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Professor Helen Hailes

6Biocatalysis and Synthetic Biology

The use of biocatalysts for the synthesis of chiral compounds and fine chemicals is of significant interest to the chemical, pharmaceutical and industrial biotechnology sectors in the search for sustainable, cost effective, synthetic strategies. The BiCE (Biocatalysis integrated with Chemistry and Engineering) research programme with an internationally recognised multidisciplinary research team was established in 2004 with the overall aim to develop a framework and tools for constructing multi-step enzymatic processes rapidly and efficiently, ultimately for industrial synthesis. The BiCE multidisciplinary research group involves Prof. G. J. Lye, Prof. J. M. Ward, Prof. P. A. Dalby, Dr. F. Baganz, Dr M. Micheletti, Dr N. Szita.

The BiCE programme focused initially on the linkage of advanced bioconversions capable of stereoselective carbon-carbon bond formation and amine bond formation. Two enzymes transketolase (TK) and transaminase (TAm) were used as model systems, and when used sequentially as an early synthetic biology example they generated 2-amino-1,3-diols. Using an integrated strategy we investigated new chemistries and assays, identified novel biocatalysts and used directed evolution strategies, together with automated microscale experimentation and process modelling, to achieve rapid and predictive process scale-up. We have also investigated the use of other biocatalytic enzymes including cytochrome P450s, Baeyer-Villiger monooxygenases, and more recently norcoclaurine synthases.

Biocatalysis

Transketolases (TKs)

Using wild-type E. coli TK a structural and phylogenetic approach was used to identify key residues for saturation mutagenesis and generate the first library of single point active site mutants. A colorimetric assay was then developed for high throughput screening of the mutants against different substrates and a biomimetic TK reaction was identified. Chiral assays were established (derivatisation + GC, HPLC, NMR Mosher’s ester analysis) and the absolute stereochemistry of selected reactions confirmed via chemical correlation. From the initial library set, S- and R- selective TKs were identified, and strategies for the generation of stable double/triple mutants were established. We now have mutants that can accept a wide range of aliphatic aldehydes as well as aromatic substrates in high yields. 

Transketolases
Use of colorimetric assay to identify new active TK mutants

Transaminases (TAms)

New w-transaminases were obtained by BLAST searching with the reported V. fluvialis w-TAm sequence. Iteration of approach has generated >100 new TAms. The TAms have been screened to biotransform TK products and other substrates together with different amine donors. Several TAm assays have also been developed. The conversion of glycolaldehyde using TK + a TAm cloned from P. aeruginosa in a dual plasmid E.coli strain gave 21% of the aminobutanetriol, an example of an early synthetic biology pathway. Other two step TK + TAm processes have been established to aliphatic and aromatic 2-amino-1,3-diols. Selected TAms have also been used in recycling cascade reactions to aminoalcohols. 

TAms - Tk-mutant

Norcoclaurine Synthases (NCSs)

In plant biosynthesis the first committed step to the benzylisoquinoline alkaloids (BIAs), a large group of bioactive plant secondary metabolites, is catalysed by NCS. The 22 kDa enzyme couples dopamine with 4-hydroxyphenylacetaldehyde (4-HPAA) to yield (S)-norcoclaurine. We have been investigating the NCS family of enzymes including their characterisation, substrate tolerance, and biocatalytic applications. Several NCSs were cloned and over-expressed including C. japonica NCS2 and T. flavum NCS and a new high-throughput fluorescence-based assay was developed and used for screening a library of nearly forty aldehydes and amines. Many aldehydes (aryl, heteroaromatic, aliphatic) were accepted in moderate to good yields by CjNCS2. Amines required a meta-hydroxyl group. A biomimetic reaction was also established, together with chiral assays (HPLC, Mosher’s derivatisation), and absolute stereochemistries were confirmed. Some reactions were scaled-up. Screening data and molecular docking calculations suggested an alternative substrate-binding model to that reported which is currently under investigation.

Norcoclaurie Synthases

BiCE Team Awards: 2010 Royal Society of Chemistry Rita & John Cornforth Award from the Chemical Biology Interface Forum for the use of key chemical biology and synthetic skills, interfaced with molecular biology and process engineering to achieve stereoselective enzymatic synthesis with potential for industrial scale-up. 2010 Institution of Chemical Engineers (IChemE) Innovation and Excellence Award for Bioprocessing.

Novel lipid synthesis and nanoparticles for delivery and imaging applications

We have been investigating the structural requirements for lipids in a range of nanoparticles including ternary delivery systems comprised of a lipid (L), targeting peptide (P) and DNA or RNA (D or R). This work is being carried out in collaboration with Prof. A. B. Tabor (Chemistry), Prof. M. J. Lawrence (Institute of Pharmaceutical Science, King’s College London) and Dr. L. Kudsiova (Institute of Pharmaceutical Science, King’s College London). The ternary LPD lipopolyplex systems have displayed high transfection efficiencies and low toxicity in vitro and in vivo, transfected non-dividing cells efficiently, and were well tolerated with low immunogenicity in vivo. We are currently tackling issues related to vector efficacy including targeting accessibility and the influence of the structure of the lipid on particle stability. We have established that shorter unsaturated lipid chains lead to enhanced transfection in several cell-types, most likely due to better membrane disruption or enhanced targeting. We have also designed and synthesised several series of novel lipids, which enhance transfection efficacy due to better particle stability. In addition, we have studied the structure and fate of the nanoparticles formed using confocal microscopy and via the synthesis of specifically labelled lipids. For in vivo applications, as well as incorporating PEG linkers to aid particle stability we have also introduced cleavable moieties to enhance release from the endosome. We are currently investigating LPR knockdown efficacies using selected lipids, peptides, and formulation approaches

As part of the multidisciplinary King's College London and UCL Cancer Imaging Centre with Prof T. Ng (Randall Division, King’s College, London) and others scientists, we are developing new strategies for in vivo cancer imaging. The main clinical objective of the EPSRC/CRUK funded CCIC is to develop and provide a comprehensive range of imaging technologies which, when combined with molecular and genetic information, will begin to accurately characterize individual patients for the ultimate goal of personalized therapy. Here we have synthesized a series of metal-chelating lipid conjugates as multimodal liposomes for use as functional MR contrast agents as well as radionuclide tracers for SPECT. These were combined with shielding lipids and tuned for in vivo applications.

Lipid synthesis

Tuberculosis Therapies

We have investigated the synthesis and biological activity of a series of calixarenes and novel small molecule analogues with anti-TB activities. To date, we have established efficient synthetic methodologies to prepare a range of novel calix[8]arenes with PEG moieties attached at the lower rim via ether linkages. Testing in vivo established preliminary structure–activity relationships, with some compounds exhibiting significant anti-mycobacterial activity. The calixarenes appear to act via a novel host macrophage mediated mechanism, involving the up-regulation of arginase and functioning iNOS. In current studies we are synthesising a series of novel isoquinolines with anti-tuberculosis properties, and investigating the mode of action in collaboration with Dr Sanjeeb Bhakta (Birbeck, University of London)

Design and Synthesis of Novel Hydrogen Bonding Arrays

We are interested in the synthesis of novel ureidopyrimidinones, incorporating functionalities to enhance hydrogen-bonding in the dimerization motifs generated. Hydrogen-bonding cytosine-based motifs have also been designed and synthesised and the dimerisation constants studied using NMR techniques with Dr. A. E. Aliev (Chemistry). This module, which can readily be difunctionalised, has been used in the synthesis of a range of novel polymers with interesting properties.

Model showing hydrogen bonding array

Organic Synthesis in Water

For a number of years we have been interested in performing carbon-carbon bond forming reactions in aqueous media. We reported the first Diels-Alder reaction in homochiral aqueous micellar media where enantioselectivities were observed. More recently we have reported the first acid catalysed aqueous Baylis-Hillman reaction and a novel mimetic of the enzymatic transketolase reaction in water. The mechanism of the TK biomimetic reaction was investigated using labelled precursors and an asymmetric version of the reaction established. We have also described a Pictet-Spengler biomimetic reaction, catalysed by phosphate.

Links:

Editorial Board member for Journal of Chemical Biology

30 year anniversary banner for New Journal of Chemistry

 Editorial Board Member of New Journal of Chemistry