Genome mining and characterisation of a novel transaminase with remote stereoselectivity

Microbial enzymes from pristine niches can potentially deliver disruptive opportunities in synthetic routes to Active Pharmaceutical Ingredients and intermediates in the Pharmaceutical Industry. Advances in green chemistry technologies and the importance of stereochemical control, further underscores the application of enzyme-based solutions in chemical synthesis. The rich tapestry of microbial diversity in the oceanic ecosystem encodes a capacity for novel biotransformations arising from the chemical complexity of this largely unexplored bioactive reservoir. Here we report a novel ω-transaminase discovered in a marine sponge Pseudovibrio sp. isolate. Remote stereoselection using a transaminase has been demonstrated for the first time using this novel protein. Application to the resolution of an intermediate in the synthesis of sertraline highlights the synthetic potential of this novel biocatalyst discovered through genomic mining. Integrated chemico-genomics revealed a unique substrate profile, while molecular modelling provided structural insights into this ‘first in class’ selectivity at a remote chiral centre.

. Biochemical characterisation of P-ω-TA representing a Enantioselectivity, b pH, and c Temperature optima. pH data is presented for assays performed at the optimum temperature of 40 o C, while temperature optima data is presented for assays performed at the pH optimum of 10.
Data is normalised to the average of the optimum value in both cases. Data presented is the average of three independent biological replicates and variation is presented as standard error of the mean. Statistical analysis was performed using One way ANOVA with Dunnett's multiple comparison corrective testing to the optimum activity (* p < 0.05, ** p < 0.01, *** p < 0.001).
S5 Figure S3. Solvent tolerance of P-ω-TA in the presence of a range of DMSO concentrations.

Synthesis of model amine compounds
General procedure for reductive amination for preparation of racemic amines 1 A mixture of ketone (5.0 mmol), titanium(IV) ethoxide (10.0 mmol, 2 eq.) and ammonia (2 M in methanol, 25.0 mmol, 5 eq.) was stirred under nitrogen, at ambient temperature for 15 h. Sodium borohydride (7.5 mmol, 1.5 eq.) was added and the resulting mixture was stirred at room temperature for an additional 5 h. The reaction was quenched by pouring onto ammonium hydroxide (2 M, 12.5 ml), the resulting inorganic precipitate was removed by filtration, and washed with ethyl acetate (2 × 15 ml). The layers were separated, and the aqueous solution was extracted with ethyl acetate (2 × 15 ml). The combined organic layers were extracted with aq. HCl (10%, 15 ml). The acidic aqueous extracts were washed with ethyl acetate (25 ml), then treated with aq. NaOH (2 M) to pH 10-12 and extracted with ethyl acetate (3 × 25ml). The combined organic extracts were washed with brine (25 ml), dried (Na2SO4), filtered and concentrated to give the primary amine, which needed no further purification. is in agreement with previously reported data. 3 Melting point was not previously reported. S10 14. 2, 44.2, 52.6, 60.6, 126.2, 127.4, 128.6, 144.7, 172.1 ppm; data is in agreement with previously reported data. 7
1, .0]undec-7-ene (1.85 mmol, 1.2 equiv.) was added dropwise over 20 mins. Upon addition, the orange solution turned cloudy. It was stirred at 0 °C for 2 hr, and then allowed to warm to room temperature and stirred for a further 12 hr. The solvent was removed in vacuo and the oily residue was passed through a silica plug to give a viscous, colourless oil. This was dissolved in THF (5 ml) and PPh3 Tetrahedron 63, 6755-6763, (2007).