Engineering Hydroxylase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of a Key Intermediate to Belzutifan.

Biocatalytic oxidations are an emerging technology for selective C-H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non-heme iron and α-ketoglutarate-dependent dioxygenase for the direct stereo- and regio-selective hydroxylation of a non-native fluoroindanone en route to the oncology treatment belzutifan, replacing a five-step chemical synthesis with a direct enantioselective hydroxylation.

Scalable Asymmetric Synthesis of MK-8998, a T-Type Calcium Channel Antagonist.

Two scalable and efficient synthetic routes for the synthesis of a T-type calcium channel antagonist MK-8998 were developed from a simple pyridine building block. The key step to set the stereochemistry relied on either chiral rhodium catalyst-mediated asymmetric hydrogenation of an enamide or transamination of an arylketone that provided the corresponding product in high enantioselectivity and high yield.

Recent preparative applications of redox enzymes.

Redox enzymes offer many powerful transformations for the efficient industrial-scale synthesis of diverse chemicals desired by society. Here we survey recent preparative applications of redox enzymes, highlighting both mature enzyme platforms and promising technologies for future applications. While in some cases commercial enzymes can be employed directly on industrial scales, in other cases protein engineering is necessary to evolve an enzyme fit for non-biological substrates and conditions.

Synthesis of Vibegron Enabled by a Ketoreductase Rationally Designed for High pH Dynamic Kinetic Reduction.

Described here is an efficient stereoselective synthesis of vibegron enabled by an enzymatic dynamic kinetic reduction that proceeds in a high-pH environment. To overcome enzyme performance limitations under these conditions, a ketoreductase was evolved by a computationally and structurally aided strategy to increase cofactor stability through tighter binding.

Synthesis of the GPR40 Partial Agonist MK-8666 through a Kinetically Controlled Dynamic Enzymatic Ketone Reduction.

A scalable and efficient synthesis of the GPR40 agonist MK-8666 was developed from a simple pyridine building block. The key step to set the stereochemistry at two centers relied on an enzymatic dynamic kinetic reduction of an unactivated ketone. Directed evolution was leveraged to generate an optimized ketoreductase that provided the desired trans alcohol in >30:1 dr and >99% ee.

Synthesis of bis-macrocyclic HCV protease inhibitor MK-6325 via intramolecular sp²-sp³ Suzuki-Miyaura coupling and ring closing metathesis.

A practical asymmetric synthesis of the complex fused bis-macrocyclic HCV protease inhibitor MK-6325 (1) is described. Through the combination of a high yielding and low catalyst loading ring-closing metathesis (RCM) to forge the 15-membered macrocycle with an intramolecular sp(2)-sp(3) Suzuki-Miyaura cross-coupling to append the 18-membered macrocycle, multikilogram access to the unique and challenging architecture of MK-6325 (1) has been achieved.

Discovery of MK-7246, a selective CRTH2 antagonist for the treatment of respiratory diseases.

In this manuscript we wish to report the discovery of MK-7246 (4), a potent and selective CRTH2 (DP2) antagonist. SAR studies leading to MK-7246 along with two synthetic sequences enabling the preparation of this novel class of CRTH2 antagonist are reported. Finally, the pharmacokinetic and metabolic profile of MK-7246 is disclosed.

Resolution of 2,2-disubstituted epoxides via biocatalytic azidolysis.

A practical procedure for the enzymatic resolution of 2-alkyl-2-aryl-disubstituted epoxides using the Codex HHDH P2E2 enzyme and sodium azide is reported. This method allowed the synthesis of novel regio- and enantioselective 1-azido-2-arylpropan-2-ols in excellent yields. Furthermore, these intermediates were used for the preparation of enantiomerically enriched amino alcohols and aziridines containing a tertiary center.

Advances in the enzymatic reduction of ketones.

Historically, biocatalytic ketone reductions involved the use of Baker's yeast. Within the last five years, a significant and growing number of isolated ketoreductases have become available that have rendered yeast-based reductions obsolete. The broad substrate range and exquisite selectivities of these enzymes repeatedly outperform other ketone reduction chemistries, making biocatalysis the general method of choice for ketone reductions.

Chemo- and enantioselective routes to chiral fluorinated hydroxyketones using ketoreductases.

Chiral fluorinated hydroxyketones were synthesized with excellent ee (>98%) and yield by a chemo- and stereoselective reduction of prochiral methyl/trifluoromethyl diketones using commercially available ketoreductase enzymes. By using p- and m-trifluoroacetyl substituted acetophenones, we demonstrate that ketoreductases can selectively differentiate between methyl and trifluoromethyl ketones within the same molecule. As a result, useful catalysts were identified that eliminated the need for costly and time-consuming protection/deprotection of the ketone moiety, enabling a more convergent synthesis of hydroxyketones.

Purification and characterization of a chemotolerant alcohol dehydrogenase applicable to coupled redox reactions.

The purification and characterization of an organic solvent tolerant, NADH-dependent medium-chain secondary alcohol dehydrogenase (denoted sec-ADH "A") from Rhodococcus ruber DSM 44541 is reported. The enzyme can withstand elevated concentrations of organic solvents, such as acetone (up to 50% v/v) and 2-propanol (up to 80% v/v). Thus, it is ideally suited for the preparative-scale enantioselective oxidation of sec-alcohol and the asymmetric reduction of ketones, using acetone and 2-propanol, respectively, as cosubstrates for cofactor-regeneration via a coupled-substrate approach.

On the organic solvent and thermostability of the biocatalytic redox system of Rhodococcus ruber DSM 44541.

The sec-alcohol dehydrogenase activity of whole cells of Rhodococcus ruber DSM 44541 has been employed as an efficient biocatalytic redox system due to the use of acetone and 2-propanol at elevated concentrations for cofactor regeneration in the oxidation and reduction mode, respectively, and external addition of NADH/NAD(+) can be omitted. The operational half-life time of the redox system is 29 hours in 20% v/v acetone and 37 hours in 30% v/v 2-propanol. The Redox system allows the enantioselective oxidation of sec-alcohols and the asymmetric reduction of ketones to furnish (S)-configurated alcohols in high optical purity.

Biocatalytic asymmetric hydrogen transfer employing Rhodococcus ruber DSM 44541.

Nonracemic sec-alcohols of opposite absolute configuration were obtained either by asymmetric reduction of the corresponding ketone using 2-propanol as hydrogen donor or by enantioselective oxidation through kinetic resolution of the rac-alcohol using acetone as hydrogen acceptor employing whole lyophilized cells of Rhodococcus ruber DSM 44541. The microbial oxidation/reduction system exhibits not only excellent stereo- and enantioselectivity but also a broad substrate spectrum. Due to the exceptional tolerance of the biocatalyst toward elevated concentrations of organic materials (solvents, substrates and cosubstrates), the process is highly efficient.