James Finnigan - Academia.edu (original) (raw)

Papers by James Finnigan

Research paper thumbnail of Chemoenzymatic Cascades for the Enantioselective Synthesis of β‐Hydroxysulfides Bearing a Stereocentre at the C−O or C−S Bond by Ketoreductases

Research paper thumbnail of Multifunctional biocatalyst for conjugate reduction and reductive amination

Research paper thumbnail of A Coupled Ketoreductase‐Diaphorase Assay for the Detection of Polyethylene Terephthalate‐Hydrolyzing Activity

ChemSusChem, 2022

In the last two decades, several PET-degrading enzymes from already known microorganisms or metag... more In the last two decades, several PET-degrading enzymes from already known microorganisms or metagenomic sources have been discovered to face the growing environmental concern of polyethylene terephthalate (PET) accumulation. However, there is a limited number of high-throughput screening protocols for PET-hydrolyzing activity that avoid the use of surrogate substrates. Herein, we describe a microplate fluorescence screening assay based on the coupled activity of ketoreductases (KREDs) and diaphorase to release resorufin in the presence of the products of PET degradation. Six KREDs were identified in a commercial panel that are able to use the PET building block, ethylene glycol, as substrate. The most efficient KRED, KRED61, was combined with the diaphorase from Clostridium kluyveri to monitor the PET degradation reaction catalyzed by the thermostable variant of the cutinase-type polyesterase from Saccharomonospora viridis AHK190. The PET degradation products were measured both fluorimetrically and by HPLC, with excellent correlation between both methods.

Research paper thumbnail of An Efficient Synthesis of the Bicyclic Darunavir Side Chain Using Chemoenzymatic Catalysis

Herein, we describe a chemoenzymatic synthesis of the bicyclic fragment of Darunavir. A ketoreduc... more Herein, we describe a chemoenzymatic synthesis of the bicyclic fragment of Darunavir. A ketoreductase was identified using metagenomic mining to catalyze a highly enantio- and diastereoselective dynamic kinetic resolution of a 𝛽-ketolactone. Subsequent lactone reduction with diisobutylaluminium hydride and phase transfer cyclization affords the bicyclic acetal fragment in 39% yield over four steps.

Research paper thumbnail of Engineering a Cytidine Aminotransferase for Biocatalytic Production of the Antiviral Molnupiravir

The COVID-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufact... more The COVID-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue currently in phase 3 clinical trials as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of a cytidine aminotransferase for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >100,000 turnovers in less than 30 minutes, operates at 180 g/L substrate loading and benefits from in situ crystallization of the N-hydroxy-cytidine product (>90% yield), which can be converted to Molnupiravir by a selective 5’-acylation using Novozym® 435.

Research paper thumbnail of Synthesis of stereoenriched piperidines via chemo-enzymatic dearomatization of activated pyridines

The development of efficient and sustainable methods for the synthesis of nitrogen heterocycles i... more The development of efficient and sustainable methods for the synthesis of nitrogen heterocycles is an important goal for the chemical industry. In particular, substituted chiral piperidines are prominent targets due to their prevalence in medicinally relevant compounds and their precursors. A potential biocatalytic approach to the synthesis of this privileged scaffold would be the asymmetric dearomatization of readily assembled activated pyridines. However, nature has yet to yield a suitable biocatalyst specifically for this reaction. Here, by combining chemical synthesis and biocatalysis, we present a general chemo-enzymatic approach for the asymmetric dearomatization of activated pyridines for the preparation of substituted piperidines with precise stereochemistry. The key step involves a stereoselective one-pot amine oxidase/ene imine reductase cascade to convert N-substituted tetrahydropyridines to stereo-defined 3- and 3,4-substituted piperidines. This chemo-enzymatic approach ...

Research paper thumbnail of Cytochromes P450 (P450s): A review of the class system with a focus on prokaryotic P450s

Advances in Protein Chemistry and Structural Biology, 2020

Cytochromes P450 (P450s) are a large superfamily of heme-containing monooxygenases. P450s are fou... more Cytochromes P450 (P450s) are a large superfamily of heme-containing monooxygenases. P450s are found in all Kingdoms of life and exhibit incredible diversity, both at sequence level and also on a biochemical basis. In the majority of cases, P450s can be assigned into one of ten classes based on their associated redox partners, domain architecture and cellular localization. Prokaryotic P450s now represent a large diverse collection of annotated/known enzymes, of which many have great potential biocatalytic potential. The self-sufficient P450 classes (Class VII/VIII) have been explored significantly over the past decade, with many annotated and biochemically characterized members. It is clear that the prokaryotic P450 world is expanding rapidly, as the number of published genomes and metagenome studies increases, and more P450 families are identified and annotated (CYP families).

Research paper thumbnail of Synthesis of pharmaceutically relevant 2‐aminotetralin and 3‐aminochroman derivatives via enzymatic reductive amination

Angewandte Chemie, 2021

2-Aminotetralin and 3-aminochroman derivatives are key structural motifs present in a wide range ... more 2-Aminotetralin and 3-aminochroman derivatives are key structural motifs present in a wide range of pharmaceutically important molecules. Herein, we report an effective biocatalytic approach towards these molecules through the enantioselective reductive coupling of 2-tetralones and 3-chromanones with a diverse range of primary amine partners. Metagenomic imine reductases (IREDs) were employed as the biocatalysts, obtaining high yields and enantiocomplementary selectivity for >15 examples at preparative scale, including the precursors to Ebalzotan, Robalzotan, Alnespirone and 5-OH-DPAT. We also present a convergent chemo-enzymatic total synthesis of the Parkinson's disease therapy Rotigotine in 63% overall yield and 92% ee.

Research paper thumbnail of A Biocatalytic Approach to a Key Intermediate for the Synthesis of the COVID-19 Experimental Drug Molnupiravir

Herein we report the conversion of cytidine 2 to N-hydroxycytidine 7 catalysed by cytidine deamin... more Herein we report the conversion of cytidine 2 to N-hydroxycytidine 7 catalysed by cytidine deaminase (CD). The wild-type enzyme operates efficiently at high sustrate loadings and hydroxylamine concentrations to favor N-hydroxy-cytidine formation over uridine. Although the wild-type enzyme demonstrated good activity, we were able to further enhance the ratio of N-hydroxycytidine to uridine produced through directed evolution of CD. In particular, a T123G mutation close to the active site dramatically reduces cytidine hydrolysis activity whilst preserving desired amination activty. The approach reported provides a new route to a key intermediate for the COVID-19 experimental drug Molnupiravir 1.

Research paper thumbnail of Cytochromes P450

Insights into Enzyme Mechanisms and Functions from Experimental and Computational Methods, 2016

Cytochromes P450, a family of heme-containing monooxygenases that catalyze a diverse range of oxi... more Cytochromes P450, a family of heme-containing monooxygenases that catalyze a diverse range of oxidative reactions, are so-called due to their maximum absorbance at 450nm, ie, "Pigment-450nm," when bound to carbon monoxide. They have appeal both academically and commercially due to their high degree of regio- and stereoselectivity, for example, in the area of active pharmaceutical ingredient synthesis. Despite this potential, they often exhibit poor stability, low turnover numbers and typically require electron transport protein(s) for catalysis. P450 systems exist in a variety of functional domain architectures, organized into 10 classes. P450s are also divided into families, each of which is based solely on amino acid sequence homology. Their catalytic mechanism employs a very complex, multistep catalytic cycle involving a range of transient intermediates. Mutagenesis is a powerful tool for the development of improved biocatalysts and has been used extensively with the archetypal Class VIII P450, BM3, from Bacillus megaterium, but with the increasing scale of genomic sequencing, a huge resource is now available for the discovery of novel P450s.

Research paper thumbnail of Screening and characterization of a diverse panel of metagenomic imine reductases for biocatalytic reductive amination

Research paper thumbnail of Enantioselective Synthesis of α‐Thiocarboxylic Acids by Nitrilase Biocatalysed Dynamic Kinetic Resolution of α‐Thionitriles

Chemistry – A European Journal, 2020

Abstract The enantioselective synthesis of α‐thiocarboxylic acids by biocatalytic dynamic kinetic... more Abstract The enantioselective synthesis of α‐thiocarboxylic acids by biocatalytic dynamic kinetic resolution (DKR) of nitrile precursors exploiting nitrilase enzymes is described. A panel of 35 nitrilase biocatalysts were screened and enzymes Nit27 and Nit34 were found to catalyse the DKR of racemic α‐thionitriles under mild conditions, affording the corresponding carboxylic acids with high conversions and good‐to‐excellent ee. The ammonia produced in situ during the biocatalytic transformation favours the racemization of the nitrile enantiomers and, in turn, the DKR without the need of any external additive base.

Research paper thumbnail of Chemoenzymatic Synthesis of Substituted Azepanes by Sequential Biocatalytic Reduction and Organolithium-Mediated Rearrangement

Journal of the American Chemical Society, 2018

Imidazolidinone derivatives of a range of aromatic -amino acids, on treatment with phosgene and ... more Imidazolidinone derivatives of a range of aromatic -amino acids, on treatment with phosgene and potassium iodide, undergo a mild Bischler-Napieralski-style cyclocarbonylation reaction that generates a tricyclic lactam by insertion of a C=O group between amino acid nitrogen and the ortho position of the aryl substituent. Regio-and diastereoselective functionalization of the lactam generates a library of substituted dihydroisoquinolinones and their congeners in enantioenriched form.

Research paper thumbnail of nanoDSF as screening tool for enzyme libraries and biotechnology development

Research paper thumbnail of Role of toll-like receptors in systemic sclerosis

Expert Reviews in Molecular Medicine, 2013

Accumulative evidence demonstrates the crucial role of evolutionary conserved Toll-like receptors... more Accumulative evidence demonstrates the crucial role of evolutionary conserved Toll-like receptors (TLRs) in identifying microbial or viral compounds. TLRs are also able to recognise endogenous molecules which are released upon cell damage or stress and have been shown to play a key role in numerous autoimmune diseases including systemic sclerosis (SSc). A classic feature of SSc, is vascular injury manifested as Raynaud's phenomenon and ischaemia of the skin, resulting in the release of endogenous TLR ligands during inflammation and local tissue damage. These locally released TLR ligands bind TLRs possibly complexed to autoantibodies, and initiate intracellular signalling pathways and may be one of the mechanisms that initiate and drive autoimmunity and subsequent fibrosis. Activation of the immune system results in interferon (IFN) sensitive gene transcription. There is also an IFN gene signature in SSc peripheral blood. TLRs may represent the link between immune activation, com...

Research paper thumbnail of Serum amyloid A induces interleukin-6 in dermal fibroblasts via Toll-like receptor 2, interleukin-1 receptor-associated kinase 4 and nuclear factor-κB

Immunology, 2014

Systemic sclerosis is an autoimmune idiopathic connective tissue disease, characterized by vascul... more Systemic sclerosis is an autoimmune idiopathic connective tissue disease, characterized by vasculopathy, inflammation and fibrosis. There appears to be a link between inflammation and fibrosis, although the exact nature of the relationship is unknown. Serum amyloid A (SAA) is an acute-phase protein that is elevated up to 1000-fold in times of infection or inflammation. This acute-phase reactant, as well as being a marker of inflammation, may initiate signals in a cytokine-like manner, possibly through toll-like receptors (TLRs) promoting inflammation. This study addressed the role of SAA in initiating interleukin-6 (IL-6) production in dermal fibroblasts and the role of TLR2 in this system. We show that SAA induces IL-6 secretion in healthy dermal fibroblasts and that blockade of TLR2 with a neutralizing antibody to TLR2 or specific small interfering RNA attenuated the SAA-induced IL-6 secretion and that this was also mediated through the TLR adaptor protein IL-1 receptor-associated kinase 4. The effect is nuclear factor-jB-mediated because blockade of nuclear factor-jB reduced the induction. We also demonstrate that dermal fibroblasts express TLR2; this is functional and over-expressed in the fibroblasts of patients with systemic sclerosis. Taken together these data suggest that SAA is a danger signal that initiates IL-6 signalling in systemic sclerosis via enhanced TLR2 signalling.

Research paper thumbnail of Discovery of a multi-functional biocatalyst for asymmetric conjugate reduction-reductive amination

A major challenge in chemical synthesis is to develop catalytic systems that convert simple molec... more A major challenge in chemical synthesis is to develop catalytic systems that convert simple molecules to complex high-value products. Often these valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Of great interest are enantioenriched amine diastereomers, which are prevalent in pharmaceuticals and agrochemicals,1 yet their preparation often relies on low-efficiency multi-step synthesis.2 Herein, we report the discovery and characterisation of a multi-functional biocatalyst, which operates using a previously unreported conjugate reduction-reductive amination mechanism. This enzyme (pIR-120), identified within a metagenomic imine reductase (IRED) collection3 and originating from an unclassified Pseudomonas species, possesses an unusual active site architecture that facilitates an amine-activated conjugate alkene reduction followed by reductive amination. This enzyme enables the coupling of a broad ...

Research paper thumbnail of Chemoenzymatic Cascades for the Enantioselective Synthesis of β‐Hydroxysulfides Bearing a Stereocentre at the C−O or C−S Bond by Ketoreductases

Research paper thumbnail of Multifunctional biocatalyst for conjugate reduction and reductive amination

Research paper thumbnail of A Coupled Ketoreductase‐Diaphorase Assay for the Detection of Polyethylene Terephthalate‐Hydrolyzing Activity

ChemSusChem, 2022

In the last two decades, several PET-degrading enzymes from already known microorganisms or metag... more In the last two decades, several PET-degrading enzymes from already known microorganisms or metagenomic sources have been discovered to face the growing environmental concern of polyethylene terephthalate (PET) accumulation. However, there is a limited number of high-throughput screening protocols for PET-hydrolyzing activity that avoid the use of surrogate substrates. Herein, we describe a microplate fluorescence screening assay based on the coupled activity of ketoreductases (KREDs) and diaphorase to release resorufin in the presence of the products of PET degradation. Six KREDs were identified in a commercial panel that are able to use the PET building block, ethylene glycol, as substrate. The most efficient KRED, KRED61, was combined with the diaphorase from Clostridium kluyveri to monitor the PET degradation reaction catalyzed by the thermostable variant of the cutinase-type polyesterase from Saccharomonospora viridis AHK190. The PET degradation products were measured both fluorimetrically and by HPLC, with excellent correlation between both methods.

Research paper thumbnail of An Efficient Synthesis of the Bicyclic Darunavir Side Chain Using Chemoenzymatic Catalysis

Herein, we describe a chemoenzymatic synthesis of the bicyclic fragment of Darunavir. A ketoreduc... more Herein, we describe a chemoenzymatic synthesis of the bicyclic fragment of Darunavir. A ketoreductase was identified using metagenomic mining to catalyze a highly enantio- and diastereoselective dynamic kinetic resolution of a 𝛽-ketolactone. Subsequent lactone reduction with diisobutylaluminium hydride and phase transfer cyclization affords the bicyclic acetal fragment in 39% yield over four steps.

Research paper thumbnail of Engineering a Cytidine Aminotransferase for Biocatalytic Production of the Antiviral Molnupiravir

The COVID-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufact... more The COVID-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue currently in phase 3 clinical trials as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of a cytidine aminotransferase for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >100,000 turnovers in less than 30 minutes, operates at 180 g/L substrate loading and benefits from in situ crystallization of the N-hydroxy-cytidine product (>90% yield), which can be converted to Molnupiravir by a selective 5’-acylation using Novozym® 435.

Research paper thumbnail of Synthesis of stereoenriched piperidines via chemo-enzymatic dearomatization of activated pyridines

The development of efficient and sustainable methods for the synthesis of nitrogen heterocycles i... more The development of efficient and sustainable methods for the synthesis of nitrogen heterocycles is an important goal for the chemical industry. In particular, substituted chiral piperidines are prominent targets due to their prevalence in medicinally relevant compounds and their precursors. A potential biocatalytic approach to the synthesis of this privileged scaffold would be the asymmetric dearomatization of readily assembled activated pyridines. However, nature has yet to yield a suitable biocatalyst specifically for this reaction. Here, by combining chemical synthesis and biocatalysis, we present a general chemo-enzymatic approach for the asymmetric dearomatization of activated pyridines for the preparation of substituted piperidines with precise stereochemistry. The key step involves a stereoselective one-pot amine oxidase/ene imine reductase cascade to convert N-substituted tetrahydropyridines to stereo-defined 3- and 3,4-substituted piperidines. This chemo-enzymatic approach ...

Research paper thumbnail of Cytochromes P450 (P450s): A review of the class system with a focus on prokaryotic P450s

Advances in Protein Chemistry and Structural Biology, 2020

Cytochromes P450 (P450s) are a large superfamily of heme-containing monooxygenases. P450s are fou... more Cytochromes P450 (P450s) are a large superfamily of heme-containing monooxygenases. P450s are found in all Kingdoms of life and exhibit incredible diversity, both at sequence level and also on a biochemical basis. In the majority of cases, P450s can be assigned into one of ten classes based on their associated redox partners, domain architecture and cellular localization. Prokaryotic P450s now represent a large diverse collection of annotated/known enzymes, of which many have great potential biocatalytic potential. The self-sufficient P450 classes (Class VII/VIII) have been explored significantly over the past decade, with many annotated and biochemically characterized members. It is clear that the prokaryotic P450 world is expanding rapidly, as the number of published genomes and metagenome studies increases, and more P450 families are identified and annotated (CYP families).

Research paper thumbnail of Synthesis of pharmaceutically relevant 2‐aminotetralin and 3‐aminochroman derivatives via enzymatic reductive amination

Angewandte Chemie, 2021

2-Aminotetralin and 3-aminochroman derivatives are key structural motifs present in a wide range ... more 2-Aminotetralin and 3-aminochroman derivatives are key structural motifs present in a wide range of pharmaceutically important molecules. Herein, we report an effective biocatalytic approach towards these molecules through the enantioselective reductive coupling of 2-tetralones and 3-chromanones with a diverse range of primary amine partners. Metagenomic imine reductases (IREDs) were employed as the biocatalysts, obtaining high yields and enantiocomplementary selectivity for >15 examples at preparative scale, including the precursors to Ebalzotan, Robalzotan, Alnespirone and 5-OH-DPAT. We also present a convergent chemo-enzymatic total synthesis of the Parkinson's disease therapy Rotigotine in 63% overall yield and 92% ee.

Research paper thumbnail of A Biocatalytic Approach to a Key Intermediate for the Synthesis of the COVID-19 Experimental Drug Molnupiravir

Herein we report the conversion of cytidine 2 to N-hydroxycytidine 7 catalysed by cytidine deamin... more Herein we report the conversion of cytidine 2 to N-hydroxycytidine 7 catalysed by cytidine deaminase (CD). The wild-type enzyme operates efficiently at high sustrate loadings and hydroxylamine concentrations to favor N-hydroxy-cytidine formation over uridine. Although the wild-type enzyme demonstrated good activity, we were able to further enhance the ratio of N-hydroxycytidine to uridine produced through directed evolution of CD. In particular, a T123G mutation close to the active site dramatically reduces cytidine hydrolysis activity whilst preserving desired amination activty. The approach reported provides a new route to a key intermediate for the COVID-19 experimental drug Molnupiravir 1.

Research paper thumbnail of Cytochromes P450

Insights into Enzyme Mechanisms and Functions from Experimental and Computational Methods, 2016

Cytochromes P450, a family of heme-containing monooxygenases that catalyze a diverse range of oxi... more Cytochromes P450, a family of heme-containing monooxygenases that catalyze a diverse range of oxidative reactions, are so-called due to their maximum absorbance at 450nm, ie, "Pigment-450nm," when bound to carbon monoxide. They have appeal both academically and commercially due to their high degree of regio- and stereoselectivity, for example, in the area of active pharmaceutical ingredient synthesis. Despite this potential, they often exhibit poor stability, low turnover numbers and typically require electron transport protein(s) for catalysis. P450 systems exist in a variety of functional domain architectures, organized into 10 classes. P450s are also divided into families, each of which is based solely on amino acid sequence homology. Their catalytic mechanism employs a very complex, multistep catalytic cycle involving a range of transient intermediates. Mutagenesis is a powerful tool for the development of improved biocatalysts and has been used extensively with the archetypal Class VIII P450, BM3, from Bacillus megaterium, but with the increasing scale of genomic sequencing, a huge resource is now available for the discovery of novel P450s.

Research paper thumbnail of Screening and characterization of a diverse panel of metagenomic imine reductases for biocatalytic reductive amination

Research paper thumbnail of Enantioselective Synthesis of α‐Thiocarboxylic Acids by Nitrilase Biocatalysed Dynamic Kinetic Resolution of α‐Thionitriles

Chemistry – A European Journal, 2020

Abstract The enantioselective synthesis of α‐thiocarboxylic acids by biocatalytic dynamic kinetic... more Abstract The enantioselective synthesis of α‐thiocarboxylic acids by biocatalytic dynamic kinetic resolution (DKR) of nitrile precursors exploiting nitrilase enzymes is described. A panel of 35 nitrilase biocatalysts were screened and enzymes Nit27 and Nit34 were found to catalyse the DKR of racemic α‐thionitriles under mild conditions, affording the corresponding carboxylic acids with high conversions and good‐to‐excellent ee. The ammonia produced in situ during the biocatalytic transformation favours the racemization of the nitrile enantiomers and, in turn, the DKR without the need of any external additive base.

Research paper thumbnail of Chemoenzymatic Synthesis of Substituted Azepanes by Sequential Biocatalytic Reduction and Organolithium-Mediated Rearrangement

Journal of the American Chemical Society, 2018

Imidazolidinone derivatives of a range of aromatic -amino acids, on treatment with phosgene and ... more Imidazolidinone derivatives of a range of aromatic -amino acids, on treatment with phosgene and potassium iodide, undergo a mild Bischler-Napieralski-style cyclocarbonylation reaction that generates a tricyclic lactam by insertion of a C=O group between amino acid nitrogen and the ortho position of the aryl substituent. Regio-and diastereoselective functionalization of the lactam generates a library of substituted dihydroisoquinolinones and their congeners in enantioenriched form.

Research paper thumbnail of nanoDSF as screening tool for enzyme libraries and biotechnology development

Research paper thumbnail of Role of toll-like receptors in systemic sclerosis

Expert Reviews in Molecular Medicine, 2013

Accumulative evidence demonstrates the crucial role of evolutionary conserved Toll-like receptors... more Accumulative evidence demonstrates the crucial role of evolutionary conserved Toll-like receptors (TLRs) in identifying microbial or viral compounds. TLRs are also able to recognise endogenous molecules which are released upon cell damage or stress and have been shown to play a key role in numerous autoimmune diseases including systemic sclerosis (SSc). A classic feature of SSc, is vascular injury manifested as Raynaud's phenomenon and ischaemia of the skin, resulting in the release of endogenous TLR ligands during inflammation and local tissue damage. These locally released TLR ligands bind TLRs possibly complexed to autoantibodies, and initiate intracellular signalling pathways and may be one of the mechanisms that initiate and drive autoimmunity and subsequent fibrosis. Activation of the immune system results in interferon (IFN) sensitive gene transcription. There is also an IFN gene signature in SSc peripheral blood. TLRs may represent the link between immune activation, com...

Research paper thumbnail of Serum amyloid A induces interleukin-6 in dermal fibroblasts via Toll-like receptor 2, interleukin-1 receptor-associated kinase 4 and nuclear factor-κB

Immunology, 2014

Systemic sclerosis is an autoimmune idiopathic connective tissue disease, characterized by vascul... more Systemic sclerosis is an autoimmune idiopathic connective tissue disease, characterized by vasculopathy, inflammation and fibrosis. There appears to be a link between inflammation and fibrosis, although the exact nature of the relationship is unknown. Serum amyloid A (SAA) is an acute-phase protein that is elevated up to 1000-fold in times of infection or inflammation. This acute-phase reactant, as well as being a marker of inflammation, may initiate signals in a cytokine-like manner, possibly through toll-like receptors (TLRs) promoting inflammation. This study addressed the role of SAA in initiating interleukin-6 (IL-6) production in dermal fibroblasts and the role of TLR2 in this system. We show that SAA induces IL-6 secretion in healthy dermal fibroblasts and that blockade of TLR2 with a neutralizing antibody to TLR2 or specific small interfering RNA attenuated the SAA-induced IL-6 secretion and that this was also mediated through the TLR adaptor protein IL-1 receptor-associated kinase 4. The effect is nuclear factor-jB-mediated because blockade of nuclear factor-jB reduced the induction. We also demonstrate that dermal fibroblasts express TLR2; this is functional and over-expressed in the fibroblasts of patients with systemic sclerosis. Taken together these data suggest that SAA is a danger signal that initiates IL-6 signalling in systemic sclerosis via enhanced TLR2 signalling.

Research paper thumbnail of Discovery of a multi-functional biocatalyst for asymmetric conjugate reduction-reductive amination

A major challenge in chemical synthesis is to develop catalytic systems that convert simple molec... more A major challenge in chemical synthesis is to develop catalytic systems that convert simple molecules to complex high-value products. Often these valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Of great interest are enantioenriched amine diastereomers, which are prevalent in pharmaceuticals and agrochemicals,1 yet their preparation often relies on low-efficiency multi-step synthesis.2 Herein, we report the discovery and characterisation of a multi-functional biocatalyst, which operates using a previously unreported conjugate reduction-reductive amination mechanism. This enzyme (pIR-120), identified within a metagenomic imine reductase (IRED) collection3 and originating from an unclassified Pseudomonas species, possesses an unusual active site architecture that facilitates an amine-activated conjugate alkene reduction followed by reductive amination. This enzyme enables the coupling of a broad ...