Biological, thermal and photochemical transformation of 2-trifluoromethylphenol

Article abstract of DOI:10.1023/A:1008394115008

This is the first report on the metabolism of trifluoromethyl aromatics in a thermophilic bacterium. Bacillus thermoleovorans A2. Enzymes of the phenol degradation pathway are induced when cultivating Bacillus thermoleovorans A2 on complex medium. Direct measurements of fluorinated xenobiotics in cell suspensions using ¹⁹F-NMR made it possible to follow quantitatively the biotransformation routes. During the biotransformation of 2-CF₃-phenol by B. thermoleovorans A2, a fluorinated metabolite, 2-hydroxy-6-oxo-7,7,7,-trifluorohepta-2,4-dienoate (7-TFHOD), accumulated. This metabolite is transformed non-enzymatically when exposed to sunlight. The accumulation of 7-TFHOD as an intermediate in the 2-CF₃-phenol pathway was rationalized by calculating molecular properties of a series of meta-cleavage products.

Full text of DOI:10.1023/A:1008394115008

Biodegradation 9: 487–499, 1998.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
487
Biological, thermal and photochemical transformation of
2-trifluoromethylphenol
Uwe Michael Reinscheid1,2, Han Zuilhof3, Rudolf Müller2 & Jacques Vervoort1,∗
1Department of Biochemistry, Wageningen Agricultural University, Dreijenlaan 3, NL-6708 HA Wageningen,
The Netherlands; 2Department of Biotechnology and Technical Biochemistry, Technical University Hamburg-
3
Harburg, Denickestrase 15, D-21073 Hamburg, Germany; Department of Organic Chemistry, Wageningen
Agricultural University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands (∗author for correspondence)
Key words: Bacillus, biotransformation, NMR, thermophilic, trifluoromethylphenol
Abstract
This is the first report on the metabolism of trifluoromethyl aromatics in a thermophilic bacterium, Bacillus
thermoleovorans A2. Enzymes of the phenol degradation pathway are induced when cultivating Bacillus ther-
moleovorans A2 on complex medium. Direct measurements of fluorinated xenobiotics in cell suspensions using
19F-NMR made it possible to follow quantitatively the biotransformation routes. During the biotransformation
of 2-CF3-phenol by B. thermoleovorans A2, a fluorinated metabolite, 2-hydroxy-6-oxo-7,7,7,-trifluorohepta-2,4-
dienoate (7-TFHOD), accumulated. This metabolite is transformed non-enzymatically when exposed to sunlight.
The accumulation of 7-TFHOD as an intermediate in the 2-CF3-phenol pathway was rationalized by calculating
molecular properties of a series of meta-cleavage products.
Abbreviations: CF3 – trifluoromethyl; CHF2 – difluoromethyl; CH2F – monofluoromethyl; 7-TFHOD – 2-
hydroxy-6-oxo-7,7,7,-trifluorohepta-2,4-dienoic acid; HOD – 2-hydroxy-6-oxo-hepta-2,4-dienoic acid; HMS –
2-hydroxymuconic acid semialdehyde; LUMO – lowest unoccupied molecular orbital; LB – Luria Bertani, NMR
- nuclear magnetic resonance; MTBSTFA – N(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide
Introduction
extensive overlap of the xenobiotic signals with those
from endogenous metabolites (Bollard et al. 1996).
Fluorinated hydrocarbons are widely used as her-
bicides, fungicides and human medicines. Fluorine
remains frequently an outsider among the halogens, as
its apparent relatively simple electronic structure nev-
ertheless leads to a wide variety of reactivity patterns.
These patterns – both the influence of C-F moieties on
other parts of a molecule as well as the reactivity of
the C-F bond itself – are often not well understood.
Yet, this chemistry becomes of increasing importance
as more than 10% of all pesticides contain fluorine,
in most cases as a trifluoromethyl (CF3) group. CF3
groups have been the subject of significant interest by
both synthetic (Banks & Tatlow 1994), mechanistic
(Zuilhof et al. 1997; Koch et al. 1998) and medicinal
chemists (Edwards 1994). Despite the fact that most of
The use of NMR spectroscopy to characterise and
quantify xenobiotic metabolites in biological fluids is
well established. 19F-NMR is of particular value in
studies on fluorinated xenobiotics since concentrations
as low as 0.1 µM have been detected and quantified in
biological samples with typically no interference from
endogenous compounds (Parisot et al. 1991; Serre
et al. 1997). Minor structural changes occurring far
away from the fluorine atom in a molecule can be de-
tected because of the large chemical shift range, thus
providing a subtle probe of metabolic transformation
1
reactions (Mabury & Crosby 1995). H-NMR spec-
troscopy is generally much less useful for the direct
analysis of drug metabolites in biofluids because of

488
the fluorinated agrochemicals contain CF3 groups (e.g.
trifluralin, a large-scale herbicide used to control a va-
riety of grasses and broadleaf weeds) there are only a
few reports on their microbial degradation pathways
(Golab et al. 1979; Zeyer & Kearney 1983; Gennari
et al. 1991; Kulowski et al. 1997; Zayed et al. 1983;
Abernethy & Walker 1993) .
Generally, tests with agrochemicals in laboratory
and ecosystems revealed incomplete degradation with
still intact CF3 groups (Key et al 1997). On the other
hand, this metabolic deactivating effect is a useful
property for some pharmaceuticals e.g. mefloquine, an
antimalaria agent with half-lives in the range of weeks
in the human body. Eventually, upon release into the
environment an accumulation of CF3 parent molecules
and/or metabolites may occur.
evance. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, for ex-
ample, has a very low water solubility, volatility and
is thermally stable. However, in the presence of sur-
factants it undergoes photo reduction with UV light.
Many photo-products are formed, with salicylic acid
reported as the final product. Taylor et al. (1993)
measured a partial release of fluorine anions using an
ion-sensitive electrode when irradiating an aqueous
solution of 7-TFHOD, an intermediate in the degra-
dation pathway of fluorinated benzoates and fluori-
nated catechols (Engesser et al. 1988a; Engesser et al.
1988b; Engesser et al. 1990). In the study of Taylor et
al. (1993) it remained however unclear to what extent
defluorination of 7-TFHOD occurred. In the present
study purified 7-TFHOD obtained by anion-exchange
chromatography was irradiated.
It was postulated that many of the CF3-containing
aromatics are biologically metabolised to CF3-
catechols by bacterial enzymes (Engesser et al. 1988a;
Engesser et al. 1988b). This raised the question about
the occurrence and fate of these CF3-catechols. Poly-
merisation of catechols at elevated temperatures is a
possible non-biological reaction. Under thermophilic
conditions the rate of polymerisation of chlorocate-
chols has been studied (Reinscheid et al. 1997). With
an initial concentration of 0.1 mM the concentration of
3-chlorocatechol had decreased by 3% after 20 min at
As model compounds the series of CF3-phenol
isomers was selected. The model organism is a ther-
mophilic and metabolically versatile Bacillus species
that has been isolated by Mutzel et al. (1996). The
degradation pathway for phenol and substituted phe-
nols has been investigated (Reinscheid et al. 1997;
Duffner & Müller 1998). Data on the taxonomical po-
sition revealed that the organism of this study, Bacillus
thermoleovorans A2, is a typical representative of
the phenol-degrading group of thermophilic Bacilli
(Duffner et al. 1997). The widespread occurrence
of these spore-forming bacteria, especially dominat-
ing self-heated compost (Strom 1985), leads directly
to questions about their metabolic potential against
CF3 aromatics. As a valuable tool for investigating
the metabolism of fluorinated compounds 19F-NMR
permits a direct monitoring without sample clean-
up. In this study we focus on the biological, thermal
and photochemical transformation characteristics of
2-CF3-phenol.
◦
60 C representing a promising method to treat solid
waste.
Our decision to study the different aspects of the
transformation reactions of CF3 organics in the en-
vironment was mainly based on two facts: the mass
production of CF3 aromatics as agrochemicals and
the strong resistance of the CF3 group to biological
processes involving enzymes (Engesser et al. 1990).
This combination directly leads to their world-wide
abundance in high concentrations in the environment
(Franklin 1993). A recent review article about fluo-
rinated organics in the biosphere summarises that in
a number of key areas major uncertainties exist con-
cerning the distribution, fate, and biological behaviour
of these compounds in the environment (Key et al.
1997). This situation deserves a thorough study of
the environmentally relevant biological, chemical and
photochemical reaction types.
Materials and methods
Bacteria, media and culture conditions
The phenol-degrading Bacillus thermoleovorans A2
(Mutzel et al. 1996) was maintained at 60 ◦C on solid-
ified, chloride-free medium containing phenol as sole
carbon and energy source (Reinscheid et al. 1997).
Growth was measured photometrically at 540 nm. For
the induction experiments and the production of in-
duced cells a Luria-Bertani (LB) medium (Sambrook
et al. 1989) was used. All incubations were car-
Many environmental modifications of agrochemi-
cals may be ascribed to photochemical reactions (Lod-
der & Cornelisse 1995). The photo-degradation of
these toxic, halogenated compounds is in aqueous
solutions one of the factors determining their persis-
tence, and is as such a topic of environmental rel-
◦
ried out at 60 C. Screw-capped vials equipped with

489
a Teflon-lined rubber septum were used to prevent
evaporation.
withdrawn and analysed using HPLC, 19F-NMR and
1H-NMR.
Test for growth-substrates and cometabolic
transformation by resting cells
Purification of 7-TFHOD by anion-exchange
chromatography
The fluorinated substrates 2-trifluoromethylphenol (2-
CF3-phenol), 3-CF3-phenol and 4-CF3-phenol were
tested as sole carbon and energy source by incubat-
A sample of the cometabolic transformation of 2-CF3-
phenol was centrifuged and the supernatant brought
to 0.1 M KCl. This solution was applied to a MonoQ
column (bed volume 1 ml). After washing the column
with phosphate buffer plus 0.1 M KCl, the meta-
cleavage product was eluted with a linear gradient of
0.1 M to 2 M KCl and appeared at 0.8 M KCl. Detec-
tion wavelengths were set at 380 nm and 270 nm. The
salt gradient was followed by conductivity measure-
ments. Fractions containing the meta-cleavage product
were collected.
◦
ing the organism in chloride-free medium at 60 C.
Sterile controls permitted the determination of abiotic
substrate loss due to elevated temperatures. After 48
hours of incubation, the optical densities were deter-
mined. Substrate concentrations were measured using
19F-NMR.
For the resting cells experiments LB-grown cells
in the late-exponential growth phase were centrifuged,
washed with sodium phosphate buffer (0.1 M; pH =
7.2) and resuspended to a final cell concentration of
1 × 109/ml. The above mentioned fluorinated com-
pounds and salicylic acid were added to a final con-
centration of 1 mM each. The vials were incubated
and samples were withdrawn for direct 19F-NMR
measurements. For HPLC and GC-MS analysis the
supernatant obtained after centrifugation of the sam-
ples was used. After 20 min of incubation an aliquot
of the 2-CF3-phenol containing cell suspension was
withdrawn for the anion-exchange chromatography.
Photolysis and temperature stability of the
meta-cleavage product
Photolysis was examined by exposure of solutions of
HMS or 7-TFHOD in sodium phosphate buffer (0.1
M; pH = 7.2) to full sunlight (between 10:00 a.m. and
noon) in covered quartz cuvettes. To test for the in-
fluence of oxygen, solutions were flushed with air or
argon for 20 min. During the light exposure temper-
◦
atures never exceeded 20 C. Control cuvettes were
kept in the dark. Temperature stability was tested at
60 ◦C in sodium phosphate buffer (0.1 M; pH = 7.2).
After appropriate time intervals the solutions were
analysed photometrically and, in case of 7-TFHOD
solutions, by 19F-NMR.
Whole-cell enzyme activity measurements
For the induction experiments the cells were cen-
trifuged and resuspended in 0.1 M sodium phosphate
buffer (pH = 7.2). Catechol 2,3-dioxygenase activity
was assayed by measuring the increase in absorbance
due to the formation of the yellow meta-cleavage prod-
uct HMS formed with catechol as substrate using
Analytical methods
2-CF3-phenol, 3-CF3-phenol, 4-CF3-phenol, salicylic
acid and 2,3-dihydroxybenzoic acid were analysed
by HPLC using a photo diode-array-detector (Wa-
ters 996). Identification was done by comparing UV
spectra and retention times with those of reference
substances. Reversed-phase HPLC was carried out on
a RP18 column (Nucleosil 5µ, 4.6 mm × 150 mm)
using methanol/water/acetic acid (600/400/10, v/v/v)
as eluent with a flow rate of 1 ml/min.
ꢀ375 nm = 33,400 cm−1
M
−1. Phenol hydroxylase ac-
tivity was detected by observing the formation of HMS
with phenol as substrate in the presence of catechol
2,3-dioxygenase activity in the intact cells. Both sub-
strates were added to a final concentration of 1 mm.
Specific enzyme activities in whole cells are expressed
as activity per unit OD540nm (1OD375nm × min−1
×
−1
OD
).
540nm
7-TFHOD and HMS concentrations were calcu-
lated from their molar extinction coefficients (En-
gesser et al. 1988b; Bayly et al. 1966).
Temperature stability of 2-trifluoromethylphenol
A 1 mm solution of 2-CF3-phenol in sodium phos-
phate buffer (0.1 M; pH = 7.2) was incubated at
60 C. After appropriate time intervals samples were
For the identification with the GC-MS system the
same sample clean-up procedure was used as already
described (Reinscheid et al. 1997). The residues were
◦

490
redissolved in 50 µl ethyl acetate and 10 µl of deriva-
tizing reagent (MTBSTFA; Aldrich) that introduces
tert-butyldimethylsilyl groups were added. After 5
minutes of heating the cooled mixture was concen-
trated by evaporation under a stream of argon. The
analyses were performed on a HP 5890 GC System
connected to a mass selective detector (5970 series)
equipped with a capillary column (HP5) of 30 m
× 0.25 mm dimensions at a helium flow rate of 20
ml/min. Injector and detector temperatures were 150
◦C and 280 ◦C, respectivel◦y, and the column temper-
From the thus calculated wavefunction the three
dimensional density of electrons can be computed, but
it is impossible to uniquely partition this among the
atomic centers in a totally unarbitrary manner. We
therefore calculated the sum of two electrostatic po-
tential charges as relevant charge indicator. This sum
is indicative of the charge felt by approaching nu-
cleophiles and suffers relatively little from arbitrary
demarcations of the electron clouds, and is thus used
throughout as an charge indicator.
◦
ature was raised from 80 C to 250 C at a rate of
Chemicals
◦
5 C/min with an initial holding time of 4 min. The
Phenol, salicylic acid (Fluka, Buchs, CH) and 2,3-
dihydroxybenzoic acid (Acros Chimica’s-Hertogen-
bosch, The Netherlands) were used. All trifluo-
romethylphenol isomers were obtained from Fluo-
rochem Limited (Derbyshire, UK). Stock so◦lutions of
the trifluoromethylphenols were kept at 4 C in the
dark.
mass spectra were obtained at 70 eV.
19F-NMR spectra were obtained on a Bruker
DPX 400 spectrometer equipped with a 10 mm flu-
orine probehead operating at 376.498 MHz. To sam-
2
ples of cell suspensions (1.9 ml) 100 µl H2O was
added as a field-frequency lock. For quantification p-
fluorobenzoate was used as internal standard. Chem-
ical shifts are reported relati◦ve to CFCl3. Generally,
spectra were acquired at 25 C with 5 µs pulses (30
degree flip angles), sweep width of 100 ppm, 32 K data
points, line broadening of 1 Hz, and between 3000
and 8000 scans for each experiment depending on
the concentration of the compounds. Selectivity tests
were performed with cell suspensions of metabolically
inactive B. thermoleovorans A2 cells (cell density =
1.0 × 1010/ml) in sodium phosphate buffer (0.1 M;
Results
Direct measurement of CF3-phenol in cell
suspensions using 19F-NMR
19F-NMR was used to investigate the biological trans-
formation of CF3-phenols. Direct measurements of
cell suspensions without any extraction procedure
could be done because of the high selectivity of
19F-NMR.
Under our experimental conditions we could eas-
ily measure fluorinated metabolites in the µM range
within 30–60 min. We determined the chemical shift
values for all three CF3-phenols to test for peak reso-
lution capacity. We observed clearly distinct chemical
shift values for all three isomers with 1δ values of at
least 0.4 ppm.
pH = 7.2). For routine 19F NMR measurements H
1
couplings were eliminated using broad-band proton
decoupling using a Waltz-16 pulse sequence. To test
1
for the formation of CF2H or CFH2 groups no H
decoupling was used. 1H-NMR spectra were mea-
sured on the same spectrometer as described above,
now using a 5 mm dual 13C/1H probe operating at
400.13 MHz. Samples were prepared in phosphate-
2
buffer with H2O as solvent. Generally, spectra were
◦
acquired at 7 C, sweep width of 12 ppm, 32 K data
points and line broadening of 0.5 Hz. The water signal
was presaturated during the relaxation delay.
Cell growth
Expression of the phenol hydroxylase and the cate-
chol 2,3-dioxygenase does not require the addition of
phenol in the growth medium. During growth on LB
medium the catechol 2,3-dioxygenase activity could
be observed after 13 h in the exponential phase (Fig-
ure 1). Three hours later phenol hydroxylase activity
was detected. Both activities decreased immediately
when the cells had reached the stationary phase. It is
therefore of crucial importance to harvest the cells at a
Computational chemistry
Molecular orbital calculations were carried out using
Spartan 4.1.1 (Wavefunction Inc., Irvine, CA) with the
default convergence criteria implemented in there. Re-
stricted Hartree-Fock computations were performed
within the framework of semi-empirical computations
using the AM1 Hamiltonian (Dewar et al. 1985).

491
senting [M - t-butyl]+ and [M – t-butyl-Si(CH3)2]+,
respectively.
These data matched up with the masses obtained
by analysis of derivatized salicylic acid as a refer-
ence compound. Quantitative measurements by HPLC
revealed that 34% of the initital 2-CF3-phenol was
◦
hydrolysed to salicylic acid within 20 min at 60 C.
The same type of experiment at 25 ◦C showed no
detectable decomposition of substrate within 2 hours.
◦
However 19F NMR measurements at 37 C showed
that decomposition of the substrate already can be
detected within this timeframe.
Biotransformation of 2-CF3-phenol by resting cells of
B. thermoleovorans A2
19F NMR experiments showed that 2-CF3-phenol was
transformed biologically by resting cells. However,
neither a decrease in concentration nor the occurrence
of additional peaks in the 19F spectra due to biolog-
ical processes could be observed for the other two
isomers. Therefore, the biological transformation of
2-CF3-phenol was further examined.
Figure 1. Growth and enzyme activities in whole-cells of B. ther-
moleovorans A2 with LB medium as substrate: #, OD
; ꢀ,
540nm
phenol hydroxylase activity; 3, catechol 2,3-dioxygenase activity.
well defined time point. For the resting cells transfor-
mation of 2-CF3-phenol cells were harvested after 16
h of incubation.
Biotransformation of 2-CF3-phenol resulted in the
accumulation of a yellow metabolite. The UV/VIS
analysis of this product revealed an absorption max-
imum at 387 nm at neutral pH which shifted to 325
nm after acidification to pH 2. This spectral transition
has an apparent pka value of 5.75. These data are in
accordance with a meta-cleavage product of the cor-
responding 3-trifluoromethylcatechol (Engesser et al.
1988a; Engesser et al. 1988b; Engesser et al. 1990).
The resting cells suspension was measured by 19F-
NMR without any extraction procedure (Figure 3).
The peak at −66.14 ppm is due to the fluorinated sub-
strate and the major metabolite signal is observed at
−78.73 ppm.
Tests for CF3-phenols as growth substrates –
temperature stability of 2-CF3-phenol
None of the tested three CF3-phenols could serve as
sole carbon and energy source under the conditions
used as judged from growth tests in the chloride-free
medium. However, a temperature-dependent chemical
process that led to the release of fluorine anions with
a concomitant decrease in the substrate concentration
was observed in 19F-NMR measurements in case of
2-CF3-phenol and 4-CF3-phenol.
The 1H-NMR spectrum of 2-CF3-phenol is shown
in Figure 2A. Figure 2B shows the 1H NMR spectrum
of 2-CF3-phenol after incubation for 20 min at 60◦C
without bacterial cells. The new signals correspond
to salicylic acid by comparison with the reference
compound.
The signals at −114.17 ppm and −123.0 ppm
are ascribed to the internal standard p-fluorobenzoate
and fluorine anions. NMR experiments without de-
coupling revealed the absence of CHF2 and CH2F
groups because no splitting of the signal at −78.73
ppm was observed which would arise from geminal
proton-fluorine interactions. The chemical shift value
of −78.73 agrees with literature data of a CF3 group at
an α-position to a carbonyl function (Wray 1983). Ad-
ditionally, after purification by anion-exchange chro-
matography no aldehydic proton was detectable in 1H-
NMR indicating that the catechol derivative is cleaved
between the trifluoromethyl substituent and the adja-
cent hydroxyl function. GC-MS analysis allowed the
19F-NMR measurements of these samples clearly
demonstrated the temperature-induced release of fluo-
rine anions. At every time interval the molar amount
of fluorine anions was three times the molar amount
of 2-CF3-phenol being transformed indicating the re-
lease of all three fluorine atoms of the CF3 group. No
fluorinated intermediates could be detected.
Upon GC-MS analysis after extraction and deriva-
tization the following data were determined retention
time = 29.1 min, masses at 309 u and 251 u repre-

492
1
1
Figure 2. (A) H-NMR spectrum of 2-trifluoromethylphenol in phosphate buffer; (B) H-NMR spectrum of 2-trifluoromethylphenol after
partial hydrolysis to salicylic acid.
identification of this metabolite as 7-TFHOD. At a re-
tention time of 21.0 min a peak was detected with the
following masses: 381 u, 353 u and 324 u, indicating
[M – tert-butyl]+, [M - tert-butyl – CO]+ and [M –
tert-butyl – tert-butyl]+ ions, respectively (Figure 4).
The derivatisation produced the tert-butyl-
dimethylsilyl derivative of 7-TFHOD. Due to the oc-
currence of two functional groups the mass of the
product (210 u) will be raised by 2∗114u = 228 u.
The spectra of TBDMS derivatives are normally dom-
inated by [M – 57]+ ions, in this case leading to
the fragment ion of 381 u. By HPLC and GC-MS
analysis of the resting cells suspension no catecholic
intermediates, 3-CF3-catechol and/or after hydrolysis
of the CF3 group 2,3-dihydroxybenzoic acid, could be
found. Moreover, no conversion of salicylic acid was
observed.
Purification of 7-TFHOD
Anion-exchange chromatography allowed the direct
application of a centrifuged sample of the resting cells
mixture. Stability problems did not occur as judged by
19F-NMR analysis of the sample before and after the
chromatographic separation. Simultaneous detection
at 270 nm and 380 nm revealed the clear separation
of a product with an absorbance at 380 nm and no
absorption at 270 nm (7-TFHOD) from the contam-
inating compounds, 2-CF3-phenol and salicylic acid,
only absorbing at 270 nm.
Temperature- and photochemical stability of
7-TFHOD
◦
Upon incubation at 60 C without exposure to light
7-TFHOD remained stable for at least 4 hours. The
absorption value at 387 nm remained constant and
no additional absorption band occurred. No fluorine
release was measured by 19F-NMR, indicating that

493
19
Figure 3. F-NMR spectrum of a resting cells incubation of Bacillus thermoleovorans A2 with 2-trifluoromethylphenol as substrate.
not only the chromophore remained intact, but that
also the trifluoromethyl group was not hydrolysed.
However, exposure to sunlight of oxic solutions of 7-
TFHOD and HMS-used as a reference-led to a rapid
decrease in absorption (Figure 5). Within the first 20
min 28% of the 7-TFHOD was decomposed. First-
order decay kinetics were followed at low substrate
concentrations. Both compounds behaved similar in
this way. The decomposition rates were equal for the
experiments with anoxic solutions.
Molecular properties of a series of meta-cleavage
products
Semiemperical AM1 molecular orbital properties
computations were performed to obtain information
on the molecular properties of a number of semi-
aldehydes in their dianionic, monoanionic and neutral
form (Table 1). These calculations revealed that within
this series the lowest LUMO energy was found for
7-TFHOD (neutral form: −1.74 [eV]). This LUMO
orbital is mainly located on the carbonyl carbon. In
comparison to the derivative 2-hydroxy-6-oxo-hepta-
2,4-dienoic acid (HOD) this property could lead to
a facilitated nucleophilic attack of water or an OH-
group of an enzyme. Generally, the alkyl deriva-
tives form a clear distinct group of semialdehydes
in comparison to 7-TFHOD. As expected from the
strong electron-withdrawing effect of fluorine atoms,
the sum of electrostatic potential charges on C6 and
C7 – see experimental section for details – increases
monotonically with an increasing number of fluorine
atoms in going from HOD (+0.33) via 2-hydroxy-6-
oxo-7,7,-difluoromethyl-hepta-2,4-dienoic acid and 2-
19F-NMR measurements revealed a decreasing
concentration of 7-TFHOD (−78.73 ppm) while no
increase of the fluorine anion concentrations was
detected. The photolytical breakdown of the chro-
mophore is therefore not accompanied by a defluori-
nation reaction leading to fluoride anions. No other
signals appeared in the 19F NMR spectrum resulting
in a negative fluorine balance.

494
Figure 4. Mass spectrum of the t-butyldimethylsilyl derivative of 7-TFHOD. The spectra of t-butyldimethylsilyl derivatives are normally
+
dominated by large [M-57] ions, in this case leading to the fragment ion of 381 u.
hydroxy-6-oxo-7-fluoromethyl-hepta-2,4-dienoic acid
to 7-TFHOD (+1.00). Substituent effects on the
LUMO energy differences remained almost constant
for the dianionic, monoanionic and neutral form.
Table 2 shows the calculated heat of formation for
the monoanionic form and the corresponding hydrate.
The differences in energy of the two aldehydes HMS
and 5-methyl-HMS are similar to the calculated value
for 7-TFHOD (−73.9 kcal/mol). The alkyl-substituted
ketones show energy differences above −70 kcal/mol.
The introduction of a fluorine atom in the substituent
on C6 is lowering the energy difference below −70
kcal/mol.
Discussion
Biotransformation of 2-CF3-phenol
Figure 5. Decomposition of 7-TFHOD and HMS in phos-
phate-buffer solution (pH 7.2) during exposure to sunlight:
7-TFHOD; ꢀ HMS.
3
The regulatory system of the o-cresol catabolism of
Pseudomonas CF600 has been investigated. It has
been reported by Chau Sze et al. (1996) that a
growth phase-dependent response, mediated by the
σ
54-dependent DmpR activator, was only a property of
medium. Under these conditions, the response to o-
fast-growing cultures cultivated on carbon-rich (LB)
cresol is suppressed until the exponential-to-stationary

495
Table 1. Calculations of LUMO energies and electrostatic potential charges
LUMO energy (eV)
Compound
Dianion
Neutral
Sum of electrostatic potential charges
on C6 and C7 for the dianionic form
HMS
+7.84
+7.79
+7.75
+7.69
+7.66
+7.66
+7.52
+7.40
+7.14
−1.32
−1.29
−1.28
−1.30
−1.08
−1.07
−1.26
−1.48
−1.74
–
5-methyl-HMS
HOD
–
+0.33
+0.41
+0.37
+0.39
+0.58
+0.79
+1.00
7-ethyl-HMS
7-isopropyl-HMS
7-propyl-HMS
7-fluoromethyl-HOD
7-difluoromethyl-HOD
7-trifluoromethyl-HOD
Table 2. Calculated heat of formation of meta-cleavage products
Heat of formation (kcal/mol)
Compound
Monoanionic form
Hydrate
Difference
HMS
−167.9
−174.1
−171.1
−176.8
−182.4
−184.9
−220.5
−266.9
−321.5
−240.9
−247.2
−241.2
−246.4
−249.3
−252.3
−291.6
−342.6
−359.4
−73.0
−73.1
−70.1
−69.6
−66.9
−67.4
−71.1
−75.7
−73.9
5-methyl-HMS
HOD
7-ethyl-HMS
7−isopropyl-HMS
7-propyl-HMS
7-fluoromethyl-HOD
7-difluoromethyl-HOD
7-trifluoromethyl-HOD
phase transition. Obviously, the expression of the first
two enzymes in the phenol catabolism of B. ther-
moleovorans A2 when growing on LB medium, is also
suppressed until the cells enter the late-exponential
phase. However, at this moment it is still unclear if
only a growth phase-dependent regulation controls the
expression of the aromatic degradation pathway or if
this is a fortuitous induction, i.e., a component of the
LB ingredients is utilized in the late-exponential phase
controlled by a regulatory system also operating on the
phenol pathway.
Analogously to ortho-substituted monohalophe-
nols (Reinscheid et al. 1997) we propose a path-
way for 2-CF3-phenol metabolism in B. thermoleovo-
rans A2 (Figure 6). After hydroxylation to 3-CF3-
catechol, a meta-cleavage produces 2-hydroxy-6-oxo-
7,7,7-trifluorohepta-2,4-dienoic acid (7-TFHOD).
Since catechol 2,3-dioxygenase is converting 3-
CF3-catechol to 7-TFHOD and also since the hydrol-
ysis of the initial substrate 2-CF3-phenol is lower-
ing the total amount of 3-CF3-catechol formed, it
seems polymerisation and/or hydrolysis of 3-CF3-
catechol is of importance in our system. Consequently,
we could not detect neither 3-CF3-catechol nor 2,3-
dihydroxybenzoic acid. Nevertheless, under real envi-
ronmental conditions it is possible that polymerisation
comes into play due to thermal enzyme inactivation.
The thermal stability of the catechol 2,3-dioxygenase
is currently under investigation in our laboratory.
During our work it became clear that the analysis
of semialdehydes by GC-MS poses a problem. Higson
& Focht (1992) could not detect the methyl derivative
of 5-chloro-HMS with an EI detector the ion energy
set at the typical value of 70 eV. In this case the detec-
tion was achieved by lowering the ion energy to 20 eV
or using a chemical ionization method. Even with a 20
eV setting Koh et al. (1997) detected a methyl deriv-
ative of 3,5-dichloro-HMS only in the selective ion

496
Figure 6. Overview of biological and non-biological transformations of 2-trifluoromethylphenol.
monitoring mode with an estimated 500fold increased
sensitivity. With a possible initial concentration in the
range of 0.1 M this difficulty cannot be due to initially
small amounts of analyte. More likely, in the course
of extraction, derivatisation, separation and detection,
the instability of at least the methyl derivatives of the
semialdehydes is leading to these analytical problems.
In our study the GC-MS detection of 7-TFHOD
was achieved with the tert-butyldimethylsilyl deriva-
tive. Two factors might have been important:
(b) The derivatisation solution was again evaporated to
dryness and the analyte redissolved in only 5 µl of
solvent. This last step increased the concentration
by a factor of 200 compared to the ethyl acetate
extract. A clear GC signal for the 7-TFHOD deriv-
ative was obtained at 70 eV and without a selective
ion monitoring mode.
Thermal transformation of 2-CF3-phenol
The detection of salicylic acid as a major chemical
degradation product of 2-CF3-phenol was very sur-
prising since it is known that the hydrolysis of a CF3
group attached to an aromatic nucleus frequently re-
quires rather energetic conditions, e.g., heating in
concentrated sulphuric acid or strongly basic condi-
tions (12N NaOH; Jones 1947) (Figure 6). In our study
the critical step, the fission of the halogen-carbon
bond, occurred under mild conditions. This process
(a) These derivatives are expelling a tert-butyl moiety
very easily, thereby reducing the excess energy of
the molecular ion and preventing further fragmen-
tation. Further support of the idea that the kind of
derivative is important for successful analysis by
GC-MS is given by Sung Bae et al. (1996). They
were able to analyse the trimethylsilyl derivative of
5-chloro-HMS at 70 eV ion energy.
◦
is a topic of further investigation. At 60 C the hy-

497
drolysis is competing with the enzymatic formation of
7-TFHOD via 3-CF3-catechol.
C6. Interestingly, this enol-keto tautomer [(E)-2,6-di-
oxo-hex-4-enoic acid] depicted in Figure 6 would be
analogous to the first step in the enzymatic hydrolysis
mechanism proposed by Engesser et al. (1988b).
In a recent study the effect of incubation tem-
perature on the reductive dechlorination of 2,3,4,6-
tetrachlorobiphenyl was investigated (Wu et al. 1997).
Temperature affected the lag time and the preferred
route of dechlorination. The studied temperature range
was between 4 ◦C and 66 ◦C, therefore real ther-
mophilic conditions have been applied.
Molecular orbital calculations
Growth tests with all three isomers of CF3-phenol
turned out to be unsuccessful indicating no produc-
tive pathway in the organism. A possible explana-
tion is the observation of Engesser et al. (1990)
that semialdehyde-hydrolases are not converting 7-
TFHOD as a substrate.
Experimental data about the enzymatic hydroly-
sis of semialdehydes in meta-pathways of catechol
degradation are scarce (Nordlund & Shingler 1990;
Duggleby & Williams 1986; Bayly et al. 1987; Horn et
al. 1991). Horn et al. (1991) suggested that the hydro-
lase of P. putida mt-2 may possess a serine hydrolytic
enzymatic mechanism. Under these conditions mole-
cular properties calculations can be helpful for testing
the above mentioned hypotheses made on common
grounds of organic chemistry.
Molecular orbital calculations have already been
introduced in the field of phenol degradation (Cozza
& Woods 1992; Peelen et al. 1995). It becomes clear
that on the enzyme level turnover rates of a series of
compounds can be correlated to computated molec-
ular properties. The LUMO energy of the series of
semialdehydes fit the proposed enzyme mechanism of
Engesser et al. (1988) for the semialdehyde-hydrolase.
The LUMO energy is of importance for the nucle-
ophilic attack by the enzyme which results in the
formation of a bounded semiketal (Fleming 1985).
Kinetic measurements of the hydrolase encoded
by the TOL plasmid pWW0 from P. putida mt-2
showed that the higher catalytic efficiency with ke-
tone substrates over aldehydes was the result of higher
Vmax values rather than lower Km values (Duggleby
& Williams 1986). Bayly & Di Berardino (1978) re-
ported on the hydrolase activity of a Pseudomonas
putida strain against different meta-cleavage products.
Methyl substitution in the 7 position of 7-TFHOD
resulted in reduced activity in comparison with the na-
tive compound. These results are in accordance to the
calculated similar LUMO energy values for the alkyl
derivatives which would result in similar Km values
Photochemical decompositon of 7-TFHOD
Using 19F-NMR and photometrical analysis it was es-
tablished that decomposition of 7-TFHOD involves
both the disappearance of the chromophore and the
loss of the 19F NMR signal. The latter process is,
however, not accompanied with the formation of flu-
oride anion, and a negative fluorine mass balance is
observed. This can be explained via an α-cleavage
reaction of the photo-excited molecule, yielding a con-
jugated acyl radical and a trifluoromethyl radical. It
has been shown that α-cleavage can occur in conju-
gated systems, e.g., CH2 = CH-C(= O)H (Klessinger
& Mich 1989). We expect α-cleavage to occur more
easily in our conjugated system than in, e.g., CH2
= CH-C(= O)H (where α-cleavage was shown to oc-
cur) because the loss of a CF3 radical is much less
endothermic than the loss of a H radical in CH2 = CH-
C(= O)H, the difference being more than 25 kcal/mol
according to PM3 calculations.
When the CF3 radical is liberated, this species
can abstract a hydrogen atom from water to form
trifluoromethane (fluoroform). This component has a
low solubility in water, which in combination with
◦
its low boiling point (−84 C) will lead to loss of
fluorine atoms from the aqueous solution. A second
independent photochemical process that can occur is
cis-trans isomerisation around the C(4)–C(5) bond,
to relieve some of the steric strain that exists in the
depicted isomer. From the computed heats of forma-
tion (AM1, using COSMO with ꢀ = 78 to simulate
solvation in water) this is estimated to yield about 1–
5 kcal/mol. But neither of these processes explains
the disappearance of the chromophore. However, loss
of absorption at 387 nm could be due to enol-keto
tautomerisation which yields about 2 kcal/mol. The
driving force of this process is the intrinsic higher
stability of the keto-form in comparison to the enol-
tautomer, in combination with the observation of a
non-planar π-system, which diminished the stabil-
isation offered by through-conjugation from C1 to
but would not automatically affect Vmax
.
According to the Curtin-Hammett principle (Carey
& Sundberg 1990) the energies for the different ionic
forms had to be calculated. The rationale for this is that

498
under conditions where these forms have low inter-
conversion energy barriers compared to the following
reaction i.e. hydration, it is not possible to state which
form is the starting compound for the reaction. Since
proton transfer reactions belong to the fastest reactions
in chemistry the corresponding activation energies are
in fact very low. Supporting the idea that the tri-
fluoromethyl substituent is facilitating a nucleophilic
attack irrespective of the degree of ionisation (neutral
species, mono-anion or dianion) 7-TFHOD displays
the lowest LUMO energy in all three species under
study.
In line with these calculated data about LUMO en-
ergies, the CF3-substituted compound also possesses
the highest positive charge at the center of attack, also
facilitating nucleophilic attack (Fleming 1985). Ad-
ditionally, differences in the reactivity of 7-TFHOD
in comparison with alkylated analogous compounds
(e.g., HOD) can also be related to the larger driving
force for semi-ketal formation for the CF3 compound
(Table 2). The larger (>4 kcal/mol) driving force for
the hydration of the CF3-substituted compounds itself,
however, directly suggests a reason for the observa-
tion that 7-TFHOD is not a substrate for the hydrolase
(Duggleby & Williams 1986). The occurrence of a
stabilised hydrate as intermediate might in fact ham-
per subsequent enzymatic steps in a thermodynamic
fashion.
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Acknowledgement
This work was supported by an EU Training Mobil-
ity and Research grant ERBFMGE-CT95-0066 (ac-
cess to large scale facility Wageningen NRM Centre).
U.M. Reinscheid wishes to acknowledge the LSF for
provision of research facilities.
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F