Biotransformations Employing Nitrile Hydrolysing Enzymes Towards The Enantioselective Synthesis of β-amino Acids

Tatenda Mareya

    Research output: Types of ThesisDoctoral Thesis


    Nitrile hydrolysing enzymes continue to be of great interest particularly for their pharmaceutical applications. This work set out to utilise novel bacterial isolates containing nitrile-metabolising enzyme systems in the synthesis of a series of chiral β-amino acids and amines with key goals to achieve high enantioselectivity and reaction efficiency. Biocatalysis offers many advantages as an alternative to traditional chemical synthetic methods employed in the production of fine chemicals such as pharmaceuticals with improved productivity, higher yields, shortened synthetic sequences, and reduced costs. In addition, the use of biocatalysts in synthesis adheres to many of the “green chemistry principles” which have become core values of pharmaceutical production. Initial work focussed on further assessing the functional group tolerance and mechanistic action of bacterial isolate SET1 with a view to the production of β-amino acids. A series of model β-aminonitriles, structurally related to the β-hydroxynitriles previously studied were synthesised and evaluated. Whilst the synthesis of the aliphatic unprotected nitrile, 3-aminobutyronitrile, was unsuccessful and led to its purchase, the remaining seven nitriles were successfully synthesised. The corresponding acid and amide standards were also synthesised. Additionally, work was carried out to develop analytical methods to assess yield and purity of the reaction products. Some of this work entailed derivatisation of the products to allow for successful analysis. Bacterial isolate SET1 unfortunately has shown to be disappointingly poor at selectively hydrolysing the unprotected β-amino nitriles, 3-aminobutyronitrile and 3-amino-3-phenylpropionitrile. The acid yields were extremely low, with the highest being <1% at pH 7 and steadily decreased as pH increased. The ee results again were very low, with the highest ee being 29% at pH 7 and this steeply dropped again as pH increased, and by pH 9 the ee was 5%. Studies on protected variants of 3-aminobutyronitrile gave more promising results, in particular with the N-Benzyl group which gave the overall best result of acid product with 75% ee and 6% yield. Screening was carried out on five bacterial isolates from the PMBRC bank to identify possible other isolates to work with. They were screened on 3-aminobutyronitrile and 3-phenyl-3-propionitrile at pH 7 and 9. The best results were seen from bacterial isolates 6 and 39. Both isolates 6 and 39 gave excellent ee’s of 99% at pH 9 and ee’s of 89% and 87% respectively at pH 7. For 3-phenyl-3-propionitrile, isolate 39 was the only one to show activity giving an ee of >99% at pH 7 with no activity at pH 9. Bacterial isolate 39 was thus selected for further investigations and to screen it against the N-protected amino nitriles. Contamination was unfortunately discovered with isolates 6 and 39, and this placed on hold further work with these isolates. A novel nitrilase enzyme Nit1, was then acquired from the PMBRC isolate bank. Nit1 showed improved results with 3-ABN from the initial screen with 76% yield and 24 % (S) ee being observed. As for the N-protected variants, it only showed activity with the benzyl aliphatic nitrile. It encouragingly achieved ee’s of 43% (S) at pH 7 and 4% (S) at pH 9 for this substrate. Some issues were experienced however with the presence of solvent and buffer from the biotransformations, possibly interfering with the HPLC results and extensive method development had to be carried. More robust HPLC methods were successfully developed which allowed for high throughput screening and would enable future work to be carried out with Nit1 more efficiently.
    Original languageEnglish
    Awarding Institution
    • Lennon, Claire, Supervisor
    • Coffey, Lee V., Supervisor
    • Kinsella, Michael, Supervisor
    Publication statusUnpublished - 2020


    • Nitrile Hydrolysing Enzymes, β-Amino acids, Enantioselective synthesis


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