Dependence of in vitro enzymic hydrolysis of alkyl(2-benzoyl-oxyethyl)dimethylammonium bromides on the alkyl length

Article abstract of DOI:10.1135/cccc19980245

Microsomal esterases were used in (he study of the m vitro enzymic hydrolysis of the ester bond in alkyl(2-benzoyloxyethyl)dimethylammonium bromides. These compounds are potential "soft" disinfectants, easily biodegradable to nontoxic biologically inactive hydrolytic products, namely substituted choline and benzoic acid. Formation of the latter product was used to monitor the kinetics of the reaction. It has been found that the rate of enzymic hydrolysis is substantially influenced by different length of the alkyl chain on the ammonium nitrogen. At the same time, interspecies (rat-mouse) and interorgan (liver-kidney) variability has been observed.

Full text of DOI:10.1135/cccc19980245

In vitro Enzymic Hydrolysis
245
DEPENDENCE OF in vitro ENZYMIC HYDROLYSIS OF ALKYL(2-BENZOYL-
OXYETHYL)DIMETHYLAMMONIUM BROMIDES ON THE ALKYL
LENGTH
a1
a2
a3
b1
Eva OLASZOVA , Ingrid PAULIKOVA , Otto HELIA , Emil SVAJDLENKA ,
Ferdinand DEVINSKYb2 and Ivan LACKO
b3
a Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University,
832 32 Bratislava, Slovak Republic; e-mail: 1 olaszova@kbm.fpharm.uniba.sk,
2 paulikova@kbm.fpharm.uniba.sk, 3 helia@kbm.fpharm.uniba.sk
b Department of Chemical Theory of Drugs, Faculty of Pharmacy, Comenius University,
832 32 Bratislava, Slovak Republic; e-mail: 1 svajdlenka.kchtl@fpharm.uniba.sk,
2 devinsky@fpharm.uniba.sk, 3 lacko.kchtl@fpharm.uniba.sk
Received September 5, 1996
Accepted June 19, 1997
Microsomal esterases were used in the study of the in vitro enzymic hydrolysis of the ester bond in
alkyl(2-benzoyloxyethyl)dimethylammonium bromides. These compounds are potential “soft” disin-
fectants, easily biodegradable to nontoxic biologically inactive hydrolytic products, namely sub-
stituted choline and benzoic acid. Formation of the latter product was used to monitor the kinetics of
the reaction. It has been found that the rate of enzymic hydrolysis is substantially influenced by dif-
ferent length of the alkyl chain on the ammonium nitrogen. At the same time, interspecies (rat–
mouse) and interorgan (liver–kidney) variability has been observed.
Key words: Quaternary ammonium salts; Soft drugs; Enzymic hydrolysis; Microsomal esterases.
Due to their high environmental adaptability, microorganisms are able to acquire resist-
ance against antimicrobial agents. Therefore, research efforts are constantly devoted to
a search for effective disinfectants with suppressed side effects.
At present, efforts concentrate on synthesis of the so-called “soft” drugs. Their mole-
cules contain groupings similar to biomolecules and acceptable to biological systems
(e.g., ester bonds). After their therapeutic effect, the “soft” drugs are quickly decom-
posed (by hydrolysis, in most cases) to metabolites with low biological activity and toxi-
city. Typically, the deactivation of “soft” compounds is easily predicted and controlled.
The quaternary ammonium salts (QAS), synthesized by Csiba et al.1 also belong to
the soft drug class. These compounds, derived from 2-(dimethylamino)ethyl benzoate
acid, are isosteric and/or isoelectronic with the common “hard” surface-active quater-
nary ammonium disinfectants. They are characterized by the general formula C6H5–
COO(CH2)2–N+(CH3)2R Br– (R = straight-chain alkyl with n = 1,… carbon atoms). In
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

246
Olaszova, Paulikova, Helia, Svajdlenka, Devinsky, Lacko:
further text, we will designate these molecules with symbol BCHn (the parent structure
of the homologous series is benzoylcholine).
The antimicrobial effect of QAS is based on their physicochemical properties. Their
ammonium functional groups bind to the negatively charged phosphate groups of phos-
pholipids, whereas their nonpolar parts penetrate into the hydrophobic membrane core.
This results in a loss of membrane semipermeability, leakages of intracellular compo-
nents, inhibition of metabolic reactions as glucolysis or oxidative phosphorylation,
changes in protein structure and final cell death. Thus, QAS could be characterized as
membrane-active compounds2, and an optimum balance of their hydrophobic and hy-
drophilic characteristics is substantial for the desired effect3.
The antimicrobial activity of the newly synthesized QAS was investigated in a pre-
vious paper1. The results show a high activity against the microorganisms tested
(S. aureus, E. coli, C. albicans). An activity increase with the alkyl length was found.
A good activity was in this case associated with alkyl chains ranging from decyl to
octadecyl. The most sensitive organism was S. aureus and the most resistant one E. coli.
For E. coli, the antimicrobial activity was nonlinearly dependent on the alkyl length,
whereas for the Gram-positive S. aureus and C. albicans, the authors1 observed in
general a high activity (with the exception of hexyl and octyl derivatives), not much
influenced by the alkyl length. In all the cases studied, the most effective were the
dodecyl and tetradecyl derivatives (n = 12, 14).
For a complete characterization of new compounds, knowledge of their behaviour in
living systems and of their interaction with enzyme systems is necessary. The structure
of model substrates can be influenced by biotransformation at several sites. The most
sensitive site is probably the ester bond.
Helia and coworkers4 studied the hydrolytic activity of microsomal esterases isolated
from different organs of two animal species using (2-benzoyloxyethyl)trimethyl-
ammonium bromide as substrate.
In this work, we have examined the reaction rate of in vitro enzymic hydrolysis in
dependence on the alkyl length in alkyl(2-benzoyloxyethyl)dimethyl bromides by
microsomal fractions of murine and rat livers and kidneys.
EXPERIMENTAL
Model Substrates
The alkylammonium salts have been synthesized in the laboratory of F. Devínsky at the Faculty of
Pharmacy, Comenius University, Bratislava. All substrates were white crystalline substances, chro-
matographically pure, well soluble in polar and insoluble in nonpolar solvents. They have been stored
in a vacuum desiccator above P4O10. The substrate solutions were freshly prepared by dissolution in
distilled water at laboratory temperature. For BCH14, BCH16 and BCH18 heating to 40–60 °C and
intense shaking was necessary. Basic characteristics of the substrates used in this study are sum-
marized in Table I.
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In vitro Enzymic Hydrolysis
247
Reagents
4-Iodobenzoic acid was a product of BDH, England, methanol of HPLC grade was purchased from
Sigma Chemie, Germany. All the other reagents were obtained from Lachema, Brno, Czech Republic
and were of reagent grade purity (with the exception of benzoic acid, which had the sL 3 purity).
Preparation of Microsomal Fractions
The liver and kidney tissue was obtained from male Wistar rats (weighing 150–180 g) or male Balb
mice (approximately 20 g). Microsomal fractions were isolated by differential centrifugation of a
20% homogenate of the particular organ tissue in 0.25 mol l–1 saccharose according to Remmer and
coworkers5. The protein concentration was determined by the method of Lowry et al.6.
Conditions of Incubation
The incubation was done under aerobic conditions in a Dubnoff incubator at 37 °C with intense mix-
ing and aeration. First, the reaction mixture containing 0.1 mol l–1 potassium phosphate (pH 7.4)
buffer, 1 mmol l–1 substrate and 40 µmol of Mg2+ ions was preincubated for 5 min. Then, the reac-
tion was started by addition of the microsomal fraction in an amount corresponding to 15 or 5 mg of
protein for rats or mice, respectively. Total volume of the incubation mixture was 3.5 ml. After the
specified incubation period, 1 ml of incubation mixture was removed and the reaction in this sample
stopped by cooling to 0 °C and by denaturation using 100 µl of 1 mol l–1 HCl.
Isolation and Determination of the Hydrolytic Product (Benzoic Acid)
To the sample, the internal standard of 4-iodobenzoic acid in reagent grade methanol was added in
an amount corresponding to the amount of liberated benzoic acid. The acidity of the resulting reac-
tion mixture was adjusted to pH 2.3–3.0 with 1 mol l–1 HCl and benzoic acid was extracted three
TABLE I
Characteristics of C6H5COO(CH2)2N+(CH3)2(CH2)n–1CH3Br–
a
Code
n
M.w.
cc , mol–1
BCH1
BCH2
BCH3
BCH4
BCH6
BCH8
BCH10
BCH12
BCH14
BCH16
BCH18
1
288.18
302.21
316.23
330.26
358.34
286.39
414.44
442.49
470.55
498.60
526.66
–
2
3
–
–
4
–
6
1.51 . 10–1
4.82 . 10–2
1.22 . 10–2
2.31 . 10–3
7.93 . 10–4
8.96 . 10–5
1.50 . 10–4
8
10
12
14
16
18
a cc Critical micellar concentration.
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

248
Olaszova, Paulikova, Helia, Svajdlenka, Devinsky, Lacko:
times with 5 ml of chloroform. The collected extracts were evaporated to dryness on a rotatory
evaporator, dissolved in methanol (HPLC grade) and analyzed with HPLC as described in an earlier
paper4.
Enzyme Kinetics
Enzyme activity of microsomal esterase was expressed as the quantity of liberated benzoic acid in
nmol min–1 mg–1 of protein. For evaluation, we used calibration curves correlating the benzoic acid
concentration with the peak areas for benzoic and 4-iodobenzoic acids. The calibration curves were
analyzed with linear regression, giving the linear correlation coefficient r = 0.997.
RESULTS AND DISCUSSION
We have studied the in vitro enzymic hydrolysis of the ester bond in model QAS sub-
strates in both liver and kidney microsomes of rats and mice. The rate of hydrolysis of
homologues with alkyls of different lengths was also compared for different species
and organs. As shown in Fig. 1, in all cases, there are obtained nonlinear relations
between the reaction rate and alkyl length, showing maxima for different substrates,
most often for BCH2 and BCH8.
The rates of enzymic hydrolysis in rat liver (Fig. 1a) vary for different substrates
between 0.24 and 3.42 nmol min–1 mg–1. The curve has alternating maxima and
minima, with maxima found for BCH2 > BCH4 > BCH8 and minima for BCH3 and
BCH6. After the last maximum for BCH8, the rate of enzymic hydrolysis decreases in
the order BCH10 > BCH12 > BCH14, and becomes roughly stabilized of the minimum
value for BCH16 and BCH18.
In rat kidneys, the rates of enzymic hydrolysis range from 0.07 to 0.52 nmol min–1 mg–1
with maxima for substrates BCH2 > BCH8 > BCH4. From BCH10 to BCH16, the rates
gradually decrease, with an interesting increase for BCH18 (Fig. 1b).
The enzymic hydrolysis rates in murine liver have similar values around 70 nmol
min–1 mg–1 for BCH1–BCH10, with low maxima for BCH8 > BCH2 (see Fig. 1c).
Starting from BCH10, the rates drop rapidly to the value of 7.09 nmol min–1 mg–1 for
the BCH18 substrate.
In murine kidney, the observed relationhip is bell-shaped (Fig. 1d) with maximum
value found for BCH10 and with an increase in hydrolysis rate for BCH18. The reac-
tion rates vary between 0.35 and 3.03 nmol min–1 mg–1.
A comparison of curves obtained for rat and murine liver and for rat kidney shows
several common features. In all three cases, there are more or less pronounced maxima
for substrates BCH2, BCH4 and BCH8. However, the relative intensity of these three
maxima is different. Substrates BCH1, BCH3 and BCH6 lie at the minima. For both rat
liver and kidney, the reaction rates in the maxima decrease in the mentioned order,
whereas for murine liver they increase. After reaching a maximum for BCH8, the rate
of hydrolysis gradually decreases in the order BCH10, BCH12, BCH14, BCH16 and
BCH18, with an exception of the increase for BCH18 in rat kidney. In the case of
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In vitro Enzymic Hydrolysis
249
murine kidney, the curve has a shape differing from all the three above mentioned. The
only resemblance is the increase in hydrolysis rate for BCH18. This can be used as a
basis for evaluation of interpecies variability in kidney.
The obtained results were subjected to a statistical analysis using the Student t-test.
In microsomal fractions of rat liver and kidney and of murine liver, the overall shape of
the curves obtained (Fig. 1a–1c) roughly corresponds to a hyperbolic decrease with the
alkyl length. The reaction rate decreases/increases between neighboring homologues
are not statistically significant on the P = 0.05 level, but the differences were confirmed
experimentally. In murine kidney, the maximum for BCH10 is statistically significant
when compared with substrates BCH1 and BCH16–BCH18.
0.6
0.4
4
3
a
b
2
1
0
v
v
0.2
0.0
BCHn
BCHn
90
60
3.5
2.5
c
d
1.5
0.5
v
v
30
0
BCHn
BCHn
FIG. 1
Influence of the alkyl length in the substrate on the rate of enzymic hydrolysis in: a rat liver, b rat
kidney, c murine liver, d murine kidney. The values are averages from 3–5 experiments. In each
experiment, microsomes isolated from at least five animals were used. The standard deviation is in-
dicated by the bar
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

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Olaszova, Paulikova, Helia, Svajdlenka, Devinsky, Lacko:
A comparison for all studied organs shows that the overall rate of enzymic hydrolysis
is highest in murine liver. In rat liver and murine kidney, the reaction rates are about
20–30 times slower compared to murine liver. Ester bonds of model substrates were
cleaved with lowest velocity in murine kidney preparations.
Comparing different organs, the enzymic hydrolysis has been found faster in liver.
On an interspecies level, it has higher reaction rates for mice.
These conclusions correlate with kinetic studies of Helia and coworkers4, who have
determined KM and Vmax for enzymic hydrolysis of BCH1 by microsomal fractions from
different rat and murine organs. On the basis of these KM values, one can conclude that
the affinity to BCH1 follows the order: rat kidney (KM = 0.7 mM) = (murine kidney?) >
rat liver (KM = 1.09 mM) > murine liver (KM = 1.58 mM). Interestingly, the affinity to
BCH1 substrate decreases with increasing rate of hydrolysis of this substrate.
We expect that in rat liver and kidney and in murine liver we deal with microsomal
esterases differing in affinity to particular alkylammonium salts, but with comparable
properties. Similar conclusions are found in literature4. The enzymic hydrolysis of
model substrates in murine kidney indicates the presence of an esterase with different
properties. A similar anomalous behaviour was noted previously also for KM of BCH1
hydrolysis4.
The rate of enzymic splitting of the ester bond is influenced also by the general
amphiphilicity of the molecule, not solely by the presence of the lipophilic alkyl chain.
We can expect, on the basis of structural resemblance of higher homologues to the
phosphatidylcholine membrane components, that the lipophilic part of the molecule
penetrates into the microsomal membrane. Also, the antimicrobial effect of this group
of QAS has a similar basis. Such penetration, as well as possible formation of micelles,
can influence the enzymic hydrolysis considerably. (Similar phenomena were quoted
by Arndt and Krisch7, who found a decrease in the carboxylesterase-catalyzed hydro-
lysis of aliphatic esters with increasing alkyl length.)
Micelle formation strongly inhibits enzymic hydrolysis (in contrast to chemical
hydrolysis). Abrupt changes in the observed curves suggest micelle formation as one of
the causes of the rapid decrease of hydrolysis, catalyzed by microsomal cholinesterase
(CHE), E.C.3.1.1.7./8. (ref.8). Taking into account the known structure of the active site
of CHE and chemical mechanisms of ester hydrolysis9, we can conclude that the mi-
celle dimensions are too large to allow the micelle to react in a complementary way with
the enzyme active site. This makes formation of a stabilized transition state impossible.
The catalytic triad of the active site cannot function as proton pendulum and, conse-
quently, the ester bond degradation in these substrates slows down dramatically.
This work was supported by the grant GAV 1/1223/94 of the Ministry of Education of the Slovak
Republic.
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REFERENCES
1. Csiba I., Devinsky F., Lacko I., Mlynarcik D.: Cesk. Farm. 1986, 35, 61.
2. Herold F. M. in: Advances in Microbial Physiology (A. H. Rose and J. E. Wilkinson, Eds), p. 455.
Academic Press, London 1970.
3. Devinsky F., Lacko I., Mlynarcik D., Racansky V., Krasnec E.: Tenside Detergents 1985, 22, 10.
4. Helia O., Paulikova I., Svajdlenka E., Devinsky F., Lacko I., Olaszova E.: Pharmazie 1995, 50,
705.
5. Remmer M., Greim H., Schenkamn J. B., Estabrook V.: Methods Enzymol. 1955, 10, 706.
6. Lowry O. H., Rosenbrough N. J., Randall R. J., Farr A. L.: J. Biol. Chem. 1951, 193, 265.
7. Arndt R., Krisch K.: Eur. J. Biochem. 1973, 36, 129.
8. Jbilo O., Lhermite V., Talesa V., Toutant J. P., Chatonnet A.: Eur. J. Biochem. 1994, 225, 115.
9. Voet D., Voet G. J.: Biochemistry, p. 373. Wilye, New York 1990.
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