Microbial biotransformation of aryl sulfanylpentafluorides
Emma Kavanagh,1 Michael Winn,1 Cliona Nic Gabhann,1 Neil K. O’Connor,1 Petr Beier,2
Cormac D. Murphy1*
1. School of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical
Biology, Ardmore House, University College Dublin, Dublin 4, Ireland
2. Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech
Republic, Flemingovo nam. 2, 166 10 Prague 6, Czech Republic
*Corresponding author Fax: +353 (0)1 716 1183, Telephone: +353 (0)1 716 1311, email:
Cormac.d.murphy@ucd.ie
Keywords: Fluorine; emerging pollutant; N-acetylation
Abstract
We report for the first time the biotransformation of potential pollutants bearing the
pentafluorosulfanyl (SF5-) functional group in a fungus and bacteria. Cunninghamella
elegans transformed p-methoxy phenyl SF5 via demethylation; Pseudomonas knackmussii
and P. pseudoalcaligenes KF707 transformed amino-, hydroxyamino- and diaminosubstituted phenyl SF5, forming the N-acetylated derivatives as the main product. Cell free
extract of Streptomyces griseus transformed 4-amino-3-hydroxy-phenyl SF5 to the Nacetylated derivative in the presence of acetyl CoA, confirming that an N-acetyltransferase is
responsible for the bacterial biotransformations. Approximately 25 % of drugs and 30 % of
1
agrochemicals contain fluorine, and the trifluoromethyl group is a prominent feature of many
of these since it improves lipophilicity and stability. The pentafluorosulfanyl moiety is seen
as an improvement on the trifluoromethyl group and research efforts are underway to develop
synthetic methods to incorporate this moiety into biologically active compounds. It is
important to determine the potential environmental impact of these compounds, including the
potential biotransformation reactions that may occur when they are exposed to
microorganisms.
Introduction
Despite the relative abundance of fluorine in the terrestrial environment, there are very few
naturally produced fluorinated compounds. This can be accounted for by the biological
unavailability of fluorine, e.g. the most abundant form of fluorine is insoluble CaF2, and its
physicochemical properties, such as the high solvation and redox potential energies of the
fluoride ion (Harper and O’Hagan 1994). Nevertheless, owing to fluorine’s unique
characteristics, in particular its high electronegativity, small atomic radius and the strength of
the carbon-fluorine bond, fluorinated organic compounds have a large number of applications
including pharmaceuticals, agrochemicals, refrigerants, solvents, degreasers and aerosol
propellants (Dolbier, 2005). Thus, organofluorine compounds are ubiquitous in the
environment, where they may be transformed by organisms that they come into contact with,
possibly generating highly toxic metabolites (Key et al. 1997).
Some microorganisms are capable of transforming, and indeed degrading,
organofluorine compounds (Murphy 2010). Only one specific carbon-fluorine bondhydrolysing enzyme is known from microorganisms that can employ the most abundant
fluorinated natural product, fluoroacetate, as a carbon and energy source (Donnelly and
Murphy, 2009). Many microorganisms can partially or completely degrade fluorinated
2
aromatic compounds, such as fluorophenol (Ferreira et al. 2008), fluorobenzoate (Boersma et
al. 2004) and fluorobiphenyl (Hughes et al. 2011), via the classical oxidative pathways, since
fluorine’s small size means that there is no steric hindrance to binding to the enzymes’ active
site. However, the transformation of trifluoromethyl-substituted aryls is much less well
known (Engesser et al. 1988a; Engesser et al. 1988b; Engesser et al. 1990); the
trifluoromethyl (CF3) group is very common in pharmaceuticals and agrochemicals, where it
improves stability and lipophilicity. The pentafluorosulfanyl (SF5) group is currently
considered a replacement for CF3, owing to its stability and electrical properties (Alomonte
and Zanda 2012). Recently, several SF5-analogues of drugs and pesticides that contain the
CF3 functional group have been synthesised (Fig 1), including the antimalarial agent
mefloquin, the selective serotonin re-uptake inhibitor fluoxetine and the herbicide trifluralin
(Welch and Lim 2007; Wipf et al. 2009; Lim et al. 2007). Initial biological assays indicated
that the derivatives are at least as effective as the parent compounds.
Currently, nothing is known about the microbial metabolism of compounds bearing
the SF5 moiety, which are emerging pollutants (Jackson and Mabury 2009). Thus, in order to
enable prediction of the likely microbial metabolites upon widespread exposure of aryl-SF5
compounds, we conducted a series of transformation experiments with compounds 1-6 (Fig
2) in three bacteria and a fungus.
Materials and Methods
Materials
Compounds 1-6 were synthesised previously (Beier and Pastyrikova 2011; Beier et al. 2011;
Pastyrikova et al. 2012). N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), acetyl
CoA, Sabouraud dextrose agar and broth were purchased from Sigma. Peptone was
purchased from Fisher Scientific and Meat extract was from Merck. Cunninghamella
3
elegans DSM1908, Pseudomonas knackmussii B13 and Pseudomonas pseudoalcaligenes
were acquired from DSMZ, Germany. Streptomyces griseus ATCC13273 was obtained from
LGC Standards, UK.
Culture conditions
C. elegans was cultured in 250 ml Erlenmeyer flasks containing Sabouraud dextrose broth as
described previously (Amadio and Murphy, 2010). After 72 h growth the substrate (3-5 mg)
was added and incubation continued for a further 72 h. At the end of the experiment the
culture was sonicated (U200S, IKA Labortechnik) for 5 min and the sonicate extracted with
ethyl acetate.
P. knackmussii and P. pseudoalcaligenes were cultivated in 250 ml Erlenmeyer flasks
containing either 50 ml of nutrient broth (5g L-1 peptone, 3g L-1 meat extract, pH 7.0) or
minimal medium (2.44 g L-1 Na2HPO4, 1.52 g L-1 KH2PO4, 0.5 g L-1 (NH4)2SO4, 0.2 g L-1
MgSO4.7H2O, 0.05 g L-1 CaCl2.2H2O, pH 7), supplemented with either benzoate or 4fluorobenzoate (25 mg) for 24-72 h at 28 °C; cells were harvested and substrate (5 mg) was
added and the incubation continued for a further 48-72 h. The supernatant was harvested by
centrifugation and extracted with ethyl acetate; the organic solvent was removed by rotary
evaporation.
Streptomyces griseus was grown in 250 ml Erlenmeyer flasks containing 50 ml of
tryptone soya broth. The cultures were incubated for 96 h at 30 °C in an orbital incubator;
compound 3 (2.5 mg) was added to the flasks after 24 h. At the end of the incubation period
cells were harvested by centrifugation, and the supernatant extracted with ethyl acetate as
described.
4
Control experiments were conducted in which the microorganism was incubated in
the absence of aryl-SF5 compound and the compounds 1-6 were incubated in the absence of
microorganism.
Preparation of cell free extract
S. griseus cells were cultivated as described, harvested by centrifugation, washed with water
and resuspended in phosphate buffer (20 mM, pH 7). The cells (0.1 g/ml) were disrupted by
sonication (5 min in total, bursts of 1 s with 1 s interval) using an ice bath to prevent
overheating. The sonicate was clarified by centrifugation and the supernatant was used as the
cell free extract. Compound 3 (1 mM) was incubated with cell free extract (240 µl) and
acetyl CoA (1 mM) for 16 h at 30 °C to assay N-acetyl transferase activity. Experiments
without cell extract or acetyl CoA were conducted as controls. The products were extracted
twice with ethyl acetate (250 µl), and the solvent was removed over a stream of N2. The
residue was derivatised with MSTFA and analysed by GC-MS.
Analysis of metabolites
Culture extracts were evaporated to dryness and silylated by addition of MSTFA and
heating at 100 °C for 1 h. The derivatised samples were analysed by gas chromatographymass spectrometry (GC-MS) on an Agilent 6890 gas chromatograph coupled to a 5973 mass
selective detector, by injection of the analytes (1 µl) onto a HP-5MS column (30 m x 0.25
mm x 0.25 μm) either employing a 20:1 split ratio and raising the temperature from 150 to
300 °C at 10 °C per min, or splitless and raising the temperature from 120-300 °C. Nonderivatised culture extracts redissolved in ethyl acetate were also analysed, employing oven
conditions starting at 70 °C and rising to 250 °C. The mass spectrometer was operated in the
scan mode.
5
To confirm metabolite identity multiple culture flasks containing P. knackmussii were
incubated with 4 (40 mg in total) for 72 h. Cultures were extracted as described and the
products isolated using semi-preparative high performance liquid chromatorgraphy (HPLC)
on a Varian Prostar system fitted with a Supelco Ascentis C18 (15 cm x 10 mm x 5 μm)
column. Compounds were eluted using H2O/0.1% TFA (A) versus acetonitrile/0.1% TFA (B)
(4 ml/min; 0-30 min, 20-95% B; 30-35 min, 95-20% B; 35-40 min, 20% B). Fractions were
lyophilised, yielding a crystalline solid. 1H NMR (CDCl3): δ ppm 2.29 (s, 3H), 7.024 (d, J =
8.8, 1H), 7.416 (s, 1H), 7.508 (d, J = 6.4, 1H).
Results and discussion
Transformation in bacteria.
P. knackmussii B13 is a very well-studied microorganism that will use a range of halogenated
xenobiotics as sole carbon and energy sources (Stolz et al. 2007), including fluorobenzoate
(Misiak et al. 2011). P. pseudoalcaligenes KF707 is a well-known polychlorinated biphenyl
(PCB)-degrader that can also employ fluorinated biphenyls as sole carbon and energy sources
(Murphy et al. 2008). Both organisms employ dioxygenases to activate aromatic compounds
for further degradation via ortho- (P. knackmussii) and meta- (P. pseudoalcaligenes)
cleavage pathways. Thus it was expected that hydroxylated metabolites were the most likely
transformation products upon incubation of the substituted (pentafluorosulfanyl)benzenes.
However, while biotransformation of compounds 3-6 was observed with Pseudomonas spp.,
the masses of the products did not correspond to those expected for hydroxylation. In each
case a mass difference of 42 between the products and the substrate was observed. An
example of the observations is shown in Fig 3, which illustrates the product formed upon
incubation of 3 with Pseudomonas knackmussii. The data from the biotransformation
6
experiments are summarised in Table 1 and the chromatograms and mass spectra are shown
in the Supplemental Information. These data indicated a modification of the amino group by
acetylation, and to confirm this a scaled-up biotransformation experiment was conducted with
P. knackmussii incubated with 4, and the product isolated by HPLC. The 1H NMR spectra
confirmed the presence of a methyl group (δ ppm 2.29 (s, 3H)) not present in the starting
compound. O-acetylation could be excluded since such a transformation would result in a
product that could not be further derivatised by silylation, with a consequent mass
implication. In the case of the diamine 6, two products were detected, both of which had
identical mass spectra (Fig S9). The presence of isomers as biotransformation products of 6
indicated that either of the amino groups could be acetylated (Fig 4a); however, no diacetylated product was detected.
It is most likely that an N-acetyltransferase (NAT) is responsible for the
transformation of these compounds. Such enzymes have been identified from a range of
microorganisms, including Streptomyces griseus, which expresses an arylamine NAT that is
involved in grixazone biosynthesis and catalyses the acetylation of 2-aminophenols (Suzuki
et al. 2007). In the present study the degree of transformation ranged from < 10 % for the
aniline 5 to >90 % for 3, suggesting that the presence of a hydroxyl ortho to the amino group
has an effect on the transformation efficiency. Interestingly, a similar effect was observed for
the S. griseus NAT, which readily transformed 3-amino-4-hydroxybenzoic acid, but not 3- or
4-aminobenzoic acid (Suzuki et al. 2007). To investigate if S. griseus could transform aryl
SF5 compounds, 3 was incubated with growing S. griseus resulting in the production of the
same compound detected in the Pseudomonas cultures. To confirm that the transformation
was as a consequence of NAT activity, S. griseus cell free extract was incubated with 3 plus
acetyl CoA. The expected acetylated product was detected by GC-MS; it was not present
either in the absence of acetyl CoA, or cell free extract (supplemental information, Fig S11).
7
Transformation in C. elegans
This fungus is well known for its ability to transform xenobiotics and was previously shown
to transform other fluorinated aromatics (Cerniglia et al. 1984; Amadio and Murphy 2010).
However, no transformation product was observed by GC-MS analysis of silylated extracts of
C. elegans cultures that were incubated with compounds 2-6. The ethyl ether 2 was not
detectable in the culture after 72 h incubation, but this was due to evaporation rather than
metabolism. The absence of any transformation products from 3-6 suggests that there is no
NAT activity, which is consistent with previous findings (Zhang et al. 1996).
(Pentafluorosulfanyl)anisole 1 also evaporated from the control flask (no fungus), but in the
flask with the fungus, some of it was transformed to a compound with the expected mass
spectrum of a hydroxylated (pentafluorosulfanyl)benzene (Table 1 and Supplemental
Information), which was most likely a consequence of biological demethylation (Fig 4b).
Demethylation of xenobiotics has been observed with this fungus (Bright et al. 2013), but it
was expected that the cytochrome P450 (CYP) enzymes present in C. elegans would have
oxidised the aromatic ring, which is the most well-established transformation described in the
species. However, the electron-withdrawing effect of the SF5 moiety probably deactivates
the ring making monooxygenase attack impossible.
Conclusions
The use of organofluorine compounds in a range of applications means that biological
systems are exposed to these compounds, and far from being inert, fluorinated compounds
are transformed, possibly to toxic metabolic intermediates. Compounds bearing the
pentafluorosulfanyl functional group are predicted to increase in number as the advancements
in synthetic chemistry are made. This prompted us to investigate the possible microbial
8
transformation of these compounds, by three bacteria and a fungus that are known to
metabolise organofluorine compounds, as an initial study as to the likely biotransformation
products that would occur in the environment. The aromatic rings of the compounds tested
were stable to microbial attack, possibly because of deactivation due to the SF5 substituent.
In contrast, oxidative attack on the ring of aryl CF3 compounds has been observed in bacteria,
for example 3-trifluoromethyl benzoate was transformed via benzoate dioxygenase of
Pseudomonas putida, yielding the corresponding catechol that was subsequently cleaved by
catechol 2,3-dioxygenase (Engesser et al. 1988a). Nevertheless, other substituents of the aryl
SF5 compounds tested were susceptible to transformation, thus demethylation of the methyl
ether and N-acetylation of the amino groups were observed. Acetylation of anilines has been
recognised as a mechanism for detoxification of xenobiotics (Tweedy et al. 1970), and Nacetyl transferases have been identified in a range of microorganisms that are
environmentally and clinically relevant (Wright, 1999; Takenaka et al. 2009; Kubiak et al.
2012). Broadening this study to include a larger range of pentafluorosulfanylbenzenes with
modified substituents to a greater number of environmentally present microorganisms would
enable a more comprehensive understanding of these potential future pollution risks.
Acknowledgments
This work was conducted with financial assistance from the Society for General
Microbiology (UK), Science Foundation Ireland, Irish Research Council, Grant Agency of
the Czech Republic (P207/12/0072) and the Academy of Sciences of the Czech Republic
(RVO: 61388963).
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12
Table 1. GC-MS data of the products from microbial biotransformation of various PhSF5
compounds.
tR (min)
m/z M+
1
7.9a
234
3.0
292b
2
9.0a
248
-
-
3
6.1
379
6.65
421c,d
4
5.9
379
6.87
421c,d
5
4.0
291
4.39
333c
6
7.15
378
7.88, 8.2
420c
Substrate
Product tR (min)
a
The retention time (tR) is of the underivatised compound.
b
Product only observed with C. elegans
c
Product observed with P. knackmussii
d
Product observed with P. pseudalcaligenes
Product m/z M+
13
Figure legends
Fig 1. Structures of mefloquin, fluoxetine and trifluralin and their corresponding SF5
analogues.
Fig 2. Structures of the aryl sulfanylpentafluorides used in this study.
Fig 3. Transformation of 3 by P. knackmussii. A, total ion chromatogram (TIC) of 3
incubated in absence of microorganism, with a retention time of 6.1 min; B, TIC of
biotransformation product, indicated by an arrow, after incubation with P. knackmussii; C,
TIC of bacterium cultured without 3; D, mass spectrum of silylated biotransformation
product.
Fig 4. Biotransformation of 3,4-diamino-( pentafluorosulfanyl)benzene 6 by Pseudomonas
(A) and (pentafluorosulfanyl)anisole 1 by C. elegans (B).
14
Fig 1. Kavanagh et al.
Mefloquine
Fluoxetine
Trifluralin
F5
F5
F5
15
Fig 2. Kavanagh et al.
1,
2,
3,
4,
5,
6,
R1=OMe, R2=H
R1=OEt, R2=H
R1=NH2, R2=OH
R1=OH, R2=NH2
R1=H, R2=NH2
R1=NH2, R2=NH2
16
Fig 3. Kavanagh et al.
17
Fig 4. Kavanagh et al.
A
Pseudomonas spp.
B
C. elegans
18