Biphenyl dioxygenase-catalysed cis-dihydroxylation of tricyclic azaarenes: chemoenzymatic synthesis of arene oxide metabolites and furoquinoline alkaloids

Biotransformation of acridine, dictamnine and 4-chlorofuro[2,3-b ]quinolone, using whole cells of Sphingomonas yanoikuyae B8/36, yielded five enantiopure cyclic cis -dihydrodiols, from biphenyl dioxygenase-catalysed dihydroxylation of the carbocyclic rings. cis -Dihydroxylation of the furan ring in dictamnine and 4-chlorofuro[2,3-b ]quinoline, followed by ring opening and reduction, yielded two exocyclic diols. The structures and absolute configurations of metabolites have been determined by spectroscopy and stereochemical correlation methods. Enantiopure arene oxide metabolites of acridine and dictamnine have been synthesised, from the corresponding cis -dihydrodiols. The achiral furoquinoline alkaloids robustine, c -fagarine, haplopine, isohaplopine-3,3 9 -dimethylallylether and pteleine have been obtained, from either cis -dihydrodiol, catechol or arene oxide metabolites of dictamnine.


Introduction
Polycyclic azaarenes are ubiquitous in the environment as atmospheric pollutants, resulting from incomplete combustion of nitrogen-containing molecules present in fossil fuels or tobacco and also as plant alkaloids.1a,b Some larger members of the family of aza-polycyclic aromatic hydrocarbons (APAHs) present a significant hazard to human health, resulting from the mutagenicity/carcinogenicity of their mammalian metabolites.1c-e The mineralization of APAHs and alkaloids containing azaaromatic rings by soil bacteria, via non-mutagenic/noncarcinogenic metabolites can, therefore, play a useful role in reducing this problem.Earlier bacterial studies from these laboratories have focused on the toluene dioxygenase (TDO)catalysed biodegradation of bicyclic heterocycles including quinolines, 2a,b benzo [b]thiophenes 2c and benzo [b]furans, 2c using the UV4 mutant strain of Pseudomonas putida (Schemes 1a and 1b).Regioselective cis-dihydroxylation of the carbocyclic and the heterocyclic rings in the quinolines (5,6 and/or 7,8 and/or 2,3 bonds), benzo [b]thiophenes (4,5 and/or 2,3 bonds) and benzo [b]furans (6,7 and/or 2,3 bonds), occurred to give the corresponding cis-dihydrodiol metabolites.The 3-hydroxyquinoline and anthranilic acid metabolites of quinoline were assumed to be derived from the undetected heterocyclic cis-3,4-dihydrodiol intermediate (Scheme 1a).2a,b   Further metabolism of the benzo [b]furan 2,3-cis-diols involved spontaneous ring opening and enzyme-catalysed carbonyl reduction to give exocyclic phenolic diol products (Scheme 1b).2c  Dihydroxylation of the 3,4-bond in the electron-deficient pyridine ring of the quinoline substrates was found to yield only minor metabolites in comparison with its carbocyclic 5,6and 7,8-bonds.However, when benzo [b]thiophene and benzo [b]furan substrates, containing electron-rich heterocyclic rings, were used as substrates, dihydroxylation of the 2,3bond revealed a more favourable metabolic route (Schemes 1a and 1b).
The steric dimensions of the active site in TDO, expressed in P. putida UV4, limited the acceptable size of substrates to mono-or bi-cyclic arenes (Schemes 1a and 1b).However, the biphenyl dioxygenase (BPDO) enzyme, present in the B8/36 mutant strain of Sphingomonas yanoikuyae, has a larger active site and was able to accept tri-, and tetra-cyclic arenes (e.g.benzo[f]quinoline, benzo[h]quinoline, phenanthridine, 3a benzo [c]phenanthridine, 3b Scheme 2) as substrates.It is noteworthy that in these examples a marked regioselective preference for cis-dihydroxylation was found at a bond within the bay-region.
As part of an earlier programme 3b,c to investigate the ability of BPDO to catalyse the cis-tetrahydroxylation of larger polycyclic aromatic rings, it was found that bis-cis-dihydrodiols were formed as further metabolites of the initial cis-dihydrodiols derived from larger carbocyclic (e.g.anthracene, chrysene, benz [a]anthracene) and heterocyclic (e.g.acridine, phenazine, benzo [b]naphtha [2,1-d]thiophene) substrates.The similarity in size and shape of the linear tricyclic arenes, anthracene and acridine, and their acceptability as substrates for the BPDO enzyme, 3c,d prompted this comparative biotransformation study of acridine with furo [2,3-b]quinoline substrates.Following our earlier reports on the isolation and synthesis 4a-e of quinoline alkaloids, from plants of the Rutaceae family, e.g.Choisya ternata, and Skimmia japonica, linear furoquinolines (4-chlorofuro [2,3-b]quinoline and dictamnine) were briefly examined as potential substrates, using whole cells of S. yanoikuyae B8/36 expressing BPDO enzyme.4d In our preliminary studies of the biotransformations of acridine and dictamnine, using S. yanoikuyae B8/36, we had reported 3d,4d the presence of the corresponding cis-dihydrodiol metabolites.This comprehensive study now provides full structural and stereochemical characterization of all new bacterial metabolites and shows how they can be utilized in the chemoenzymatic synthesis of a wider range of animal and plant metabolites, e.g.arene oxides and furoquinoline alkaloids.
The absolute configuration of cis-dihydrodiol 4 was initially assigned as (1R,2S), based on the well established 1 H-NMR pattern previously observed for MPBA derivatives from other polycyclic arene cis-dihydrodiol metabolites (e.g. from naphthalene, anthracene, phenanthrene and their aza-analogues).2a,b,3a,b The observation of a larger chemical shift value (d H 3.18) for the MeO group protons, using the (R)-(+)-MPBA compared with the value obtained using (S)-( 2)-MPBA (d H 3.11), was again assumed to be consistent with a benzylic (R) and an allylic (S) configuration for cis-dihydrodiol 4. The reliability of the MPBA method for the linear azaarene cisdihydrodiol 4 was confirmed by an unequivocal stereochemical correlation sequence similar to that used for other polycyclic arene cis-dihydrodiols (Scheme 4).3a,6 The sequence involved a catalytic hydrogenation (H 2 ,Pd/C) to yield cistetrahydrodiol 5 followed by bis-acetylation (Ac 2 O, pyridine) to give cis-diacetate 6.In the final step, an oxidative ring opening reaction (RuO 2 /NaIO 4 ) gave a mixture of dicarboxylic acid products (7/8).It was assumed that the bicyclic dicarboxylic acid 7 was formed initially and then a part of it degraded to acyclic dicarboxylic acid 8 via a further oxidative ring opening reaction.The mixture of dicarboxylic acids 7 and 8 was methylated (CH 2 N 2 ) to yield dimethylesters 9 and 10 which were separated by column chromatography.The minor component, dimethyl (2,3-diacetoxy)adipate 10 ([a] D 214) was of established (2S,3S) configuration 6 and thus the (1R,2S) configuration was unequivocally assigned to (+)-cis-dihydrodiol 4.
It has been proposed that the mutagenicity/carcinogenicity associated with some larger PAHs and APAHs results from: (i) a monooxygenase-catalysed epoxidation of a carbocyclic ring to yield an arene oxide (cf.compound 2), (ii) an epoxide hydrolase-catalysed hydrolysis of the arene oxide to yield a trans-dihydrodiol (cf.compound 3), (iii) a monooxygenasecatalysed epoxidation of the alkene bond in the transdihydrodiol to yield diastereoisomeric trans-diol epoxides and (iv) nucleophilic attack of DNA on the epoxide ring within a bay region to yield a covalent adduct.1b-f Although the corresponding acridine trans-diol epoxides from metabolite 3 could, in principle, also be mutagens, their synthesis and mutagenicity has not yet been reported.
(ii) Biotransformation of furoquinolines [11][12][13] In common with acridine 1, the mammalian metabolism and mutagenicity of dictamnine 12 and other furoquinoline alkaloids, e.g.c-fagarine, had been reported earlier.7a-e In a more recent study, from these laboratories, the furoquinoline alkaloid skimmianine 13 was found to be the major compound present in C. ternata, 4a and was thus available as a potential substrate for the current biotransformation studies.However, dictamnine 12, another furoquinoline alkaloid required as a potential substrate, was not isolated from C. ternata.Thus, a five-step chemical synthesis of dictamnine 12 was carried out, starting from aniline and using the literature procedure 8a-c which involved 4-chlorofuroquinoline 11 as precursor.Furoquinolines 11 and 12 were thus also available as possible substrates for BPDO.
While cis-dihydroxylation had occurred exclusively at the 1,2 bond of acridine 1, similar regioselectivity for the equivalent Scheme 4 Scheme 5 (5,6) bond in furoquinolines 11 and 12 was not found.A modest preference (38% yield) was observed for BPDOcatalysed cis-dihydroxylation at the 7,8-bond to form cis-diol 15 compared with the 5,6-bond (10% yield) to give cis-diol 14, when 4-chlorofuro [2,3-b]quinoline 11 was the substrate.A stronger preference for oxidation of the 7,8-bond was found with dictamnine 12 as the substrate, which resulted in cis-diol 17 being the major metabolite (20-30% yield) relative to cisdiol 16 (1-3% yield).The combined isolated yields (21-33%) of dictamnine cis-dihydrodiols (16 and 17) were slightly lower than 4-chlorofuroquinoline cis-dihydrodiols (14 and 15, 48%); no cis-dihydrodiol metabolites were detected from skimmianine 13 as substrate.These observations suggest that the presence of substituents at C-4, C-7 and C-8 and the overall steric requirements of the substrate within the active site of the BPDO enzyme are important factors.Based on isolated yields, it appears that cis-dihydroxylation occurred preferentially at the less sterically hindered 7,8-bond and that the best yields resulted from the use of the smaller substrates (11 and 12).As the largest substrate, skimmianine 13, did not yield cisdiol metabolites, this is consistent with its failure to be accommodated within the BPDO active site.However, alternative factors, including aqueous solubility, toxicity and further metabolism, could influence the isolated yields of bioproducts.
The most polar metabolites, formed from 4-chlorofuro [2,3b]quinoline 11 and dictamnine 12, were found to be exocyclic diols (compounds 20 and 23 The origin of mutagenicity associated with dictamnine 13 has not yet been rigorously established.7a-e However, it has been proposed that, in common with other naturally occurring mutagenic furans, e.g.aflatoxin B1 and 8-methoxypsoralen, the corresponding transient furan epoxides, 7f formed as initial mammalian metabolites via monooxygenase-catalysed epoxidation, e.g.arene oxide 24 (Scheme 5) may be responsible for their mutagenicity.It has been proposed that the mutagenicity results from the ability of furan epoxides to form covalently bound adducts following nucleophilic ring-opening reactions with DNA.7e,f (iii) Application of acridine cis-dihydrodiol 4 in the synthesis of arene oxide 2 As part of an earlier study of the mammalian metabolism and mutagenicity/carcinogenicity of PAHs and APAHs, (1R,2S)arene oxide 2 was obtained via an eight stage chemical synthesis, involving a chemical resolution of MTPA esters, with an overall yield of ca.13%. 9 Alkaline hydrolysis (KOH, t-BuOH) of (1R,2S)-arene oxide 2 gave the mammalian metabolite (1R,2R)-trans-1,2-dihydroacridine-1,2-diol 3. 9 In the current study, the possibility of a much shorter synthesis of acridine 1,2-oxide 2 was examined (Scheme 6), using the readily available bacterial metabolite, (1R,2S)-cis-1,2-dihydroacridine-1,2-diol 4. Treatment of diol 4 with 1-bromocarbonyl-1methylethyl acetate, in acetonitrile solution, gave a mixture of bromoacetates 25/26 whose structures were determined from 1 H-NMR and MS data.Due to their instability, during attempted separation, the mixture of bromoacetates 25 and 26 in Et 2 O solution was reacted directly with NaOMe.Using this two step method, the relatively stable (1R,2S)-arene oxide 2 was synthesised from cis-dihydrodiol 4 in 66% yield.Despite the stability of arene oxide 2, it was not detected during mammalian metabolism, probably due to its further metabolism via a rapid epoxide hydrolase-catalysed conversion to the corresponding trans-dihydrodiol 3. 5a-e A preliminary study 3d later showed that when the stable acridine cis-dihydrodiol 4 was used as a substrate for S. yanoikuyae B8/36, it was also further metabolised and formed a bis-cis-dihydrodiol bioproduct.
(iv) Application of dictamnine cis-dihydrodiols 16 and 17 as precursors in the synthesis of furoquinoline alkaloids The potential of dictamnine cis-dihydrodiol metabolites 16 and 17 in the biomimetic synthesis of furoquinoline alkaloids, including the proposed arene oxide intermediate 27, was of biosynthetic interest (Schemes 7 and 8).Possible biosynthetic pathways to furoquinoline alkaloids occurring in Rutaceaeous plants, e.g.Skimmia japonica and Choisya ternata, have been studied using 14 C-labelled precursors.10a These labelling studies showed that enzyme-catalysed hydroxylation could occur on the benzene ring of dictamnine 12 to yield a wider range of furoquinoline alkaloids e.g.skimmianine 13 and possibly also robustine 30 and c-fagarine 31 (Scheme 7).It was proposed that skimmianine 13 could be formed via a monooxygenase-catalysed epoxidation of dictamnine 12, to yield the transient arene oxide 27, followed by epoxide hydrolase-catalysed hydrolysis to yield trans-dihydrodiol 28.10a The possibility of an alternative dioxygenase-catalysed cis-dihydroxylation of dictamnine 12 to yield cis-dihydrodiol 17 was also discussed.10a The enzyme-catalysed oxidations of trans-and cis-dihydrodiols, to yield catechols followed by O-methylation, are well established metabolic steps 1a and, when allied to the earlier labelling studies, 10a either type of enzymatic oxidation could account for the formation of catechol 29 and skimmianine 13.To date, none of the potential biosynthetic intermediates 17, 27-29 have been detected by the labelling studies using Choisya ternata 10a or found among the furoquinoline alkaloids recently isolated from this 4a or other plants in the Rutaceae family.10b  As expected, the B8/36 mutant strain of S. yanoikuyae did not yield catechol metabolites e.g.compound 29 from dictamnine 12 (Scheme 7).The biphenyl cis-diol dehydrogenase (DD) activity required to catalyse the dehydrogenation of cisdihydrodiols to yield catechols, was blocked in the B8/36 strain.However, when the wild type strain of S. yanoikuyae (B1), expressing both BPDO and DD enzymes, was used with dictamnine 12, the only metabolite identified and isolated was cis-dihydrodiol 16, albeit in low yield (8%).This observation is consistent with both cis-dihydrodiols 16 and 17 being formed but the major metabolite (17) being further metabolized preferentially.
Our attempt to synthesise the proposed dictamnine arene oxide metabolite 27 from cis-dihydrodiol 17, via a two-step process similar to that used earlier for acridine arene oxide 2 (Scheme 6), was unsuccessful.This was due to compound 17 being less stable under the reaction conditions and more readily dehydrated under acid conditions to yield phenols (e.g.robustine 30).An alternative approach (Scheme 8) was adopted involving the catalytic hydrogenation (H 2 , Pd-C) of compound 17 to yield the stable cis-tetrahydrodiol 32 (76% yield).Treatment of diol 32 with 1-bromocarbonyl-1-methylethyl acetate gave trans-bromoacetate 33 in good yield (90%).Benzylic bromination of bromoacetate 33 (NBS, CCl 4 ) gave an inseparable mixture of diastereoisomers 34 which was immediately treated with sodium methoxide in THF, to yield the proposed dictamnine arene oxide metabolite 27 (60% yield from compound 33).Initial attempts to purify this elusive arene oxide by PLC resulted in its aromatization to give the furoquinoline alkaloid robustine 30.Purification of (7S,8R)dictamnine oxide 27 was achieved by careful crystallization.A sample of oxide 27 was found to survive in CDCl 3 solution without decomposition, at ambient temperature over a 24 h period.
In the final phase of this study, cis-dihydrodiols 16 and 17, arene oxide 27 and catechol 29, as confirmed or proposed metabolites of dictamnine 12, were utilized as synthetic precursors of other furoquinoline alkaloids, using biomimetic methods (Scheme 8).While robustine 30 was obtained by isomerisation of arene oxide 27 under acidic conditions, the acid-catalysed dehydration of cis-dihydrodiol 17 was the preferred route.Methylation of robustine 30 with diazomethane yielded the alkaloid c-fagarine 31.Under similar conditions, methylation of catechol 29 occurred mainly at C-8, to yield the alkaloid haplopine 36.Treatment of catechol 29 in acetone with 1-chloro-3-methylbut-2-ene in presence of K 2 CO 3 resulted in the preferential prenylation at C-8 to yield phenol 37, which on methylation yielded the alkaloid, isohaplopine-3,39-dimethylallylether 38.Acid-catalysed dehydration of cisdihydrodiol 16, to form phenol 39, followed by methylation, yielded the furoquinoline alkaloid pteleine 40.
Experimental 1 H and 13 C NMR spectra were recorded on Bruker Avance 400, DPX-300 and DRX-500 instruments.Chemical shifts (d) are reported in ppm relative to SiMe 4 and coupling constants (J) are given in Hz.Mass spectra were run at 70 eV, on a VG Autospec Mass Spectrometer, using a heated inlet system.Accurate molecular weights were determined by the peak matching method, with perfluorokerosene as the standard.CD spectra were recorded in spectroscopic grade acetonitrile using a JASCO J-720 instrument.A PerkinElmer 341 polarimeter was used for optical rotation ([a] D ) measurements (ca.20 uC).Flash column chromatography and preparative layer chromatography (PLC) were performed on Merck Kieselgel type 60 (250-400 mesh) and PF 254/366 plates respectively.Merck Kieselgel type 60F 254 analytical plates were employed for TLC.Authentic samples of 4-chlorofuro [2,3-b]quinoline 11 and skimmianine 13 were available from earlier studies.4a-e As reported, 2b Sphingomonas yanoikuyae B8/36 was grown on minimal salts medium with 0.5% sodium succinate and 0.5 g L 21 of yeast extract.Biphenyl dioxygenase (BPDO) was induced, during the exponential phase of growth, by the addition of m-xylene (1 cm 3 L 21 ) every 0.5 h for 7 h.Substrate concentration was 0.5 mg cm 23 .