Novel Reductive Aminases for the Preparation of Chiral Amines

  • Ryan Palmer

Student thesis: Phd


Biocatalytic imine reductions and reductive aminations are an important tool to access high value primary, secondary, and tertiary chiral amines. Chiral amines are a prevalent functional group that is found in around 40% of pharmaceuticals and 20% of agrochemicals. Imine reductases, and the more recently discovered reductive aminases, are two valuable classes of biocatalysts that have emerged in recent years. They both possess a wide substrate scope of ketones and amines, therefore making them suitable candidates for reductive aminations, as well as a wide scope of imines for imine reductions. Despite the promise that these two enzymes show over other relevant biocatalysts for accessing chiral amines such as transaminases and monoamine oxidases, IREDs and RedAms have their limitations. The activity and stereoselectivity of these enzymes have been shown to be very substrate dependent. Even small changes in substituents, e.g. in steric demands or electronic properties, can result in drastically reduced, or improved, activity, as well as invert the enantiopreference. This report looks at exploring the activity and stereoselectivity of a reductive aminase from Ajellomyces dermatitidis (AdRedAm) towards previously untested carbonyl acceptors that showed interest as pharmaceutical building block precursors. Of the 7 carbonyl acceptors chosen for investigation, only one showed conversion to the desired amine product which was 1-methylpiperidine-2,3-dione. This achieved conversions of up to 80% and 78.6% with allylamine and trifluoroethylamine as the amine donors respectively. A secondary peak in GC traces was observed with this reactions which corresponding to the intermediate enamine compound that formed through tautomerization after the in situ formation of the imine intermediate. Through enzyme and no enzyme control reactions it was observed that this in situ imine formation and enamine tautomerization was not enzyme catalysed and reach 100% conversion to the enamine after 1 hour. The poor feasibility of the chosen substrates as carbonyl acceptors for reductive amination lead to a rational engineering investigation into a selection of active site residues. A total of six residues (N93, Y177, W208, H215, M237, and Q238) were selected in AdRedAm based on crystallographic data previously obtained from the reductive aminase from Aspergillus oryzae (AspRedAm)90 and screed against 2-phenylpyrroline and other cyclic imines with an aromatic substituent in the 2-position. Of the seven variants generated, W208A and M237G stood out as the best performing due to showing either an improvement in activity or stereoselectivity, or an inversion in stereoselectivity. AdWT showed a conversion of 19% for 2-phenylpyrroline and was 92% (S)-selective whereas W208A and M237G showed conversions of 100% and 54% with ee values of >99% (S) and 88% (R) respectively. Addition of a para-chloro group onto the aromatic resulted in a change of stereoselectivity of AdWT, N94A, Y177F, and H215A to the (R)-enantiomer. This substrate was more accepted by AdWT with conversion of 39% to the amine after 3 hours. W208A and M237G showed conversions of 99% and 25% with ee values of 74% (S) and 88% (R) respectively. A para-methoxy substituent on the aromatic ring resulted in 99% conversion and was >99% (S)-selective. Upon changing the ring size to a 6-membered ring, W208A and M237G both showed 100% conversion and ee >99% (R) compared to the AdWT with a conversion of 30% and 72% (R)-selective. This shows that the stereoselectivity of this reductive aminase is also substrate dependent as well as the shape and chemical nature of the active site. An in-silico docking was carried out of 2-phenylpyrroline into AspWT as well as the corresponding mutations of W208A and M237G from AdRedAm (W210A and M239G). It was seen in both variants, a domain shift around positions 238 and 239 had occurred which appears to push the su
Date of Award31 Dec 2022
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorSabine Flitsch (Supervisor) & Nicholas Turner (Supervisor)

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