Microbial degradation of fluorinated drugs: biochemical pathways, impacts on the

environment and potential applications

Cormac D. Murphy

UCD School of Biomolecular and Biomedical Science, University College Dublin, Belfield,

Dublin 4, Ireland

Email: Cormac.d.murphy@ucd.ie

Phone: +353 (0)1 7162572

Abstract

Since the discovery over 60 years ago of fluorocortisone’s biological properties [Fried J, Sabo

EF (1954) 9-α-fluoro derivatives of cortisone and hydrocortisone. J Am Chem Soc 76: 1455-

1456], the number of fluorinated drugs has steadily increased. With the improvement in

synthetic methodologies, this trend is likely to continue and will lead to the introduction of

new fluorinated substituents into pharmaceutical compounds. Although the

biotransformation of organofluorine compounds by microorganisms has been well studied,

specific investigations on fluorinated drugs are relatively few, despite the increase in the

number and variety of fluorinated drugs that are available. The strength of the carbon-

fluorine bond conveys stability to fluorinated drugs, thus they are likely to be recalcitrant in

the environment, or may be partially metabolized to a more toxic metabolite. This review

examines the research done on microbial biotransformation and biodegradation of

fluorinated drugs and highlights the importance of understanding how microorganisms

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interact with this class of compound from environmental, clinical and biotechnological

perspectives.

Keywords: Organofluorine; biotransformation; fluorometabolite; pollutant

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Introduction

Fluorine in drugs

Approximately 25 % of drugs, either currently available or in the pipeline, are fluorinated,

and typically contain fluorophenyl or trifluromethylphenyl groups (Fig 1). Six new

fluorinated drugs were approved by the FDA in 2014 (Jarvis 2015) and it is expected that the

number of fluorinated drugs will increase year-on-year. Fluorine’s unique physicochemical

properties, in particular the van der Waals radius (1.35 Å), which is between that of

hydrogen (1.20 Å) and the oxygen of hydroxyl (1.40 Å), electronegativity and stability of the

C-F bond can enhance certain properties of drugs such as lipophilicity, metabolic stability

and potency (Murphy and Sandford 2015). Whilst regio- and stereo-selective fluorinations

are chemically challenging, synthetic methodologies have been developed that enable

fluorination of a wide variety of drug-like molecules and building blocks. Enzymatic

methods are now part of the toolbox to enable specific fluorination of pharmaceutically

relevant molecules, for example, the fluorinase from Streptomyces cattleya can be used to

incorporate radioactive F-18 into molecules that can be employed in positron emission

tomography (PET) (Onega et al. 2010); Rentmiester et al. (2009) combined cytochrome P450

hydroxylation and deoxofluorination with DAST (diethyl aminoethyl trifluoride) to produce

fluorinated ibuprofen. The continued and increasing use of fluorine in drug manufacture

has implications for the environment that urgently require attention.

In addition to pharmaceuticals, fluorine is used in the manufacture of agrochemicals

and industrial chemicals such as aerosols and refrigerants; naturally produced

organofluorines are rare (O'Hagan and Deng 2015). Consequently, anthropogenic

fluorinated compounds are commonly found in the environment. Whilst some can be

readily degraded, others, in particular perfluorinated compounds, are recalcitrant and

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accumulate in the environment. PFOA (perfluoro-octanoic acid), for example, is used in the

preparation of fluoropolymers and has been detected in the bloodstream of humans and

animals (Fromme et al. 2009). Chronic exposure to high concentrations of this compound in

animals results in increased incidence of tumors (Biegel et al. 2001). Thus, such compounds

are an environmental and health concern.

Pharmaceutical compounds and their metabolites have been detected in sewage and

their removal rates are a focus of current research efforts (Gurke et al. 2015). Fluorinated

compounds are a significant subset of pharmaceuticals and contamination of the

environment by such compounds, in particular fluoroquinolone antibiotics, is worthy of

separate study. Microorganisms can degrade myriad xenobiotic compounds, including

those that are fluorinated, thus have the potential to bioremediate contaminated

environments containing fluorinated pharmaceuticals. In this paper the bacterial and fungal

biotransformation of fluorinated drugs will be reviewed and the possible applications

discussed.

Monitoring fluorinated drug biotransformation

A convenient technique for assessing the biotransformation of fluorinated compounds is

fluorine-19 nuclear magnetic resonance spectroscopy (F-19 NMR), which enables the

observation of newly formed fluorometabolites without the need for purification. The

sensitivity of F-19 NMR is comparable to that of H-1 NMR, thus µM concentrations of

compounds can be detected, and even minor structural modifications to will lead to

significant chemical shift changes. Corcoran et al. (2001) demonstrated that F-19 NMR was

an effective tool for screening fluorinated drug metabolism. The biotransformation of three

fluorinated drug-like compounds by 48 microbial strains was assessed by cultivating the

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microorganisms in 24-well plates, incubating with the fluoro-drug (1 mM) for 10 days before

quenching with acetonitrile:water (4:1) and recording the F-19 NMR spectra of the culture

supernatants.

Whilst the technique has been applied in the study of microbial biotransformation of

other fluorinated drugs, limited structural information regarding the products is provided,

thus other techniques, such as HPLC, GC- and LC-MS and ion selective electrodes, are often

applied in conjunction with F-19 NMR. Qin et al. (2012) developed a LC- Continuum Source

Molecular Absorption Spectrometry (CS-MAS) technique that can be applied to fluorinated

compounds and has a detection limit in the nM range, thus is highly applicable for

environmentally relevant fluorinated drugs and their metabolites.

Overview of microbial biotransformation of organofluorine compounds

Bacteria and fungi are known to transform and degrade model aromatic and aliphatic

organofluorine compounds and this topic has been thoroughly reviewed recently (Kiel and

Engesser 2015). While the focus of the current review is on fluorinated drugs, many of the

biochemical pathways revealed through the study of model organofluorine compounds are

relevant, thus a brief summary of the key findings is given below. For mineralization of

organofluorine compounds, cleavage of the highly stable C-F bond is necessary. This can be

achieved either directly via the actions of a specific dehalogenase enzyme, or indirectly as a

consequence of the formation of an unstable metabolic intermediate. Fluoroacetate

dehalogenase is the only class of enzyme known to specifically hydrolyse C-F, yielding

fluoride ion and glycolate, and the enzyme has been isolated from a number of bacterial

species (Goldman 1965; Kurihara et al. 2003; Donnelly and Murphy 2009). In addition to

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being used as a rodenticide in some countries, fluoroacetate is the most abundant naturally

produced organofluorine compound (O'Hagan and Deng 2015), thus microorganisms have

evolved to employ this compound as a carbon and energy source (Camboim et al. 2012).

Aromatic compounds such as fluorophenols, fluorobenzene and fluorobenzoic acids

can be biotransformed by microorganisms through the established catabolic pathways

(Boersma et al. 2004; Carvalho et al. 2008; Kim et al. 2010; Duque et al. 2012). The

oxygenase enzymes that are involved in these pathways in aerobic microbes will accept

fluorinated derivatives of their natural substrates, owing to the limited steric impact of the

small fluorine atom. Depending on the position of the fluorine, formation of the oxidized

intermediates can result in the spontaneous elimination of fluoride ion, or formation of a

dead-end fluorometabolite (Harper and Blakley 1971; Clarke et al. 1975; Schreiber et al.

1980; Engesser et al. 1990). For example, 3-fluorobenzoic acid is a sole carbon and energy

source for Sphingomonas sp. HB1, and is catabolized via the benzoate-degrading pathway

(Boersma et al. 2004). The first enzyme of this pathway, benzoate dioxygenase,

hydroxylates the substrate at either C-1/C-2 or C1/C6, yielding both 3-fluoro- and 5-fluoro-

fluorocyclohexadiene cis,cis-1,2-diol-1-carboxylate, which are respectively transformed to 3-

and 4-fluorocatechol (Figure 2). The latter is completely degraded releasing fluoride ion, but

3-fluorocatechol is catabolised to the dead-end metabolite 2-fluoro-cis,cis-muconic acid.

Most other reports of fluoroaromatic biodegradation via 3-fluorocatechol observe the same

accumulation of the fluorinated muconate. However, a notable exception is Burkholderia

fungorum FLU100, which mineralises fluorobenzene principally via 3-fluorocatechol and 2-

fluoro-cis,cis-muconic acid, yielding fluoride ion (Strunk and Engesser 2013).

The biodegradation of trifluoromethyl-substituted aromatic compounds is less well

studied, yet the trifluoromethyl phenyl group is present in a large number of drugs,

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including the blockbuster selective serotonin reuptake inhibitor Prozac (fluoxetine) and the

COX-2 inhibitor Celecoxib. Engesser et al. (1988a and 1988b) studied the co-metabolism of

3- and 4-trifluoromethyl benzoates in Pseudomonas putida strains and observed limited

biodegradation and the accumulation of 2-hydroxy-6-oxo-7,7,7-trifluorohepta-2,4-dienoate

(7-TFHOD, Figure 3). In a separate study with a marine bacterium Taylor et al. (1993)

demonstrated mineralization of trifluoromethylbenzoates via a combination of

biodegradation to 7-TFHOD and photodegradation. The thermophilic bacterium Bacillus

thermolevorans biotransformed 2-trifluoromethylphenol to 7-TFHOD and by F-19 NMR it

was possible to observe that the isolated metabolite was degraded by sunlight. However,

no fluoride ion was detected and it was reasoned that the photoexcited molecule was a

trifluoromethyl radical that abstracted a proton from water yielding volatile fluoroform that

was readily lost to the atmosphere. Yano et al. (2015) isolated a bacterial strain,

Rhodococcus sp. 065240, that degrades benzotrifluoride yielding trifluoroacetic acid. A gene

cluster (btf) was identified as being necessary for the biodegradation, and sequence analysis

revealed homology with the ipb cluster enabling isopropylbenzene biodegradation in

Rhodococcus erythropolis BD2.

Fluoroquinolone antibiotics

Fluoroquinolone antibiotics (Figure 4), such as ciprofloxacin and norfloxacin, are important

human and veterinary drugs widely used in the treatment of serious bacterial infections.

Quinolone antibiotics inhibit DNA synthesis targeting both DNA gyrase and topoisomerase

IV. Of a major concern is the development of resistance to these antibiotics, and the

extensive use of fluoroquinolones results in many microorganisms in the wider environment

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coming into contact with them. Understanding microbial biotransformation of these drugs

is crucial in predicting the likely mechanism of resistance.

Kim et al. (2011) were first to isolate a bacterium that could biotransform a

fluoroquinolone antibiotic. Using wastewater as an inoculum and N-phenylpiperazine as a

carbon source, a Microbacterium sp. was enriched that was insensitive to norfloxacin. The

bacterium could not grow on norfloxacin, but biotransformed it to four metabolites: 8-

hydroxynorfloxacin, 6-defluoro-6-hydroxynorfloxacin, desethylene norfloxacin and N-

acetylnorfloxacin (Figure 5). The latter required the presence of Casamino acids in the

medium and a glutamine synthase was subsequently isolated that catalyzed this reaction

(Kim et al. 2013). The production of the hydroxylated metabolites was inhibited by free

radical scavengers, thus it was inferred that these metabolites were formed from hydroxyl

radicals.

The bacterium Labrys portucalensis F11, which was originally isolated by enrichment

on fluorobenzene as a sole carbon and energy source, can co-metabolise fluoroquinolone

antibiotics (Amorim et al. 2014). Whilst the antibiotic effects of ciprofloxacin, norfloxacin

and ofloxacin were observed at concentrations above 3.5 µM, the bacterium could

transform all three of the fluoroquinolones, either separately or as a mixture. Complete

defluorination was not observed, but reached approx. 60 % for ofloxacin (2 µM) when

incubated with cells supplemented with 5.9 mM acetate. Detection of metabolites was

afforded by LC-MS and indicated several biotransformation reactions that were similar to

those reported in Microbacterium N2-2 (Kim et al., 2011) and included

hydroxylation/defluorination, demethylation, decarboxylation and piperzine ring cleavage.

Fungal biotransformation of fluoroquinolones has been studied in a number of

species (Table 1). Mineralisation of the antibiotics, as determined by measuring 14CO2

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emissions from radiolabeled starting material, was observed in some basidiomycetes

(Wetzstein et al. 1999; Marengo et al. 2001). Numerous metabolites have been identified,

most notably from Gloeophyllum striatum, which are thought to arise through reaction with

extracellularly generated hydroxyl radicals produced by lignin-degrading enzymes (Karl et al.

2006). Commonly detected transformations reported are similar to those observed in

bacteria and include modification of the piperazinyl ring (deethylation, N-acetylation, N-

formylation), decarboxylation and hydroxylation. Wetzstein et al. (2012) demonstrated for

the first time the formation of a catechol-type metabolite from pradofloxacin, and a late

stage ring-opened metabolite (Figure 6), suggesting a mechanism for complete degradation

of this class of antibiotic.

Despite the extensive effort in determining the intermediates arising from microbial

biotransformation of fluoroquinolone antibiotics, little is known about the enzymes

involved. In some fluoroquinolone resistant bacteria, an N-acetyl transferase can

biotransform the antibiotics, thereby eliminating bioactivity (Adjei et al. 2006; Jung et al.

2009). The biotransformation of flumequine to diastereomers of 7-hydroxyflumequine in

Cunninghamella elegans (Williams et al. 2007) is likely to be catalyzed by cytochrome P450.

Prieto et al. (2011) demonstrated that laccase degraded ciprofloxacin in vitro, and inferred

from inhibitor studies that cytochrome P450 is also involved in the biodegradation of

fluoroquinolone antibiotics in Trametes versicolor. Cvancarova et al. (2015) conducted

Principle Component Analysis of the ligninolyitc enzyme activities (laccase, manganese

peroxidase and manganese independent peroxidase) of several fungi in the presence of

ciprofloxacin, norfloxacin and ofloxacin. Manganese peroxidase correlated with the

degradation of the three antibiotics, suggesting a role for this enzyme in the degradation

process.

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Antidepressants

Fluoxetine (Prozac) is a widely prescribed antidepressant that contains a

trifluoromethylphenyl moiety (Figure 7). It is recalcitrant and has been detected in

waterways where it exerts measureable biological effects on the aquatic animals (Silva et al.

2015). Rodarte-Morales et al. (2011) studied the degradation of fluoxetine and another

fluorinated antidepressant, citalopram (Figure 7) by three white rot fungi: an anamorph of

Bjerkandera sp. R1, Bjerkandera adjusta and Phanerochaete chrysosporium. Whilst

citalopram (1 mg/l) was completely degraded by all three species after 14 days, fluoxetine

was comparatively poorly degraded, to a maximum of 46 % in culture of the anamorph of

Bjerkandera sp. R1. No intermediates were determined in the study, and although MnP

activity was measured, no in vitro experiments with the enzyme were reported.

The bacterium Labrys portucalensis F11 can degrade fluoxetine, releasing

stoichiometric amounts of fluoride ion from low concentrations (2 µM) of substrate

(Moreira et al. 2014). In the presence of another, more readily useable carbon source, such

as acetate, higher concentrations of fluoxetine could be degraded. Furthermore, the

authors demonstrated that the R-enantiomer was preferentially degraded by the bacterium.

No intermediates were detected, which is frustrating since mineralization of compounds

containing trifluoromethylphenyl via biotransformation is very rare. Nevertheless, this

bacterium might enable further understanding of the metabolism and enzymology of

xenobiotics containing trifluoromethyl substituted aromatic systems.

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Flurbiprofen

Biphenyl structures are common in pharmaceuticals and microbial degradation of this

moiety is known. Biotransformation of fluorinated biphenyls to hydroxylated products has

been reported in fungi (Green et al. 1999; Amadio and Murphy 2010), although full

biodegradation has not been observed. The biphenyl-degrading bacterium Pseudomonas

pseudoalcaligenes KF707 can grow on mono-, di- and poly-fluorinated biphenyls (Murphy et

al. 2008; Hughes et al. 2011), employing the upper biphenyl degrading pathway.

The fluorobiphenyl-containing drug flurbiprofen can be biotransformed to various

oxidised fluorometabolites (Figure 8) by fungi belonging to the genus Cunninghamella and

Streptomyces bacteria (Amadio et al. 2010; Bright et al. 2011). These organisms are

producers of cytochromes P450 and are established models of mammalian xenobiotic

metabolism (Murphy 2015). E. coli expressing cyanobacterial cytochrome P450 (CYP110E1)

transformed flurbiprofen methyl ester into two hydroxylated products at very low (<5 %)

yield (Makino et al. 2012). Such biotransformations can be useful in identifying

metabolically labile sites on drug molecules so that more stable/potent derivatives can be

prepared (Shaughnessy et al. 2014). Racemic flurbiprofen can be resolved by incubation of

the compound with dried mycelium of Aspergillus oryzae suspended in an organic solvent

(e.g. toluene) with ethanol (Spizzo et al. 2007). The fungus enantioselectively esterifies R-

flurbiprofen.

Flutamide

Flutamide (Figure 9) is a widely used nonsteroidal, antiandrogen drug for the treatment of

prostate cancer. In vivo it is extensively transformed to bioactive metabolites, notably 2-

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hydroxyflutamide and 4-nitro-3-(trifluoromethyl) phenylalanine. Two studies have

examined the microbial biotransformation of this drug. Herath and Khan (2010) screened

40 microorganisms for the ability to transform flutamide and observed the fungi

Rhodotorula mucilaginosa (ATCC20129) and Beauveria bassiana (ATCC 7159) and the

bacterium Nocardia sp. (NRRL 5646) produced three metabolites via hydrolysis of the

isobutanoyl group, nitroreduction and N-acetylation (Table 2). In a second study Amadio

and Murphy (2011) demonstrated the production of hydroxylated metabolites from

flutamide incubated with Cunninghamella elegans, which has the necessary cytochrome

P450 activity to catalyse such biotransformations. When the drug was incubated with

biofilms of C. elegans, a substantial decrease in biomass was observed strongly suggesting

that the compound is highly toxic and may be concentrated in the biofilm (Quinn et al.

2015), which has implications in the wider environment where biofilms are the dominant

mode of growth of microorganisms.

Fluorouracil

5-Fluorouracil (5FU) is a very common anticancer compound first introduced in the 1950s

and is detected in hospital and municipal wastewaters (Kosjek et al. 2013). Its

biodegradation has not been well studied, although Lutterbeck et al. (2015) demonstrated

that in closed bottle tests, using the final effluent from a municipal sewage treatment plant

as an inoculum, 5FU was not biodegraded over 28 days. However, in the same study

biodegradation of transformation products arising from UV/H2O2 treatment of 5FU could be

inferred from Biochemical Oxygen Demand measurements.

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Atorvastatin

The cholesterol-lowering drug atorvastatin (Fig 1) is one of the best selling drugs of all time.

Despite its wide use, studies on its biodegradation are few. No studies on the bacterial

degradation of this compound are reported, but Rodriguez-Rodriduez et al. (2012, 2011)

have studied the application of the white-rot fungus Trametes versicolor for pharmaceutical

removal, including atorvastatin. They reported a reduction from 38 to 5 ng g-1 atorvastatin

when the fungus was grown on sterile sewage sludge for 42 days (Rodriguez-Rodriduez et al.

2011). A similar reduction was observed when non-sterile sludge was augmented with the

fungus (Rodriguez-Rodriduez et al. 2012). Laccase activity was proposed as a significant

factor in the biodegradation of the pharmaceuticals, although no in vitro experiments were

conducted. No analysis of the metabolites was conducted, but the toxicity of the treated

sludge was significantly reduced.

SF5-containing drugs

Pentafluorosulfanyl (-SF5) is a potential replacement for trifluoromethyl (-CF3) and with

easier access to synthetic building blocks, such as SF5-nitrobenzene, several drug analogues

have been synthesized containing this functional group (Figure 10), such as the antimalarial

agent mefloquine and the SSRI blockbuster drug fluoxetine (Prozac) (Welch and Lim 2007;

Mo et al. 2010). The substantial interest in drug molecules bearing the SF5 moiety derives

from its greater electronegativity (3.65 vs 3.36 for CF3) and the improvement in stability and

hydrophobicity of molecules bearing this substituent in comparison to those with a

trifluoromethyl group (Bowden et al. 2000; Reddy 2015; Sheppard 1962). With the

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potentially large number of molecules of this type that might be applied as drugs and

agrochemicals, it is crucial that the impacts on the environment be considered. However,

only two studies investigating the transformation of aryl-SF5 compounds have been

conducted. Jackson and Mabury (2009) demonstrated that aryl-SF5 compounds such as SF5-

phenol, were photolytically labile and released fluoride when exposed to actinic light;

however, they were stable when kept in the dark. Kavanagh et al. (2014) demonstrated that

SF5-substituted anilines could be biotransformed by some bacteria and a fungus to the N-

acetyl derivatives, and demonstrated that in Streptomyces griseus an N-acetyl transferase

catalyzed the reaction.

Outlook

Fluorine is an important element in the pharmaceutical industry and as synthetic methods

are developed to make fluorinated compounds more easily, for example, using continuous

flow methods with fluorine gas (McPake and Sandford 2012), the number and diversity of

fluorinated drugs will increase. In this context, microorganisms may provide important

alternatives to chemical catalysis, either through direct C-F bond formation, via fluorinase

enzymes, or through the introduction of fluorinated building blocks into complex natural

products (Clark et al. 2011; O’Connor et al. 2014; O’Hagan and Deng 2015). Furthermore,

microorganisms can help direct fluorine incorporation into drug compounds to improve

their metabolic stability (Shaughnessy et al 2014).

Whilst fluorine can convey useful properties to drugs, the stability of the C-F bond

often makes these compounds recalcitrant. Once released into the environment, through

wastewater, sludge and directly from farm animals, fluorinated drugs will encounter

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microorganisms. The microorganisms might be inhibited by the drug, evolve resistance (if it

is an antibiotic) or biotransform it, potentially to a more toxic compound. It is clear from a

review of the literature that only a small portion of the available fluorinated drugs currently

in use have been investigated with respect to their biodegradation, and that complete

mineralization of any fluorinated drug, by either an isolated microorganism or in a mixed

culture, is rare. A handful of enzymes have been identified thus far that will biotransform

fluorinated drugs, thus much work remains to identify others. Should this area of research

be ignored and effective remediation solutions are not developed, of which microorganisms

would likely play a part, there is a risk that regulatory authorities will impose restrictions on

the use of the drugs. In particular, this applies to widely prescribed drugs such as fluoxetine,

which are not eliminated by conventional waste water treatment and are known to have an

impact on aquatic life.

The development of pharmaceuticals with new fluorinated substituents is exciting,

but brings with it additional challenges with respect to the eventual fate of the compounds

and their broader environmental effects. On the other hand, a greater understanding of

microbial biotransformation will also enable the discovery of novel enzyme activities that

may have potential as industrial biocatalysts for the production of new fluorinated

compounds.

Conflict of Interest: The author declares no conflict of interest.

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Figure legends

Figure 1. Examples of fluorinated drugs

Figure 2. Biodegradation of 3-fluorobenzoic acid by Sphingomonas sp. HB-1. Enzymes: i)

benzoate 1,2-dioxygenase; ii) reductase; iii) catechol 1,2-dioxygenase

Figure 3. Biotransformation of trifluoromethylbenzoic acid by Ps. putida via benzoate

dioxygenase (i) and catechol 2,3-dioxygenase (ii) yielding the metabolite 7-TFHOD.

Figure 4. The general structure of fluoroquinolone antibiotics.

Figure 5. Biotransformation of norfloxacin by Microbacterium sp. 4N2-2

Figure 6. Biodegradation of pradofloxacin in G. striatum. The intermediates between the

catechol-type metabolite and the ring-open metabolite have not been isolated; it is thought

that pyrogallol- and cis, cis-muconate-type metabolites are likely intermediates.

Figure 7. Structures of the antidepressants citalopram and fluoxetine

Figure 8. Biotransformation of flurbiprofen (FBp) by bacteria and fungi. E. coli CYP110E1-

Red transforms the methyl ester of the drug.

Figure 9. The structure of the anti-cancer drug flutamide.

Figure 10. Structures of pentafluorosulfanyl derivatives of the drugs fluoxetine and

mefloquine

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Table 1. Overview of fungal degradation of fluoroquinolone antibiotics; X indicates the

antibiotic transformed (CIP=ciprofloxacin; NOR=norfloxacin; OF=ofloxacin;

PRA=pradofloxacin; DAN=danofloxacin; SAR=sarafloxacin; ENR=enrofloxacin).

Fungus Fluoroquinolone ReferenceCIP NOR OF PRA FLU DAN SAR ENR

Cunninghamella elegans X (Williams et al.

2007)

Gloeophyllum striatum X X X

(Wetzstein et al. 1999; Karl et al. 2006; Wetzstein et al. 2012)

Irpex lacteus X X X (Cvancarova et al. 2015)

Mucor ramannianus X X X

(Parshikov et al. 1999; Parshikov et al. 2001); (Parshikov et al. 2000)

Panus tigrinus X X X (Cvancarova et al. 2015)

Pestalotiopsis guepini X X (Parshikov et al. 2001)

Phanerochaete chrysosporum X (Marengo et al.

2001)

Pleurotus ostreatus X X X (Cvancarova et al. 2015)

Trametes versicolor X X X

(Cvancarova et al. 2015); (Prieto et al. 2011)

Xylaria longipes X (Rusch et al. 2015)

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Table 2. Summary of microbial fluorometabolites produced from flutamide

MetaboliteMicroorganism

R. mucilaginosa B. bassiana Nocardia sp.

C. elegans

X

X

X X X X

X X X X

X X X

23

Fig 1

24

Fig 2

25

Fig 3

26

Fig 4

27

Fig 5

28

Fig 6

29

Fig 7

30

Fig 8

31

Metabolite Strain Substitution4'-hydroxyflurbiprofen Cunninghamella, Streptomyces,

E. coli CYP110E1-RedR1, R4=OH; R2, R3=H

3',4'-hydroxy-FBp Cunninghamella, Streptomyces R1, R2, R4=OH; R3=H3'-methoxy,4'-hydroxy-FBp Cunninghamella, Streptomyces R1, R4=OH, R2=OMe; R3=HFlurbiprofenamide Streptomyces R1, R2, R3=H; R4=NH2

7-hydroxyflurbiprofenamide Streptomyces R1, R2=H; R3=OH; R4=NH2

7-hydroxyFBp methylester E. coli CYP110E1-Red R1, R2=H; R3=OH, R4=OMeFlurbiprofen ethylester Aspergillus oryzae R1, R2, R3=H; R4=OEt

Fig 9

32

Fig 10

33