Benzoate dioxygenase from Ralstonia eutropha B9 – unusual regiochemistry of dihydroxylation permits rapid access to novel chirons†

Of the various oxygenases that have found use in biocatalysis, it is arguably the arene dioxygenases which add the most value in terms of the synthetic versatility of the products they produce. The direct transformation of an aromatic ring into a dearomatised cyclohexadiene diol (Scheme 1) is a reaction that has very little precedent in organic chemistry and is therefore appealing to access rapidly uncharted chemical space. In wildtype organisms, these arene cis-diols are usually fleeting metabolic intermediates. However, mutants in which the subsequent enzyme in the metabolic pathway is blocked are able to accumulate these diols and they can be isolated in synthetically useful quantities. The densely-packed, diverse functionality in these chirons finds ready application in different areas such as synthesis of natural products, pharmaceuticals, carbohydrates, polymers and dyes. To date, in excess of 400 arene cis-diol products have been reported. The majority of these are produced by organisms expressing toluene dioxygenase (TDO), naphthalene dioxygenase (NDO) and biphenyl dioxygenase (BPDO) enzymes, which are Rieske type non-heme iron oxygenases. These metabolise substituted aromatic substrates in a regioand stereoselective fashion. A robust predictive model has been developed for such transformations, with the sense of enantioinduction being consistent across organisms and substrates (Scheme 1a, ortho,meta oxygenation). However, organisms expressing benzoate dioxygenase (BZDO) enzymes dihydroxylate benzoic acids in a process that proceeds with both different regioselectivity and also the opposite absolute sense of enantioinduction. For example, Ralstonia eutropha B9 (formerly known as Alcaligenes eutrophus B9), Pseudomonas putida U103 and Pseudomonas putida KTSY01 (pSYM01) oxidise benzoic acid to benzoate 1,2-cis dihydrodiol 4 (Scheme 1b, ipso,ortho oxygenation). Diol acid 4 is a highly versatile chiral pool starting material and many transformations of this building block can be envisaged (Fig. 1). Despite this, 4 has been comparatively underutilised to date in synthesis, in comparison with arene cis-diols of


Introduction
Of the various oxygenases that have found use in biocatalysis, it is arguably the arene dioxygenases which add the most value in terms of the synthetic versatility of the products they produce. 1 The direct transformation of an aromatic ring into a dearomatised cyclohexadiene diol (Scheme 1) is a reaction that has very little precedent in organic chemistry 2 and is therefore appealing to access rapidly uncharted chemical space. In wildtype organisms, these arene cis-diols are usually fleeting metabolic intermediates. 3 However, mutants in which the subsequent enzyme in the metabolic pathway is blocked are able to accumulate these diols and they can be isolated in synthetically useful quantities.
The densely-packed, diverse functionality in these chirons finds ready application in different areas such as synthesis of natural products, 4 pharmaceuticals, 5 carbohydrates, 6 polymers 7 and dyes. 8 To date, in excess of 400 arene cis-diol products have been reported. The majority of these are produced by organisms expressing toluene dioxygenase (TDO), naphthalene dioxygenase (NDO) and biphenyl dioxygenase (BPDO) enzymes, which are Rieske type non-heme iron oxygenases. 1e,9 These metabolise substituted aromatic substrates in a regioand stereoselective fashion. A robust predictive model has been developed for such transformations, 10 with the sense of enantioinduction being consistent across organisms and substrates (Scheme 1a, ortho,meta oxygenation). However, organisms expressing benzoate dioxygenase (BZDO) enzymes dihydroxylate benzoic acids in a process that proceeds with both different regioselectivity and also the opposite absolute sense of enantioinduction. For example, Ralstonia eutropha B9 11 (formerly known as Alcaligenes eutrophus B9), Pseudomonas putida U103 12 and Pseudomonas putida KTSY01 ( pSYM01) 13 oxidise benzoic acid to benzoate 1,2-cis dihydrodiol 4 (Scheme 1b, ipso,ortho oxygenation).
Diol acid 4 is a highly versatile chiral pool starting material and many transformations of this building block can be envisaged (Fig. 1). Despite this, 4 has been comparatively underutilised to date in synthesis, in comparison with arene cis-diols of Scheme 1 Regio-and stereoselectivity of dioxygenases. type 2. 4i,q,t,5h,i,6a,e,o,14-17 In the current work, we describe the synthesis of a library of cyclohexyl chirons from 4, both minimally and more extensively functionalised. This serves to showcase further the versatility of 4 and we anticipate these new building blocks will find diverse applications in synthesis and catalysis. With regards to handling and storage, it should be noted that although 4 is prone to exothermic decomposition by rearomatisation, as are all arene cis-diols, it may be stored in pure form in excess of a year at −78°C without appreciable decomposition occurring. Additionally, storage of 4 as its mixed sodium/potassium salt has been described and reportedly leads to enhanced stability. 16b Production of 4 on a multihundred gram scale is possible without recourse to specialised equipment. 15 The absolute configuration and enantiopurity of 4 have been demonstrated through chemical correlation and by X-ray crystallographic analysis of a derivative. 14,15 Results and discussion

Ring-saturated derivatives
The diene in 4 readily undergoes hydrogenation over palladium on carbon to give saturated cyclohexane diol acid 5 (Scheme 2). Perhaps surprisingly, this compound has not been reported previously, although the diastereoisomeric trans-diol is known. 18 Saturation of ortho,meta arene cis-diols of type 2 to give 3-substituted cyclohexane-1,2-diols and applications of these in catalysis have been reported; 19 the analogous approach has not previously been applied to ipso,ortho-arene cis-diols of type 4, however. We wished to target derivatives of 5 with the diol protected, but direct acetonide introduction was surprisingly unsuccessful. To circumvent this, esterification of 5 was carried out prior to ketalisation. Acetonide protection of 6 was successful, albeit with traces of 8 being formed through competing transesterification. Subsequent hydrolysis of ester 7 did indeed give desired acetonide acid 9, but appreciable acetonide migration and deprotection were also observed.
The structure of 10 was assigned on the basis of its polarity and of the 13 C resonance for the ketal carbon (δ = 110.1 ppm, consistent with a five-membered cyclic ketal as opposed to sixmembered 20 ), as well as 2D NMR spectroscopic data. In view of the difficulties associated with accessing 9 by means of base-mediated ester cleavage, we instead implemented an approach employing a benzyl ester (Scheme 3). It was found that reliable production of 9 in good yield was best achieved through purification of the final product by dry column chromatography. 21 Other chiral acid building blocks were also targeted; to this end, the diol in benzyl ester 11 was protected as dioxasilole 13 prior to hydrogenation to give 14 (Scheme 4). This last step proved capricious, however, with undesired desilylation also occurring to a varying extent. A bis(ether) derivative was also accessed by permethylation of 5. With a slight excess of alkylating agent incomplete ether formation led to an inseparable mixture of bis(ether) 15 and monoether 16, which required silylation to allow separation to be effected. However, a greater excess of alkylating agent led to clean formation of 15 in good yield and hydrolysis gave bis(ether) acid 18.
Diol protection as a benzylidene acetal was explored and a moderate (3 : 2) diastereoselectivity was observed for formation of 19 over 20. Structures were assigned on the basis of NOESY correlations (see ESI †); careful chromatography allowed for isolation of both diastereoisomers in pure form. We next sought to access novel ketones bearing an adjacent quaternary centre. One such target, 21, was available simply by oxidation of byproduct 10. A second such ketone was accessed by a multistep procedure involving silyl protection of the secondary alcohol in 4. Thus, ester 6 was silylated and reduced to give monoprotected triol 23. Choice of reductant proved crucial, since with LiAlH 4 , yields of 23 were low, with appreciable silyl migration observed, giving rise to 24. In contrast, NaBH 4 cleanly effected reduction to 23 only, in good yield. Ketalisation, desilylation and oxidation then gave target ketone 27, a reduced analogue of 21 (Scheme 5).
These comparatively minimally functionalised cyclohexyl chirons that all bear a quaternary stereocentre are synthetically valuable insofar as it is difficult to conceive of other means of accessing them as easily, in enantiopure form. We anticipate their finding diverse uses in synthesis and catalysis.

Highly oxygenated derivatives
The structures of arene cis-diols such as 2 and 4 are highly suggestive of applications in the synthesis of cyclitols such as inositols. 22 A particular advantage of their use for synthesis of novel inositol derivatives is that they provide ready access to C-substituted derivatives. 6d,23 In contrast, use of natural inositols or other carbohydrates as starting materials lends itself to synthesis of O-substituted derivatives, but C-substituted derivatives are accessible only by means of more involved synthetic sequences.
The ortho,meta diols of type 2 have been extensively exploited in this context 6i,j,p,r,7a,24-27 but no inositol derivatives have been synthesised to date from 4. To access rapidly such a species from 4, we opted to introduce oxygenation into the diene by means of a singlet oxygen photocycloaddition. Use of singlet oxygen to access novel cyclitols from starting materials other than arene cis-diols has previously proven to be a very successful strategy. 28 Thus, known silyl ether 28 was transformed to endoperoxide 29 and reduced to protected pentaol 30 by our reported procedure. 4i Protection of free hydroxyl groups as methoxymethyl ethers gave 31, which underwent osmium-catalysed dihydroxylation to afford 32 as a single isomer; regiochemistry of osmylation in such systems is well precedented. 6y,24d,f,l,25c,l,27d,29 Global deprotection with aqueous acid (and an organic wash to remove silanol) furnished the desired product, but in impure form; attempts using acidic resins also gave impure product. Its purification necessitated exhaustive acetylation to 33, chromatography and ammonolysis to give 34, a C-hydroxymethyl-muco-inositol. C-Hydroxymethyl derivatives of other isomeric inositols are extremely rare 30 and those of muco-inositol are wholly unknown (Scheme 6).
Endoperoxides derived from diene-1 O 2 photocycloaddition are versatile intermediates and in addition to reductive cleavage, they are capable of undergoing several other synthetically useful transformations. For example, they undergo basemediated Kornblum-DeLaMare fragmentation to afford γ-hydroxyenones; we have previously demonstrated such transformations for endoperoxides derived from 4. 4i In addition, they may be isomerised to the corresponding bis(epoxides) upon treatment with cobalt-tetraphenylporphine complex. 31 This latter transformation has not previously been applied to an endoperoxide derived from 4. To this end, known alcohol 35 was acetylated and subjected to the photocycloaddition. In addition to the expected endoperoxide 37, epoxide 38 was also isolated in small amounts. The structure of 38 was assigned on the basis of its spectroscopic data in comparison with those for the previously reported 39. 15 As regards the mechanism of formation of 38, analogous epoxide byproducts of singlet oxygen photocycloaddition have been described previously for other substrates and are believed to arise via a radical pathway. 32 Upon treatment with CoTPP, 37 underwent facile isomerisation to bis(epoxide) 40. These epoxides proved resistant to opening with ammonia, with ammonolysis instead cleanly removing the acetate to give 41 (Scheme 7).

Conclusions
We have described synthetic sequences which allow for the functionalization of every position on the cyclohexadiene ring of 4. As the stereocentres in 4 are in close proximity to the diene, it has proven possible to introduce additional stereocentres in a highly selective fashion under substrate control. We anticipate that the novel chirons described here may find use in the synthesis of more complex targets.

General procedures
Reactions were carried out under an atmosphere of nitrogen. In most cases, solvents were obtained by passing through anhydrous alumina columns using an Innovative Technology Inc. PS-400-7 solvent purification system. All other solvents were purchased as "anhydrous" grade from Fisher Scientific. "Petrol" refers to petroleum spirit b.pt 40-60°C. TLC was performed using aluminium backed plates precoated with Alugram® SIL G/UV 254 nm. Visualization was accomplished by UV light and/or KMnO 4 followed by gentle warming. Organic layers were routinely dried with anhydrous MgSO 4 and evaporated using a Büchi rotary evaporator. When necessary, further drying was facilitated by high vacuum. Flash column chromatography was carried out using Davisil LC 60 Å silica gel (35-70 micron) purchased from Fisher Scientific. IR spectra were recorded on a Perkin-Elmer 1600 FT IR spectrometer with only selected absorbances quoted as ν in cm −1 . NMR spectra were run on Bruker Avance 250, 300, 400 or 500 MHz instruments at 298 K. A micrOTOF electrospray timeof-flight (ESI-TOF) mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) was used; this was coupled to an Agilent 1200 LC system (Agilent Technologies, Waldbronn, Germany). The LC system was used as an autosampler only. 10 μL of sample was injected into a 30 : 70 flow of water-acetonitrile at 0.6 mL min −1 to the mass spectrometer. For each acquisition 10 μL of calibrant of 5 mM sodium formate was injected after the sample. The observed mass and isotope pattern matched the corresponding theoretical values as calculated from the expected elemental formula.