COPE elimination reaction observed in the biodegradation of quaternary
ammonium surfactants
Shaun F. Clancy,*a† Michael Thiesb,c and Henrich Paradies*b,c‡
a Safety, Health and Environmental Affairs, Witco Corporation, One American Lane, Greenwich, CT 06831-2559, USA
b Ma¨rkische Fachhochschule, Biotechnology and Physical Chemistry, D-58590 Iserlohn, PO Box 2061, Germany
c Universita¨t Paderborn, Fachbereich Chemie, Institut fu¨r Physikalische Chemie and Chemietechnik, D-33095 Paderborn,
Germany
The biodegradation of dialkyldimethylammonium com-
pounds in aqueous solutions indicates that part of the
degradation pathway includes the Cope elimination reac-
tion, which does not occur under mild conditions in the
absence of microorganisms.
washed with 0.1 m KH2PO4–K2HPO4, pH 7.5 and stored at
210 °C. The intracellular contents were purified by (NH4)2SO4
fractionation (25–65% of a saturated solution at 5 °C). The
portions containing the monooxygenase activity were found in
the fractions collected in 48–60% saturated (NH4)2SO4 solu-
tions. These fractions were used to prepare the S-100 super-
natant at 5 °C. The supernatant was used as a crude enzyme
preparation and stored in 10% (v/v) glycerol in the presence of
This report details the first report of the Cope reaction mediated
by microorganisms and starting with long-chained quaternary
ammonium compounds. This reaction appears to be a key step
in the biodegradation of quaternary ammonium compounds
(‘quats’). The quats which are the subject of this study are either
dialkylethoxymethylammonium, dialkylamidoammonium or
dialkylmethylimidazolinium quats.
0.01
m KH2PO4–K2HPO4, 5 mm MgCl2 and 1 mm
b-mercaptoethanol (pH 7.8, 20 °C). The S-100 fraction
containing 10–15 mg ml21 protein was used in the enzymatic
experiments in the presence of glycerol¶ and the distearyl-
dimethylammonium salt (DSDMA OH; 5 mm) at pH 7.8
(37–40 °C) for studying enzyme mediated olefin formation,
eqn. (1), where R = CnH2n+1 and n = 8, 10, 12, 14, 16
Long-chained quaternary ammonium compounds have found
use in a variety of products and sometimes end their life-cycle
by disposal into wastewater or surface waters. Thus the
biodegradation of these compounds has been the subject of
many studies. It has been found that a wide variety of
microorganisms can utilise quats as energy sources. It is also
known that many microorganisms can utilise alkylamines as a
substrate and use an inducible monooxygenase (EC 1.14.13.8)
which is capable of oxidising the substrate to the corresponding
N-oxide.1 We have discovered that a key step in the biodegrada-
tion of these quats is the formation of an N-oxide which is
formed once the quats have first degraded to an alkyldimethyla-
mine. During our investigations of the biodegradation of
dialkydimethylammonium salts (DADMA X; X = F, Cl, Br, I,
OH), and subsequent determination of the metabolic pathway
by fermentation of these compounds, we isolated the corre-
sponding N-oxides and cleavage products of these N-oxides,
e.g. the C18 alkene and the corresponding hydroxylamine. The
N-oxide is converted to the corresponding trialkylamine and
formaldehyde (which are mineralised to CO2 and H2O) by an
amine N-oxide aldolase (EC 4.1.2-) functioning as a demethy-
lase.2 The degradation of the quats was done using acclimated
bacterial strains, fungi and yeasts for fermentation, typical of
those known to be present in the environment.
The Cope reaction is the cleavage of dialkylamine N-oxides
to produce alkenes and hydroxylamines.3 Mild conditions are
used to reduce side reactions, and the generated olefins do not
normally rearrange. In this report we show the transformation of
dialkyldimethylammonium salts to N-oxides, followed by
cleavage of the N-oxides to the corresponding alkenes in a
Cope-like process using a crude enzymatic preparation from
microorganisms of environmental origin. We also show an
enhancement of the kinetics of olefin formation due to the
presence of the DADMA X micelles (primarily for X = OH).
Fermentation was performed with 12 strains of microorgan-
ism§ known to be present in soil and aquatic systems, including
waste treatment (Xanthomonas comprestis, salt regulative
#33951, Bacillus subtilis vulgatus #6984, Torulopsis
spp. #34356, together with aerobic and faculative anaerobic
bacterial genera e.g. Actinobacter, Agrobacterium, Alcaligenes,
Nocardia, Arthrobacter, Cellulomonas, Mycobacterium and
Candida albicans). The cells were harvested, centrifuged,
+
?
(R)2N CH3?O — (R)CH3NOH + CH2NCH(CH2)15CH3
(1)
or, as in the example given here, 18. The formation of the
(R)2N(CH3)?O compounds have also been studied at pH 6.0 in
0.01 m borate buffer at a protein concentration of 1 mg ml21, in
+
the presence of 5 mm NADH2 (or 3.5 mm NAD+), and a
NADH2+ (NAD+) regenerating system at 37 °C.∑ Formation of
the olefins has not been observed in the absence of the crude,
enzymatic mixture at 37 °C under the same conditions.
However, by refluxing the N-oxides in xylene in the presence of
1 mm NaOH for two hours the corresponding olefin can be
obtained in an almost quantitative yield. Since the formation of
N-oxides operates only at pH 6.0–6.5 (37 °C) and the olefin
formation is seen mainly in the presence of 10% (v/v) glycerol
and pH 7.5–8.0, both pathways can be studied separately. The
N-oxides studied have also been prepared chemically by
reacting the appropriate amine with 30% hydrogen peroxide at
0 °C for 10 min and characterised using standard methods. The
N-oxides obtained from the enzymatic reactions were charac-
terised in the same fashion.
Fig. 1 shows the yields of the olefin CH2NCHC16H33
generated from distearylmethylamine N-oxide (10 mg ml21
)
using the crude enzymatic fraction in the presence of 5 mm
DSDMA X (X = OH, F, Cl, Br, I) (pH 7.8, 37 °C) and 10%
(v/v) glycerol. The most effective yield is obtained in the
presence of 5 mm DSDMA OH [10% (v/v) glycerol], whereas at
pH 6.5 the yield is very low as it was for the Br. Furthermore,
addition of 1–10 mm NaCl to the reaction medium reveals
inhibition of olefin generation (Ci = 6 mm NaCl, 50%
inhibition). The influence of NaCl can be related to: (i) the salt
induced change of the colloidal state of DSDMA OH from
vesicle to micelle,4,5 and (ii) the shifting of the equilibrium of
DSDMA OH + Cl2 /? DSDMA Cl + OH2, toward the
insoluble DSDMA Cl and colloidal state of both the hydroxide
and chloride.
The biotransformation of DADMA X is strongly dependent
on the critical micelle concentration (CMC) pH, temperature
and ionic strength.6 The initial rate constants for the anion
?
exchange step where DSDMA X — DSDMA OH (X = Cl,
Chem. Commun., 1997 2035