Hydrates of quaternary ammonium aldehydes as potential reactivators of sarin-inhibited acetylcholinesterase

Article abstract of DOI:10.1135/cccc19981021

Synthesis of 2-(5a), 3-(5b) and 4-formyl-1-methylpyridinium iodides (5c) and (2,2-dihydroxyethy)trimethylammonium chloride (6a) is described. In aqueous solution aldehydes 5 exist predominantly as hydrates - geminal diols 7. The ability of the geminal diols 6a and 7 to reactivate acetylcholinesterase inhibited by isopropyl methylflourophosphonate (Sarin) was tested in vitro. Although compounds 6a and 7 exhibit an affinity towards the acetylcholinesterase bonding site comparable with that of the natural substrate acetylcholine, their reactivation ability was negligible. Second-order rate constants for cleavage of 4-nitrophenyl diphenyl phosphate (PNPDPP) with anions of the studied geminal diols were measured in order to determine their nucleophilicity.

Full text of DOI:10.1135/cccc19981021

Hydrates of Quaternary Ammonium Aldehydes
1021
HYDRATES OF QUATERNARY AMMONIUM ALDEHYDES AS POTENTIAL
REACTIVATORS OF SARIN-INHIBITED ACETYLCHOLINESTERASE
a1
b1
b2
a2
b3
Jiri CABAL , Frantisek HAMPL , Frantisek LISKA , Jiri PATOCKA , Frantisek RIEDL
b4,
and Katerina SEVCIKOVA
*
a Department of Toxicology, J. E. Purkyne Military Medical Academy, 500 01 Hradec Kralove, Czech
Republic; e-mail: 1 cabal@pmfhk.cz, 2 patocka@pmfhk.cz
b Department of Organic Chemistry, Prague Institute of Chemical Technology, 166 28 Prague 6,
Czech Republic; e-mail: 1 hamplf@vscht.cz, 2 liskaf@vscht.cz, 3 riedlf@vscht.cz,
4 sevcikok@vscht.cz
Received January 8, 1998
Accepted March 18, 1998
Synthesis of 2- (5a), 3- (5b) and 4-formyl-1-methylpyridinium iodides (5c) and (2,2-dihydroxy-
ethyl)trimethylammonium chloride (6a) is described. In aqueous solution aldehydes 5 exist predomi-
nantly as hydrates – geminal diols 7. The ability of the geminal diols 6a and 7 to reactivate
acetylcholinesterase inhibited by isopropyl methylfluorophosphonate (Sarin) was tested in vitro. Al-
though compounds 6a and 7 exhibit an affinity towards the acetylcholinesterase bonding site com-
parable with that of the natural substrate acetylcholine, their reactivation ability was negligible.
Second-order rate constants for cleavage of 4-nitrophenyl diphenyl phosphate (PNPDPP) with anions
of the studied geminal diols were measured in order to determine their nucleophilicity.
Key words: Acetylcholinesterase; Organophosphorus inhibitors; Reactivators of phosphonylated
acetylcholinesterase; Pyridinium salts; Sarin; Phosphonates.
Many derivatives of organic phosphorus acids, particularly some alkyl phosphates and
alkyl phosphonates, are highly active irreversible inhibitors of acetylcholinesterase1 (ACE).
In addition to the generally known poisonous compounds such as isopropyl fluoromethyl-
phosphonate (Sarin), Soman, Tabun, VX, etc., many compounds of this group, e.g. para-
oxon, parathion, dimefox, metrifonat, are used as insecticides and veterinary preparations.
Their inhibitory effect is based on phosphonylation (or phosphorylation) of the hydroxy
group in serine at the so-called esteratic site of the active center of the enzyme2 (as exem-
plified in Scheme 1 by inhibition of ACE with Sarin). Acetylcholinesterase plays an im-
portant physiological role in the cholinergic nervous system and therefore its inhibition is a
life-endangering factor. For this reason, there is a constant quest for ways of reactivating
* The author to whom correspondence should be addressed.
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

1022
Cabal, Hampl, Liska, Patocka, Riedl, Sevcikova:
the phosphonylated (or phosphorylated) ACE and thus restoring its physiological func-
tion. This can be done by use of suitable nucleophilic reagents (Scheme 1).
O
O
Nu
P
O
3
iPr
F
P
O
3
iPr
Nu
F
CH
CH
O
CO
CO
CO
CHCH O
2
CHCH O
2
P
O
3
iPr
CHCH O
2
NH
NH
CH
NH
Sarin-inhibited ACE
ACE
ACE
(Ser-200)
(Ser-200)
SCHEME 1
Very soon after discovery of toxic effects of organophosphates, the reactivation
ability of quaternary pyridinium aldoximes has been found3 and these compounds still
represent the leading structure of all currently used ACE reactivators, such as 2-PAM
(pralidoxim) 1, TMB-4 (trimedoxim) 2a, toxogonin (obidoxim) 2b, and HI-6 3 (ref.4).
The high effectivity of the mentioned compounds is due to several factors: (i) the
presence of easily ionizable hydroxyimino group (7 < pKa < 8) ensuring a high concen-
tration of the nucleophilic oximate anion even at the physiological pH, (ii) the high
nucleophilicity of the oximate anion (compared with O-nucleophiles of comparable ba-
sicity), caused by the so-called α-effect of the hydroxyimino nitrogen lone electron
pair4a,5, and (iii) high affinity of pyridinium salts towards ACE as a result of Coulombic
attraction between the positively charged quaternary nitrogen of the reactivator and the
so-called anionic center of the enzyme2.
CH=NOH
CONH
2
2 X
2 Cl
N
CH=NOH
N
N
O
CH
I
3
Y
Y
N
N
CH=NOH
HON=CH
X
1
2
a
b
Br CH
2
3
Cl
O
In spite of the existence of the ACE reactivators, many attempts to find new and
more effective compounds have been made4b,6, however, the search consists mostly in
structural variations of the leading structure, quaternary pyridinium aldoximes. In the
present study, we decided to investigate the reactivation ability of (2-oxoethyl)tri-
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

Hydrates of Quaternary Ammonium Aldehydes
1023
methylammonium chloride (4a), 2-, 3-, and 4-formylpyridinium iodides (5a–5c), and
their hydrates 6a and 7. Although these compounds have already been described7,8
,
they were not tested as ACE reactivators. We have been inspired by studies of Menger et
al.9 published in the eighties. They found9a that organophosphates are rapidly hydro-
lysed in homomicelles of a cationic tenside (2-oxoethyl)dimethyltetradecylammonium
CH
N
3
CH=O
R
CH CH=O
2
N
CH
3
Cl
I
CH
3
R
4
a
b
Position
5
a
b
c
CH
3
2
3
4
CH (CH )
2 13
3
OH
CH
OH
OH
CH CH Cl
CH
3
R
N
2
N
CH
OH
3
I
CH
3
R
6
a
b
Position
7
a
b
c
CH
3
2
3
4
CH (CH )
2 13
3
chloride (4b). The organophosphate phosphorus atom is attacked by the nucleophilic
anion which is formed by deprotonation of hydroxy group in geminal diol 6b arising by
addition of water molecule to the carbonyl group of aldehyde 4b, activated by the
electron-accepting quaternary ammonium group. The nucleophilic group in tenside 4b
is recovered in a catalytic cycle (Scheme 2).
The reactivation of ACE is also based on cleavage of a phosphate or phosphonate
and one can therefore assume that geminal diols of the type 6a and 7 could reactivate
ACE. Whereas classical reactivators of the aldoxime type (such as 1) dephosphorylate
ACE in the stoichiometric ratio, geminal diols 6a and 7 should act in a catalytic process
(Scheme 2). A necessary condition for reactivators is their sufficient affinity to ACE.
The structure of hydrate 6a resembles that of choline, a natural product arising by
action of ACE on acetylcholine, and geminal diols 7 have some features similar to
those of “classical” reactivators, aldoximes 1–3. Therefore, we assumed that the studied
compounds could exhibit sufficient affinity to ACE. The only weakness of the geminal
diols was the pKa value published9a for diol 6b (10.9). However, in spite of this high
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

1024
Cabal, Hampl, Liska, Patocka, Riedl, Sevcikova:
value we decided to study the reactivation ability of compounds 6a and 7 towards ACE
inhibited by phosphonylation.
CH
N
CH
N
CH
N
OH
OH
O
3
3
3
+ H O
2
- H
R
CH CH=O
R
CH CH
2
R
CH CH
2
2
- H O
2
+ H
OH
CH
CH
CH
3
3
3
Cl
Cl
Cl
6
4
O
RO P(OR)
2
O
O
O
O
P(OR)
2
RO
CH
N
CH
3
OP(OR)
OP(OR)
3
2
2
R
CH CH
2
R
N
CH CH
2
O
OH
CH
CH
3
3
Cl
Cl
H O
2
OH
SCHEME 2
EXPERIMENTAL
Melting points were measured on a Boetius block and are uncorrected. 1H NMR spectra were taken
on a Varian Gemini 300 (300 MHz) and a Bruker AM 400 (400 MHz) instruments. Chemical shifts
are given in ppm and are referenced to tetramethysilane, coupling constants J are in Hz.
Chemicals
Chloroacetaldehyde diethyl acetal (Aldrich), 2-, 3-, and 4-pyridinecarbaldehyde (Merck), acetylcholine
iodide (ACI) (Lachema), isopropyl methylfluorophosphonate (VOZ 072 Zemianske Kostol’any), 3-cyclo-
hexylaminopropane-1-sulfonic acid (Sigma). 4-Nitrophenyl diphenyl phosphate (PNPDPP) was pre-
pared and purified by a described procedure10. Solvents were purified and dried by usual
procedures11. The enzyme was obtained from brains of male white laboratory Wistar rats (Velaz,
Prague). The animals in light ether narcosis were killed by cutting the cervical artery. After opening
the calva, the brain was taken out, washed with cold physiological solution, and subjected to three
freeze (to –35 °C)–thaw cycles. Homogenization in distilled water using a Ultra-Turrax instrument
(Janke–Kunkel, Germany) at 20 000 r.p.m. for 1 min yielded a 10% (w/v) homogenate. Aliquots (2 ml)
of the homogenate were placed into small test tubes and stored at –35 °C in a freezer and defrozen
immediately prior the use.
Formyl-1-methylpyridinium Iodides 5a–5c (General Procedure)
A mixture of pyridinecarbaldehyde (1.0 g, 9.8 mmol), methyl iodide (4.1 g, 28 mmol) and acetone
(2.4 ml) was heated at 60 °C for 8 h. After cooling, the separated crystals were collected, washed
with dry ether (20 ml) and dried in vacuo over phosphorus pentoxide.
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

Hydrates of Quaternary Ammonium Aldehydes
1025
2-Formyl-1-methylpyridinium Iodide (5a)
1
Yield 1.6 g (68%), m.p. 181–182 °C (ref.8 gives m.p. 180–183 °C). H NMR spectrum ((CD3)2SO):
4.66 s, 3 H (CH3); 8.35 m, 1 H (H-5); 8.53 d, 1 H, J(3,4) = 7.8 (H-3); 8.79 t, 1 H, J(4,3) = J(4,5) =
1
7.8 (H-4); 9.15 d, 1 H, J(6,5) = 5.9 (H-6); 10.29 s, 1 H (CH=O). H NMR spectrum (D2O): 4.33 s,
3 H (CH3); 6.33 s, 1 H (CH(OD)2); 7.97 m, 1 H (H-5); 8.26 d, 1 H, J(3,4) = 7.9 (H-3); 8.50 dd, 1 H,
J(4,3) = 7.8, J(4,5) = 7.9 (H-4); 8.70 d, 1 H, J(6,5) = 6.0 (H-6).
3-Formyl-1-methylpyridinium Iodide (5b)
1
Yield 2.0 g (83%), m.p. 172–176 °C (ref.8 gives m.p. 173–175 °C). H NMR spectrum ((CD3)2SO):
4.50 s, 3 H (CH3); 8.35 dd, 1 H, J(5,4) = 7.8, J(5,6) = 6.2 (H-5); 9.00 d, 1 H, J(4,5) = 8.0 (H-4);
1
9.22 d, 1 H, J(6,5) = 6.0 (H-6); 9.55 s, 1 H (H-2); 10.20 s, 1 H (CH=O). H NMR spectrum (D2O):
4.33 s, 2.7 H (CH3 of 7b); 4.41 s, 0.3 H (CH3 of 5b); 6.14 s, 0.9 H (CH(OD)2); 7.97 dd, 0.9 H, J(5,4) =
7.7, J(5,6) = 6.4 (H-5 of 7b); 8.18 t, 0.1 H, J(5,4) = 5.4, J(5,6) = 6.9 (H-5 of 5b); 8.53 d, 0.9 H,
J(4,5) = 8.02 (H-4 of 7b); 8.68 d, 0.9 H, J(6,5) = 6.0 (H-6 of 7b); 8.84 s, 0.9 H (H-2 of 7b); 8.91 d,
0.1 H, J(4,5) = 7.4 (H-4 of 5b); 8.95 d, 0.1 H, J(6,5) = 5.9 (H-6 of 5b); 9.30 s, 0.1 H (H-2 of 5b);
10.07 s, 0.1 H (CH=O).
4-Formyl-1-methylpyridinium Iodide (5c)
1
Yield 2.3 g (94%), m.p. 106–109 °C (ref.8 gives m.p. 105–106 °C). H NMR spectrum ((CD3)2SO):
4.45 s, 3 H (CH3); 8.49 d, 2 H, J(3,2) = J(5,6) = 5.4 (H-3,5); 9.25 d, 2 H, J(2,3) = J(6,5) = 5.9
1
(H-2,6); 10.24 s, 1 H (CH=O). H NMR spectrum (D2O): 4.30 s, 3 H (CH3); 6.12 s, 1 H (CH(OD)2);
8.03 d, 2 H, J(3,2) = J(5,6) = 6.41 (H-3,5); 8.70 d, 2 H, J(2,3) = J(6,5) = 6.6 (H-2,6).
(2,2-Diethoxyethyl)trimethylammonium Chloride (8)
A mixture of chloroacetaldehyde diethyl acetal (12.0 g, 78.7 mmol), anhydrous trimethylamine (9.2 g,
155 mmol) and benzene (14.7 g) was heated in a sealed tube at 100 °C for 51 h. The unreacted
trimethylamine was distilled off and the residue was neutralized with HCl (1 : 1). The solvent was
evaporated and the crude product was crystallized from acetone. Yield 0.40 g (2.4%), m.p. 131–142 °C.
1H NMR spectrum ((CD3)2SO): 1.16 t, 6 H, J(2′,1′) = 6.9 (CH3); 3.14 s, 9 H (CH3N); 3.50 d, 2 H,
J(1,2) = 4.9 (CH2N); 3.63 m, 4 H (CH2O); 5.02 t, 1 H, J(2,1) = 5.2 (CH).
(2,2-Dihydroxyethyl)trimethylammonium Chloride (6a)
Acetal 8 (0.40 g, 1.9 mmol) was refluxed with 5.6% HCl (3.2 ml) under argon for 20 min. After
evaporation of the solvent, the product was washed with acetone and dried in vacuo over phosphorus
pentoxide. Yield 0.28 g (96%), m.p. 133–140 °C (ref.7 gives m.p. 123–124 °C; ref.12 gives m.p. 142–144 °C).
1H NMR spectrum ((CD3)2SO): 3.14 s, 9 H (CH3); 3.27 d, 2 H, J(1,2) = 5.0 (CH2); 5.26 t, 1 H,
J(2,1) = 5.2 (CH); 6.75 s, 2 H (OH).
Determination of pKa (ref.13)
Acid constants pKa of compounds 6a and 7a were determined by potentiometric titration of their 5.0 . 10–4
M
aqueous solutions with 0.01 M sodium hydroxide at 25 °C at constant ionic strength I ≈ 0.25 mol kg–1
rendered with potassium chloride. The titrations were performed using an RTS 822 (RADIOMETER,
Kobenhaven) titrator. The acid constants were calculated using the Henderson–Hasselbach equation (1)
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

1026
Cabal, Hampl, Liska, Patocka, Riedl, Sevcikova:
pKa = pH – log ([A–]/[HA]) ,
(1)
where [A–] is the concentration of the geminal diol anion and [HA] is the concentration of undisso-
ciated geminal diol.
The pKa values of compounds 7b and 7c were determined spectrophotometrically by measuring the
dependence of absorbance A on the pH of their 1.0 . 10–4 M aqueous solutions at 25 °C at the absorp-
tion maxima of the dissociated and undissociated forms. The measurements were carried out on a
Specord M42 (Carl Zeiss, Jena) instrument. The pH values were adjusted with 0.1 M phosphate buf-
fers. The pKa values were calculated by Eq. (2).
pKa = pH + log [(Amax – Aobs)/(Aobs – Amin)]
(2)
Determination of Reactivation Effectivity of Geminal Diols
The reactivation effectivity was calculated from the measured activities of the intact enzyme (A0),
Sarin-inhibited enzyme (AI), and reactivated enzyme (AR), using Eq. (3).
% R = [1 − (A0 – AR)/(A0 – AI)] . 100
(3)
Determination of Activity of Intact ACE (A0)
Rat brain homogenate (0.5 ml) was added to a 0.3 M sodium chloride solution (20 ml). Then a 0.02 M
solution of ACI (2.0 ml) was added and the system was adjusted with 0.3 M sodium chloride solution
to a total volume of 25.0 ml. The liberated acetic acid was titrated with 0.01 M sodium hydroxide on
an RTS 822 titrator in the pH-stat mode (pH 8.0). The ionic strength of the solution was rendered
with sodium chloride at constant value I ≈ 0.25 mol l–1. The slope of the linear part of the time
dependence of the sodium hydroxide used represents the activity (A0) of the intact enzyme (in fact,
the initial rate v0 of the enzymatic reaction).
Determination of Activity of Inhibited ACE (AI)
Rat brain homogenate (0.5 ml) was treated with 5 . 10–8 M aqueous solution of Sarin (0.5 ml) for 30 min,
which resulted in about 85% inhibition of the enzyme. A 0.02 M solution of ACI (2.0 ml) was added
and the mixture was adjusted to 25.0 ml with 0.3 M sodium chloride solution. The activity of the in-
hibited enzyme (AI) was immediately determined analogously as described in the preceding experiment.
Determination of Activity of Reactivated ACE (AR)
Reactivation of the enzyme inhibited in the above experiment was performed immediately after the
inhibition. A solution (1.0 ml) of the reactivator was added to the enzyme, the resulting reactivator
concentration being 1.0 . 10–3 mol l–1. After 10 min of reactivation at 25 °C, a 0.02 M solution of ACI
(2.0 ml) was added. The mixture was adjusted to 25.0 ml with 0.3 M sodium chloride solution and
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

Hydrates of Quaternary Ammonium Aldehydes
1027
immediately afterwards the activity of the reactivated enzyme (AR) was determined analogously as
described in the preceding experiments.
Determination of Dissociation Constants Kdis of Enzyme–Reactivator Complexes
Appropriate volume of 0.01 M solution of the geminal diol was added to rat brain homogenate (0.5 ml) so
as to achieve the desired diol concentration (CD) in the interval 5.0 . 10–5 to 5.0 . 10–3 mol l–1. Then
a 0.02 M solution of ACI (2.0 ml) was added, the mixture was adjusted to 25.0 ml with 0.3 M sodium
chloride and the enzyme activity (AINH) was immediately determined analogously as in the preceding
experiments. The dissociation constant (Kdis) was obtained by nonlinear regression14 from the de-
pendence of AINH on CD (Eq. (4), see ref.15).
AINH = Amax CS/[CS + KM(1 + CD/Kdis)],
(4)
where, in addition to the above-defined symbols, Amax denotes the maximum (limiting) rate of the
enzymatic reaction, CS is substrate concentration and KM is the Michaelis constant for hydrolysis of
acetylcholine with ACE (1.9 . 10–4 mol l–1).
Kinetics of Cleavage of PNPDPP
Solutions of the geminal diols of the desired concentration were prepared in 0.05 M CAPS buffer (pH 10.4).
The reaction was followed at 25.0 ± 0.1 °C using an HP 8452A spectrophotometer (diode array,
Hewlett–Packard) equipped with a thermostatted multicell transport cell holder HP89075C. The reac-
tion was initiated by addition of 5.0 . 10–4 M solution of the substrate in acetonitrile (20 µl) into the
spectrophotometric cell containing a buffered solution (1.98 ml) of the geminal diol; in all cases, the
resulting concentration of the substrate was 5.0 . 10–6 mol l–1. The concentration of the formed 4-nitro-
phenoxide ion was monitored at 400 nm. The reactions followed the first-order konetics up to 100%
conversion. The rate constants were obtained by nonlinear regression of the dependence of absorb-
ance on time using program Enzfitter (R. J. Leatherbarrow, Elsevier–Biosoft, Amsterdam 1987), the
error of the interpolation being not higher than 0.6%.
RESULTS AND DISCUSSION
Synthesis of Reactivators
(2,2-Dihydroxyethyl)trimethylammonium chloride (6a) was prepared by alkylation of
trimethylamine with chloroacetaldehyde diethyl acetal and subsequent hydrolysis of the
obtained (2,2-diethoxyethyl)trimethylammonium chloride (8). The synthesis was analo-
gous to that of the corresponding bromide7. The respective quaternary pyridinium car-
baldehydes 5a–5c were obtained by alkylation of 2-, 3-, and 4-pyridinecarbaldehydes
with methyl iodide8. The obtained salts were identified by their melting points and 1H NMR
spectra. The assumption that in aqueous solution aldehydes 5 exist predominantly in the
1
form of hydrates was confirmed by their H NMR spectra in deuterium oxide. The
spectra of compounds 5a and 5c exhibited no aldehyde proton signals but, instead, they
contained signals at 6.33 and 6.12 ppm due to –CH(OD)2 groups in the respective hy-
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

1028
Cabal, Hampl, Liska, Patocka, Riedl, Sevcikova:
drates 7a and 7c. In aqueous solution of salt 5b, the ratio aldehyde : hydrate was ap-
proximately 10 : 90, as follows from the integrated signals of the groups –CH=O (10.07 ppm)
and –CH(OD)2 (6.14 ppm).
Affinity of Geminal Diols to ACE and Their Ability to Reactivate
the Sarin-Inhibited Enzyme
The affinity of the prepared compounds to ACE and their ability to reactivate the phos-
phonylated enzyme was studied by in vitro experiments with rat brain ACE, using iso-
propyl methylfluorophosphonate (Sarin). The activity of the geminal diols towards
intact ACE was expressed by values of dissociation constants of the ACE–diol com-
plexes (Kdis). The values of Kdis were derived from the assumption that geminal diols
behave as competitive inhibitors of cleavage of the natural substrate acetylcholine by
ACE. The relation between the dissociation constant Kdis and the experimentally ac-
cessible quantities is given by Eq. (4). The obtained values of Kdis are shown in Table
I. It is obvious that the affinity of the geminal diols to ACE is comparable with that of
the natural substrate acetylcoline; in some cases (6a and 7b) is even higher.
Neither of the studied compounds exhibited reactivation effects. The measured
values of % R ranged in the interval 0–2%, i.e. within the range of experimental error
(Table I). This failure of the geminal diols to reactivate Sarin-inhibited ACE can be
explained by their low acidity. Their pKa values are 11.0–13.8 (Table I) and thus at the
physiological pH only a negligible fraction of the diols exists in the ionized form.
Reactivity of Geminal Diols Towards 4-Nitrophenyl Diphenyl Phosphate
(PNPDPP)
These experiments were aimed at assessing the nucleophilicity of anions of the geminal
diols. For the cleavage of PNPDPP under pseudo-first order reaction conditions, the
slope of the plot of the observed rate constant (kobs) vs concentration of the geminal diol
TABLE I
Acidity, affinity toward ACE, and reactivation ability of the prepared compounds
Compound
pKa
Kdis . 104
% R
6a
11.2
11.0
13.3
13.8
1.3
3.8
1.1
2.6
1.9
0.3
<0.1
<0.1
2.0
7a
7b
7c
Acetylcholine
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

Hydrates of Quaternary Ammonium Aldehydes
1029
−
anion (cA ) represents the second order rate constant (k2) for this reaction. The rate
constant k2, which is a measure of nucleophilicity of anions of geminal diols, can thus
−
−
be obtained from the relation kobs vs cA by linear regression. The cA values are calcu-
lated from the total concentrations of the geminal diols (cHA) using Eq. (5).
cA = cHA Ka/([H+] + Ka)
(5)
−
The rate constants kobs were determined at 25 °C and pH 10.4 (0.05 M CAPS buffer).
Under the described conditions, compounds 7 decomposed, probably as a result of nu-
cleophilic attack of the pyridine nucleus by hydroxide ion16. The dependence of kobs on
concentration cHA could be measured only for the geminal diol 6a (Fig. 1). From this
dependence we calculated the reaction rate k2 = 5.4 . 10–2 l mol–1 s–1 (standard deviation
4.3 . 10–3 l mol–1s–1).
It appears that the nucleophilicity of the anion of geminal diol 6a is very low com-
pared with that, e.g., of the hydroxide ion (k2 = 0.4 l mol–1 s–1, ref.17a), choline anion (k2 =
3.1 l mol–1 s–1, ref.17b) or oximate anion of 1 (k2 = 1.0 l mol–1 s–1, ref.16). Thus, the low
reactivity of the hydroxylic anion in the geminal diols also obviously contributes to
their observed low reactivation ability.
CONCLUSIONS
Hydrates of aldehydes with the aldehyde group activated by the electron-accepting qu-
aternary ammonium group are not suitable for reactivating phosphonylated or phospho-
rylated ACE. Although they exhibit affinity toward the enzyme comparable to that of
the natural substrate, the concentration of nucleophilic species under physiological con-
ditions and their reactivity are low. The surprisingly low reactivity of the geminal diol
6a toward the model substrate PNPDPP raises the question whether the reported9 high
hydrolytic activity of the homologous compound 6b is caused by micellar medium
rather than by the catalytic cycle suggested by the authors.
5.0
4
–1
k
. 10 , s
obs
4.0
3.0
2.0
FIG. 1
Dependence of observed rate constant for cleav-
age of PNPDPP, kobs, on concentration of geminal
diol 6a, cHA
0
1
2
3
2
–1
c
. 10 , mol l
HA
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

1030
Cabal, Hampl, Liska, Patocka, Riedl, Sevcikova:
The authors are indebted to the Grant Agency of the Czech Republic for financial support (grant No.
203/96/1585).
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1969, 25, 236.
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Collect. Czech. Chem. Commun. (Vol. 63) (1998)