Drug Intermediates Research Articles

Rhodium-Catalyzed Homogeneous Asymmetric Hydrogenation of Naphthol Derivatives.

Due to their strong aromaticity and difficulties in chemo-, regio-, and enantioselectivity control, asymmetric hydrogenation of naphthol derivatives to 1,2,3,4-tetrahydronaphthols has remained a long-standing challenge. Herein, we report the first example of homogeneous asymmetric hydrogenation of naphthol derivatives catalyzed by tethered rhodium-diamine catalysts, affording a wide array of optically pure 1,2,3,4-tetrahydronaphthols in high yields with excellent regio-, chemo-, and enantioselectivities (up to 98% yield and >99% ee). Mechanistic studies with experimental and computational approaches reveal that fluorinated solvent 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) plays vital roles in the control of reactivity and selectivity, and 1-naphthol is reduced via a cascade reaction pathway, including dearomative tautomerization, 1,4-hydride addition, and 1,2-hydride addition in sequence. A novel synergistic activation mode was proposed in which HFIP assists a synergistic activation of both the hydrogen molecule and naphthol in the presence of a base, and the in situ-generated fleeting keto tautomer is immediately trapped and reduced by the Rh(III)-H species before it escapes from the solvent cage. This protocol provides a straightforward and practical pathway for the synthesis of key intermediates for several chiral drugs. Particularly, optically pure Nadolol, a drug for the treatment of hypertension, angina pectoris, congestive heart failure, and certain arrhythmias, is enantioselectively synthesized for the first time.

Application of Biocatalysis in the Synthesis of Bioactive Compounds

Biocatalysis has emerged as a transformative technology in pharmaceutical synthesis, enabling efficient and sustainable production of chiral bioactive compounds. This review focuses on recent advancements in biocatalytic methods, highlighting key enzyme systems, such as ketoreductases, aminotransferases, and imine reductases, which facilitate the synthesis of chiral alcohols, amines, and other pharmaceutical intermediates with high enantiomeric purity. Directed evolution and protein engineering have significantly enhanced enzyme stability, activity, and substrate specificity, broadening their applications in industrial-scale drug production. For example, ketoreductases have been successfully applied in the synthesis of intermediates for drugs, such as enalapril and duloxetine, while engineered transaminases enable efficient production of sitagliptin. Beyond asymmetric reduction and transamination, advancements in C-H bond activation using enzymes like P450 monooxygenases and unspecific peroxygenases provide new opportunities for regioselective functionalization. Additionally, multienzyme cascade reactions and chemo-biocatalytic approaches integrate multiple catalytic steps into single processes, reducing reaction complexity and waste while enhancing efficiency. These innovations highlight the growing role of biocatalysis in green chemistry, offering a sustainable alternative to traditional chemical synthesis methods. As the field advances with contributions from bioinformatics, artificial intelligence, and high-throughput techniques, biocatalysis is poised to drive further innovation in pharmaceutical manufacturing, fostering environmentally friendly processes and scalable solutions for complex drug synthesis. This review underscores the critical impact of biocatalysis in shaping the future of sustainable chemistry and its pivotal role in addressing the growing demand for greener pharmaceutical technologies.

Exploring a metal/base-free porphyrin involving a carboxyl-functionalized pyridine moiety for photocatalytic N-arylation of benzamide validated using RSM.

A porphyrin comprising a carboxyl-functionalized pyridine moiety was synthesized and characterized using 1H NMR, 13C NMR, FT-IR, powder-XRD, BET, ICP-MS, SEM and EDAX. The proton level (H0 = 1.19) and energy band gap (1.39 eV) were determined via UV-Vis spectrophotometry. The UV-visible and fluorescence emission spectra indicated the absorption window of the porphyrin photocatalyst with a distinct Soret band at 424 nm and four Q-bands at 517, 558, 595, and 649 nm. The existence of four Q-bands, the powder XRD data and the ICP-MS analysis supported the absence of metal in the porphyrin photocatalyst. The best photocatalytic conditions generated using Box-Behnken design of RSM (0.2 mol% PcCFP, 5 W LED, 1 : 1.2 ArX : ArCONH2, 24 h) were confirmed through the model reaction of benzamide and 1-bromo-4-nitrobenzene. The N-arylation of benzamide was achieved in a custom-built photoreactor at ambient conditions under exposure to 5 W LED light. Different ArX compounds comprising electron-repelling and electron-attracting groups were assessed to test the potential of the photocatalyst. The porphyrin was found to exhibit significant catalytic activity for C-N bond formation, resulting in 21-73% yields of the substituted benzanilide products. The N-arylated benzamide formation was confirmed using 1H NMR, 13C NMR, HR-MS and SC-XRD. Additionally, heteroaryl halides such as 2-bromo-, 3-bromo-, and 4-bromo-pyridine, as well as 2-chloro-4-methylpyridine, were also found to be compatible and provided admirable yields (28-67%). The stability and heterogeneous nature of the porphyrin photocatalyst were confirmed using FT-IR. The stability of the photocatalyst after the sixth run was demonstrated by the slight decline in the yield of the product from 71 to 67%. The formation of an aryl radical was detected using the scavenger TEMPO, which led to the achievement of N-arylated benzamides containing intermediates of industrial drugs.

Structure-guided engineering an (R)-transaminase from Mycobacterium neoaurum for efficient synthesis of chiral N-heterocyclic amines.

(R)-selective amine transaminases (R-ATAs) show considerable potential for the asymmetric synthesis of chiral drug intermediates. However, the low catalytic efficiency of natural R-ATAs toward bulky ketone substrates, such as N-heterocyclic compounds, severely limits its industrial application. In this study, five putative (R)-ATAs were mined from NCBI database, among which MnTA showed the highest activity for N-Boc-3-pyrrolidinone (1a) and N-Boc-3-piperidone (2a), and its crystal structure was performed. Furthermore, a structure-guided engineering strategy combined with directed evolution and in silico design was executed. Four key sites for substrate binding were identified based on alanine scanning. Then, a saturated mutation library was constructed, and residues G66 and F127 were found to be the key sites affecting substrate binding. By further combining mutation and iterative saturation mutation, variants with markedly improved activity were obtained. The optimal mutant MnTA-M1 (F127M) and MnTA-M5 (G66L/H67N/F127M/L160I) also displayed significantly enhanced activity toward various cyclic ketones or bulky N-heterocyclic ketone analogs. Finally, the gram-scale synthesis of (R)-3-amino-N-Boc-pyrrolidin (1b) and (R)-3-amino-N-Boc-piperidine (2b) was performed by the best mutants, achieving the space-time yields (STY) of 108 and 214g/L·d, respectively. This research provides efficient biocatalysts for the synthesis of various chiral N-heterocyclic amines, along with a structural insight into the molecular mechanism for enhanced catalytic performance.

Salen-Pd(II)-Modified Stereoregular Polyisocyanides for Efficient Cooperative Catalysis of Suzuki Coupling Reaction.

The development of high activity catalysts is crucial for improving industrial efficiency and mitigating environmental pollution. Polyisocyanides, with their pendant groups capable of forming ordered adjacent structures, offer a promising framework for designing cooperative catalysts that mimic the functionality of bimetallic centers. This unique structural arrangement is anticipated to significantly enhance catalytic activity in cooperative reactions. A novel approach to enhance the Suzuki coupling reaction using polymer-supported catalysts is presented. In this study, stereoregular polyisocyanides with Salen-Pd are functionalized to produce the Pd(II) metalized polyisocyanide (P1-Pd). The rigid backbone of the polymer facilitates the parallel alignment of Salen-Pd pendants, enabling double activation of the two substrates at an average distance of ≈1.2nm. Catalytic efficiency is evaluated through Suzuki coupling reactions using various aryl halides. P1-Pd demonstrates high activity, yielding the desired products with excellent conversion rates. Conversely, the irregular polymer counterpart P2-Pd. P3-Pd and the small molecule control C1-Pd exhibit lower performance due to the absence of cooperative catalysis. To showcase the applicability of this strategy, Suzuki coupling is successfully conducted with outstanding yields for key drug intermediates, while also offering innovative insights for conjugated polymer synthesis.

Ancestor-originated engineering of medium-chain alcohol dehydrogenase enhances its catalytic compatibility by balancing activity, stability, and selectivity trade-offs

The role of medium-chain alcohol dehydrogenases (MDR) in the preparation of chiral drug intermediates is indispensable; however, limited activity and stability towards unnatural substrates are crucial factors that restrict their application. Further, research on the ancestral MDRs remains unclear. Therefore, enhancing the activity, selectivity, and stability of MDRs is necessary. In this study, we resurrected ancestors of the MDR family using ancestral sequence reconstruction with descendant GcADH as a probe. Among these ancestors, ancestor 136 exhibited increased thermal stability (ΔTm = 19.5 °C) and had a comparable conversion and selectivity as GcADH to the selected substrates. After in-silico screening using the Funclib tool, we obtained the mutant anc136-R2, which exhibited strengthened selectivity and activity towards N-Benzyl-3,3-difluoropiperidin-4-one (1a). Moreover, the mutations of anc136-R2 were simultaneously mapped to GcADH, resulting GcADH-R2. Based on the results of substrate spectrum characterisation, anc136 was more receptive to mutations. Specifically, for the substrates N-Boc-3-piperidone (4a) and N-boc-3-oxoazepane (7a), the conversion rates of anc136-R2 were 29 and 57 times higher, respectively, than those of GcADH-R2, without compromising selectivity. In conclusion, this work provides guidance for exploring the evolutionary trajectory of the MDR family and further illustrates the superior balanced “trade-off” of activity–selectivity–stability on ancestral enzymes.

An Enzymatic Method to Obtain Enantiopure 3-Pyridyl andSubstituted Phenyl Alanine.

Chiral phenylalanine derivatives are important raw materials and building blocks for the synthesis of peptides and drug molecules. Enantiomerically pure D/L-3-pyridyl- and phenylalanine has shown wide application potential in the synthesis of various drug intermediates. This article focuses on two synthetic routes from different feedstocks. The first approach is an Erlenmeyer-Plöchl route study using N-acetylglycine as starting material, whereas the second is an alkylation route study using diethyl acetamidomalonate as starting material. The key step is the resolution of N-acetamido-alanine esters using different quantities of fairly inexpensive Protamex proteinase to obtain pure enantiomeric D/L-3-pyridyl- and substituted phenylalanine or its derivative, with the ee value and purity of all products exceeding 99%. The different chiral arylalanine derivatives that can be prepared using the above two methods have good versatility.

Ru-Catalyzed Asymmetric Hydrogenation of α,β-Unsaturated γ-Lactams.

A highly efficient Ru-catalyzed asymmetric hydrogenation of α,β-unsaturated γ-lactams has been developed by using a C2-symmetric ruthenocenyl phosphine-oxazoline as the chiral ligand. This method achieves the enantioselective synthesis of chiral β-substituted γ-lactams in high yields and with excellent enantioselectivities (up to 99% yield with 99% ee). Mechanistic studies based on detailed control experiments and computational investigation revealed that the cationic Ru-complex acts as the active catalytic species; the protonation process of the oxa-π-allyl-Ru complex, which is formed by the migratory insertion of the C=C double bond to the Ru-H bond (the stereocontrolling step) followed by an isomerization process, is the rate-determining step, and the existence of PPh3 is crucial for the highly efficient catalytic behavior. The protocol provides a straightforward and practical pathway for the synthesis of key intermediates for several chiral drugs and bioactive compounds, particularly for the 150 kg-scale industrial production of Brivaracetam, an antiepileptic drug that shows 13-fold more potent binding to the synaptic vesicle protein 2A compared with the well-known Levetiracetam.

Making Global Pharma Supply Chain Resilient: Will the PLI Scheme of India make it a Reliable Alternative to China for the Supply of APIs?

The COVID-19-induced disruptions in the global supply chains and growing geopolitical sensitivities of governments and multinational corporations (MNCs) are increasingly resulting in China plus one strategy in order to ensure supply chain resilience. This strategy is expected to benefit countries like India in sectors such as pharmaceuticals. Having faced the brunt of excessive reliance on a single country for supplies during the pandemic, India launched a product linked incentive (PLI) scheme in July 2020, covering 41 products, to incentivise domestic production of key starting materials (KSMs), drug intermediates (DIs) and active pharmaceutical ingredients (APIs) with the objective of reducing dependence on China. Launching at a time when countries and firms were actively considering the strategy of supply chain diversification, this scheme was perceived to be perfectly timed. However, the response of the industry was not on the expected lines. This article analyses the early trends coming from the implementation of the scheme and looks into the reasons for the lukewarm response of the industry. It identifies three areas—creating confidence among the investors, accommodating micro, small and medium enterprises (MSMEs) and involving public sector enterprises—where more attention is needed to make India self-reliant in APIs and their DIs and KSMs. JEL Codes: F02, F13, L24, L65, L78, O30

Structure-based reshaping of a new ketoreductase from Sphingobacterium siyangense SY1 toward α-haloacetophenones

Ketoreductases play an indispensable role in the asymmetric synthesis of chiral drug intermediates, and an in-depth understanding of their substrate selectivity can improve the efficiency of enzyme engineering. In this endeavor, a new short-chain dehydrogenase/reductase (SDR) SsSDR1 identified from Sphingobacterium siyangense SY1 by gene mining method was successfully cloned and functionally expressed in Escherichia coli. Its activity against halogenated acetophenones has been tested and the results illustrated that SsSDR1-WT exhibits high activity for 3,5-bis(trifluoromethyl)acetophenone (1f), an important precursor in the synthesis of aprepitant. In addition, SsSDR1-WT showed obvious substrate preference for acetophenones without α-halogen substitution compared to their α-halogen analogs. To explore the structural basis of substrate selectivity, the X-ray crystal structures of SsSDR1-WT in its apo form and the complex structure with NAD were resolved. Taking 2-chloro-1-(3, 4-difluorophenyl) ethanone (1i) as the representative α-haloacetophenone, the key sites affecting substrate selectivity of SsSDR1-WT were identified and through the rational remodeling of the cavities C1 and C2 of SsSDR1, an excellent mutant I144A/S153L with significantly improved activity against α-halogenated acetophenones was obtained. The asymmetric catalysis of 1f and 1i was performed at the scale of 50 mL, and the space-time yields (STY) of the two were 1200 and 6000 g/L∙d, respectively. This study not only provides valuable biocatalysts for halogenated acetophenones, but also yields insights into the relationship between the substrate-binding pocket and substrate selectivity.

X-Ray-Sensitizers: Organic Pharmaceutical Drug Intermediates Activated Directly by X-Rays to Efficiently Populate Triplet Excitons for Cancer Treatment

Radiotherapy is an important treatment for cancer, but it is associated with major side effects due to the high dose of radiation (generally more than 50 Gy). Because radiation’s low acute and late toxicity, many tumors are treated with fractionated radiation in small doses (< 2 Gy). Scintillator X-ray-induced photodynamic therapy is an efficient methodology for cancer management that employs small doses of X-ray irradiation (< 2 Gy) in a complex process. Here we screened pharmaceutical drug intermediates that are derivatives of thioxanthone (TX) and investigated TX-derived organic pharmaceutical molecules that efficiently undergo X-ray-sensitization to populate triplet excitons (singlet oxygen) for cancer therapy when exposed to low-dose X-ray irradiation. By modifying alkoxy side chain substitutions at the 2-position to tune the molecular packing and intermolecular interactions, the fluorescence and room-temperature phosphorescence of a series of TX derivatives were assessed under X-ray irradiation. The ability of these derivatives to generate singlet oxygen and their potential for treating tumors provide new opportunities for developing organic molecules with simple chemical structures, in which large numbers of triplets can be populated directly under ultralow-dose X-ray irradiation.

Application of Directed Evolution and Machine Learning to Enhance the Diastereoselectivity of Ketoreductase for Dihydrotetrabenazine Synthesis.

Biocatalysis is an effective approach for producing chiral drug intermediates that are often difficult to synthesize using traditional chemical methods. A time-efficient strategy is required to accelerate the directed evolution process to achieve the desired enzyme function. In this research, we evaluated machine learning-assisted directed evolution as a potential approach for enzyme engineering, using a moderately diastereoselective ketoreductase library as a model system. Machine learning-assisted directed evolution and traditional directed evolution methods were compared for reducing (±)-tetrabenazine to dihydrotetrabenazine via kinetic resolution facilitated by BsSDR10, a short-chain dehydrogenase/reductase from Bacillus subtilis. Both methods successfully identified variants with significantly improved diastereoselectivity for each isomer of dihydrotetrabenazine. Furthermore, the preparation of (2S,3S,11bS)-dihydrotetrabenazine has been successfully scaled up, with an isolated yield of 40.7% and a diastereoselectivity of 91.3%.

Rerouting phytosterol degradation pathway for directed androst-1,4-diene-3,17-dione microbial bioconversion

Steroid-based drugs are now mainly produced by the microbial transformation of phytosterol, and a two-step bioprocess is adopted to reach high space–time yields, but byproducts are frequently observed during the bioprocessing. In this study, the catabolic switch between the C19- and C22-steroidal subpathways was investigated in resting cells of Mycobacterium neoaurum NRRL B-3805, and a dose-dependent transcriptional response toward the induction of phytosterol with increased concentrations was found in the putative node enzymes including ChoM2, KstD1, OpccR, Sal, and Hsd4A. Aldolase Sal presented a dominant role in the C22 steroidal side-chain cleavage, and the byproduct was eliminated after sequential deletion of opccR and sal. Meanwhile, the molar yield of androst-1,4-diene-3,17-dione (ADD) was increased from 59.4 to 71.3%. With the regard of insufficient activity of rate-limiting enzymes may also cause byproduct accumulation, a chromosomal integration platform for target gene overexpression was established supported by a strong promoter L2 combined with site-specific recombination in the engineered cell. Rate-limiting steps of ADD bioconversion were further characterized and overcome. Overexpression of the kstD1 gene further strengthened the bioconversion from AD to ADD. After subsequential optimization of the bioconversion system, the directed biotransformation route was developed and allowed up to 82.0% molar yield with a space–time yield of 4.22 g·L−1·day−1. The catabolic diversion elements and the genetic overexpression tools as confirmed and developed in present study offer new ideas of M. neoaurum cell factory development for directed biotransformation for C19- and C22-steroidal drug intermediates from phytosterol.Key points• Resting cells exhibited a catabolic switch between the C19- and C22-steroidal subpathways.• The C22-steroidal byproduct was eliminated after sequential deletion of opccR and sal.• Rate-limiting steps were overcome by promoter engineering and chromosomal integration.

Direct α-Arylation of Benzo[b]furans Catalyzed by a Pd3 Cluster.

As an interim paradigm for the catalysts between those based on more conventional mononuclear molecular Pd complexes and Pdn nanoparticles widely used in organic synthesis, polynuclear palladium clusters have attracted great attention for their unique reactivity and electronic properties. However, the development of Pd cluster catalysts for organic transformations and mechanistic investigations is still largely unexploited. Herein, we disclose the use of trinuclear palladium (Pd3Cl) species as an active catalyst for the direct C-H α-arylation of benzo[b]furans with aryl iodides to afford 2-arylbenzofurans in good yields under mild conditions. With this method, broad substrate adaptability was observed, and several drug intermediates were synthesized in high yields. Mechanistic studies indicated that the Pd3 core most likely remained intact throughout the reaction course.

Harnessing alcohols as sustainable reagents for late-stage functionalisation: synthesis of drugs and bio-inspired compounds.

Alcohol is ubiquitous with unparalleled structural diversity and thus has wide applications as a native functional group in organic synthesis. It is highly prevalent among biomolecules and offers promising opportunities for the development of chemical libraries. Over the last decade, alcohol has been extensively used as an environmentally friendly chemical for numerous organic transformations. In this review, we collectively discuss the utilisation of alcohol from 2015 to 2023 in various organic transformations and their application toward intermediates of drugs, drug derivatives and natural product-like molecules. Notable features discussed are as follows: (i) sustainable approaches for C-X alkylation (X = C, N, or O) including O-phosphorylation of alcohols, (ii) newer strategies using methanol as a methylating reagent, (iii) allylation of alkenes and alkynes including allylic trifluoromethylations, (iv) alkenylation of N-heterocycles, ketones, sulfones, and ylides towards the synthesis of drug-like molecules, (v) cyclisation and annulation to pharmaceutically active molecules, and (vi) coupling of alcohols with aryl halides or triflates, aryl cyanide and olefins to access drug-like molecules. We summarise the synthesis of over 100 drugs via several approaches, where alcohol was used as one of the potential coupling partners. Additionally, a library of molecules consisting over 60 fatty acids or steroid motifs is documented for late-stage functionalisation including the challenges and opportunities for harnessing alcohols as renewable resources.

Photocatalytic oxidative coupling of terminal alkynes with p-benzoquinone to diaryl ketones by the Cu(I)-PS-POM assembling system through synergistic effect

Photocatalytic C–C bonds formation is an effective approach to synthesize the significant drug intermediates diaryl ketones. Herein, a new polyoxometalate-based metal–organic framework (POMOF), CuW6–TPT, was synthesized by assembling an oxidation catalyst [W6O19]2− into photoactive MOF constructed by photosensitizer (PS) 4,6-tri(4-pyridyl)-1,3,5-triazine (TPT) and binuclear Cu(I) units. CuW6–TPT exhibits excellent light absorption ability and unique conductivity due to the coordination bond, strong π∙∙∙π interactions and H-bonding among Cu(I) ions, TPT moieties and [W6O19]2− in the defined spatial. The abundant exposed Cu(I) catalytic centres are conductive to activate phenylacetylene in situ formation Cu(I)-phenylacetylide, which then undergoes a reductive single electron transfer (SET) process to obtain the key intermediate Cu(II)-phenylactetylide for the Paterno-Buchi (2+2) cycloaddition with benzoquinone to form the labile Cu(II)-oxetane ring. It is a direct route to synthesize diaryl ketones with a hydroxyl group from simple starting materials such as terminal alkynes with p-benzoquinone.

Enzymatic Catalysts for Hydroxamic Acid Formation: A Mini‐Review

AbstractIn recent years, biocatalysts have emerged as crucial tool in organic synthesis, particularly for the production of drug intermediates and precursors, e.g., the synthesis of hydroxamic acids. Traditionally, hydroxamic acids were synthesized using organic chemistry methods. However, with the growing emphasis on sustainable and environment‐friendly practices, the chemical industry has increasingly turned towards green synthesis approaches. The significance of hydroxamic acids in medicinal chemistry has also contributed to the changing trends. Following the approval of certain hydroxamic acids as histone deacetylase (HDAC) inhibitors for cancer treatment by the Food and Drug Administration (US‐FDA), there has been a renewed focus on their synthesis and the development of derivatives with improved properties. As an alternative route, amidases have emerged as promising biocatalysts for hydroxamic acid synthesis through their acyltransferase activity. Recent advancements in the synthesis approaches for hydroxamic acids are reviewed. The biocatalytic routes are explored, emphasizing the use of amidases and their acyltransferase activity. The scope and potential applications of this chemoenzymatic approach in synthesizing various hydroxamic acids and their derivatives are discussed. Such advancements have the potential to revolutionize the production of these important compounds, making the synthesis process more sustainable, efficient, and economically viable.

Adaptation of the electron transport chain improves the biocatalytic efficiency of progesterone 17α hydroxylation

17α hydroxylase is a key enzyme for the conversion of progesterone to prepare various progestational drug intermediates. To improve the specific hydroxylation capability of this enzyme in steroid biocatalysis, the CYP260A1 derived from cellulose-mucilaginous bacteria Sorangium cellulosum Soce56 and the Fpr and bovine adrenal-derived Adx4-108 derived from Escherichia coli str. K-12 were used to construct a new electron transfer system for the conversion of progesterone. Selective mutation of CYP260A1 resulted in a mutant S276I with significantly enhanced 17α hydroxylase activity, and the yield of 17α-OH progesterone reached 58% after optimization of the catalytic system in vitro. In addition, the effect of phosphorylation of the ferredoxin Adx4-108 on 17α hydroxyl activity was evaluated using a targeted mutation technique, and the results showed that the mutation Adx4-108T69E transferred electrons to S276I more efficiently, which further enhanced the catalytic specificity in the C17 position of progesterone, and the yield of 17α-OH progesterone was eventually increased to 74%. This study provides a new option for the production of 17α-OH progesterone by specific transformation of bacterial-derived 17α hydroxylase, and lays a theoretical foundation for the industrial production of progesterone analogs using biotransformation method.

Phytosterol conversion into C9 non-hydroxylated derivatives through gene regulation in Mycobacterium fortuitum.

Androst-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) are important drug intermediates that can be biosynthesized from phytosterols. However, the C9 hydroxylation of steroids via 3-ketosteroid 9α-hydroxylase (KSH) limits AD and 4-HBC accumulation. Five active KshAs, the oxidation component of KSH, were identified in Mycobacterium fortuitum ATCC 35855 for the first time. The deletion of kshAs indicated that the five KshA genes were jointly responsible for C9 hydroxylation during phytosterol biotransformation. MFKDΔkshA, the five KshAs deficient strain, blocked C9 hydroxylation and produced 5.37 g/L AD and 0.55 g/L 4-HBC. The dual function reductase Opccr knockout and 17β-hydroxysteroid dehydrogenase Hsd4A enhancement reduced 4-HBC content from 8.75 to 1.72% and increased AD content from 84.13 to 91.34%, with 8.24 g/L AD being accumulated from 15 g/L phytosterol. In contrast, hsd4A and thioesterase fadA5 knockout resulted in the accumulation of 5.36 g/L 4-HBC from 10 g/L phytosterol. We constructed efficient AD (MFKDΔkshAΔopccr_hsd4A) and 4-HBC (MFKDΔkshAΔhsd4AΔfadA5) producers and provided insights for further metabolic engineering of the M. fortuitum ATCC 35855 strain for steroid productions. KEY POINTS: • Five active KshAs were first identified in M. fortuitum ATCC 35855. • Deactivation of all five KshAs blocks the steroid C9 hydroxylation reaction. • AD or 4-HBC production was improved by Hsd4A, FadA5, and Opccr modification.

Designing a novel (R)-ω-transaminase for asymmetric synthesis of sitagliptin intermediate via motif swapping and semi-rational design

The application of (R)-ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by inadequate stereoselectivity and narrow substrate spectrum. Herein, an effective evolution strategy for (R)-ω-transaminase designing for the asymmetric synthesis of sitagliptin intermediate is presented. Since natural transaminases lack activity toward bulky prositagliptin ketone, transaminase scaffolds with catalytic machinery and activity toward the truncated prositagliptin ketone were firstly screened based on substrate walking principle. A transaminase chimera was established synchronously conferring catalytic activity and (R)-selectivity toward prositagliptin ketone through motif swapping, followed by stepwise evolution. The process resulted in a “best” engineered variant MwTAM8, which exhibited 79.2-fold higher activity than the chimeric scaffold MwTAMc. Structural analysis revealed that the heightened activity is mainly due to the enlarged and adaptive substrate pocket and tunnel. The novel (R)-transaminase exhibited unsatisfied industrial operation stability, which is expected to further modify the protein to enhance its tolerance to temperature, pH, and organic solvents to meet sustainable industrial demands. This study underscores a useful evolution strategy of engineering biocatalysts to confer new properties and functions on enzymes for synthesizing high-value drug intermediates.