Origin of the slow-binding inhibition of aldolase by D-glycero-tetrulose 1-phosphate (D-erythrulose 1-phosphate) from the comparison with the isosteric phosphonate analog

Article abstract of DOI:10.1002/(sici)1099-0690(199911)1999:11<2853::aid-ejoc2853>3.0.co;2-0

The mechanistic reaction pathway for the slow-binding inhibition of rabbit muscle aldolase by D-glycero-tetrulose 1-phosphate (D-erythrulose 1- phosphate) was investigated through the use of its phosphonomethyl isoster 4 which was synthezised for this stu

Full text of DOI:10.1002/(sici)1099-0690(199911)1999:11<2853::aid-ejoc2853>3.0.co;2-0

FULL PAPER
Origin of the Slow-Binding Inhibition of Aldolase by
D
-glycero-Tetrulose
1-Phosphate (
D
-Erythrulose 1-Phosphate) from the Comparison with the
Isosteric Phosphonate Analog
[a]
[a]
´ ´[a]
Patrick Page, Casimir Blonski,* and Jacques Perie
Keywords: Bioorganic chemistry / Enzyme inhibitors / Aldolase / Structure-activity relationship / Enzyme mechanism
The mechanistic reaction pathway for the slow-binding
inhibition of rabbit muscle aldolase by -glycero-tetrulose 1-
phosphate -erythrulose 1-phosphate) was investigated
through the use of its phosphonomethyl isoster 4 which was
enamine intermediate. We show from these results that
enzyme slow-binding inhibition by -erythrulose 1-
phosphate is consistent with a phosphate β-elimination
D
D
(D
reaction through the enamine intermediate. This mechanism
synthezised for this study. The latter is not a substrate nor a takes into account the stereochemical features known for
slow-binding inhibitor but interferes in the enzyme- aldolase, the parallel between enzyme activity recovery and
catalyzed reaction with the substrate fructose 1,6-
diphosphate in a competitive manner. It was found that
phosphonate 4 forms an iminium ion with aldolase and
undergoes subsequent α-proton abstraction to form an
phosphate release after action of D-erythrulose 1-phosphate,
and also the same reaction from dihydroxyacetone
phosphate.
Fructose 1,6-diphosphate aldolases reversibly cleave the bition. This result is of interest for both a better knowledge
fructose 1,6-diphosphate (FDP) into two triose phosphates: of the class I aldolases nontypical mechanistic pathway, and
dihydroxyacetone phosphate (DHAP) and glyceraldehyde the design of a new class of inhibitors for these enzymes.[8]
3-phosphate (GAP). There are two distinct classes of aldol-
ases: Class I aldolases proceed by Schiff base formation
with the substrate, while class II require a metal ion as co-
factor and do not form a Schiff base.[1] The ordered multis-
tep reaction catalyzed by class I aldolases, typified by rabbit
muscle aldolase, shown in Scheme 1 (E1: iminium ion or
Schiff base; E2: enamine; E3: iminium ion), is supported by
several experimental corroborations.[2] There is also some
evidence that the enzyme rate is limited by the release of
products in both directions.[3] Additionally, in the absence
of GAP, the unusual reversible covalent complex, enamine-
aldehyde plus phosphate intermediate E4, is made possible
by the slow aldolase-catalyzed conversion of DHAP into
methylglyoxal and orthophosphate.[4] However, the concen-
tration of this intermediate could not exceed 6% of the total
amount of DHAP bound to aldolase.[2d]
Other data suggest that class I aldolases prefer the (S)
configuration at C-3 for their substrates.[5] Accordingly, -
glycero-tetrulose 1-phosphate (-erythrulose 1-phosphate),
having the (R) configuration at this carbon atom, is not a
substrate but interestingly leads to a time-dependent inhi-
bition (slow-binding)[6] of aldolase by formation of a stable
enzymeϪligand intermediate,[7] whereas the  derivative is
a substrate.[5c,7] We report the comparative mechanism/inhi-
bition studies of aldolase from rabbit muscle by -erythru-
lose 1-phosphate and phosphonate isoster 4 (Scheme 2); the
stabilization of an intermediate analogous to E4, for the
phosphate derivative, is consistent with the observed inhi-
Scheme 1. Reaction catalyzed by aldolase and mechanistic pathway
Results and Discussion
When aldolase (35 µ subunits) is incubated in the pres-
ence of -erythrulose 1-phosphate monosodium salt
(16 m) in D2O (pD ϭ 7.2), the reaction monitored by 31P-
NMR spectroscopy indicates the slow formation of inor-
ganic phosphate from the inhibitor catalyzed by the enzyme
[a]
with a turn-over number k ϭ 0.0022 minϪ1 [9]
In control,
.
Groupe de Chimie Organique Biologique, UMR/CNRS 5623,
´
Universite Paul Sabatier,
where β-glycerophosphate aldolase (an active-site modified
enzyme, see Experimental Section) was used instead of the
ˆ
Bat. II R1, 118 route de Narbonne, F-31062 Toulouse Cedex
4, France
Eur. J. Org. Chem. 1999, 2853Ϫ2857
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
1434Ϫ193X/99/1111Ϫ2853 $ 17.50ϩ.50/0
2853

´
´
P. Page, C. Blonski, J. Perie
FULL PAPER
native enzyme, no inorganic phosphate formation was ob- (Scheme 3; X ϭ CH2), this intermediate was also charac-
served; this suggests that the reaction does occur at the en- terized by oxidation by hexacyanoferrate(III),[2jϪ2l] di-
zyme active site. The value of the turn-over number for al- hydroxy-2,3-butadione phosphate being expected as an oxi-
dolase-catalyzed conversion of -erythrulose 1-phosphate dation product. The initial rates of oxidation were of zero-
to inorganic phosphate, is the same within the error of these order in hexacyanoferrate(III) and depended on the concen-
experiments as the the rate constant of 0.0041 minϪ1[7] for tration of 4 in accordance with Michaelis-Menten kinetics.
the slow recovery of enzyme activity from an enzyme-in- The values of kЈcat and KЈm for this reaction were deter-
hibitor complex.
mined by fitting the kinetic data to the Michaelis-Menten
equation using a nonlinear regression program and were
found to be 37 minϪ1 and 87 µ, respectively.[16] The latter
correlates with the Ki value determined above. Thus, phos-
phonate 4 has a dissociation constant value similar to those
of DHAP (60 µ[13]) and -erythrulose 1-phosphate (75
µ[7]) with aldolase, within fast equilibria.
Scheme 2. Synthetic scheme for the synthesis of phosphonate 4: (i)
(BnO)2P(O)CH3, BuLi, Et2O·BF3, THF, Ϫ70°C, 63%; (ii) DCC,
pyridine/DMSO, CF3CO2 H, benzene, 84%; (iii) a) H2, Pd/C,
MeOH/H2O, b) Ba(OH)2, 90%
To gain additional insight into the inhibition mechanism,
phosphonomethyl analog 4 of -erythrulose 1-phosphate
was synthesized (Scheme 2) starting from (2R,3S)-3,4-
epoxy-1,2-O-isopropylidenebutane-1,2-diol (1).[10] The ring
opening of the starting epoxide by dibenzylmethanephos-
[11]
phonate anion catalyzed by BF3·OEt2
afforded dibenzyl
(3S,4R)-3,4,5-trihydroxy-4,5-O-isopropylidenepentyl phos-
phonate 2. Oxidation of the latter compound according to
the Pfitzner-Moffat method[12] yielded dibenzyl (4R)-4,5-di-
Scheme 3. Proposed mechanism for aldolase inhibition by -ery-
thrulose 1-phosphate and 4
hydroxy-4,5-O-isopropylidene-3-oxopentyl
phosphonate
(3). Removal of the benzyl protecting group by catalytic
These results account for the fast formation of intermedi-
hydrogenation concomitant with ketal hydrolysis, provided ates E1Ј and E2Ј (Scheme 3; X ϭ CH2) catalyzed by aldol-
(4R)-4,5-dihydoxy-3-oxopentylphosphonic acid (4), isolated ase with the phosphonate 4 and without observable de-
as barium salt.
composition by retro-aldolization from intermediate E1Ј.
Phosphonate 4 (monosodium salt), is not a time-depen- As it can be argued that -erythrulose 1-phosphate reacts in
dent inhibitor nor a substrate for aldolase but inhibits the a similar manner to its phosphonate isoster, these resulting
enzyme-catalyzed reaction competitively with a Ki value of intermediates E1Ј and E2Ј (Scheme 3; X ϭ O) have also to
110 µ. The Schiff base intermediate E1Ј (Scheme 3; X ϭ be in fast equilibrium with free enzyme and -erythrulose 1-
CH2) was evidenced by sodium tetrahydroborate treat- phosphate; therefore, both intermediates cannot be directly
ment[2aϪ2c,13] of the aldolaseϪ4 complex leading to a sig- associated with the slow-binding inhibition process of the
nificant level of irreversible inactivation of the enzyme.[14] enzyme. On the other hand, the different inhibition patterns
The reaction performed with the parent phosphate indicates observed between these two compounds are consistent with
that this latter compound also forms a reductible intermedi- the possibility for the parent phosphate to lead to a stabil-
ate with the enzyme.[14] Aldolase catalyzes the exchange of ized intermediate enamine-hydroxy ketone plus phosphate
the (pro-S) hydrogen atom at C-3 of DHAP with solvent E4Ј, analogous to E4, formed from the enamine intermedi-
water through enamine intermediate E2 (Scheme 1).[2eϪ2h] ate E2Ј (Scheme 3; Xϭ O) with a limiting first-order rate
The enzyme (1 mg mLϪ1) was able to catalyze the H/D ex- constant of 0.28 minϪ1 previously determined.[7] This find-
change in D2O (pD ϭ 7.2) of the hydrogen atom at the ing is reminiscent of the nonenzymatic phosphate β-elimin-
corresponding carbon atom of 4 (16 m); indeed this hy- ation reaction of DHAP through an enediolate intermedi-
drogen atom occupies the equivalent position of the (pro-S) ate, similar in structure to intermediate E2 (Scheme 1).[17]
hydrogen atom in the substrate, the reaction being followed Subsequently, intermediate E4Ј undergoes the observed
by 1H-NMR spectroscopy (CHOH peak; δ ϭ 4.43).[15] The slow release of inorganic phosphate concomitant with the
observed exchange rate value v ϭ 0.144 mmol·minϪ1·unitϪ1 recovery of the enzyme activity (k ϭ 0.0041 minϪ1), while
is close to that determined with the phosphonomethyl ana- the phosphonate isoster cannot do so. This parallel, and
log of DHAP (v ϭ 0.18 mmol·minϪ1·unitϪ1).[13] To provide also the fact that the inhibition can be reversed, indicates
additional evidence for the formation of enamine E2Ј that the covalent adduct present in E4Ј is hydrolyzed and
2854
Eur. J. Org. Chem. 1999, 2853Ϫ2857

Origin of the Slow-Binding Inhibition of Aldolase by D-glycero-Tetrulose 1-Phosphate
FULL PAPER
plots; initial rates were measured at different substrate concen-
trations (10, 20, 40 and 100 µ FDP) with four different inhibitor
concentrations (50, 100, 200 and 300 µ); aldolase concentration
was 50 n in subunits.
suggests that inorganic phosphate release and enzyme ac-
tivity recovery are both under the control of slow confor-
mational change of the enzyme. It turns out that the stereo-
chemistry at C-3 controls two different mechanistic path-
ways with the substrate erythrulose 1-phosphate: (i) with
the  derivative, the formation of an enamine-hydroxy ke-
tone thermodynamically more stabilized by extra hydrogen
bonds into the enzyme active site than the equivalent alde-
hyde E4 formed from DHAP (no more than 6% of the total
DHAP bound to the enzyme),[2d] can account for the ob-
served inhibition; (ii) as indicated above, the  derivative is
substrate.[5c,7] In other words: (i) the time-dependent inhi-
bition of aldolase by -erythrulose 1-phosphate is consist-
ent with the slow transformation of the resulting enamine
into the new complex E4Ј (Scheme 3; X ϭ O); (ii) in the
absence of GAP, the formation of complex E4Ј is quantitat-
ive from E2Ј whereas this process occurs to a low extent
from DHAP-enamine intermediate E2; (iii) the decompo-
sition of this complex E4Ј leads to a second time-dependent
process, the recovery of the enzyme activity, which parallels
the slow inorganic phosphate release rate. As a conclusion,
-erythrulose 1-phosphate can also be regarded as a slow
alternative substrate for the minor and nontypical meth-
ylglyoxal synthase activity of aldolase.
Orthophosphate Formation from
D-Erythrulose 1-Phosphate Cata-
lyzed by Aldolase: The inorganic phosphate formation rate in D2O
catalyzed by aldolase was followed by 31P-NMR spectroscopy. In
a typical run, aldolase (0.70 mg) was added to -erythrulose 1-
phosphate (16 m) in D2O (0.5 mL final volume) pre-equilibrated
in the spectrometer probe (25°C). The pD ϭ 7.2 of the solution,
adjusted using small quantities of concentrated NaOD or DCl
solutions, was measured before and after each kinetic run. At inter-
vals of 24 h after the addition, 31P-NMR spectra were taken. The
series of spectra shows the progress, consistent with a zero-order
kinetic, of -erythrulose 1-phosphate peaks (δ ϭ 2.43 and 3.22,
hydrate and ketone forms, 30% and 70%, respectively) which are
diminished in size with respect to the increase of inorganic phos-
phate peak (δ ϭ 0.9). Inorganic phosphate was evidenced by 31P-
NMR spectroscopy by adding Na2HPO4 to the solution under
study. In controls where the enzyme was omitted or replaced by β-
glycerophosphate aldolase derivative, or -erythrulose 1-phosphate
used instead of the  isomer, no inorganic phosphate formation
was detected.
Reduction by Sodium Tetrahydroborate: Sodium tetrahydroborate
treatments of the enzyme-inhibitor (or substrate) complex were
performed according to a previously described technique.[13][23]
Isotope Exchange and Rate Measurements: The deuteration rate of
1
4 in D2O catalyzed by aldolase was monitored by H-NMR spec-
troscopy (CHOH peak; δ ϭ 4.43), as described previously.[13][15] In
a typical run, solid aldolase (0.5 mg) was added to 4 (16 m) in
D2O (0.5 mL) pre-equilibrated in the spectrometer probe. No ad-
ditional buffer was used. The pD ϭ 7.2 of the solution, adjusted
using small quantities of concentrated NaOD or DCl solutions,
was measured before and after each kinetic run. At timed intervals
after the addition, 64 scans (lasting about 1.5 min) were taken and
transformed separately. In a control run without enzyme, no deut-
eration was observed.
Experimental Section
General: FDP, NADH, glycerol-3-phosphate dehydrogenase, triose-
phosphate isomerase, rabbit muscle aldolase were purchased from
Boerhinger Mannheim, DHAP (lithium salt) from Sigma. Aldolase
(10 U/mg) was dialyzed overnight against the appropriate buffer
prior to use. For isotope exchange and inorganic phosphate forma-
tion studies aldolase was freed of triose-phosphate isomerase ac-
tivity as previously described.[18] β-Glycerophosphate aldolase de-
rivative was obtained by sodium tetrahydroborate treatment of a
solution of aldolase and DHAP as previously described.[19] The
enzymatic activity of β-glycerophosphate aldolase was 1% of the
initial value. -Erythrulose 1-phosphate was obtained by aldol reac-
tion beetween DHAP (formed in situ) and formaldehyde catalyzed
by rabbit muscle FDP aldolase.[20] Chemicals and solvents were
reagent grade. Ϫ The NMR spectra were recorded in CDCl3 or
D2O with a Bruker AC80 (80 MHz 1H NMR), a Bruker AC200
Carbanion Assay: The carbanion (or enamine) can be oxidized by
a variety of oxidants, for example, hexacyanoferrate(III). The rate
of hexacyanoferrate(III) reduction was monitored by the decrease
in A420 difference between sample and reference cuvettes as de-
scribed previously.[2k,13] In a typical run, both cuvettes (final vol-
ume 1 mL) contained 1 m hexacyanoferrate(III) in triethanolam-
ine buffer (0.1 , 50 m NaCl, pH ϭ 7.6) and 0.03 mg of aldolase
(0.19 µ); in addition, the sample cuvette contained 4 (10Ϫ400 µ).
After 5 min of preincubation, the enzyme was added to start the
reaction and the decrease in absorbance was measured within the
first 60 s at 25°C. The molar activity (kЈcat) of aldolase for oxi-
dation of 4 by hexacyanoferrate(III) and the substrate concen-
tration at which the initial rate of hexacyanoferrate(III) reduction
is half-maximal (KЈm, which is a measure of aldolase affinity for
4), were determined by fitting the data to the Michaelis-Menten
equation.
1
(200 MHz H NMR, 50 MHz 13C NMR and 81 MHz 31P NMR)
or a Bruker ARX400 (400 MHz 1H NMR and 162 MHz 31P
NMR) spectrometer. All chemical shifts are reported in ppm with
1
respect to TMS for H- and 13C-NMR spectra and H3PO4 for 31P-
NMR spectra as internal standards. Ϫ Elementary analyses were
performed by the Ecole Nationale de Chimie de Toulouse, France.
Activity Assays: Aldolase activity was measured using a coupled
assay system by monitoring NADH oxidation at 340 nm, with de-
tection by a PerkinϪElmer Lambda 2 spectrophotometer thermo-
stated at 25°C.[21] Assays were initiated by the addition of substrate Dibenzyl (3S,4R)-(3,4,5-Trihydroxy-4,5-O-isopropylidenepentyl)phos-
(FDP, 1 m final concentration) to a final volume of 1 mL of solu-
tion containing aldolase made up in triethanolamine buffer
phonate (2): (2R,3S)-3,4-Epoxy-1,2-O-isopropylidenebutane-1,2-
diol (1) was synthesized as previously described.[10] A 1.6  solu-
(100 m triethanolamine/HCl, pH ϭ 7.6, 50 m NaCl, 1 m tion of nBuLi in hexane (7.3 mL, 11.6 mmol) was added dropwise
EDTA), 0.42 m NADH and coupling enzymes (10 µg/mL gly-
cerol-3-phosphate dehydrogenase; 1 µg/mL triose-phosphate iso-
merase). The aldolase concentration was determined spectrophoto-
to a stirred solution of dibenzylmethylphosphonate (3.2 g,
11.6 mmol) in dry THF (60 mL) at Ϫ80°C under nitrogen. After
30 min of stirring, the mixture was added dropwise to a stirred
metrically from ε280 ϭ 0.91 mL mgϪ1 cmϪ1 [22] Inhibition constant solution of 1 (0.56 g, 3.89 mmol) in dry THF (40 mL) at Ϫ80°C.
.
value (Ki) for 4 was determined on the basis of double-reciprocal
After 15 min of stirring, BF3·Et2O (1.4 mL, 11.6 mmol) was slowly
Eur. J. Org. Chem. 1999, 2853Ϫ2857
2855

´
´
P. Page, C. Blonski, J. Perie
FULL PAPER
introduced. The reaction mixture was stirred for 2 h at Ϫ70°C, then dЈInterface Chimie-Biologie du Centre National de la Recherche
overnight at room temperature. Saturated NH4Cl solution (20 mL)
was added, the solvents were removed under reduced pressure and
the remaining residue was dissolved in 100 mL of ethyl acetate. The
organic solution was washed with brine, dried with MgSO4, then
concentrated and purified by flash chromatography (CH2Cl2/
MeOH, 96:4) to provide 2 as a colourless oil (1.03 g, 63%). Ϫ
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3947Ϫ3952. Ϫ
1
[2f]
4 H), 7.34 (s, 10 H). Ϫ 13C NMR (CDCl3): δ ϭ 22.5 (d, JCP
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141 Hz), 25.3, 26.0, 26.6, 66.0, 67.4, 71.6 (d, JCP ϭ 12 Hz) 78.3,
109.2, 128.4, 128.6, 136.3. Ϫ 31P NMR (CDCl3): δ ϭ 34.5. Ϫ
C22H29O6P: calcd. C 62.86, H 6.9, O 22.86; found C 63.0, H 6.8;
O 22.7.
[2h]
80, 5835Ϫ5836. Ϫ
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Dibenzyl (4R)-(4,5-Dihydroxy-4,5-O-isopropylidene-3-oxopentyl)phos-
phonate (3): To a stirred mixture of dicyclohexylcarbodiimide
(0.74 g, 3.6 mmol) in dry pyridine (0.2 mL, 2.5 mmol) or dry ben-
zene (20 mL) was added dropwise 2 (0.5 g, 1.2 mmol) in dry DMSO
(2.5 mL) under nitrogen. Trifluoroacetic acid (0.1 mL, 1.2 mmol)
was added and the mixture was stirred overnight at room tempera-
ture. The solvent was removed under reduced pressure and the re-
maining residue dissolved in 60 mL of ether. The suspension (di-
cyclohexylurea) was filtered and the filtrate washed with brine
(3 ϫ 30 mL). The organic layer was dried with MgSO4 and concen-
trated under reduced pressure. The residue was chromatographed
(CH2Cl2/MeOH, 96:4) to yield 3 as a colourless oil (0.41 g, 82.4%).
[3]
[4] [4a]
E. Grazi, G. Trombetta, Biochem. J. 1978, 175, 361Ϫ365.
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E. Grazi, G. Trombetta, V. Lanzara, Biochemistry 1983,
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25
Aldolases are considered as targets for the development of new
Ϫ [α]D ϭ ϩ18.36 (c ϭ 3.65 mg/mL, EtOH). Ϫ IR (neat): ν˜ ϭ
antiparasitic drugs since glycolysis is the only source of energy
1
1720, 1240 cmϪ1. Ϫ H NMR (CDCl3): δ ϭ 1.35 (s, 3 H), 1.40 (s,
[8a]
for parasites, such as trypanosome:
F. R. Opperdoes, Ann.
[8b]
3 H), 1.90Ϫ2.25 (m, 2 H), 2.70Ϫ2.90 (m, 2 H), 3.80Ϫ4.45 (m, 3
Rev. Microbiol. 1987, 41, 127Ϫ151. Ϫ
T. Gefflaut, C. Blon-
H), 5.0 (m, 4 H), 7.30 (s, 10 H). Ϫ 13C NMR (CDCl3): δ ϭ 19.2
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301Ϫ340. Ϫ [8c] J. Perie, I. Riviere-Alric, C. Blonski, T. Gefflaut,
1
(d, JCP ϭ 145 Hz), 25.0, 26.0, 31.8, 66.5, 67.5, 80.0, 111.0, 128.0,
N. Lauth de Viguerie, M. Trinquier, M. Willson, F. R. Opper-
3
128.5, 136.2, 208.2 (d, JCP ϭ 14 Hz). Ϫ 31P NMR (CDCl3): δ ϭ
[8d]
does, M. Callens, Pharmac. Ther. 1993, 60, 347Ϫ365. Ϫ
C.
32.6. Ϫ C22H27O6P: calcd. C 63.15, H 6.46, O 23.0; found C 63.4,
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[9]
Turn-over number k ϭ V/E, where V represents the ortho-
phosphate formation rate (mmol·hϪ1) and E the amount of en-
zyme subunit (µmol) used in the assay.
(4R)-(4,5-Dihydroxy-3-oxopentyl)phosphonic Acid, Barium Salt (4):
To a suspension of Pd/C (10%, 100 mg) in a solution of water/
MeOH (1:1, 10 mL) was added 3 (0.38 g, 0.9 mmol). The mixture
was degassed and hydrogenated for 12 h at atmospheric pressure.
The catalyst was filtered off and water (20 mL) was added to the
filtrate. The pH value was adjusted to 7.6 with saturated Ba(OH)2.
The solution was freeze-dried, and the residue dissolved in 5 mL
of distilled water. The suspension was discarded by centrifugation,
barium salt 4 was precipitated by addition of ethanol (15 mL) and
the resulting mixture was kept at 0°C for 3 h. The salt was collected
by centrifugation, washed twice with ethanol (80%, then absolute),
diethyl ether and dried in vacuo to yield 4 (0.27 mg, 90%). Ϫ
[10]
E. Abushanab, P. Vemishetti, R. W. Leiby, H. K.Singh, A. B.
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Concentrations of 4 and aldolase were 1 m and 5 µ repec-
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(1 m) used instead of compound 4, the resulting losses of en-
zyme activity were 45% and 34%, respectively. In other controls
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performed with -erythrulose 1-phosphate (1 m), the sample
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ible loss of enzyme activity was 53%. In controls where -er-
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ted, the loss of enzyme activity was 10%.
25
[α]D ϭ ϩ16.0 (c ϭ 3.50 mg/mL, H2O). Ϫ IR (KBr): (nu)tilde ϭ
1706, 1234 cmϪ1. Ϫ 1H NMR (D2O): δ ϭ 1.60Ϫ2.05 (m, 2 H),
2.70Ϫ2.90 (m, 2 H), 3.80Ϫ4.05 (m, 2 H), 4.5 (m, 1 H). Ϫ 13C NMR
1
(D2O): δ ϭ 24.6 (d, JCP ϭ 134 Hz), 36.1, 65.3, 80.3, 216.2 (d,
3JCP ϭ 12 Hz). Ϫ 31P NMR (D2O): δ ϭ 23.2. Ϫ C5H9BaPO6: calcd.
C 18.0, H 2.7, O 28.8; found C 18.2, H 2.5, O 28.6.
[15] [15a]
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Attempts to characterise an enamine intermediate formed be-
tween aldolase and -erythrulose 1-phosphate by 1H/D ex-
Acknowledgments
1
change in D2O followed by H-NMR spectroscopy, or by oxi-
dation by hexacyanoferrate(III), failed due possibly to the en-
zyme inhibition.
Dimethylketal bis(cyclohexylammonium) salt of -erythrulose 1-
phosphate was kindly provided by Professor C. E. Ballou. We are
[17] [17a]
[17b]
R. Iyengar, I. A. Rose, J. Am. Chem. Soc. 1983, 105, 330. Ϫ
J. P. Richard, J. Am. Chem. Soc. 1984, 106, 4926Ϫ4936. Ϫ
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[17c]
´
grateful for financial support for this work from the Comite
2856
Eur. J. Org. Chem. 1999, 2853Ϫ2857

Origin of the Slow-Binding Inhibition of Aldolase by
D
-glycero-Tetrulose 1-Phosphate
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Received December 7, 1998
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2857