An investigation of nitrile transforming enzymes in the chemo-enzymatic synthesis of the taxol sidechain.

Paclitaxel (taxol) is an antimicrotubule agent widely used in the treatment of cancer. Taxol is prepared in a semisynthetic route by coupling the N-benzoyl-(2R,3S)-3-phenylisoserine sidechain to the baccatin III core structure. Precursors of the taxol sidechain have previously been prepared in chemoenzymatic approaches using acylases, lipases, and reductases, mostly featuring the enantioselective, enzymatic step early in the reaction pathway. Here, nitrile hydrolysing enzymes, namely nitrile hydratases and nitrilases, are investigated for the enzymatic hydrolysis of two different sidechain precursors. Both sidechain precursors, an openchain α-hydroxy-β-amino nitrile and a cyanodihydrooxazole, are suitable for coupling to baccatin III directly after the enzymatic step. An extensive set of nitrilases and nitrile hydratases was screened towards their activity and selectivity in the hydrolysis of two taxol sidechain precursors and their epimers. A number of nitrilases and nitrile hydratases converted both sidechain precursors and their epimers.


Introduction
2][3][4] Taxol was first isolated from the bark of the pacific yew Taxus brevifolia, following an initiative of the US National Cancer Institute (NCI), screening for antineoplastic activity of new substances from various origins. 1,57][8] Despite its known limitations, e.g., poor solubility, toxicities and emerging drug resistance, taxol is still widely used in cancer therapy.0][11] Numerous efforts have been made to determine the structure activity relationship of taxol. 12In recent years, the role of the (2R,3S)-N-benzoyl-3-phenylisoserine C-13 sidechain had been confirmed as essential for the biological activity of taxol (Fig. 1). 13arious sidechain precursors have been prepared in asymmetric chemical syntheses 14 and in chemoenzymatic approaches 15 using acylases, 16 lipases 17 and reductases. 18n this work, nitrile transforming enzymes, namely nitrilases and nitrile hydratases, are investigated for the synthesis of the taxol sidechain.Nitrilases (EC 3.5.5.1) and nitrile hydra- tases (EC 4.2.1.84)are attractive biocatalysts for the fine chemicals and pharmaceutical industries.Nitrilases catalyse the cleavage of nitriles to the corresponding carboxylic acids and ammonia and have been shown to catalyse the hydrolysis of a variety of nitriles, including the enantioselective synthesis of β-amino acids from β-amino nitriles. 19However, several nitrilases were reported to convert nitriles to both, acid and amide products (Scheme 1). 20Nitrile hydratases are Fe-or Co-metalloenzymes that catalyse the hydration of nitriles to their corresponding amides. 21To the best of our knowledge, nitrilases have not yet been investigated in the synthesis of the taxol sidechain.Previously, a nitrile hydratase from Debaryomyces hansenii DSM 3428 was used in whole cell experiments for the hydration of (±)-trans-3-phenyloxirane-2-carbonitrile.The reaction proceeded with low enantioselectivity (ee-value <36%), despite stopping the reaction shortly before 50% conversion was achieved.15b Here, two different taxol sidechain precursors were prepared in chemical synthesis, an openchain α-hydroxy-β-amino nitrile and a cyanodihydrooxazole.A set of 24 nitrilases and four nitrile hydratases was investigated for the biotransformation of these taxol sidechain precursors and their epimers.The enzymatic hydrolysis of these nitrile containing sidechain precursors applies the stereoselective, enzyme-catalysed reaction as the last step in the synthesis.

Results and discussion
Two different taxol sidechain precursors were prepared in chemical synthesis, as depicted in Scheme 2. The corresponding acids and amides were prepared in chemical synthesis as reference materials for the biotransformation reactions. 22In the first synthetic step, benzaldehyde was transformed to a mixture of (±)-cis-and (±)-trans-3-phenyloxirane-2-carbonitrile in a Darzens reaction. 23The epimers were separated by column chromatography and used separately to synthesise the dihydrooxazoles (±)-trans-1 and (±)-cis-1 in a Rittertype reaction. 24Ring opening under acidic conditions gave the openchain precursors (±)-syn-2 and (±)-anti-2.
All fungal nitrilases used here were previously characterised as arylacetonitrilases with preference for phenylacetonitrile and (R,S)-mandelonitrile as substrates.Their similar substrate specificities correspond with considerable identities of their amino acid sequences (mostly over 50%).Nevertheless, differences have previously been observed between their specific activities, enantioselectivities and chemoselectivities for (R,S)mandelonitrile. 28,32 The ee-values obtained in the nitrilase catalysed reactions of (±)-trans-1 were below 80%. 33The moderate ee-values might be explained by a number of reasons.Racemisation and/or epimerisation of the compounds might occur during or after the biotransformation.Stopping the reaction by precipitating the enzyme might not have been efficient enough, as the protein might not have been quantitatively precipitated.Reactions could be most efficiently stopped by using immobilized enzyme which can be easily removed from the reaction mixture.In commercially available enzymes, additives in the enzyme preparation might influence enantioselectivity and ratio of the acid and amide products.The additive dithiothreitol (DTT) is added to nitrilases to prevent disulphide bond formation of the catalytically active cysteine, though it has been previously proven to catalyse the non-stereoselective hydrolysis of nitriles to amides. 34In recent examples, the presence of organic solvents has been shown to enhance activity and stereoselectivity in nitrilase catalysed biotransformations. 35he influence of organic solvents on enzymatic nitrile hydrolysis is poorly studied so far, especially compared to other hydrolytic enzymes, such as lipases and esterases. 36Influences on the stereoselectivity of the nitrilase and nitrile hydratase catalysed reactions need to be further investigated to achieve a reliable nitrile transforming biocatalyst for the synthesis of the taxol sidechain.

Conclusions
In this work, an extensive set of nitrilases and nitrile hydratases was screened towards their activity and selectivity in the hydrolysis of two taxol sidechain precursors and their epimers.Both sidechain precursors were designed to utilize the enzymatic step as final step in the synthesis.A number of nitrilases and nitrile hydratases catalysed the biotransformation of both sidechain precursors and their epimers.
Fungal nitrilases, first of all arylacetonitrilases, have almost been neglected until recently due to their low production in wild-type strains, but genome mining has enabled the discovery of the first enzymes of this type.Their biocatalytic potential was confirmed with a new type of nitrile substrates in this work, and indicated further screening of this enzyme group would be promising.
This work presents the first investigation of nitrilases as tools for the chemo-enzymatic synthesis of the taxol sidechain.Nitrile hydratases (especially Co-type nitrile hydratases) and arylacetonitrilases (both, bacterial and fungal) were found to be suitable catalysts for one or both taxol sidechain precursors tested.The stereoselectivity of the enzyme catalysed reactions will need to be further investigated and improved for a possible application of these nitrile transforming enzymes in an enantioselective, chemo-enzymatic synthesis of the taxol sidechain.

Biotransformation reactions
Biotransformation reactions with commercially available nitrile hydratases.Nitrile hydratases were obtained from Prozomix Ltd (PRO-E0256 to PRO-E0259).Screening reactions were done in 1.5 mL microcentrifuge tubes using the following conditions and concentrations: nitrile hydratase suspension (50 µL-200 µL), and substrate in DMSO (10 µL of a 20 mM stock solution, end concentration of substrate 0.4 mM, 2% v/v DMSO), buffer (50 mM K 2 HPO 4 , pH 8) to achieve a total volume of 500 µL.Blank reactions contained substrate in DMSO (0.4 mM, 2% v/v DMSO), and buffer (50 mM K 2 HPO 4 , pH 8).The screening reactions were incubated on a thermomixer at 22 °C and 500 rpm, unless stated otherwise.The reactions were stopped by adding methanol (290 µL).The protein was precipitated by centrifugation and the supernatant was analysed by HPLC-MS.
Preparative scale biotransformations.Preparative scale biotransformations were carried out with whole cells of E. coli expressing the nitrilase from Neurospora crassa OR74A.Five parallel biotransformations were run, each in a 250 mL Erlenmeyer flask, containing 100 mL of 50 mM Tris/HCl buffer with 150 mM NaCl, pH 8.0 and (±)-trans-2,4-diphenyl-4,5-dihydrooxazole-5-carbonitrile, (±)-trans-2 (100 mg, 0.40 mmol) in DMSO (30% v/v), optical density of the cells approximately 2. The reactions were stopped after three hours reaction time by addition of 0.5 M HCl (dropwise addition until pH 3), representing approximately 50% conversion of the nitrile substrate.The cells were removed by centrifugation.The supernatant was extracted with DCM (4 × 50 mL).The combined organic layers were washed with brine (2 × 100 mL), dried over sodium sulphate and reduced in vacuum until dryness.Residual DMSO was removed by lyophilisation overnight.Yield (crude product, containing a mixture of acid product and unreacted starting material) 70 mg.The crude product was purified by column chromatography using a gradient of chloroform to 15% v/v methanol in chloroform as eluents.The product was isolated as an off-white solid (15.6 mg, 10%, purity 71%, er 37 1/1.6 (4S,5R)-acid/enantiomer). 33 1H NMR and 13 C NMR data were found in accordance with the reference acid.