Expanding the synthetic scope of biocatalysis by enzyme discovery and protein engineering

This paper is dedicated to the memory of Jon Williams who was a pioneer of chemo-enzymatic catalysis, a field which has grown significantly thanks to his vision and ideas.Despite the growing impact of enzyme catalysis in industrial chemistry, the full potential of this technology is yet to be unlocked. Accessing new chemistries and expanding the scope of existing reactions is necessary in order to make biocatalysis a pivotal technology in the manufacturing of chemicals across the whole industrial spectrum. This review highlights how the biocatalytic toolbox for synthetic chemistry has recently been expanded by extending the scope of industrially relevant reactions, and the addition of new reactions via enzyme discovery or protein engineering.We would like to thank the Industrial Biotechnology Innovation Centre (IBioIC) and Biotechnology and Biological Sciences Research Council (BBSRC) for the awarding the CASE studentship to J.R.M from Prozomix Ltd. J.M − S thanks the ARAID Foundation for personal funding and the Research Group E07_20R for scientific support. N.J.T is grateful to the ERC for the award of an Advanced Grant (Grant number 742987).Peer reviewe

Controlling Regioseletivity and Enantioselectivity in C–H activation

Abstract The direct functionalisation of C–H bonds has emerged in recent years as an efficient and atom-economic alternative to the traditional cross coupling reaction. One of the challenges towards this goal is the selective transformation of a particular C–H bond amongst many other C–H bonds. This thesis describes studies on the use of CO2 as a traceless promoter for controlling meta-regioselective arylation of phenols and a separate investigation into an enantioselective arylation of pre-chiral η6-arene tricarbonyl chromium complexes. The introduction provides a general review of recent advances in the use of coordinating moieties as directing groups for selective activation of aromatic C–H bonds. The challenge of meta-selective C–H activation and the direct functionalisation of phenols is assessed and discussed. To overcome the scarce methods for accessing meta-arylated phenols, our objective was to develop an efficient methodology for the meta-arylation of phenols. Our strategy includes an ortho-carboxylation of the phenol followed by ortho-arylation of the salicylic acids and the subsequent protodecarboxylation to afford meta-arylphenols is proposed. A step-wise approach towards our aim is applied. First, a convenient and efficient method for the carboxylation of phenols is presented in Chapter 2. Then, a tandem arylation/decarboxylation reaction of salicylic acids is described in Chapter 3. Last, an efficient methodology for the meta-arylation of phenols via carboxylation/arylation/ decarboxylation processes in one-pot is presented in the following section in Chapter 3. To demonstrate the utility of this powerful methodology, an efficient synthesis of ϒ-secretase inhibitor and the further transformation of the meta-arylphenols are shown. Furthermore, an alternative method is described for the synthesis of meta-arylphenols via a tandem oxidation/arylation/decarboxylation reaction of salicylaldehydes. In Chapter 4, an approach towards the development of enantioselective C–H arylation leading to planar chirality is stated. Preliminary results are presented for this ongoing project.China Scholarship Council and Queen Mary University of Londo

Approaches towards C-10-hydroxylated analogues of narciclasine

Discussed in this thesis is the synthesis of a C10-benzyloxy unnatural derivative of narciclasine. The described approach involves the use of homochiral cyclohexadiene diols, products of the biocatalytic transformation of aromatic compounds, as precursors to ring C, and of highly oxygenated aromatic molecules to construct ring A. The document also reports a detailed account of the protocols studied for the intramolecular formation of ring B. Experimental data and spectral data are provided for the novel compounds

Expanding the repertoire of enzymatic C-C bond formation with one-carbon units

C-C bonds are the basis for virtually all organic molecules on Earth. In nature, hundreds of different enzymes catalyze reactions in which C-C bonds are formed. A major task of these enzymes is the fixation of carbon, i.e. a C-C bond formation with at least one-carbon (C1) molecule. Most of these enzymes utilize electrophilic C1 species to fix carbon, while only very few use nucleophilic or radical C1 species. In this work the repertoire of enzymatic C-C bond formation was expanded by 4 new examples, 3 of which are based on a C1 nucleophile and one on a C1 radical. The thiamine diphosphate (ThDP)-dependent enzyme oxalyl-CoA decarboxylase (OXC) generates a highly reactive carbanion/enamine intermediate that is protonated and released as formyl-CoA. Here it was shown that this intermediate can also undergo C-C bond formation with an electrophilic carbonyl center. This insight allowed to establish three novel C-C bond formation reactions. First, it was demonstrated that benzaldehyde serves as an excellent electrophile, giving rise to mandelyl-CoA. In combination with oxalyl-CoA synthetase and a thioesterase this enabled the one-pot synthesis of aromatic (S)-α-hydroxy acids with enantiomeric excess up to 99%. Second, it was found that OXC can also generate the carbanion/enamine intermediate from formyl-CoA. By coupling to exergonic reactions at high CO2 concentrations, OXC was shown to be reversible, that is, it can carboxylate formyl-CoA to oxalyl-CoA. Third, OXC was engineered to accept the C1 molecule formaldehyde as substrate, producing glycolyl-CoA. Through directed evolution the catalytic efficiency was improved by a factor of ~200 and the resulting variant was successfully employed in a whole-cell biocatalyst for the production of glycolate from formaldehyde. The glycyl radical enzyme pyruvate formate-lyase (PFL) can abstract a hydrogen atom from formate, thereby generating a highly reactive formyl radical that undergoes C-C bond formation with an acetyl moiety stemming from acetyl-CoA. Here it was shown that PFL exhibits promiscuous activity with glycolyl-CoA. Based on this activity, a pathway was established in vitro that converts glycolate and formate to glycerate. These additions to the toolbox of enzymatic C-C bond formation could contribute to achieve synthetic carbon fixation pathways in the future. Such pathways are thought to be instrumental in achieving a carbon neutral economy

Synthetic Enzyme-Catalyzed CO2 Fixation Reactions.

In recent years, (de)carboxylases that catalyze reversible (de)carboxylation have been targeted for application as carboxylation catalysts. This has led to the development of proof-of-concept (bio)synthetic CO2 fixation routes for chemical production. However, further progress towards industrial application has been hampered by the thermodynamic constraint that accompanies fixing CO2 to organic molecules. In this Review, biocatalytic carboxylation methods are discussed with emphases on the diverse strategies devised to alleviate the inherent thermodynamic constraints and their application in synthetic CO2 -fixation cascades

Earth-Abundant Transition Metal-Catalyzed Electro-Reductive Cross-Electrophile Coupling Reactions and C–H Activation by Cobaltaelectro-Catalysis

Within the last decades, immense progresses have been made with transition metal-catalyzed cross-electrophile reactions and C–H activation reactions. The merger of electrocatalysis with such powerful methods have further enabled a series of more sustainable synthetical protocols for tackling issues such as, the alarming proliferation of CO2 in the atmosphere and the increased demand for more effective pharmaceuticals. This thesis illustrates the electro-catalytic use of earth-abundant and cost-effective cobalt catalyst through the efficient Carbon Capture and Utilization (CCU) strategy for the carboxylation reactions of allylic chlorides with atmospheric CO2. In addition, nickelaelectro-catalytic thiolation of alkyl bromides with electrophilic thiosulfonates have also been successfully described.2021-12-2

Preparation of added-value molecules derived from 5-hmf by reduction or oxidation using continuous flow synthesis

Treball Final de Màster Universitari en Química Sostenible (Pla de 2015). Codi: SJE020. Curs acadèmic: 2019/2020The use of biomass as a carbon source in order to create platform molecules that serve as a shuttle for the synthesis of high value-added products and thus abandoning the use of fossil resources is a reality. Molecules such as 5-hydroxymethylfurfural contain a great application potential, since it can lead to the formation of numerous products of interest in numerous sectors, such as polymers or biofuels. In turn, the manufacture of HMF from continuous flow technologies for the scaling-up of the production to an industrial level could be made possible, despite the challenges that still need to be overcome in terms of production and scalability. However, it is a promising path that, combined with the right technology or strategy that makes separation and purification economically viable processes, can be disruptive

Evaluation of the Immunoprotective Potential of Recombinant Paraflagellar Rod Proteins of Trypanosoma evansi in Mice

Trypanosomosis, caused by Trypanosoma evansi, is an economically significant disease of livestock. Systematic antigenic variation by the parasite has undermined prospects for the development of a protective vaccine that targets the immunodominant surface antigens, encouraging exploration of alternatives. The paraflagellar rod (PFR), constituent proteins of the flagellum, are prominent non-variable vaccine candidates for T. evansi owing to their strategic location. Two major PFR constituent proteins, PFR1 (1770bp) and PFR2 (1800bp), were expressed using Escherichia coli. Swiss albino mice were immunized with the purified recombinant TePFR1 (89KDa) and TePFR2 (88KDa) proteins, as well as with the mix of the combined proteins at equimolar concentrations, and subsequently challenged with virulent T. evansi. The PFR-specific humoral response was assessed by ELISA. Cytometric bead-based assay was used to measure the cytokine response and flow cytometry for quantification of the cytokines. The recombinant TePFR proteins induced specific humoral responses in mice, including IgG1 followed by IgG2a and IgG2b. A balanced cytokine response induced by rTePFR 1 and 2 protein vaccination associated with extended survival and improved control of parasitemia following lethal challenge. The observation confirms the immunoprophylactic potential of the covert antigens of T. evansi

New CO₂ chemistry for fine chemical synthesis

Includes abstract.~Includes bibliographical references (leaves 89-96).There is a great need in the chemical industry for developing CO2 as a C1 building block as an important step towards "green chemistry". CO2 is also attractive as a chemical feedstock because it is readily available, inexpensive, nontoxic and it can replace toxic building blocks such as phosgene and CO. Industrially, megatons of CO2 are used each year for the production of urea, inorganic carbonates, salicylic acid, and polycarbonates, yet this is still miniscule compared to the immense potential that is still yet to be harnessed in using this versatile building block. This thesis discusses how a novel methodology was developed for synthesising a benzotriazole (Bt) urea directly from CO2 in a two-step, one-pot synthesis. The procedure involves trapping CO2 with a primary or secondary amine in the presence of DBU, and reaction of the resultant carbamate salt with triphenylphosphine and chlorobenzotriazole (BtCl) to produce Bt ureas in moderate to high yields. The Bt group may serve as a leaving group in nucleophilic substitution reactions, therefore it is also shown here how the Bt urea presents itself as a precursor for an array of useful organic intermediates. These intermediates include ureas, amides, S-thiocarbamates and sulfonylureas