The ability of microbial cells to synthesize highly reactive nanoscale functional materials provides the basis for a novel synthetic biology tool for developing the next generation of multifunctional industrial biocatalysts. Here, we demonstrate that aerobic cultures of Escherichia coli, genetically engineered to overproduce a recombinant monoamine oxidase possessing high enantioselectivity against chiral amines, can be augmented with nanoscale Pd(0) precipitated via bioreduction reactions. The result is a novel biometallic catalyst for the deracemization of racemic amines. This deracemization process is normally achieved by discrete sequential oxidation/reduction steps using a separate enantiomer-specific biocatalyst and metal catalyst, respectively. Here, use of E. coli cultures carrying the cloned monoamine oxidase gene and nanoscale bioreduced Pd(0) particles was used successfully for the conversion of racemic 1-methyltetrahydroisoquinoline (MTQ) to (R)-MTQ, via the intermediate 1-methyl-3,4-dihydroisoquinoline, with an enantiomeric excess of up to 96%. There was no loss of catalyst activity following the five rounds of oxidation and reduction, and importantly, there was minimal loss of palladium into the reaction supernatant. This first demonstration of a whole cell biometallic catalyst mixture for “single-pot”, multistep reactions opens up the way for a wide range of integrated processes, offering a scalable and highly flexible platform technology.

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A simple protocol for generating a highly stable and active surface plasmon resonance (SPR) sensor surface of recombinant human hexahistidine cyclophilin A (His-CypA) is described. The sensor surface was sensitive and stable enough to allow, for the first time, the screening and ranking of several novel small-molecule (Mr approximately 250-500 Da) ligands in a competition binding assay with cyclosporin A (CsA). It also allowed us to accurately determine the kinetic rate constants for the interaction between His-CypA and CsA. His-CypA was first captured on a Ni2+-nitrilotriacetic acid (NTA) sensor chip and was then briefly covalently stabilized, coupling via primary amines. The significant baseline drift observed due to dissociation of weakly bound His-CypA from the Ni2+-NTA moiety was eliminated, resulting in a surface that was stable for at least 36 h. In addition, immobilized protein activity levels were high, typically between 85 and 95%, assayed by the interaction between His-CypA and CsA. The mean equilibrium dissociation constant for CsA (K(dCsA)) binding to the immobilized His-CypA was 23+/-6 nM, with on and off rates of 0.53+/-0.1 microM(-1) s(-1) and 1.2+/-0.1 (x 10(-2)) s(-1), respectively. These values agree well with the values for the corresponding binding constants determined from steady-state and kinetic fluorescence titrations in solution.

We have previously reported a general method for the deracemisation of racemic chiral amines (primary, [1] secondary [2] and tertiary [3] ) by using variants of the enzyme monoamine oxidase N (MAO-N) from Aspergillus niger. This deracemisation process employs a combination of an enantioselective enzymatic oxidation of the amine to afford the corresponding imine or iminium ion, together with a non-selective chemical reduction of the imine or iminium ion back to the racemic starting material (Scheme 1). The use of an (S)-selective MAO-N enzyme leads to accumulation of the (R)-amine in high enantiomeric excess and yield through several rounds of oxidation and reduction.Previously, we have applied this chemo-enzymatic approach to the deracemisation of the alkaloid (AE)-crispine A. [4] Crispine A (1) was first isolated from extracts of the plant Carduus crispus (welted thistle), along with the cytotoxic crispine B (2, Figure 1) and three other bicyclic isoquinoline alkaloids. [5] Although the deracemisation of (AE)-crispine A resulted in the generation of the R enantiomer in > 97 % ee, the reaction required 40 h to proceed to completion with a MAO-N-5 variant in an overall yield of 48 %. This previous study also highlighted that less-functionalised analogue 3 was more reactive with the same variant, taking only 6 h to reach completion (> 97 % ee), thus indicating that the two methoxy groups of racemate (AE)-1 resulted in lower activity. We reasoned that this drop in activity was due to steric interference between these two groups and residues within the active site of the enzyme. As a result, we employed a combination of molecular modelling and a rational re-design of the MAO-N-5 variant to identify and develop potential new MAO-N variants that could have enhanced activity towards enantiomer (S)-1. In addition, we sought to improve the efficiency of the overall synthesis of enantiomer (R)-1 by utilising the microwave synthesis of the racemic amine coupled with the enhanced deracemisation that is brought about by changes to the MAO-N enzyme.To identify MAO-N variants that have improved activity towards compound (AE)-1, we modelled (S)-crispine A into the active site of the MAO-N-5 enzyme (PDB code 2VVM). [6] Four residues (Phe210, Leu213, Met242 and Met246), which are located at the entrance to the active site channel, were identified as providing possible steric interactions with the methoxy groups of crispine A (1). To optimise these residues, two randomised libraries were created: The first library targeted amino acids Phe210 and Leu213 (library A) and the second library targeted Met242 and Met246 (library B). Both libraries were screened against compound (AE)-1 by using our previously reported solid-phase assay [7] (Figure 2); sixteen active "hits" were collected from each of the two libraries (A and B), which were then subjected to a second round of screening to eliminate any false positives. After the second round of screening, the two MAO-N variants from each library that had the highest activity, as judged by t...

Background Leishmania species are parasitic protozoa that have a tightly controlled cell cycle, regulated by cyclin-dependent kinases (CDKs). Cdc2-related kinase 3 (CRK3), an essential CDK in Leishmania and functional orthologue of human CDK1, can form an active protein kinase complex with Leishmania cyclins CYCA and CYC6. Here we describe the identification and synthesis of specific small molecule inhibitors of bacterially expressed Leishmania CRK3:CYC6 using a high throughput screening assay and iterative chemistry. We also describe the biological activity of the molecules against Leishmania parasites.Methodology/Principal FindingsIn order to obtain an active Leishmania CRK3:CYC6 protein kinase complex, we developed a co-expression and co-purification system for Leishmania CRK3 and CYC6 proteins. This active enzyme was used in a high throughput screening (HTS) platform, utilising an IMAP fluorescence polarisation assay. We carried out two chemical library screens and identified specific inhibitors of CRK3:CYC6 that were inactive against the human cyclin-dependent kinase CDK2:CycA. Subsequently, the best inhibitors were tested against 11 other mammalian protein kinases. Twelve of the most potent hits had an azapurine core with structure activity relationship (SAR) analysis identifying the functional groups on the 2 and 9 positions as essential for CRK3:CYC6 inhibition and specificity against CDK2:CycA. Iterative chemistry allowed synthesis of a number of azapurine derivatives with one, compound 17, demonstrating anti-parasitic activity against both promastigote and amastigote forms of L. major. Following the second HTS, 11 compounds with a thiazole core (active towards CRK3:CYC6 and inactive against CDK2:CycA) were tested. Ten of these hits demonstrated anti-parasitic activity against promastigote L. major.Conclusions/SignificanceThe pharmacophores identified from the high throughput screens, and the derivatives synthesised, selectively target the parasite enzyme and represent compounds for future hit-to-lead synthesis programs to develop therapeutics against Leishmania species. Challenges remain in identifying specific CDK inhibitors with both target selectivity and potency against the parasite.

This work demonstrates the first example of the immobilisation of MAO-N whole cells to produce a biocatalyst that remained suitable for repetitive use after 11 months of storage and stable up to 15 months after immobilisation. The production of Escherichia coli expressing recombinant MAO-N was scaled up to bioreactors under regulated, previously optimised conditions (10% DO, pH 7), and the amount of biomass was almost doubled compared to flask cultivation. Subsequently, pilot immobilisation of the whole-cell biocatalyst using LentiKats technology was performed. The amount of the immobilised biomass was optimised and the process was scaled up to a production level by immobilising 15 g of dry cell weight per litre of polyvinyl alcohol to produce 3 kg of whole-cell ready-to-use biocatalyst. The immobilised biocatalyst retained its initial activity over six consecutive biotransformations of the secondary amine model compound 3-azabicylo [3,3,0]octane, a building block of the hepatitis C drug telaprevir. Consecutive cultivation cycles in growth conditions not only increased the initial specific activity of biocatalyst produced on the industrial plant by more than 30%, but also significantly increased the rate of the biotransformation compared to the non-propagated biocatalyst.

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