A convenient chemical-microbial method for developing fluorinated pharmaceuticals

Article abstract of DOI:10.1039/c2ob27140k

A significant proportion of pharmaceuticals are fluorinated and selecting the site of fluorine incorporation can be an important beneficial part a drug development process. Here we describe initial experiments aimed at the development of a general method of selecting optimum sites on pro-drug molecules for fluorination, so that metabolic stability may be improved. Several model biphenyl derivatives were transformed by the fungus Cunninghamella elegans and the bacterium Streptomyces griseus, both of which contain cytochromes P450 that mimic oxidation processes in vivo, so that the site of oxidation could be determined. Subsequently, fluorinated biphenyl derivatives were synthesised using appropriate Suzuki-Miyaura coupling reactions, positioning the fluorine atom at the pre-determined site of microbial oxidation; the fluorinated biphenyl derivatives were incubated with the microorganisms and the degree of oxidation assessed. Biphenyl-4-carboxylic acid was transformed completely to 4′-hydroxybiphenyl-4-carboxylic acid by C. elegans but, in contrast, the 4′-fluoro-analogue remained untransformed exemplifying the microbial oxidation-chemical fluorination concept. 2′-Fluoro- and 3′-fluoro-biphenyl-4-carboxylic acid were also transformed, but more slowly than the non-fluorinated biphenyl carboxylic acid derivative. Thus, it is possible to design compounds in an iterative fashion with a longer metabolic half-life by identifying the sites that are most easily oxidised by in vitro methods and subsequent fluorination without recourse to extensive animal studies.

Full text of DOI:10.1039/c2ob27140k

Organic &
Biomolecular Chemistry
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A convenient chemical-microbial method for
developing fluorinated pharmaceuticals†
Cite this: Org. Biomol. Chem., 2013, 11,
1135
Tara V. Bright,a Fay Dalton,a Victoria L. Elder,b Cormac D. Murphy,*a Neil K. O’Connora
and Graham Sandford*b
A significant proportion of pharmaceuticals are fluorinated and selecting the site of fluorine incorpor-
ation can be an important beneficial part a drug development process. Here we describe initial experi-
ments aimed at the development of a general method of selecting optimum sites on pro-drug molecules
for fluorination, so that metabolic stability may be improved. Several model biphenyl derivatives were
transformed by the fungus Cunninghamella elegans and the bacterium Streptomyces griseus, both of
which contain cytochromes P450 that mimic oxidation processes in vivo, so that the site of oxidation
could be determined. Subsequently, fluorinated biphenyl derivatives were synthesised using appropriate
Suzuki–Miyaura coupling reactions, positioning the fluorine atom at the pre-determined site of microbial
oxidation; the fluorinated biphenyl derivatives were incubated with the microorganisms and the degree
of oxidation assessed. Biphenyl-4-carboxylic acid was transformed completely to 4’-hydroxybiphenyl-4-
carboxylic acid by C. elegans but, in contrast, the 4’-fluoro-analogue remained untransformed exemplify-
ing the microbial oxidation – chemical fluorination concept. 2’-Fluoro- and 3’-fluoro-biphenyl-4-car-
boxylic acid were also transformed, but more slowly than the non-fluorinated biphenyl carboxylic acid
derivative. Thus, it is possible to design compounds in an iterative fashion with a longer metabolic half-
life by identifying the sites that are most easily oxidised by in vitro methods and subsequent fluorination
without recourse to extensive animal studies.
Received 2nd November 2012,
Accepted 21st December 2012
DOI: 10.1039/c2ob27140k
www.rsc.org/obc
must be assessed to complete regulatory approval.2 Conse-
quently, the identification, synthesis and isolation of drug
Introduction
A convenient, rapid in vitro chemical-microbial method for metabolites are key stages in medicinal-chemistry campaigns
establishing the structures of oxidative metabolites of drug- and, in general, in vivo studies are used to assess metabolite
like systems and subsequent improvement of substrate oxi- identity with obvious resource implications.
dative stability by site selective fluorination at pre-identified,
oxidatively sensitive positions is reported here. This strategy mimics mammalian metabolic processes would offer an attrac-
may be of potential benefit to the drug development process. tive alternative to conventional resource-intensive bio- and
A simple in vitro method that successfully and accurately
Xenobiotics such as drugs and toxins are commonly metab- medicinal chemistry approaches. Indeed, many chemical
olised in the liver prior to excretion, and the main class of methods, such as the use of iron porphyrin systems, have been
enzyme involved is the cytochromes P450 (CYP). In humans assessed as potential biomimetic cytochrome P450 mimics
there are 57 CYP isoforms responsible for oxidative or phase I in vitro with varying degrees of success.3 In complementary
metabolism of drugs,1 and one of the key factors in drug approaches, some microorganisms can transform drugs to
design is modulating the rate of CYP-mediated transformation mammalian metabolites, since they have CYP enzymes. In par-
so that optimum efficacy can be achieved. In addition, as part ticular, the zygomycete fungus Cunninghamella elegans, which
of a drug development programme, the toxicity of metabolites has been widely studied as a model of mammalian metab-
olism of xenobiotic compounds,4 offers a non-chemical CYP
biomimic.
aUCD School of Biomolecular and Biomedical Science, Centre for Synthesis and
Chemical Biology, University College Dublin, Ardmore House, Belfield, Dublin 4,
Ireland. E-mail: cormac.d.murphy@ucd.ie
Over 30% of currently available and commercially valuable
pharmaceuticals owe their biological activity to the presence of
fluorine atoms within their structures providing an indication
of the importance of fluorine incorporation for modifying
drug stability, lipophilicity and effective pH range.5–8 Indeed, a
common strategy for increasing the half-life of a drug in vivo is
bDepartment of Chemistry, Durham University, South Road, Durham, DH1 3LE, UK.
E-mail: graham.sandford@durham.ac.uk
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c2ob27140k
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Fig. 1 Structures of lead compound SCH48461 and its fluorinated analogue, the cholesterol inhibitor Ezetimibe.
Fig. 2 Examples of biphenyl-containing pharmaceuticals.
to introduce a fluorine atom at the metabolically labile sites.9 bearing a variety of functional groups by employing C. elegans
An example of how dramatic this approach can be is observed and the bacterium Streptomyces griseus, which is known to also
in the development of the cholesterol uptake inhibitor drug mimic human drug metabolism,15,16 to determine the sites of
Ezetimibe (SCH 58325), where optimization of the lead com- CYP-catalysed oxidation. The effect of strategic fluorination on
pound (SCH4861) included fluorination of the phenyl ring, metabolism was measured by further incubation of the appro-
preventing oxidation to the corresponding phenol derivative, priately fluorinated biphenyl system with the microorganisms
and substitution of a methoxy group with fluorine preventing in an overall strategy to develop systems that are oxidatively
demethylation (Fig. 1). These changes and other structural stable to CYP. This iterative process is shown in Scheme 1
modifications reduced metabolic degradation to such levels where, ultimately, a fluorinated lead compound with appropri-
whereby the required dose for activity could be decreased by ate metabolic stability can be identified in an early stage of the
55 times, while increasing activity 400-fold.10,11 Therefore, in drug discovery process.
the sphere of fluorinated drug development, it would be very
useful to easily determine the site of CYP-catalysed oxidation
in a drug candidate in vitro, so that the incorporation of a fluo-
rine atom is then made at the optimal position for increasing
Results and discussion
Initially, we incubated a series of commercially available
model biphenyl compounds with C. elegans and S. griseus, and
ethyl acetate extracts from the cultures were analysed by
GC-MS to identify the products formed (Table 1) from these
small scale experiments.
oxidative stability as a contributing approach to improving
drug efficacy.
A common motif in several drug classes is the biphenyl
system,12 and various valuable pharmaceuticals bearing this
structural unit are given in Fig. 2. Previously, it has been
demonstrated that biphenyl and fluorinated biphenyl13,14
derivatives are metabolised by C. elegans in a similar fashion
to mammals.
In this paper, we describe proof-of-concept studies concern-
ing a microbial-chemical method of identifying the most oxi-
datively sensitive sites in a range of model biphenyl substrates
Biphenyl-4-carboxylic acids
Biphenyl-4-carboxylic acid 1a was completely transformed by
C. elegans, but remained untransformed by S. griseus. By
GC-MS, one product was detected in the C. elegans extract,
which had the expected mass spectrum of 4′-hydroxy-biphenyl-
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Scheme 1 Chemical-microbial oxidation-fluorination process for drug development.
4-carboxylic acid 2a, and this was confirmed by 1H NMR of the the lower oxidation potential of the aromatic ring upon fluo-
purified compound, which showed two diagnostic AX reson- rine incorporation. Interestingly, GC-MS analysis showed that
ances centred at 7.53 and 6.86 ppm attributed to the two 1,4- the composition of the product mixture changed over the
disubstituted aromatic ring systems. Furthermore, this is the course of the experiment; biphenyl-4-carboxylic acid 1a giving
expected site of mammalian CYP-catalysed oxidation, based on 4-biphenylmethanol, which was detected after 24 h, but was
previous observations.17
not present in the product mixture 48 h incubation (Fig. 3).
Consequently, following the strategy outlined in Scheme 1, Correspondingly, in both the fluorobiphenyl-4-carboxylic acid
the corresponding 4′-fluoro-biphenyl carboxylic acid 1b was cultures 1c and 1d, fluorinated biphenyl methanol and/or
synthesised by Suzuki–Miyaura coupling of 4-fluorobenzene fluorinated hydroxylated biphenyl methanol were detected in
boronic acid with ethyl 4-bromobenzoate, followed by acid cat- the extracts, after 24 h and 48 h incubation, but were not
alysed hydrolysis (Table 1, ESI†) following usual procedures present after 72 h, leaving the final hydroxylated carboxylic
and incubated with the microorganisms (Table 1).
acid (Table 2).
We observed that the 4′-fluoro biphenyl carboxylic acid 1b
Thus, the carboxylic acid group is reduced initially by the
is not transformed, demonstrating, in this case, that fluorine culture and then re-oxidised, possibly reflecting the reducing
substitution in a position that favoured hydroxylation by CYP power available in the cell at the various time points of the
will block further oxidation thus exemplifying our strategy experiment. It is also possible that the reduction promotes
described in Scheme 1.
hydroxylation, of 2′- and 3′-fluorobiphenyl-4-carboxylic acid on
To further examine the effect of fluorine substitution on the second ring, as the –CH2OH group is electron donating,
the oxidation of biphenyl carboxylic acid derivatives, incu- activating the ring towards oxidation processes. Interestingly,
bation of 2′- or 3′-fluoro-biphenyl-4-carboxylic acid 1c and 1d 4′-fluoro-biphenyl-4-carboxylic acid 1b was almost completely
with C. elegans was carried out, resulting in 100% conversion converted to the biphenyl methanol 3c after 48 h, before being
(by GC-MS) to hydroxylated products 2b and 2c, respectively, re-oxidised back to the acid 1b. The sequences of these trans-
whose structure could not be identified with certainty. Never- formations are shown in Scheme 2 and two possible pathways,
theless, the experiment demonstrated that the 4′-position A and B, can be envisaged.
must bear a fluorine atom to prevent oxidative transformation.
Whilst transformation of the acid directly to the hydroxyl-
The observation that 2′- and 3′-fluoro-biphenyl-4-carboxylic ated acid (Path A) cannot be ruled out, the observation of the
acid 1c and 1d were completely transformed prompted experi- methanol and hydroxyl methanol derivatives within the cul-
ments to determine if there was any difference in the rate of tures suggest a step-wise process (Path B) where the reduction
oxidation at the 4′-positions compared with the non-fluori- of the acid group precedes hydroxylation. In the case of the
nated biphenyl 1a. Biphenyl-4-carboxylic acid 1a was comple- 2′-fluoro derivatives, observation of significant (80%) quan-
tely transformed to the hydroxylated product after 48 h tities of 2′-fluorobiphenyl methanol 3a shows that the hydroxy-
incubation, whereas approximately 30% of the 2′- and 3′- lation of the aromatic ring is a slower process than oxidation
fluorobiphenyl carboxylic acid 1c and 1d were converted to the of the CH2OH group whereas for the 3′-fluoro derivative, the
corresponding fluoro-hydroxy-biphenyl-4-carboxylic acids 2b methanol system is not observed indicating rapid hydroxy-
and 2c in the same time frame, indicating that the presence of lation, reflecting an increased electron donating effect of the
fluorine on the aryl ring slowed the transformation, reflecting CH2OH group on the 3′-fluoro system. Consequently, these
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Table 1 Products formed upon incubation of substrate with C. elegans and S.
Table 1 (Contd.)
griseus
Products after 72 h
incubation
Yield (%)
C. elegans S. griseus
Yield (%)
Substrate
Products after 72 h
incubation
Yield (%)
C. elegans S. griseus
Yield (%)
Substrate
30
42
25
100
100
—
2
No products detected
—
—
29
61
—
1
30
1
100
—
22
95
—
—
—
99
69
21
in vitro studies provide more information regarding possible
metabolite intermediates that may be generated compared to
in vivo studies in which only the final metabolite is typically
observed upon excretion.
The study was expanded by synthesising a range of fluoro-
biphenyl derivatives with varying functional groups by Suzuki–
Miyaura coupling reactions (Table 2, ESI†) and incubating
these with the microorganisms as described above (Table 1).
100
22
16.5
4-Methyl biphenyl 1e
Incubation of 4-methyl biphenyl 1e with C. elegans resulted in
almost complete transformation to 4′-hydroxybiphenyl-4-car-
boxylic acid 2a, indicating both oxidation of the methyl group
and CYP-mediated hydroxylation. In S. griseus, two products
were detected, 4-biphenyl methanol 2d and biphenyl-4-car-
boxylic acid 1a, demonstrating that the oxidation of the sub-
strate most likely occurs in a stepwise fashion; however, no
hydroxylation of the rings occurs, which is consistent with our
observations described above. When 4′-fluoro-4-methyl biphe-
nyl 1f was incubated with the microorganisms, the transform-
ation stopped at the corresponding carboxylic acid 1b with no
ring oxidation, again consistent with results described above.
—
22.5
91
40
15
—
4-Methoxybiphenyl 1g
Incomplete demethylation of 4-methoxybiphenyl 1g to 2e was
observed in C. elegans and S. griseus; however, in the
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Fig. 3 Gas chromatograms showing the appearance of 4-biphenylmethanol 2e (retention time, 9 min) after 24 h incubation of biphenyl-4-carboxylic acid 1a
(retention time, 9.3 min) with C. elegans (A), and its subsequent disappearance after 48 h with the formation of 4’-hydroxy-biphenyl-4-carboxylic acid 2a (retention
time 12.8 min) (B). Panel C shows the chromatogram from an uninoculated control flask.
bacterium, a highly unusual metabolite, 4-hydroxy-4-phenyl product was detected from 4′-fluoro-4-methoxybiphenyl and is
cyclohexane 2f, was also detected by GC-MS and the structure consistent with the previous experiments with 4′-fluoro-substi-
was identified by comparison with data in the NIST MS data- tuted biphenyls. Interestingly, in the S. griseus cultures, a
base. A sequence of intermediates for this unusual transform- metabolite with the expected mass spectrum of fluorinated
ation is suggested in Fig. 4 and is consistent with a related 4-hydroxy-4-phenyl cyclohexane (2n, 2k, 2h) was detected from
pathway described by Gopishetty et al.18 in which S. griseus 2′, 3′ and 4′-fluoro-4-methoxybiphenyl.
transforms naphthalene to 4-hydroxy-1-tetralone, via
1-naphthol (Fig. 4).
Nitrobiphenyl
Incubation of fluorinated 4-methoxybiphenyl 1h–1j also No transformation of nitrobiphenyl was observed, probably as
resulted in demethylation in both microorganisms, and in a consequence of the very strong electron withdrawing effect of
C. elegans hydroxylated 2′- and 3′-fluoro-4-methoxybiphenyl the nitro group which reduces the oxidation potential of the
derivatives 2j and 2m were detected, but no hydroxylated biphenyl system.
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incubation of 2′- and 3′-fluoro-biphenyl-4-carboxylic acid with
C. elegans, which resulted in the production of hydroxylated
Conclusions
The incorporation of fluorine into drugs can be an important metabolites.
aspect of hit-to-lead development as incorporation of fluorine
The study also revealed the formation of intermediate/tran-
atoms can have dramatic effects on bioactivity, stability and sient metabolites arising from metabolism of the fluorinated
lipophilicity. In this paper our initial efforts to develop a biphenyl-4-carboxylic acids (biphenyl methanol). Such metab-
method for selectively fluorinating drugs to improve metabolic olites are also likely to occur in other species, including
stability, by first identifying the most likely site of oxidation mammals, in vivo, and given the significance of the relatively
using microbial transformation, then selectively fluorinating new ‘Metabolites in Safety Testing’ (MIST) guidelines from the
that position has been presented (Scheme 1). The effectiveness US Food and Drug Administration,19 microorganisms can
of this approach is most clearly demonstrated with a model provide a method for relatively easy access to biologically active
system, biphenyl 4-carboxylic acid, which was completely oxi- compounds for toxicity testing. Thus we have developed initial
dised to 4′-hydroxy-biphenyl-4-carboxylic acid by C. elegans, yet insights into
a straightforward microbiological-chemical
the corresponding system 4′-fluoro-biphenyl-4-carboxylic acid method for determining the optimum site for fluorination and
remained unoxidised. Furthermore, the importance of the for subsequent evaluation of the metabolism. Employing
regioselectivity of fluorine incorporation was illustrated by the microorganisms, which are relatively easy to handle, will
reduce the costs of using either animal models, or mammalian
cell cultures.
Table 2 Proportion of products formed upon incubation of fluorinated biphe-
nyl-4-carboxylic acid with C. elegans for 48 h, as determined by GC-MS
Experimental
Synthesis of fluorobiphenyl derivatives – general procedure
Aryl boronic acid, potassium carbonate and Pd(PPh3)4 were
placed in a round bottomed flask filled with an argon
% Product
Biphenyl
methanol
OH-biphenyl
methanol
OH-carboxylic
acid
Incubation (h) Incubation (h) Incubation (h)
Fluorine position 24
48
24
48
24
48
1d, 2′
1c, 3′
1b, 4′
—
3a, 80 3a, 53 4a, 4
4a, 3
—
2b, 29
a
—
—
4b, 65 4b, 63 2c, 30 2c, 34
3c, 69 3c, 93 4c, 2
2e, 39
4c, 3
—
—
—
—
2d, 16 2d, 100
Scheme 2 Transformation of 3’-fluoro-biphenyl carboxylic acid 1c in
C. elegans.
a 3′-Fluorobiphenylmethanol 3b could only be detected after silylation.
Fig. 4 Proposed CYP-catalysed transformation of naphthalene and methoxy biphenyl in S. griseus.
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atmosphere. A mixture of toluene (30 mL) and water (1 mL), centrifuged (3200 rpm, 15 min)to remove the cell debris. The
which had been degassed via the freeze–thaw method, and supernatant was extracted twice with ethyl acetate (30 mL).
haloarene were added to the flask and the mixture was heated
Cunninghamella elegans was cultivated on sabouraud dex-
at reflux temperature overnight. The reaction mixture was trose agar for 120 h at 27 °C. Inoculum was prepared by homo-
cooled and extracted with water (15 mL) and toluene (15 mL) genising the mycelium, including the agar in sterile 8% NaCl
and separated. The organic extracts were combined and (100 mL). The homogenate (5 mL) was then used to inoculate
washed with water and brine before being dried (MgSO4). The 250 mL Erlenmeyer flasks containing 50 mL sabouraud dex-
solution was filtered and solvents evaporated to give crude trose broth, which were incubated 72 h with rotary agitation
fluorobiphenyl product which was purified by column chrom- (150 rpm) at 27 °C. Substituted biphenyl (5 mg) solubilised in
atography on silica gel or recrystallization.
50 μL dimethylformamide was added to the flask and incu-
Ethyl 4′-fluorobiphenyl-4-carboxylate. 4-Fluorobenzene bation was continued for a further 72 h. Cultures were soni-
boronic acid (1.5 g, 10.7 mmol), potassium carbonate (2.67 g, cated on ice for 3 min at 100% amplitude with 30 s intervals
19.3 mmol), Pd(PPh3)4 (0.37 g, 3.22 mmol), ethyl-4-bromo- every minute. Sonicate was then centrifuged to remove the cell
benzoate (1.6 mL, 9.65 mmol), toluene (30 mL) and water debris and the supernatant was extracted twice with ethyl
(1 mL), after column chromatography on silica gel using ethyl acetate (50 mL).
acetate : hexane (1 : 3) as elutant, gave ethyl 4′-fluorobiphenyl-4-
carboxylate (1.473 g, 62%) as a white solid; mp 64.5–65.5 °C
(lit.,20 63.5–64 °C); (Found: C, 73.6; H, 5.4. C15H13FO2 requires:
Metabolite analysis
C, 73.8; H, 5.3%); vmax (cm−1) 2991, 1713 (CvO), 1282; δH 1.40
(3H, t, J 7.1, CH3), 4.39 (2H, q, J 7.1, CH2), 7.54–7.58 (2H, m, Organic extracts were dried and the solid redissolved in ethyl
ArH), 7.58–7.60 (4H, m, ArH), 8.08–8.11 (2H, m, ArH); δF acetate (1 mL), and analysed by gas chromatography-mass
2
−114.72 (m); δC 14.33 (CH3), 60.97 (CH2), 115.82 (d, JCF 21.5, spectrometry (GC-MS). Samples (1 μL) were injected in the
3
C-3′), 126.79 (C-3), 128.87 (d, JCF 8.1, C-2′), 130.08 (C-2), splitless mode onto a HP5MS column (30 m × 0.25 mm ×
4
136.14 (d, JCF 3.3, C-1′), 144.43 (C-4), 146.23 (C-1), 162.89 (d, 0.25 μm) and the oven temperature held at 70 °C for 3 min,
1JCF 247.8, C-4′), 166.37 (CvO); m/z (ASAP+) 244 ([M]+, 100%).
then raised to 250 °C at 10 °C min−1. For the identification of
4′-Fluorobiphenyl-4-carboxylic acid 1b. A solution of 4′- some metabolites a portion of the extract was dried under a
fluorobiphenyl-4-carboxylic acid ethyl ester (0.71 g, 2.91 mmol) stream of nitrogen and derivatised by heating with N-methyl-
in THF (8 mL) and MeOH (8 mL) was treated with 2 M NaOH N-(trimethyl-silyl) trifluoroacetamide (50 μL) at 100 °C for 1 h.
(5 mL) at room temperature overnight. The organic solvent was The silylated samples were diluted with ethyl acetate (100 μL)
evaporated leaving the aqueous layer which was acidified by and analysed by GC-MS. Extracts from cultures that were incu-
the addition of 2 M HCl. The solution was extracted using bated with fluorinated biphenyls were also analysed by fluo-
ethyl acetate and the organic layers combined and dried rine-19 nuclear magnetic resonance spectroscopy on a Varian
(MgSO4). The solution was filtered and solvent evaporated to 400 MHz spectrometer, after drying the extract in a stream of
give 4′-fluorobiphenyl-4-carboxylic acid 1b21 (0.41 g, 66%) as a nitrogen and redissolving in CDCl3.
white solid; mp 242.2–244.9 °C; (Found: C, 71.9; H, 4.4.
4′-Hydroxybiphenyl carboxylic acid was further purified
C13H9FO2 requires: C, 72.2; H, 4.2%); vmax (cm−1) 2828, 2554, from the organic extracts of C. elegans culture supernatants by
1678 (CvO), 1290; δH(DMSO-d6) 7.24–7.32 (2H, m, ArH), preparative HPLC using a Varian Prostar system equipped with
7.70–7.77 (4H, m, ArH), 7.95–8.02 (2H, m, ArH); δF(DMSO-d6) a ZORBAX SB-C18 5 μm (150 × 9.4 mm) column. Organic
2
−114.66 (s); δC(DMSO-d6) 116.31 (d, JCF 21.4, C-3′), 127.15 extract (250 μL) from eight flasks of C. elegans incubated with
3
(C-2), 129.46 (d, JCF 8.3, C-2′), 130.03 (C-3), 130.38 (C-4), biphenyl-4-carboxylic acid was injected onto the column and
4
1
135.91 (d, JCF 3.1, C-1′), 143.64 (C-1), 162.75 (d, JCF 245.6, the compound was eluted with a gradient of methanol/water
C-4′), 167.53 (CO2H); m/z (ASAP+) 216 ([M]+, 100%).
(10–90% methanol) over 38 min with a flow rate of 4 mL
min−1. GC-MS analysis of the fractions revealed that the frac-
tion containing 4′-hydoxybiphenyl-4-carboxylic acid contained
an impurity, which was removed by further HPLC purification
Culture conditions
Streptomyces griseus ATCC 13273 was maintained on agar with a modified elution gradient: methanol/water (50%) with a
slants containing ISP4 medium. The mycelia were inoculated gradient of methanol/water (10/90%) over 25 min and then to
into 250 mL Erlenmeyer flasks containing 30 mL sterile soya 90/10% for 13 min with a flow rate of 4 mL min−1. The puri-
bean media consisting of soya bean meal (5 g L−1), glycerol fied compound was characterised by 1H NMR (Varian 300 MHz
(20 g L−1), yeast extract (5 g L−1) and K2HPO4 (5 g L−1), and the spectrometer); δH 6.88 (2H, d, J 9 Hz), 7.53 (2H, d, J 9 Hz), 7.65
pH was adjusted to 7. Cultures were incubated with rotary agi- (2H, d, J 9 Hz), 8.08 (2H, d, J 9 Hz).
tation (200 rpm) at 30 °C for 72 h. Substituted biphenyl (3 mg)
Products from transformation of non-fluorinated biphenyl
solubilised in 30 μL dimethylformamide was added to the derivatives were identified based on their mass spectra
flask, and incubation was then continued for a further 72 h. employing the NIST MS library. Fluorinated derivatives could
Cultures were sonicated on ice (Sonicator U200S control, IKA be readily deduced from the change in mass arising from the
Labortechnik) for 1 min at 50% amplitude and the sonicate presence of fluorine (ESI†, Table S1).
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11 M. VanHeek, C. F. France, D. S. Compton, R. L. McLeod,
N. P. Yumibe, K. B. Alton, E. J. Sybertz and H. R. Davis,
J. Pharmacol. Exp. Ther., 1997, 283, 157–163.
Acknowledgements
We thank the Royal Society for an International Joint Project
Grant, the Irish Research Council, Durham University M.Chem 12 H. A. Lehmann-lintz Thorsten, T. Leo and M. Michael,
programme and UCD Research Demonstrator scheme.
Biphenyl Derivatives, Production Thereof and Uses as
Medicines, Boehringer Ingelheim Pharma KG, Ingelheim,
DE, United States, 2003.
13 R. H. Dodge, C. E. Cerniglia and D. T. Gibson, Biochem. J.,
1979, 178, 223–230.
14 J. Amadio and C. D. Murphy, Appl. Microbiol. Biotechnol.,
2010, 86, 345–351.
15 M. Taylor, D. C. Lamb, R. Cannell, M. Dawson and
S. L. Kelly, Biochem. Biophys. Res. Commun., 1999, 263, 838–
842.
16 T. V. Bright, B. R. Clark, E. O’Brien and C. D. Murphy,
J. Mol. Catal. B: Enzym., 2011, 72, 116–121.
17 A. Parkinson and S. Safe, Biochem. Pharmacol., 1982, 31,
1849–1856.
18 S. R. Gopishetty, J. Heinemann, M. Deshpande and
J. P. N. Rosazza, Enzyme Microb. Technol., 2007, 40,
1622–1626.
19 T. W. Robison and A. Jacobs, Bioanalysis, 2009, 1,
1193–1200.
20 K. Ueura, T. Satoh and M. Miura, Org. Lett., 2005, 7,
2229–2231.
21 X. Li, C. Abell and M. Ladlow, J. Org. Chem., 2003, 68,
4189–4194.
References
1 F. P. Guengerich, Chem. Res. Toxicol., 2008, 21, 70–83.
2 F. P. Guengerich, Chem. Res. Toxicol., 2009, 22, 237–238.
3 D. Mansuy, C. R. Chim., 2007, 10, 392–413.
4 S. Asha and M. Vidyavathi, Biotechnol. Adv., 2009, 27,
16–29.
5 C. D. Murphy, B. R. Clark and J. Amadio, Appl. Microbiol.
Biotechnol., 2009, 84, 617–629.
6 C. Isanbor and D. O’Hagan, J. Fluorine Chem., 2006, 127,
303–319.
7 K. L. Kirk, J. Fluorine Chem., 2006, 127, 1013–1029.
8 K. Muller, C. Faeh and F. Diederich, Science, 2007, 317,
1881–1886.
9 S. Purser, P. R. Moore, S. Swallow and V. Gouverneur,
Chem. Soc. Rev., 2008, 37, 320–330.
10 S. B. Rosenblum, T. Huynh, A. Afonso, H. R. Davis,
N. Yumibe, J. W. Clader and D. A. Burnett, J. Med. Chem.,
1998, 41, 973–980.
1142 | Org. Biomol. Chem., 2013, 11, 1135–1142
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