Catalytic Synthesis of Pharmaceutical Amides in Water

The amide bond is arguably the most significant in pharmaceutical chemistry, featuring in a host of important everyday pharmaceuticals for the treatment of ulcers, high cholesterol and pathogenic infections by bacteria and viruses. It is vital therefore that there exist atom efficient and sustainable green chemical methods for the synthesis of pharmaceutical amides. However, industrial synthetic methods for the preparation of amides suffer from the use of complex or hazardous reagents to accomplish their chemistry and generate a large amount of waste. Because of this lack of efficiency, industrial synthetic chemists are increasingly turning towards 'biocatalysis' or 'Industrial Biotechnology' as the preferred method of synthesising molecules for pharmaceutical production. Biocatalysts, such as enzyme or microbes, typically achieve the synthesis of chemical bonds with excellent atom efficiency and selectivity, and Nature is also expert at synthesising amide bonds, which are the major bonds that hold the structure of proteins together. Until now however, biocatalysts for the formation of amide bonds have received little attention for industrial application, even though such enzyme reactions feature at the top of the list for many chemists looking for biocatalytic solutions to synthetic problems. This is because biocatalytic methods for amide bond formation in Nature, while efficient, are often complex, and difficult to apply out of their natural context. A recently discovered group of enzymes, which we have called amide bond synthetases (ABSs), offers new and unexplored promise for biocatalytic amide bond formation, as their reaction chemistry is comparatively simple, and also because the kind of amide bonds that they form, are much more closely related to molecules of real pharmaceutical interest than has previously been the case. In this project, which is a collaboration between biochemists and synthetic chemists at York, and in association with GSK and also the University of Freiburg, we propose to thoroughly investigate the synthetic potential of the new ABS enzymes. First we will define the potential and limitations of the natural enzymes using a mixture of synthetic chemistry and biocatalysis. We will then use the recently-determined structure of the ABS enzyme McbA to engineer the enzyme, expanding its potential for the catalysis of the synthesis of a much wider range of pharmaceutically relevant molecules. We will also use contemporary protein evolution techniques to adapt the enzymes to act on alternative substrates that are of interest to industrial collaborators. We will also apply new techniques in enzyme cofactor recycling to allow us to scale up the amide bond forming reactions, and also immobilise the enzymes in order to establish a flow biocatalysis system for amide synthesis. Finally, we will combine ABSs with other enzymes to create 'cascades' for the synthesis of amides from readily available alcohol and amine substrates. Together, the project will establish a new frontier in biocatalytic amide bond formation with a view to more sustainable chemical processes for the industrial synthesis of pharmaceuticals.

The project addresses the investigation and development of new catalysts for the production of pharmaceutically relevant amides in aqueous systems, and has potential benefits therefore to synthetic chemists and biotechnologists working in academia and in industrial pharmaceutical synthesis. There are also potential benefits to the agrochemicals sector, given the demonstrated bioactivity of carboxylic amides as pesticides. The development of enzymatic routes to useful chemicals also has benefits for the wider community in industrial chemistry as such methods can supplant conventional chemical methodology, being superior in aspects such as efficiency, selectivity, but also assisting in the move towards a more sustainable platform for the chemical industry, rooted in bio-based feedstocks. The chemicals sector in the UK is estimated to contribute £15 bn per year to UK GDP (source: Chemical Industries Association, 2015) so improved processes for the chemical synthesis can make a clear contribution to economic competitiveness. Scientists will benefit from the proposed research through the development and dissemination of new methods for the ABS-catalysed formation of amides, as well as associated analytical tools and data. These benefits are delivered through publication in scientific journals, but also through presentation at conferences and academic-industrial networks such as the EPSRC-funded Catalyst Hub, and the Centre of Excellence for Biocatalysis, Biotransformations and Biomanufacture (CoEBio3), of which the University of York is a member. The PI collaborates extensively with representatives of major UK and other international chemical companies, of whom GSK, the collaborating partner, will be the most immediate beneficiary. We also have contact with Pfizer, AZ, Merck, Codexis, Syngenta and other companies both through CoEBio3 and also company-specific collaborations. Our industrial contacts are often laboratory scientists in biotransformation laboratories who will be able to quickly transfer information on new techniques and developments to their own groups.
Beyond the academic and industrial researchers that benefit most directly from new technology advances within the project, relevant UK government stakeholders also derive benefit in general from successful developments in Catalysis and Biocatalysis as these help to publicise the benefits of investing in these technologies and also inform the development of future policy. Industrial Biotechnology makes a significant contribution to the UK economy, with an estimate of 225 companies and 8,800 employees involved in generating £2.9 billion of sales in 2013-14 alone (source BBSRC). Organisations such as the BiS-funded Bioscience Knowledge Transfer Network are well-placed to employ these case studies to champion IB in the presence of companies who had not previously considered using this technology. The general benefits of IB such as those described in the project can be communicated through Bioscience KTN activities such as visits, newsletters, webinars and workshops.
Finally, the wider benefits of the work are experienced by the general public. As consumers and users of chemical products, they will benefit from their more efficient production. In the longer term, the incorporation of biocatalysis into chemicals manufacture directly benefits the public in terms of the improved environment associated with a more efficient, sustainable and environmentally benign chemicals industry. The impact of these developments on the public can be communicated at local and international level using press releases, public engagement events and articles in popular science journals.