pH 8, 50 mm)] equilibrated against a reservoir of the
same buffer. The H. pylori Fba structure was solved by mo-
lecular replacement with use of Thermus aquaticus Fba as
search model. The refined native structure was then used as
search model to determine the structure of H. pylori Fba
bound with 2 by molecular replacement.
Superposition of native Fba with bound Fba (RMSD
0.48 Š based on all Ca atoms) revealed several significant
localized conformational changes (Figure 4).
A loop (residues 177–191) in native Fba undergoes a 16 Š
displacement (calculated from Ca atoms) upon binding of 2,
allowing Gly181 and Lys184 to interact with the P1 phospho-
ryl group. The same loop also repositions His180, which
binds the catalytic zinc ion. A second, adjacent loop (resi-
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Bisphosphate Aldolase Inhibitors
FULL PAPER
Although our initial goal
was the synthesis of simplified
analogues of the substrate
FBP, the potent Ki value of
compound 2 and the planarity
of its hydroxamate moiety sug-
gests that it behaves more like
a
transition-state analogue.
One reasonable hypothesis is
that the planar geometry ena-
bles internal electronic delocal-
ization of the hydroxamic acid
function in 2 that mimics in
part the movement of electrons
during the retro-ketolic cleav-
age of FBP (Figure 7).
Figure 5. Interactions of inhibitor 2 with proximal residues in the H. pylori Fba active site (P1 and P6 phos-
phates of 2 are explicitly labelled).
Conclusion
Taking advantage of previous results on analogues of
DHAP as inhibitors of Fba, we have prepared several ana-
logues of FBP. We introduced a well-positioned chelating
function in the hope of making these inhibitors selective for
class II (zinc-dependent) Fbas. Compound 2 gave the best
results, with low Ki values (10–20 nm) and good selectivity
for class II over class I (more than 20000). The inhibition re-
sults, however, with KM/Ki >2000, indicate that 2 is not only
a simple substrate analogue, but probably acts as a transi-
tion-state analogue. This inhibitor was co-crystallized with
H. pylori Fba (representative of class II Fbas). The resolved
structure of the complex unambiguously indicates that the
inhibitor is linked to the active site around the catalytic zinc
ion. Compound 2 is weakly active against cultivated M. tu-
berculosis (MIC 1m m). The discrepancy between in vitro in-
hibition tests and biological assays can be partly explained
by the high polarity of 2, bearing four negative charges at
physiological pH, and thus unlikely to cross biological mem-
branes by simple diffusion. Compound 3, in which the non-
essential phosphoryl group is missing, is “only” ten times
less efficient than 2 in inhibition tests. It could be a lead
compound for further synthesis of lipophilic derivatives
acting as prodrugs.
Figure 6. Coordination of catalytic Zn2+ ion in H. pylori Fba structure in
binding of A) compound 2, and B) PGH. View was obtained by first
overlaying bound structures.
lation while ensuring hydroxamate coplanarity should afford
compounds with more potent Ki values.
Compound 2 differs from the aldolase substrate and
class I
aldolase
inhibitor
hexitol-bisphosphate
(Ki
ꢀ0.5 mm)[17] in the absence of hydroxyl moieties at its C4 and
C5 atoms. In class I aldolases, both C4 and C5 hydroxyls par-
ticipate directly and indirectly in hydrogen-bonding interac-
tions with active site residues.[25] We postulate that 2, be-
cause it lacks hydroxyl moieties at positions C4 and C5, is
less able to participate in hydrogen bonding interactions
with active site residues of class I aldolases and that this, to-
gether with the apparent coplanarity of the hydroxamate
moiety atoms in 2—that would further hinder optimal fit
into the class I aldolase active site—forms a basis for the
high selectivity of 2 for class II aldolases.
Experimental section
Chemical syntheses
N-(3-Hydroxypropyl)-glycolohydrazide-bisphosphate (compound 1)
Compound 1a: NaH (8 g) was added in portions to propane-1,3-diol
(60 g, 0.79 mol) dissolved in anhydrous DMF (350 mL), followed by
drop-by-drop addition of benzyl bromide (34.2 g, 0.2 mol). After 12 h at
RT with stirring, the solvent was evaporated, the residue was dissolved in
Et2O, and the organic phase was washed with water, dried and concen-
trated. The product was purified by flash chromatography (Et2O/pentane
1:1) to give 1a (25 g, 75%). 1H NMR (250 MHz, CDCl3): d=7.36 (m,
5H), 4.55 (s, 2H), 3.80 (dt, J=11 Hz, 6 Hz, 2H), 3.68 (t, J=6 Hz, 2H),
1.89 ppm (q, J=6 Hz, 2H).
Figure 7. Presumed transition state of the retro-ketolic cleavage of FBP
and mesomeric structure of compound 2.
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M. Therisod et al.
Compound 1b: PCC (19.4 g, 90.2 mmol) was suspended in anhydrous di-
chloromethane (250 mL) with vigorous stirring. Compound 1a (10 g) was
added dropwise, and the mixture was stirred overnight at RT. The black
mixture was then filtered on a column of silica gel (200 mL), washed
through with dichloromethane. After evaporation, the crude product was
purified by flash chromatography (Et2O/pentane 1:1) to give 1b (4.61g,
47%). 1H NMR (250 MHz, CDCl3): d=9.78 (s, 1H), 7.36 (m, 5H), 4.55
(s, 2H), 3.82 (t, J=6 Hz, 2H), 2.68 ppm (t, J=7 Hz, 2H); 13C NMR
(62.9 MHz, CDCl3): d=201.15, 137.99, 128.47, 127.79, 127.72, 73.21,
63.89, 43.87 ppm.
Compound 1h: Compound 1g (4.16 g, 7.29 mmol) was dissolved in etha-
nol (50 mL). Pd/C (10%, 425 mg) was added, and the suspension was
stirred overnight under dihydrogen (4 bar). The catalyst was filtered, and
the solution was concentrated to give 1h (3.38 g, 96%). 1H NMR
(250 MHz, CDCl3): d=8.07 (brs, 1H), 7.30 (m, 10H), 5.05 (m, H), 4.76
(m, 1H), 3.99 (m, 1H), 3.59 (m, 2H), 3.28 (m, 1H), 1.71 (m, 2H),
1.45 ppm (s, 9H); 13C NMR (62.9 MHz, CDCl3): d=168.85, 168.76,
129.65, 125.42, 120.08, 82.18, 65.58, 59.58, 45.92, 28.91, 28.00 ppm;
31P NMR (101.3 MHz, CDCl3): d=À11.72 ppm; HR-MS (ESI): m/z:
calcd for C22H29N2O8P [M+Na+]: 503.1554; found: 503.1561.
Compound 1i: Compound 1h was phosphorylated with diphenylphos-
phoroyl chloride as described for 1g. Yield 2.99 g (81%); 1H NMR
(250 MHz, CDCl3): d=8.21(brs, 1H), 7.24 (m, 20H), 5.28 (m, 1H), 5.03
(m, 1H), 4.31 (m, 1H), 3.88 (m, 2H), 3.88 (m, 1H), 3.40 (m, 1H), 2.01
(m, 2H), 1.48 ppm (s, 9H); 13C NMR (62.9 MHz, CDCl3): d=168.3,
154.3, 150.2, 129.70, 125.3, 119.9, 81.9, 66.7, 65.4, 45.5, 27.9, 27.2 ppm;
31P NMR (101.3 MHz, CDCl3): d=À11.76, À11.66 ppm; HR-MS (ESI):
m/z: calcd for C34H38N2O10P2 [M+Na+]: 735.1843; found: 735.1847.
Compound 1c: Boc-hydrazine (1.74 g, 13.2 mmol) was added to com-
pound 1b (2.18 g, 13.45 mmol) in THF (25 mL). The mixture was stirred
overnight at RT and was then concentrated. The residue was recrystal-
lized from anhydrous pentane, giving 1c (2.5 g, 68%). 1H NMR
(250 MHz, CDCl3): d=7.31(m, 5H), 7.25 (t, J=5 Hz, 1H), 4.51 (s, 2H),
3.67 (t, J=6 Hz, 2H), 2.60 (q, J=6 Hz, 2H), 1.48 ppm (s, 9H); 13C NMR
(62.9 MHz, CDCl3): d=152.58, 144.64, 134.07, 129.74, 128.54, 128.41,
127.69, 127.16, 81.02, 73.00, 67.59, 32.76, 28.00 ppm; HR-MS (ESI): m/z:
calcd for C15H22N2O3 [M+Na+]: 301.1523; found: 301.1524.
N-(3-Hydroxypropyl)-glycolohydrazide bisphosphate (compound 1) (tris-
cyclohexylammonium salt): Compound 1i (1.87 g, 2.63 mmol) was dis-
solved in ethanol (20 mL). PtO2 (200 mg) was added, and the suspension
was treated under dihydrogen (40 bar) overnight with vigorous stirring.
After filtration and evaporation, solid 1j (977 mg, 83%) was recovered.
The product was dissolved in a H2O/CF3COOH mixture (2:1, 10 mL),
and the solution was kept for 12 h at RT. After evaporation, the residue
was dissolved in aqueous cyclohexylamine (1m), concentrated and recrys-
tallized from EtOH. Yield 0.9 g (49%); 1H NMR (250 MHz, D2O): d=
Compound 1d: DIBAL-H (20% in toluene, 45 mL, 55 mmol) was added
dropwise under Ar at 08C over 1h to 1c (5 g, 18.3 mmol) in toluene
(100 mL). After the system had been kept for 5 h at RT, NaOH solution
(1m, 100 mL) was added slowly (exothermic reaction). After evaporation
of the toluene, the remaining aqueous phase was extracted with dichloro-
methane. The organic phase was then washed successively with NaOH
(1m) and brine. Compound 1d was purified by flash chromatography
(pentane/ethyl acetate/NEt3 30:70:0.1, 3.18 g, 62%). 1H NMR (250 MHz,
CDCl3): d=7.32 (m, 5H), 6.3 (brs, 1H), 4.52 (s, 2H), 3.56 (t, J=6 Hz,
2H), 2.96 (t, J=6 Hz, 2H), 1.79 (quint, J=6 Hz, 2H), 1.47 ppm (s, 9H);
13C NMR (62.9 MHz, CDCl3): d=156.67, 138.38, 128.27, 127.55, 127.44,
80.24, 72.88, 68.47, 49.41, 28.29, 28.04 ppm; HR-MS (ESI): m/z: calcd for
C15H24N2O3 [M+Na+]: 303.1679; found: 303.1681.
4.49 (d, JH,P =6 Hz, 2H; -CO-CH2-OPO3HÀ), 3.62 (dt, JH,P =6 Hz, JH,H
=
7 Hz, 2H; 2ÀO3PO-CH2-CH2-), 3.40 (t, J=7 Hz, 2H; -CH2-CH2-N),
1.74 ppm (m, 2H; -CH2-CH2-CH2-) [CHA+: 2.90 (m, 3H), 1.40–1.80 (m,
15H), 0.95–1.19 (m, 15H)]; 13C NMR (62.9 MHz, D2O): d=174.20,
174.09, 63.41, 48.36, 27.90, 27.82 ppm [CHA: 51.18, 31.34, 25.20,
24.7 ppm]; 31P NMR (101.3 MHz, D2O): d=À 0.46 (s, 1P), À0.17 ppm (s,
1P); HR-MS (ESI, negative): m/z: calcd for C5H13N2O9P2À: 307.01018;
found: 307.01021.
Compound 1e: Pyridine (1.19 mL, 15.7 mmol) was added to 1d (3.18 g,
11.34 mmol) in anhydrous THF (50 mL), followed dropwise by acetoxy-
acetyl chloride (2.15 g, 15.7 mL) in THF (25 mL). After the system had
been kept for 3 h at RT, water (20 mL) and dichloromethane (100 mL)
were added, and the organic phase was washed with water and brine,
dried over sodium sulfate and concentrated. Compound 1e was obtained
after flash chromatography (pentane/Et2O/Et3N 1:1:0.01, 3.38 g, 76%).
1H NMR (250 MHz, CDCl3): d=7.40 (m, 5H), 4.85 (m, 2H), 4.57 (s,
2H), 4.26 (m, 1H), 3.61 (m, 2H), 3.19 (m, 1H), 2.24 (s, 3H), 1.93 (m,
2H), 1.54 ppm (s, 9H); 13C NMR (62.9 MHz, CDCl3): d=170.39, 168.87,
154.22, 137.64, 128.23, 127.23, 81.90, 72.97, 68.35, 61.10, 46.02, 27.88,
26.64, 20.39 ppm; HR-MS (ESI): m/z: calcd for C19H28N2O6 [M+Na+]:
403.1840; found: 403.1850.
N-(3-Hydroxypropyl)-glycolohydroxamic acid bisphosphate (compound
2)
Compound 2a: O-Benzylhydroxylamine (4.63 g, 37 mmol) was added to
3-bromopropanol (2.72 g, 18 mmol) in anhydrous ethanol (30 mL), and
the mixture was heated at reflux for 45 h. After complete concentration
of the mixture, ethyl acetate (200 mL) was added, and the precipitated
O-benzylhydroxylamine hydrobromide was removed by filtration. After
repeated concentration of the mixture, the oily residue was redissolved in
Et2O. This organic solution was washed with potassium hydrogencarbon-
ate (1m), dried and concentrated. The product was purified by flash chro-
matography (Et2O/NEt3 100:0.1). Yield 2.04 g (63%); 1H NMR
(250 MHz, CDCl3): d=7.33 (m, 5H), 5.62 (brs, 1H), 4.73 (s, 2H), 3.74 (t,
J=7 Hz, 2H), 3.18 (t, J=7 Hz, 2H), 2.85 (brs, 1H), 1.76 ppm (quint, J=
7 Hz, 2H); 13C NMR (62.9 MHz, CDCl3): d=135.3, 128.5, 128.1, 76.3,
62.6, 51.0, 29.6, 25.20 ppm; HR-MS (ESI): m/z: calcd for C10H15NO2
[M+Na+]: 204.0995; found: 204.0996.
Compound 1 f: Compound 1e (3.38 g, 8.87 mmol) was stirred overnight
at RT in a methanol/Et3N/water mixture (8:2:1). After concentration, 1 f
1
was obtained (3 g, 100%). H NMR (250 MHz, CDCl3): d=7.25 (m, 5H),
4.45 (s, 2H), 4.11 (m, 2H), 4.06 (brs, 2H), 3.61 (t, J=7 Hz, 2H), 3.19 (m,
1H), 1.93 (quint, J=7 Hz, 2H), 1.45 ppm (s, 9H); 13C NMR (62.9 MHz,
CDCl3): d=174.39, 154.16, 137.48, 128.15, 127.23, 81.75, 72.91, 68.15,
59.65, 46.02, 27.88, 26.59 ppm; HR-MS (ESI): m/z: calcd for C17H26N2O5
[M+Na+]: 361.1734; found: 361.1738.
Compound 2b: Compound 2a (0.839 g, 4.64 mmol) and triethylamine
(0.563 g, 5.6 mmol) were dissolved in methanol (10 mL) at 08C. Acetoxy-
acetyl chloride (0.76 g, 5.57 mmol) in anhydrous Et2O was added drop-
wise to this solution. The reaction mixture was stirred for 15 min at RT
and was then diluted with Et2O and treated with water (30 mL). The or-
ganic phase was washed with water, dried and concentrated. The product
was purified by flash chromatography (Et2O/NEt3 100:0.1) to yield 2b
(0.914 g, 70%). 1H NMR (250 MHz, CDCl3): d=7.4 (m, 5H), 4.89 (s,
2H), 4.73 (s, 2H), 3.83 (t, J=7 Hz, 2H), 3.58 (t, J=7 Hz, 2H), 2.15 (s,
3H), 1.83 ppm (quint, J=7 Hz, 2H); 13C NMR (62.9 MHz, CDCl3): d=
170.36, 168.88, 133.55, 129.01, 128.54, 76.23, 60.81, 58.39, 42.22, 29.18,
20.21ppm; HR-MS (ESI): m/z: calcd for C14H19NO5 [M+Na+]: 304.1155;
found: 304.1156.
Compound 1g: Compound 1 f (3 g, 8.87 mmol) was dissolved in anhy-
drous pyridine (100 mL). Diphenylphosphoroyl chloride (2.8 mL,
35.5 mmol) and DMAP (0.1mmol) were added at once. The solution was
stirred overnight at RT. Water was added, and after 10 min the reaction
mixture was concentrated. Water and dichloromethane were added to
the residue. The organic phase was washed with water, dried and concen-
trated. Compound 1 f was obtained after flash chromatography (AcOEt,
4.16 g, 82%). 1H NMR (250 MHz,CDCl3): d=7.32 (m, 15H), 4.77 (m,
1H), 4.95 (s, 1H), 4.49 (s, 2H), 4.16 (m, 1H), 3.36 (t, J=6 Hz, 2H), 3.23
(m, 1H), 1.87 (dquint, J=6 Hz, 2H), 1.47 ppm (s, 9H); 13C NMR
(62.9 MHz, CDCl3): d=167.00, 168.90, 154.26, 150.27, 15.,25, 137.54,
129.5, 128.35, 127.69, 125.25, 120.08, 82.18, 73.15, 68.52, 65.42, 46.15,
27.96, 26.74 ppm; 31P NMR (101.3 MHz, CDCl3): d=À11.70 ppm; HR-
MS (ESI): m/z: calcd for C29H35N2O8P [M+Na+]: 593.2023; found:
593.2037.
Compound 2c: Compound 2b (1.38 g, 4.9 mmol) was phosphorylated
with diphenylphosphoroyl chloride as described for 1 f. Yield 2.05 g
(81%); 1H NMR (250 MHz, CDCl3): d=7.15–7.4 (m, 15H), 4.87 (s, 2H),
4.69 (s, 2H), 4.31(dt, J=12, 7 Hz, 2H), 3.73 (t, J=7 Hz, 2H), 2.15 (s,
8526
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 8521– 8529

Bisphosphate Aldolase Inhibitors
FULL PAPER
3H), 2.06 ppm (quint, J=7 Hz, 2H); 13C NMR (62.9 MHz, CDCl3): d=
170.4, 168.6, 150.3, 150.2, 133.6, 129.0, 129.2, 129.1, 128.7, 125.2, 119.9,
119.8, 76.5, 66.7, 66.6, 61.1, 42.4, 27.5, 27.4, 20.4 ppm; 31P NMR
(101.3 MHz, CDCl3): d=À12 ppm; HR-MS (ESI): m/z: calcd for
C28H28NO8P [M+Na+]: 536.1445; found: 536.1452.
J=6, 2 Hz, 2H); 13C NMR (62.9 MHz, CDCl3): d=201.3, 137.9, 128.5,
127.7, 127.6, 73.27, 63.9, 43.9 ppm.
Compound 3c: 3-Benzyloxypropionaldehyde (3b, 0.0157 mol, 2.57 g) was
dissolved in methanol (78.5 mL). O-Benzylhydroxylamine (0.024 mol,
2.89 g) and pyridine (0.145 mol, 11.72 mL) were added. The mixture was
stirred at reflux at 658C for 3.5 h and concentrated under reduced pres-
sure. The resultant product was dissolved in CH2Cl2 (50 mL), washed
with water (3”50 mL), dried with Na2SO4 and concentrated under re-
duced pressure. The product was an oily mixture of two Z/E stereoiso-
mers (together with remaining benzylhydroxylamaine). Yield 4.211 g,
Rf1 =0.94, Rf2 =0.88 (pentane/ethyl acetate 8:2). 1H NMR (250 MHz,
CDCl3): d=7.4 (m, 10H), 6.9 and 7.5 (t, J=5.5 Hz, 1H), 5.14 and 5.19 (s,
2H), 4.76 (s, 2H), 3.65 and 3.68 (t, J=6.5 Hz, 2H), 2.56 and 2.75 ppm (q,
J=6.5 Hz, 2H); 13C NMR (62.9 MHz, CDCl3): d=149.0, 137.75, 137.5,
128.5, 128.3, 128.0, 127.9, 127.7, 127.1, 75.9, 75.7, 66.7, 30.4 ppm.
Compound 2d: Compound 2c (2.2 g, 4.2 mmol) was deprotected as de-
scribed for 1e. Yield 2 g (100%); 1H NMR (250 MHz, CDCl3): d=7.15–
7.4 (m, 15H), 4.73 (s, 2H), 4.30 (dt, J=12, 7 Hz, 2H), 4.21 (s, 2H), 3.75
(t, J=7 Hz, 2H), 2.04 ppm (quint, J=7 Hz, 2H); 13C NMR (62.9 MHz,
CDCl3): d=173.66, 150.24, 150.13, 133.46, 129.61, 129.08, 128.58, 125.23,
119.81, 119.75, 76.41, 66.51, 66.43, 59.97, 42.45, 27.46, 27.36 ppm;
31P NMR (101.3 MHz, CDCl3): d=À11.98 ppm; HR-MS (ESI): m/z:
calcd for C24H26NO7P [M+Na+]: 494.1339; found: 494.1347.
Compound 2e: Compound 2d (2 g, 4.24 mmol) was phosphorylated as
described for 1 f. Yield 1.56 g (52%); 1H NMR (250 MHz, CDCl3): d=
7.15–7.4 (m, 25H), 4.83 (d, J=11 Hz, 2H), 4.76 (s, 2H), 4.33 (dt, J=7.5,
7 Hz, 2H), 3.77 (t, J=7 Hz, 2H), 2.10 ppm (quint, J=7 Hz, 2H);
13C NMR (250 MHz, CDCl3): d=167.91, 150.54, 150.42, 133.58, 129.91,
129.82, 129.44, 128.95, 125.51, 120.30, 120.23, 120.13, 76.71, 66.85, 66.77,
65.41, 42.68, 27.74, 27.65 ppm; 31P NMR (101.3 MHz, CDCl3): d=À11.98,
À11.53 ppm; HR-MS (ESI): m/z: calcd for C36H35NO10P2 [M+Na+]:
726.1628; found: 726.1628.
Compound 3d: Sodium cyanoborohydride (0.0188 mol, 1.18 g) was added
in portions, with cooling in an ice-bath, to 3c (0.0157 mol, 4.211 g) dis-
solved in a mixture of ethanol and acetic acid (1:1, 25 mL). This was
stirred at RT for 7.5 h. The solvent was then evaporated under reduced
pressure, and the resultant product was made alkaline (pH 8.00) with sa-
turated NaHCO3. The mixture was extracted with CH2Cl2 (3”150 mL),
washed with water (150 mL) and sat. NaCl (150 mL), dried with Na2SO4
and concentrated under reduced pressure. The product was purified by
column chromatography (pentane/AcOEt 9:1). Yield 2.61 g (61% from
Compound 2 f: Compound 2e (0.945 g, 1.34 mmol) was dissolved in etha-
nol (20 mL). Pd-C (10%, 100 mg) was added, and the suspension was vig-
orously stirred under dihydrogen overnight. After filtration and evapora-
tion, 2 f (0.805 g, 98%) was recovered. 1H NMR (250 MHz, CDCl3): d=
7.15–7.4 (m, 20H), 4.97 (d, J=11 Hz, 2H), 4.33 (dt, J=6 Hz, 2H), 3.67
(t, J=6 Hz, 2H), 2.10 ppm (quint, J=6 Hz, 2H); 13C NMR (62.9 MHz,
CDCl3): d=167.39, 167.28, 150.38, 150.29, 150.20, 129.85, 129.75, 125.57,
120.19, 120.13, 12.01, 119.95, 67.04, 66.37, 65.71, 65.65, 45.09, 27.42,
27.34 ppm; 31P NMR (101.3 MHz, CDCl3): d=À11.26, À11.64 ppm; HR-
MS (ESI): m/z: calcd for C29H29N2O10P2 [M+Na+]: 636.1159; found:
636.1171.
3b). Rf =0.1(pentane/Et O 9:1 );1H NMR (250 MHz, CDCl3): d=7.4 (m,
2
10H), 4.76 (s, 2H), 4.55 (s, 2H), 3.6 (t, J=6.5 Hz, 2H), 3.12 (t, J=6.5 Hz,
2H), 1.9 ppm (quint, J=6.5 Hz, 2H); 13C NMR (62.9 MHz, CDCl3): d=
138.55, 138.1, 128.55, 128.45, 128.08, 127.85, 127.7, 76.2, 73.0, 68.65, 49.65,
27.6 ppm.
Compound 3e: Compound 3d (6.47 mmol, 1.754 g), triethylamine
(9.7 mmol, 1.36 mL) and acetoxyacetyl chloride (7.764 mmol, 0.83 mL)
were dissolved in dry CH2Cl2 (10 mL). This mixture was stirred at RT for
20 minutes. The organic phase was washed with water (3”10 mL), dried
with Na2SO4 and concentrated under reduced pressure. The product was
an oil (2.167 g, 90%). Rf =0.49 (pentane/ethyl acetate 6:4); 1H NMR
(250 MHz, CDCl3): d=7.4 (m, 10H), 4.88 (s, 2H), 4.75 (s, 2H), 4.5 (s,
2H), 3.8 (brt, J=6.5 Hz, 2H), 3.55 (t, J=6.5 Hz, 2H), 2.2 (s, 3H),
2.0 ppm (quint, J=6.5 Hz, 2H); 13C NMR (62.9 MHz, CDCl3): d=170.7,
168.5, 138.4, 134.0, 129.3, 129.2, 128.9, 128.4, 127.74, 127.6, 76.5, 73.0,
67.5, 61.4, 43.45, 27.12, 20.7 ppm.
N-(3-Hydroxypropyl)-glycolohydroxamic acid bisphosphate (compound
2) tetrakis-cyclohexylammonium salt: Compound 2g (0.805 g, 1.31 mmol)
was dephenylated as described for 1i. The residue was dissolved in etha-
nol (20 mL), and an excess of cyclohexylamine was added. The solution
was evaporated to afford a white powder, which was recrystallized from
1
ethanol. Yield 0.32 g (34%); H NMR (250 MHz, D2O): d=430 (d, JH,P
=
6 Hz, 2H; -CO-CH2-OPO32À), 3.54 (dt, JH,P =JH,H =6 Hz, 2H; 2ÀO3PO-
CH2-CH2-), 3.45 (t, J=7 Hz, 2H; -CH2-CH2-N), 1.66 ppm (quint, J=
7 Hz, 2H; -CH2-CH2-CH2) [CHA+: 2.85 (m, 4H), 1.25–1.80 (m, 20H),
0.80–1.25 (m, 20H)]; 13C NMR (62.9 MHz, D2O): d=172.13, 62.30, 62.24,
62.08, 50.92, 47.00, 31.04, 28.13, 28.04, 24.99, 24.49 ppm; 31P NMR
(101.3 MHz, D2O): d=3.52, 3.65 ppm; HR-MS (ESI, negative): m/z:
calcd for C5H12NO10P2À: 307.99420; found: 307.99427
Compound 3 f: Compound 3e was dissolved in a methanol/triethylamine/
water mixture (8:2:1, 44 mL). This was stirred at RT for 18 h. After com-
plete evaporation, the product was purified by flash chromatography
(pentane/AcOEt 6:4). The product was an oil (1.305 g, 68%). Rf =0.38
(pentane/ethyl acetate 7:3); 1H NMR (250 MHz, CDCl3): d=7.4 (m,
10H), 4.82 (s, 2H), 4.52 (s, 2H), 4.25 (s, 2H), 3.85 (m, 2H), 3.55 (t, J=
6 Hz, 2H), 3.23 (brs, 1H), 2.0 ppm (quint, J=6 Hz, 2H); 13C NMR
(62.9 MHz, CDCl3): d=173.75, 138.3, 133.95, 129.35, 128.9, 128.5, 127.8,
76.6, 73.1, 67.45, 60.25, 43.7, 27.14 ppm.
N-(3-Hydroxypropyl)-phosphoglycolohydroxamic acid (compound 3)
Compound 3a: BnBr (0.1mol, 17 g) was added dropwise over 20 minutes
to a solution of propane-1,3-diol (0.3 mol, 21.67 mL) in anhydrous DMF
(100 mL). After cooling of the mixture in an ice-bath, NaH (60%, 3 g,
0.125 mol) was added in portions. This mixture was stirred under argon
overnight at RT. The solvent was concentrated under reduced pressure.
Water (75 mL) was added, and the water phase was extracted with
CH2Cl2 (2”75 mL). The combined organic phases were dried with
Na2SO4 and concentrated under reduced pressure. The product was an
oil (0.067 mol, 11.24 g, 67%). Rf =0.18 (pentane/Et2O 8:2). 1H NMR
(250 MHz, CDCl3): d=7.3–7.4 (m, 5H), 4.54 (s, 2H), 3.76 (m, 2H), 3.64–
3.67 (t, J=6 Hz, 2H), 1.9 ppm (quint, J=5.8 Hz, 2H); 13C NMR
(62.9 MHz, CDCl3): d=138.2, 128.5, 127.7, 127.6, 32.31 73.2, 68.84,
61.09 ppm.
Compound 3g: A mixture of 3 f (3.96 mmol, 1.305 g), dibenzyl N,N-diiso-
propylphosphoramidite (7.923 mmol, 2.73 g), imidazole (11.88 mmol,
0.808 g) and 1,2,4-triazole (7.92 mmol, 0.55 g) was dissolved in dry aceto-
nitrile (27 mL). This system was stirred at RT under argon for 27 h.
tBuOOH (7.92 mmol, 1.08 mL, 7.3m) and CH2Cl2 (30 mL) were then
added. This mixture was stirred for 2 h at RT. An aqueous solution of
sodium thiosulfate (1m, 75 mL) and CH2Cl2 (30 mL) were added. The
aqueous phase was extracted with CH2Cl2 (3”100 mL). The collected or-
ganic phases were washed with sat. Na2CO3 (200 mL), dried with
Na2SO4, concentrated under reduced pressure and purified by flash chro-
matography (pentane/AcOEt). The product was an oil (1.794 g, 77%).
Rf =0.67 (pentane/ethyl acetate 1:1); 1H NMR (250 MHz, CDCl3): d=7.4
(m, 20H), 5.2 (m, 4H), 4.78 (s, 2H), 4.7 (d, J=11.25 Hz, 2H), 4.5 (s, 2
H), 3.8 (brt, J=8.7 Hz, 2H), 3.5 (t, J=8.7 Hz, 2H), 2.0 ppm (quint, J=
8.75 Hz, 2H); 13C NMR (62.9 MHz, CDCl3): d=168.5, 138.4, 135.9, 133.9,
129.3, 128.9, 128.6, 128.5, 128.4, 128.0, 127.7, 127.6, 76.5, 73.0, 69.6, 67.5,
64.4, 43.5, 27 ppm; 31P NMR (101.3 MHz, CDCl3): d=0.8 ppm.
Compound 3b: PCC (0.036 mol, 7.74 g), 3-benzyloxypropan-1-ol (3a,
0.018 mol, 3 g) and powdered molecular sieves were added to dry CH2Cl2
(72 mL). This mixture was stirred under argon for 2.5 h. Et2O (500 mL)
was then added to the reaction mixture, which was filtered through a
short column of silica gel. The solvent was evaporated under reduced
pressure. The product was an oil (0.0157 mol, 2.584 g, 88%). Rf =0.77
(pentane/ethyl acetate 8:2). 1H NMR (250 MHz, CDCl3): d=9.81(t, J=
2 Hz, 1H), 7.4 (m, 5H), 4.57 (s, 2H), 3.84 (t, J=6 Hz, 2H), 2.72 ppm (dt,
N-(3-Hydroxypropyl)-phosphoglycolohydroxamic acid (compound 3) bis-
cyclohexylammonium salt: A mixture of 3g (1.5 mmol, 0.9 g), Na2CO3
Chem. Eur. J. 2008, 14, 8521– 8529
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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8527

M. Therisod et al.
(1.5 mmol, 0.15 g) and Pd/C (170 mg) in water/ethanol (1:1, 40 mL) was
stirred under hydrogen (3 bar) for 36 h. It was then filtered through
celite and concentrated under reduced pressure. The product, dissolved
in water, was then filtered through a Dowex-50 column (cyclohexylam-
monium form), and the eluate was concentrated under reduced pressure
Synchrotron Light Source (Brookhaven National Laboratory, Upton,
USA). As control, a native data set was also collected with use of beam-
line X8C. A fluorescence energy scan, collected at beamline X8C by
energy scanning about the Zn Ka edge (1.2818 Š), demonstrated that
the H. pylori Fba crystals contained zinc, although no exogenous zinc
had been added during purification or recrystallization of the protein. All
data sets were processed by use of HKL2000[27], and the results are sum-
marized in Table I in the Supporting Information.
1
to give the crystalline product (0.566 g, 86%). H NMR (250 MHz, D2O):
d=4.4 (d, JH,P =5.75 Hz, 2H; CO-CH2-OPO32À), 3.55 (t, J=6.5 Hz, 2H;
-CH2-OH), 3.5 (t, J=6.5 Hz, 2H; -CH2-N-), 1.9 ppm (quint, J=6.5 Hz,
2H; -CH2-CH2-CH2-) [CHA+: 3.0 (m, 2H), 1–2 (m, 22H)]; 13C NMR
(62.9 MHz, D2O): d=171.7, 63.3, 61.6, 58.8, 50.2, 45.3, 30.3, 28.4, 24.2,
23.8 ppm; 31P NMR (101.3 MHz, D2O): d=3.6 ppm; HR-MS (ESI, nega-
tive): m/z: calcd for C5H11NO7PÀ [MÀH]+: 228.0273; found: 228.0272.
Structure solution and refinement: A model structure of H. pylori aldo-
lase was generated by feeding to the molecular replacement (MR) pro-
gram AMORE[28] 20 comparative homology models obtained with the
program MODELLER[29] with use of the structure of class II T. aquaticus
aldolase[30] as template (PDB code 1RVG). The best solution was used as
starting point for refinement. The native structure was solved by iterative
rounds of refinement (simulated annealing and minimization) with
CNS[31] and model building.[32] Initial phases used for model building of li-
ganded structures were obtained by using the refined native structure as
template for input into the program Phenix autoMR/autoBuild.[33] The
native and PGH structures belong to the monoclinic space group P21 and
each have one aldolase homodimer in the asymmetric unit. The structure
in complexation with compound 2 belongs to space group P1and also
has one aldolase homodimer in the asymmetric unit. All reflections with
I/s(I) >1were used in refinement; however, electron density maps were
calculated to the resolution shown in Table I in the Supporting Informa-
tion and corresponded to completeness of greater than 70% in the high-
est-resolution shell. The liganded structures were subjected to iterative
rounds of refinement Phenix and model building with Coot[34] and
PyMol.[23] The Molprobity server[35] and the Coot validating tools were
used to optimize the structures during the refinement. Water molecules
were added automatically by CNS or Phenix in the initial rounds and
manually near the end of refinement. Loop regions (residues 139–153) in
each subunit were associated with regions of weak electron density.
Compound 3 is apparently subject to the following rearrangement in
acidic medium:
Biochemistry
Biochemical evaluation of inhibitors 1–3
Enzymes: Rabbit muscle Fba was a commercial preparation, available
from Sigma or Fluka.
H. pylori Fba was a recombinant enzyme expressed in E. coli strain
JM109. Yeast Fba was isolated from S. cerevisiae. Cells were disrupted in
a French press (110 psi). The enzyme was partly purified by precipitation
with ammonium sulfate.[38]
M. bovis Fba was isolated from cultivated cells by disruption in a French
press. Crude extracts were used.
Solutions
Ligand modelling was based on interpretation of electron density shapes
of 2FoÀFc and FoÀFc annealed omit maps and use of the phenix.elbow
command for generation of topology and parameters. Binding by com-
pound 2 and PGH were readily discernable and were associated with
clearly defined electron densities in the active site. Difference electron
density (FoÀFc) annealed omit maps calculated in the final round of re-
finement confirmed identical binding of ligands in both subunits. Final
model statistics, calculated with CNS and PROCHECK,[36] are shown in
Table I in the Supporting Information. The coordinates and structure fac-
tors of H. pylori aldolase, as well as those of the native enzyme in com-
plexation with PGH and with compound 2, have been deposited with the
Protein Data Bank (PDB entry codes 3C4U, 3C52 and 3C56, respective-
ly).[37] The final structure models of native aldolase, compound 2 and
PGH enzymatic complexes have Rcryst (Rfree) values of 0.185 (0.207), 0.172
(0.208) and 0.204 (0.249), respectively. The corresponding Luzzati atomic
coordinate errors were estimated at 0.20, 0.23 and 0.28 Š, respectively.
Ramachandran analysis with PROCHECK placed at least 90% of non-
glycine and non-proline residues of the four structures in the most fa-
vourable region and with the remainder found in allowed regions, attest-
ing to good model geometry in the structures.
Glycylglycine buffer (0.1m, pH 7.4, with 0.2m potassium acetate for clas-
s II aldolases).
NADH 1.41 mm in buffer.
Fructose bisphosphate 2 mm in buffer.
Glycerophosphate dehydrogenase (GPDH, 274 UmLÀ1).
Enzymatic test: Fructose bisphosphate and inhibitor at the convenient
concentration, NADH (0.12 mm), GPDH (11 U) and aldolase (4 mU)
were placed in a cuvette to give a final volume of 1.2 mL. The decrease
in absorbance of NADH at 340 nm was monitored on a spectrophotome-
ter over 1–2 min.
Purification and recrystallization of H. pylori Fba: Plasmid pKK223-3
coding for H. pylori Fba was transformed and over-expressed in E. coli
strain JM109. Recombinant H. pylori Fba was purified by a combination
of anion exchange (DEAE), hydrophobic exchange chromatography
(Phenyl-Sepharose) and size exclusion chromatography.[26] Aldolase con-
centration was determined by use of BCA Protein Assay Reagent
(Pierce) with bovine serum albumin serving as standard. Enzymatic activ-
ity was monitored by spectrophotometry with use of a coupled assay and
monitoring of NADH oxidation at 340 nm. The purified protein was
stored at 48C in 85% saturated ammonium sulfate solution (25 mm
Tris·HCl, pH 8).
Comparisons: Superpositions were performed with the program PyMOL
with use of Ca atom coordinates of identical regions of amino acid se-
quences. Root mean square (rms) deviations based on superposition of
equivalent Ca atoms are reported. The dihedral angles for the hydroxa-
mate moieties in compound 2 and PGH are the angles between the
planes defined by atoms C2, N3 and O2 and by atoms C2, N3 and O3. A
dihedral angle of 08 indicates coplanarity and has atoms O2 and O3 posi-
tioned cis to each other and coplanar with atoms C2 and N3.
H. pylori aldolase crystals were grown by vapour diffusion from a mix-
ture (1:1) of protein solution (10 mgmLÀ1 initial protein concentration
made up in 25 mm Tris·HCl pH 7.0) and precipitant buffer [PEG 1000
(12%), PEG 8000 (12%), calcium acetate (0.2m) and Tris/HOAc (pH 8,
50 mm)]; 4 m drops were equilibrated at 238C against 1mL reservoirs of
precipitant solution. Crystals grew over two weeks.
Biology
Data collection and processing: Aldolase crystals were soaked in com-
pound 2 buffer [mother liquor plus compound 2 (10 mm)] or in PGH
buffer [mother liquor plus PGH (10 mm)]. Prior to data collection, crys-
tals were cryoprotected by transfer through a cryobuffer solution [com-
pound 2 or PGH buffer plus glycerol (10%)] and immediately flash
frozen in a stream of gaseous N2 cooled to 100 K. Diffraction data were
collected from single crystals at beamlines X8C and X29 of the National
Growth inhibition of cultivated bacteria: Minimal inhibitory concentra-
tion values of compound 2 against M. tb H37Rv were determined in
7H9-ADC broth at 378C by the colorimetric resazurin microtiter assay
described by Martin and collaborators.[22] The bacteria were grown aerob-
ically under conditions compatible with class II Fba expression[9] and sim-
ilar to those used for the preparation of the M. bovis BCG extracts.
8528
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Chem. Eur. J. 2008, 14, 8521– 8529

Bisphosphate Aldolase Inhibitors
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Acknowledgements
This work was supported by a scholarship to R.D. from the Region Ile de
France. K.K. was on leave from Politechnika Warszawska in an Erasmus
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Research Council (Canada) and the Canadian Institutes for Health Re-
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Light Source, Brookhaven National Laboratory, which is supported by
the United States Department of Energy, Division of Materials Sciences
and Division of Chemical Sciences under contract DE-AC02-98CH10886.
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Received: May 6, 2008
Published online: August 7, 2008
Chem. Eur. J. 2008, 14, 8521– 8529
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8529