I03-Macromolecular Crystallography
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Qinglong
Meng
,
Caecilie
Benckendorff
,
Charlotte
Morrill
,
Ying
Zhuo
,
Annette
Egerström
,
Aisling
Ní Cheallaigh
,
Sasha R.
Derrington
,
Richard
Obexer
,
Mary
Ortmayer
,
Colin W.
Levy
,
James D.
Finnigan
,
Simon J.
Charnock
,
Nicholas J.
Turner
,
Gavin J.
Miller
,
Sarah L.
Lovelock
Diamond Proposal Number(s):
[31850]
Open Access
Abstract: The rapid emergence of RNA therapeutics has highlighted the need for more efficient, scalable and sustainable methods for their manufacture. Biocatalytic approaches hold particular promise, but rely on a secure, sustainable and low-cost supply of nucleoside triphosphate (NTP) building blocks, including those containing chemical modifications. Here we report the development of a biocatalytic approach and engineered enzymes to convert widely available nucleosides into NTPs featuring pharmaceutically relevant modifications using inexpensive phosphate donors. Importantly our strategy obviates the need for ATP as a phosphate donor that complicates NTP isolation using existing methods. To showcase the utility of our approach, we employ an engineered acid phosphatase, polyphosphate kinase and acetate kinase to produce 2′-O-methoxyethyl-ATP (2′-MOE-ATP) and 2′-fluoro-ATP, key building blocks of commercial therapeutics. Finally, we show that crude NTPs from our process can be used directly in enzymatic oligonucleotide synthesis, obviating the need for costly NTP isolation or purification steps.
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Dec 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[32736]
Open Access
Abstract: Enantioselective reduction of hydrazones provides a convergent and versatile route to synthesize hydrazine-containing motifs that are commonly found in pharmaceuticals and agrochemicals. However current methods require the use of precious metals, costly chiral ligands and/or forcing reaction conditions. Here, we report the development of a biocatalytic approach for enantioselective hydrazone reduction using engineered imine reductases. Following evaluation of an in-house panel of >400 IRED sequences, we identified a single IR361 I127F L179V variant that promotes reduction of Cbz-protected hydrazones. Introduction of an additional two mutations via directed evolution afforded HRED1.1 that is 20-fold more active than the parent template and promotes reduction of a variety of protected hydrazones in high yields and selectivities (>99% e.e.), including in preparative scale biotransformations. Structural analysis of HRED1.1 provides insights into the origins of its unique hydrazone reductase activity. This study offers a powerful biocatalytic route to synthesize valuable chiral hydrazine products and further expands the impressive range of transformations accessible with engineered imine reductases.
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Apr 2025
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I24-Microfocus Macromolecular Crystallography
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Arnau Rué
Casamajo
,
Yuqi
Yu
,
Christian
Schnepel
,
Charlotte
Morrill
,
Rhys
Barker
,
Colin W.
Levy
,
James
Finnigan
,
Victor
Spelling
,
Kristina
Westerlund
,
Mark
Petchey
,
Robert J.
Sheppard
,
Richard J.
Lewis
,
Francesco
Falcioni
,
Martin A.
Hayes
,
Nicholas J.
Turner
Open Access
Abstract: Novel building blocks are in constant demand during the search for innovative bioactive small molecule therapeutics by enabling the construction of structure–activity–property–toxicology relationships. Complex chiral molecules containing multiple stereocenters are an important component in compound library expansion but can be difficult to access by traditional organic synthesis. Herein, we report a biocatalytic process to access a specific diastereomer of a chiral amine building block used in drug discovery. A reductive aminase (RedAm) was engineered following a structure-guided mutagenesis strategy to produce the desired isomer. The engineered RedAm (IR-09 W204R) was able to generate the (S,S,S)-isomer 3 in 45% conversion and 95% ee from the racemic ketone 2. Subsequent palladium-catalyzed deallylation of 3 yielded the target primary amine 4 in a 73% yield. This engineered biocatalyst was used at preparative scale and represents a potential starting point for further engineering and process development.
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Oct 2023
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I04-Macromolecular Crystallography
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Amelia K.
Gilio
,
Thomas W.
Thorpe
,
Alex
Heyam
,
Mark R.
Petchey
,
Balázs
Pogrányi
,
Scott P.
France
,
Roger M.
Howard
,
Michael J.
Karmilowicz
,
Russell
Lewis
,
Nicholas
Turner
,
Gideon
Grogan
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Imine reductases (IREDs) catalyze the asymmetric reduction of cyclic imines, but also in some cases the coupling of ketones and amines to form secondary amine products in an enzyme-catalyzed reductive amination (RedAm) reaction. Enzymatic RedAm reactions have typically used small hydrophobic amines, but many interesting pharmaceutical targets require that larger amines be used in these coupling reactions. Following the identification of IR77 from Ensifer adhaerens as a promising biocatalyst for the reductive amination of cyclohexanone with pyrrolidine, we have characterized the ability of this enzyme to catalyze couplings with larger bicyclic amines such as isoindoline and octahydrocyclopenta(c)pyrrole. By comparing the activity of IR77 with reductions using sodium cyanoborohydride in water, it was shown that, while the coupling of cyclohexanone and pyrrolidine involved at least some element of reductive amination, the amination with the larger amines likely occurred ex situ, with the imine recruited from solution for enzyme reduction. The structure of IR77 was determined, and using this as a basis, structure-guided mutagenesis, coupled with point mutations selecting improving amino acid sites suggested by other groups, permitted the identification of a mutant A208N with improved activity for amine product formation. Improvements in conversion were attributed to greater enzyme stability as revealed by X-ray crystallography and nano differential scanning fluorimetry. The mutant IR77-A208N was applied to the preparative scale amination of cyclohexanone at 50 mM concentration, with 1.2 equiv of three larger amines, in isolated yields of up to 93%.
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Jan 2023
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I03-Macromolecular Crystallography
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Vanessa
Harawa
,
Thomas W.
Thorpe
,
James R.
Marshall
,
Jack J.
Sangster
,
Amelia K.
Gilio
,
Lucian
Pirvu
,
Rachel S.
Heath
,
Antonio
Angelastro
,
James D.
Finnigan
,
Simon J.
Charnock
,
Jordan W.
Nafie
,
Gideon
Grogan
,
Roger C.
Whitehead
,
Nicholas J.
Turner
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: 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 is 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 has proved useful for key transformations in the syntheses of antipsychotic drugs Preclamol and OSU-6162, as well as for the preparation of two important intermediates in synthetic routes of the ovarian cancer monotherapeutic Niraparib.
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Nov 2022
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I03-Macromolecular Crystallography
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Thomas W.
Thorpe
,
James R.
Marshall
,
Vanessa
Harawa
,
Rebecca E.
Ruscoe
,
Anibal
Cuetos
,
James D.
Finnigan
,
Antonio
Angelastro
,
Rachel S.
Heath
,
Fabio
Parmeggiani
,
Simon J.
Charnock
,
Roger M.
Howard
,
Rajesh
Kumar
,
David S. B.
Daniels
,
Gideon
Grogan
,
Nicholas J.
Turner
Diamond Proposal Number(s):
[9948]
Abstract: Chiral amine diastereomers are ubiquitous in pharmaceuticals and agrochemicals1, yet their preparation often relies on low-efficiency multi-step synthesis2. These valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Herein we characterize a multifunctional biocatalyst for amine synthesis, which operates using a mechanism that is, to our knowledge, previously unreported. This enzyme (EneIRED), identified within a metagenomic imine reductase (IRED) collection3 and originating from an unclassified Pseudomonas species, possesses an unusual active site architecture that facilitates amine-activated conjugate alkene reduction followed by reductive amination. This enzyme can couple a broad selection of α,β-unsaturated carbonyls with amines for the efficient preparation of chiral amine diastereomers bearing up to three stereocentres. Mechanistic and structural studies have been carried out to delineate the order of individual steps catalysed by EneIRED, which have led to a proposal for the overall catalytic cycle. This work shows that the IRED family can serve as a platform for facilitating the discovery of further enzymatic activities for application in synthetic biology and organic synthesis.
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Apr 2022
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B18-Core EXAFS
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Diamond Proposal Number(s):
[20618]
Open Access
Abstract: Bimetallic nanoparticle catalysts have attracted considerable attention due to their unique chemical and physical properties. The ability of metal‐reducing bacteria to produce highly catalytically active monometallic nanoparticles is well known; however, the properties and catalytic activity of bimetallic nanoparticles synthesized with these organisms is not well understood. Here, we report the one‐pot biosynthesis of Pd/Ag (bio‐Pd/Ag) and Pd/Au (bio‐Pd/Au) nanoparticles using the metal‐reducing bacterium, Shewanella oneidensis, under mild conditions. Energy dispersive X‐ray analyses performed using scanning transmission electron microscopy (STEM) revealed the presence of both metals (Pd/Ag or Pd/Au) in the biosynthesized nanoparticles. X‐ray absorption near‐edge spectroscopy (XANES) suggested a significant contribution from Pd(0) and Pd(II) in both bio‐Pd/Ag and bio‐Pd/Au, with Ag and Au existing predominately as their metallic forms. Extended X‐ray absorption fine‐structure spectroscopy (EXAFS) supported the presence of multiple Pd species in bio‐Pd/Ag and bio‐Pd/Au, as inferred from Pd–Pd, Pd–O and Pd–S shells. Both bio‐Pd/Ag and bio‐Pd/Au demonstrated greatly enhanced catalytic activity towards Suzuki–Miyaura cross‐coupling compared to a monometallic Pd catalyst, with bio‐Pd/Ag significantly outperforming the others. The catalysts were very versatile, tolerating a wide range of substituents. This work demonstrates a green synthesis method for novel bimetallic nanoparticles that display significantly enhanced catalytic activity compared to their monometallic counterparts.
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Mar 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9948, 13587]
Open Access
Abstract: Chiral primary amines are important intermediates in the synthesis of pharmaceutical compounds. Fungal reductive aminases (RedAms) are NADPH-dependent dehydrogenases that catalyse reductive amination of a range of ketones with short-chain primary amines supplied in an equimolar ratio to give corresponding secondary amines. Herein we describe structural and biochemical characterisation as well as synthetic applications of two RedAms from Neosartorya spp. (NfRedAm and NfisRedAm) that display a distinctive activity amongst fungal RedAms, namely a superior ability to use ammonia as the amine partner. Using these enzymes, we demonstrate the synthesis of a broad range of primary amines, with conversions up to >97% and excellent enantiomeric excess. Temperature dependent studies showed that these homologues also possess greater thermal stability compared to other enzymes within this family. Their synthetic applicability is further demonstrated by the production of several primary and secondary amines with turnover numbers (TN) up to 14[thin space (1/6-em)]000 as well as continous flow reactions, obtaining chiral amines such as (R)-2-aminohexane in space time yields up to 8.1 g L−1 h−1. The remarkable features of NfRedAm and NfisRedAm highlight their potential for wider synthetic application as well as expanding the biocatalytic toolbox available for chiral amine synthesis.
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May 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17773]
Open Access
Abstract: The reductive amination of prochiral ketones using biocatalysts has been of great interest to the pharmaceutical industry in the last decade for integrating novel strategies in the production of chiral building blocks with the intent of minimizing impact on the environment. Amongst the enzymes able to catalyze the direct amination of prochiral ketones, pyridoxal 5′-phosphate (PLP) dependent ω-transaminases have shown great promise as versatile industrial biocatalysts with high selectivity, regioselectivity, and broad substrate scope. Herein the biochemical characterization of a putrescine transaminase from Pseudomonas putida (Pp-SpuC) was performed, which showed an optimum pH and temperature of 8.0 and 60°C, respectively. To gain further structural insight of this enzyme, we crystallized the protein in the apo form and determined the structure to 2.1 Å resolution which revealed a dimer that adopts a class I transaminase fold comparable to other class III transaminases. Furthermore we exploited its dual substrate recognition for biogenic diamines (i.e., cadaverine) and readily available monoamines (i.e., isopropylamine) for the synthesis of benzylamine derivatives with excellent product conversions and extremely broad substrate tolerance.
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Dec 2018
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948]
Abstract: Reductive Aminases (RedAms) catalyze the asymmetric reductive amination of ketones with primary amines to give secondary amine products. RedAms have great potential for the synthesis of bioactive chiral amines, however, insights into their mechanism are currently limited. Comparative studies on reductive amination of cyclohexanone with allylamine in the presence of RedAms, imine reductases (IREDs) or NaBH3CN support the distinctive activity of RedAms in catalyzing both imine formation and reduction in the reaction. Structures of AtRedAm from Aspergillus terreus, in complex with NADPH and ketone and amine substrates, along with kinetic analysis of active-site mutants, reveal modes of substrate binding, the basis for the specificity of RedAms for reduction of imines over ketones, and the importance of domain flexibility in bringing the reactive participants together for the reaction. This information is used to propose a mechanism for their action and also to expand the substrate specificity of RedAms using protein engineering.
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Oct 2018
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