Biocatalysis is now a well-established branch of catalysis and the growing toolbox of natural, evolved and designer enzymes is enabling chemistry previously deemed inaccessible. However, most enzyme methodologies have been developed for functional group interconversions, such as the conversion of a ketone into an amine or alcohol, and do not result in the generation of significant 3D molecular complexity. The application of enzyme-triggered reaction cascade methodologies has the potential to transform simple substrates into complex sp3-rich molecules in one step. Herein, we describe a single-step biocatalytic route to high-value, complex indolizidine, and quinolizidine alkaloids, which relies on a transaminase-triggered double intramolecular aza-Michael reaction. This approach allows access to architecturally complex, natural-product-like N-heterocycles and reveals intriguing examples of diastereoselectivity in these enzyme-triggered reactions. Significantly, we demonstrate an elegant example of a biocatalytic cascade where the transaminase plays a dual role in generating complex N-heterocycles and where a retro-double intramolecular aza-Michael reaction mediates a dynamic kinetic resolution and enables the isolation of sp3-rich indolizidine diastereoisomers containing five stereocenters, as single isomers.

Shuttle catalysis has emerged as a useful methodology for the reversible transfer of small functional groups, such as CO and HCN, and goes far beyond transfer hydrogenation chemistry. While a biocatalytic hydrogen-borrowing methodology is well established, the biocatalytic borrowing of alternative functional groups has not yet been realized. Herein, we present a new concept of amine borrowing via biocatalytic shuttle catalysis, which has no counterpart in chemo-shuttle catalysis and allows efficient intermolecular amine shuttling to generate reactive intermediates in situ. By coupling this dynamic exchange with an irreversible downstream step to displace the reaction equilibrium in the forward direction, high conversion to target products can be achieved. We showcase the potential of this amine-borrowing methodology using a biocatalytic equivalent of both the Knorr-pyrrole synthesis and Pictet–Spengler reaction.

Here, we report a chemoenzymatic approach for the preparation of a small panel of biologically important iminosugars from readily available aldoses, employing a transaminase in combination with Gluconobacter oxydans whole cells.

An ω-transaminase-triggered intramolecular aza-Michael reaction has been employed for the preparation of cyclic β-enaminones in good yield and excellent enantio- and diastereoselectivity, starting from easily accessible prochiral ketoynones and commercially available enzymes. The powerful thermodynamic driving force associated with the spontaneous aza-Michael reaction effectively displaces the transaminase reaction equilibrium towards product formation, using only two equivalents of isopropylamine. To demonstrate the potential of this methodology, this biocatalytic aza-Michael step was combined with annulation chemistry, affording unique stereo-defined fused alkaloid architectures.

An alcohol dehydrogenase-mediated asymmetric reduction and subsequent intramolecular oxa-Michael reaction has been developed for the preparation of tetrahydropyrans (or oxanes) and tetrahydrofurans, in excellent conversion, yield and high enantiomeric and diastereomeric excess. To highlight the utility of the methodology, we report the synthesis of an analogue of the fungal antioxidant brocaketone A. Also described is the preparation of the (-)-(R,R)enantiomer of the natural product, (+)-(S,S)-(cis-6methyltetrahydropyran-2-yl)acetic acid. The intramolecular oxa-Michael reaction (IMOMR) is a direct and rapid approach for carbon-oxygen bond formation, which allows the construction of synthetically useful cyclic oxygencontaining heterocycles.[1] In particular, the IMOMR is exploited as a key step in cascade strategies for the preparation of chiral tetrahydropyrans (THPs) and tetrahydrofurans (THFs), which are prevalent in natural products (Figure 1).[1d,2] The syntheses of these motifs often commence from the chiral pool, to provide the desired enantiomer of the nucleophilic alcohol, prior to the IMOMR. This chiral centre has also been installed via asymmetric catalysis but the scope of this chemistry is limited.[1c,1g, 2d] Figure 1. A selection of tetrahydropyran and tetrahydrofuran-containing natural products. A useful approach for the synthesis of THPs/THFs is represented in Scheme 1 and involves the chemoand stereo-selective reduction of ketoenone 1, followed by a spontaneous IMOMR to afford oxa-Michael product 3. However, this strategy has a number of challenges (highlighted in blue), which would be difficult to overcome when using traditional reduction chemistry. Scheme 1. A retrosynthetic approach for the synthesis of THP/THF derivatives starting from ketoenone 1. The growing toolbox of biocatalysts, which can mediate synthetically challenging reactions and complement traditional chemical synthesis, has inspired the concept of biocatalytic retrosynthesis.[3] Incorporating enzymes into retrosynthetic design strategies enables completely new disconnections, which would not be feasible using more traditional synthetic approaches. We have previously reported a transaminase-triggered intramolecular azaMichael reaction (IMAMR) for the synthesis of chiral 2,6disubstituted piperidines,[4] and envisaged that an analogous approach could be used for the chemo-enzymatic synthesis of THPs/THFs, by employing an alcohol dehydrogenase (ADH). These enzymes have been heavily exploited for the selective reduction of prochiral ketones to afford the corresponding chiral alcohol. This methodology has been used both in vitro and in vivo, often in combination with a suitable co-factor recycling system.[5] ADHs are also used for the selective oxidation of primary or secondary alcohols, with the latter typically resulting in a kinetic resolution.[6] Herein, we report an expansion of our aza-Michael methodology to include the biocatalytic ADH reduction/IMOMR cascade on a panel of prochiral ketoenone substrates. An (R)-selective ADH from Lactobacillus kefir (LK) DSM 20587 has been selected to showcase this methodology, due to its broad substrate specificity and high enantioselectivity in the synthesis of chiral alcohols, including dicarbonyl substrates.[7] Ketoenone substrates 1a-c and 1e-i (Table 1) were prepared via oxidative cleavage of 1-methylcyclopentene or 6-methyl5-hepten-2-one, followed by reaction with a suitable phosphorus ylid. Ketoenone 1d was prepared via an Mr H. Eastman,[a] Dr J. Ryan,*[a] Dr. B. Maciá,[b] Dr V. Caprio[b] and Dr E. O’Reilly*[a,c] [a] School of Chemistry, University of Nottingham, University Park, Nottingham, NG7 2RD, UK [b] Faculty of Science & Engineering, Division of Chemistry & Environmental Science, Manchester Metropolitan University, Chester Street, Manchester M1 5GD, United Kingdom [c] School of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland (current address) E-mail: elaine.oreilly@ucd.ie James.ryan@nottingham.ac.uk Supporting information for this article is given via a link at the end of the document. O O n O

An investigational drug targeting the HIV virus is synthesized with nine enzymes

An alcohol dehydrogenase‐mediated asymmetric reduction and subsequent intramolecular oxa‐Michael reaction has been developed for the preparation of tetrahydropyrans (or oxanes) and tetrahydrofurans, in excellent conversion, yield and high enantiomeric and diastereomeric excess. To highlight the utility of the methodology, we report the synthesis of an analogue of the fungal antioxidant brocaketone A. Also described is the preparation of the (–)‐(R,R)‐enantiomer of the natural product, (+)‐(S,S)‐(cis‐6‐methyltetrahydropyran‐2‐yl)acetic acid.

The conversion of readily available monosaccharides to high value amino alcohols using a key biocatalytic step is an attractive strategy for the preparation of these chiral synthons. Here, we report a previously undescribed example of the direct amination of monosaccharides, which exist predominantly in their cyclic form at equilibrium, using amine transaminase biocatalysts, providing access to a panel of amino alcohols in moderate to high conversion and isolated yield. A recently developed high-throughput colorimetric screen, employing o-xylylenediamine, was initially used to identify amine transaminase enzymes displaying this activity toward cyclic sugars, and reactions were successfully scaled up using isopropylamine.

The “smart” amine donors o‐xylylenediamine and cadaverine were employed for the rapid screening of a large ketone library and subsequent preparative‐scale synthesis of selected compounds using a commercially available amine transaminase, ATA256. The methodology enables both screening and preparative‐scale biotransformations to be performed with a single enzyme and simplifies the generation of sp3‐rich small‐molecule libraries.

This thesis focuses on the development of new biocatalytic strategies as a contemporary solution to synthetic design. Here we have put to use the unique regio-, stereo- and/or chemoselectivity offered by biocatalysts to develop synthetically attractive routes to enantiopure materials. The first chapter discusses the development of a transaminase triggered aza-Michael cascade towards the synthesis of enantioenriched 2,6-disubstituted piperidines in good yield with >99% e.e. and >99% d.e. This methodology utilises a favourable spontaneous intramolecular aza-Michael reaction (IMAMR) to drive the reversible enzymatic transformation towards the formation of cyclic products, thus removing the need for additional approaches to displace the reaction equilibrium towards product formation. The alkaloid, (-)-pinidinone, was synthesised in three steps on a 0.5 g scale and a range of analogues was also successfully prepared to demonstrate the scope of the reaction. The reversible transamination reaction in combination with the thermodynamically favourable IMAMR, forming a stable cyclic product, results in a regioselective transamination of (3E)-dec-3-ene-2,8-dione. This inspired us to develop an amino donor and acceptor substrate that was successfully transaminated to form pinidinone with no external source of amine. The second chapter discusses the synthesis of novel bis-conjugated enones and their subsequent transamination to provide bicyclic alkaloids via double aza-Michael additions. However, under the tested reaction conditions, the TA reaction resulted in complete decomposition of all but two of the tested substrates. 1-Methyldecahydropyrrolo[1,2-a]quinolin-5(1H)-one was produced as three isomers whose relative stereochemistry was assigned by NMR. Interestingly, transamination of (2E)-1-(cyclohex-1-en-1-yl)oct-2-ene-1,7-dione provided 1-(cyclohex-1-en-1-yl)-2-[(2S,6S)-6-methylpiperidin-2-yl]ethanone as the major product and an inseparable mixture of 1-methyldodecahydro-6H-pyrido[1,2-a]quinolin-6-one isomers as the minor product. Initiating the second IMAMR of 1-(cyclohex-1-en-1-yl)-2-[(2S,6S)-6-methylpiperidin-2-yl]ethanone was attempted. The epimerisation of 1-methyldecahydropyrrolo[1,2-a]quinolin-5(1H)-one and 1-methyldodecahydro-6H-pyrido[1,2-a]quinolin-6-one proved unproductive. The third chapter investigates the synthesis of 2-alkyl-3-(4-oxopentyl)cyclohex-2-en-1-one and their subsequent use in a TA-IMAMR cascade towards the pragmatic synthesis of the natural product histrionicotoxin (HTX) and its derivatives. A Baylis-Hillman reaction was optimised for the insertion of the α-alkyl substituent into the cyclohexanone scaffolds. TA conditions were found to convert the unsubstituted scaffold to provide (2S)-2-methyl-1-azaspiro[5.5]undecan-8-one as a 1:1 mixture of diastereoisomers. The optimal epimerisation conditions found provided a 3:1 mixture of diastereoisomers, however, isolation of the compounds proved unsuccessful. Reduction of the carbonyl to provide the core HTX structure was tested. This provided an inseparable mixture of products. Under the biocatalytic reaction conditions, the IMAMR of 2-ethyl-3-(4-oxopentyl)cyclohex-2-en-1-one provided 3-[(4S)-4-aminopentyl]-2-ethylcyclohex-2-en-1-one as the major product. This is due to the (2S)-7-ethyl-2-methyl-1-azaspiro[5.5]undecan-8-one being an unfavourable product, which is in agreement with current literature.

The expanding "toolbox" of biocatalysts opens new opportunities to redesign synthetic strategies to target molecules by incorporating a key enzymatic step into the synthesis. Herein, we describe a general biocatalytic approach for the enantioselective preparation of 2,6-disubstituted piperidines starting from easily accessible pro-chiral ketoenones. The strategy represents a new biocatalytic disconnection, which relies on an ω-TA-mediated aza-Michael reaction. Significantly, we show that the reversible enzymatic process can power the shuttling of amine functionality across a molecular framework, providing access to the desired aza-Michael products.