Template-dependent DNA ligation for the synthesis of modified oligonucleotides.

Chemical modification of DNA is a common strategy to improve the properties of oligonucleotides, particularly for therapeutics and nanotechnology. Existing synthetic methods essentially rely on phosphoramidite chemistry or the polymerization of nucleoside triphosphates but are limited in terms of size, scalability, and sustainability. Herein, we report a robust alternative method for the de novo synthesis of modified oligonucleotides using template-dependent DNA ligation of shortmer fragments.

Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor.

Semi-rational enzyme engineering is a powerful method to develop industrial biocatalysts. Profiting from advances in molecular biology and bioinformatics, semi-rational approaches can effectively accelerate enzyme engineering campaigns. Here, we present the optimization of a ketoreductase from Sporidiobolus salmonicolor for the chemo-enzymatic synthesis of ipatasertib, a potent protein kinase B inhibitor.

Artificial nucleotide codons for enzymatic DNA synthesis.

Herein, we report the high-yielding solid-phase synthesis of unmodified and chemically modified trinucleotide triphosphates (dNTPs). These synthetic codons can be used for enzymatic DNA synthesis provided their scaffold is stabilized with phosphorothioate units. Enzymatic synthesis with three rather than one letter nucleotides will be useful to produce xenonucleic acids (XNAs) and for selection of modified functional nucleic acids.

Towards the controlled enzymatic synthesis of LNA containing oligonucleotides.

Enzymatic, XNA synthesis represents an alternative method for the production of long oligonucleotides containing chemical modifications at distinct locations. While such an approach is currently developed for DNA, controlled enzymatic synthesis of XNA remains at a relative state of infancy. In order to protect the masking groups of 3'--modified LNA and DNA nucleotides against removal caused by phosphatase and esterase activities of polymerases, we report the synthesis and biochemical characterization of nucleotides equipped with ether and robust ester moieties.

Mass Balance of the Indoleamine 2,3-Dioxygenase Inhibitor Navoximod (GDC-0919) in Rats and Dogs: Unexpected Cyanide Release from Imidazo[5,1-]isoindole and Species Differences in Glucuronidation.

Navoximod (GDC-0919) is a small molecule inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1) developed to reduce T cell immunosuppression associated with cancer. This study describes the absorption, metabolism, and excretion (AME) of navoximod in rats and dogs after a single oral dose of [C]-navoximod. An unexpected thiocyanate metabolite M1 and a chiral inversion metabolite M51 were captured as the major circulating metabolites in rats, accounting for 30% and 18% of 0-24 hours exposure, respectively.

Evaluation of 3'-phosphate as a transient protecting group for controlled enzymatic synthesis of DNA and XNA oligonucleotides.

Chemically modified oligonucleotides have advanced as important therapeutic tools as reflected by the recent advent of mRNA vaccines and the FDA-approval of various siRNA and antisense oligonucleotides. These sequences are typically accessed by solid-phase synthesis which despite numerous advantages is restricted to short sequences and displays a limited tolerance to functional groups. Controlled enzymatic synthesis is an emerging alternative synthetic methodology that circumvents the limitations of traditional solid-phase synthesis.

Screening methods for enzyme-mediated alcohol oxidation.

Alcohol oxidation for the generation of carbonyl groups, is an essential reaction for the preparation of fine chemicals. Although a number of chemical procedures have been reported, biocatalysis is a promising alternative for more sustainable and selective processes. To speed up the discovery of novel (bio)catalysts for industrial applications, efficient screening approaches need to be established.

Towards the enzymatic synthesis of phosphorothioate containing LNA oligonucleotides.

Therapeutic oligonucleotides require the addition of multiple chemical modifications to the nucleosidic scaffold in order to improve their drug delivery efficiency, cell penetration capacity, biological stability, and pharmacokinetic properties. This chemical modification pattern is often accompanied by a synthetic burden and by limitations in sequence length. Here, we have synthesized a nucleoside triphosphate analog bearing two simultaneous modifications at the level of the sugar (LNA) and the backbone (thiophosphate) and have tested its compatibility with enzymatic DNA synthesis which could abrogate some of these synthetic limitations.

The Swiss Industrial Biocatalysis Consortium (SIBC): Past, Present and Future.

Since its inception in 2004, the Swiss Industrial Biocatalysis Consortium (SIBC) has brought together scientists from the Pharma, Fine Chemicals, Agrochemicals and Flavor and Fragrance Industries with the goal of promoting biocatalysis inside and outside of Switzerland as well as providing mutual benefits in the form of pre-competitive knowledge sharing. One of the 'founding fathers' of the SIBC was of course Oreste Ghisalba, whom we are honoring here in this special edition. The history of the SIBC as well as current activities and future challenges will be presented.

Human Enzymes for Organic Synthesis.

Human enzymes have been widely studied in various disciplines. The number of reactions taking place in the human body is vast, and so is the number of potential catalysts for synthesis. Herein, we focus on the application of human enzymes that catalyze chemical reactions in course of the metabolism of drugs and xenobiotics.

Amine Transaminase Engineering for Spatially Bulky Substrate Acceptance.

Amine transaminase (ATA) catalyzing stereoselective amination of prochiral ketones is an attractive alternative to transition metal catalysis. As wild-type ATAs do not accept sterically hindered ketones, efforts to widen the substrate scope to more challenging targets are of general interest. We recently designed ATAs to accept aromatic and thus planar bulky amines, with a sequence-based motif that supports the identification of novel enzymes.

Protein-engineering of an amine transaminase for the stereoselective synthesis of a pharmaceutically relevant bicyclic amine.

Application of amine transaminases (ATAs) for stereoselective amination of prochiral ketones represents an environmentally benign and economically attractive alternative to transition metal catalyzed asymmetric synthesis. However, the restrictive substrate scope has limited the conversion typically to non-sterically demanding scaffolds. Recently, we reported on the identification and design of fold class I ATAs that effect a highly selective asymmetric synthesis of a set of chiral aromatic bulky amines from the corresponding ketone precursors in high yield.

Identification of (S)-selective transaminases for the asymmetric synthesis of bulky chiral amines.

The use of transaminases to access pharmaceutically relevant chiral amines is an attractive alternative to transition-metal-catalysed asymmetric chemical synthesis. However, one major challenge is their limited substrate scope. Here we report the creation of highly active and stereoselective transaminases starting from fold class I.

Human xanthine oxidase recombinant in E. coli: A whole cell catalyst for preparative drug metabolite synthesis.

Human xanthine oxidoreductase (XOR), which is responsible for the final steps of the purine metabolism pathway and involved in oxidative drug metabolism, was successfully expressed in Escherichia coli BL21(DE3) Gold. Recombinant human (rh) XOR yielded higher productivity with the gene sequence optimized for expression in E.coli than with the native gene sequence.

Human FMO2-based microbial whole-cell catalysts for drug metabolite synthesis.

Background: Getting access to authentic human drug metabolites is an important issue during the drug discovery and development process. Employing recombinant microorganisms as whole-cell biocatalysts constitutes an elegant alternative to organic synthesis to produce these compounds. The present work aimed for the generation of an efficient whole-cell catalyst based on the flavin monooxygenase isoform 2 (FMO2), which is part of the human phase I metabolism.

Expanding the Imine Reductase Toolbox by Exploring the Bacterial Protein-Sequence Space.

Recent investigations on imine reductases (IREDs) have enriched the toolbox of potential catalysts for accessing chiral amines, which are important building blocks for the pharmaceutical industry. Herein, we describe the characterization of 20 new IREDs. A C-terminal domain clustering of the bacterial protein-sequence space was performed to identify the novel IRED candidates.

Expression of recombinant human flavin monooxygenase and moclobemide-N-oxide synthesis on multi-mg scale.

A panel of human flavin monooxygenases were heterologously expressed in E. coli to obtain ready-to-use biocatalysts for the in vitro preparation of human drug metabolites. Moclobemide-N-oxide (65 mg) was the first high-priced metabolite prepared with recombinant hFMO3 on the multi-milligram scale.

Metabolite identification via LC-SPE-NMR-MS of the in vitro biooxidation products of a lead mGlu5 allosteric antagonist and impact on the improvement of metabolic stability in the series.

Detailed information on the metabolic fate of lead compounds can be a powerful tool for an informed approach to the stabilization of metabolically labile compounds in the lead optimization phase. The combination of high performance liquid chromatography (HPLC) with nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) has been used to give comprehensive structural data on metabolites of novel drugs in development. Recently, increased automation and the embedding of on-line solid-phase extraction (SPE) into a integrated LC-SPE-NMR-MS system have improved enormously the detection limits of this approach.

Asymmetric reduction of racemic sulfoxides by dimethyl sulfoxide reductases from Rhodobacter capsulatus, Escherichia coli and Proteus species.

The enantioselective reduction of racemic sulfoxides by dimethyl sulfoxide reductases from Rhodobacter capsulatus, Escherichia coli, Proteus mirabilis and Proteus vulgaris was investigated. Purified dimethyl sulfoxide reductase from Rhodobacter capsulatus catalysed the selective removal of (S)-methyl p-tolyl sulfoxide from a racemic mixture of methyl p-tolyl sulfoxide and resulted in an 88% recovery of enantiomerically pure (R)-methyl p-tolyl sulfoxide. Rhodobacter capsulatus was shown to be able to grow photoheterotrophically in the presence of certain chiral sulfoxides under conditions where a sulfoxide is needed as an electron sink.

2-phenylethylamine catabolism by Escherichia coli K-12: gene organization and expression.

A gene encoding phenylacetaldehyde dehydrogenase (PAD), the enzyme involved together with a copper-topaquinone-containing amine oxidase in the initial steps of 2-phenylethylamine catabolism, was located at 31.1 min on the Escherichia coli K-12 genetic map. It was immediately adjacent to the gene encoding the amine oxidase but transcribed in the opposite direction.