Catalyzing the future: recent advances in chemical synthesis using enzymes

Hanley D., Li Z., Gao S., Virgil S.C., Arnold F.H., Alfonzo E.

Zeng Q., Zhou Q., Calvó-Tusell C., Dai S., Zhao X., Garcia-Borràs M., Liu Z.

Efficient methods for achieving desaturation of carbonyl compounds are highly sought after in organic chemistry. In contrast to synthetic approaches, enzymatic desaturation systems offer the potential to enhance sustainability and selectivity but have remained elusive. Here we report the development of an enzymatic desaturation system based on flavin-dependent ene-reductases for desymmetrizing cyclohexanones. This platform facilitates the synthesis of a wide array of chiral cyclohexenones bearing quaternary stereocentres—structural motifs commonly present in bioactive molecules—with excellent yields and enantioselectivities. Experimental and computational mechanistic studies reveal the roles of key active-site residues that enable the formation and stabilization of an enolate intermediate in the desaturation event. Additionally, by leveraging these insights, we have devised a biocatalytic strategy for the synthesis of enones by reductively desymmetrizing cyclohexadienones. This method yields the opposite enantiomer compared to our desaturation system, underscoring the enantiodivergence and broad applicability of our flavin-based desymmetrization approaches. Biocatalytic methods for the synthesis of chiral cyclohexenones bearing quaternary stereocentres through oxidation and reduction reactions are reported. Mechanistic studies reveal the role of active-site residues in the oxidation process and inform the development of the enzymatic reduction reaction.

Ouyang Y., Page C.G., Bilodeau C., Hyster T.K.

Wang T., Mai B.K., Zhang Z., Bo Z., Li J., Liu P., Yang Y.

Photobiocatalysis—where light is used to expand the reactivity of an enzyme—has recently emerged as a powerful strategy to develop chemistries that are new to nature. These systems have shown potential in asymmetric radical reactions that have long eluded small-molecule catalysts1. So far, unnatural photobiocatalytic reactions are limited to overall reductive and redox-neutral processes2–9. Here we report photobiocatalytic asymmetric sp3–sp3 oxidative cross-coupling between organoboron reagents and amino acids. This reaction requires the cooperative use of engineered pyridoxal biocatalysts, photoredox catalysts and an oxidizing agent. We repurpose a family of pyridoxal-5′-phosphate-dependent enzymes, threonine aldolases10–12, for the α-C–H functionalization of glycine and α-branched amino acid substrates by a radical mechanism, giving rise to a range of α-tri- and tetrasubstituted non-canonical amino acids 13–15 possessing up to two contiguous stereocentres. Directed evolution of pyridoxal radical enzymes allowed primary and secondary radical precursors, including benzyl, allyl and alkylboron reagents, to be coupled in an enantio- and diastereocontrolled fashion. Cooperative photoredox–pyridoxal biocatalysis provides a platform for sp3–sp3 oxidative coupling16, permitting the stereoselective, intermolecular free-radical transformations that are unknown to chemistry or biology. We report on the oxidative cross-coupling of organoboron reagents and amino acids via pyridoxal biocatalysis to produce non-canonical amino acids, uncovering stereoselective, intermolecular free-radical transformations.

Chao T., Wu X., Fu Y., Yang L., Renata H.

Non-canonical amino acids (ncAAs) are prized building blocks in the synthesis of natural products, designer peptides and drug molecules. Despite their general utility, the structural complexity of these molecules still presents an enormous challenge for chemical synthesis. Here we develop a one-pot chemoenzymatic approach for the construction of azacyclic ncAAs with multiple substitutions and various ring sizes. A promiscuous transaminase was identified to convert a wide range of diketoacids to the corresponding α-amino acids. A spontaneous cyclic imine formation was followed by a stereocontrolled chemical reduction to generate the corresponding products in one pot with high stereoselectivity. More than 25 azacyclic ncAAs were successfully prepared using this approach. This work demonstrates the value of developing hybrid biocatalytic–chemocatalytic approaches to prepare privileged small molecule motifs. Non-canonical amino acids are important building blocks in the synthesis of natural products, peptides and drugs. Now, a one-pot chemoenzymatic approach to synthesize branched azacyclic non-canonical amino acids is reported. This method combines enzymatic transamination of 2,n-diketoacids and stereocontrolled chemical reduction to provide the desired products with high stereoselectivity.

Zhao Q., Chen Z., Soler J., Chen X., Rui J., Ji N.T., Yu Q.E., Yang Y., Garcia-Borràs M., Huang X.

In recent years there has been a surge in the development of methods for the synthesis of organofluorine compounds. However, enzymatic methods for C–F bond formation have been limited to nucleophilic fluoride substitution. Here we report the incorporation of iron-catalysed radical fluorine transfer, a reaction mechanism that is not used in naturally occurring enzymes, into enzymatic catalysis for the development of biocatalytic enantioselective C(sp3)–F bond formation. Using this strategy, we repurposed (S)-2-hydroxypropylphosphonate epoxidase from Streptomyces viridochromogenes (SvHppE) to catalyse an N-fluoroamide-directed C(sp3)–H fluorination. Directed evolution has enabled SvHppE to be optimized, forming diverse chiral benzylic fluoride products with turnover numbers of up to 180 and with excellent enantiocontrol (up to 94% enantiomeric excess). Mechanistic investigations showed that the N–F bond activation is the rate-determining step, and the strong preference for fluorination in the presence of excess NaN3 can be attributed to the spatial proximity of the carbon-centred radical to the iron-bound fluoride. Methods for enzymatic C–F bond formation are rare. Now an enzymatic method for enantioselective C(sp3)–F bond formation is reported, through reprogramming non-haem iron enzyme (S)-2-hydroxypropylphosphonate epoxidase. Mechanistic studies reveal that the process proceeds through an iron-mediated radical fluorine transfer process.

Cheung-Lee W.L., Kolev J.N., McIntosh J.A., Gil A.A., Pan W., Xiao L., Velásquez J.E., Gangam R., Winston M.S., Li S., Abe K., Alwedi E., Dance Z.E., Fan H., Hiraga K., et. al.

AbstractBiocatalytic 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. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high‐yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan.

Sarai N.S., Fulton T.J., O’Meara R.L., Johnston K.E., Brinkmann-Chen S., Maar R.R., Tecklenburg R.E., Roberts J.M., Reddel J.C., Katsoulis D.E., Arnold F.H.

Volatile methylsiloxanes (VMS) are man-made, nonbiodegradable chemicals produced at a megaton-per-year scale, which leads to concern over their potential for environmental persistence, long-range transport, and bioaccumulation. We used directed evolution to engineer a variant of bacterial cytochrome P450 BM3 to break silicon-carbon bonds in linear and cyclic VMS. To accomplish silicon-carbon bond cleavage, the enzyme catalyzes two tandem oxidations of a siloxane methyl group, which is followed by putative [1,2]-Brook rearrangement and hydrolysis. Discovery of this so-called siloxane oxidase opens possibilities for the eventual biodegradation of VMS.

Rapp J.T., Bremer B.J., Romero P.A.

AbstractProtein engineering has nearly limitless applications across chemistry, energy and medicine, but creating new proteins with improved or novel functions remains slow, labor-intensive and inefficient. Here we present the Self-driving Autonomous Machines for Protein Landscape Exploration (SAMPLE) platform for fully autonomous protein engineering. SAMPLE is driven by an intelligent agent that learns protein sequence–function relationships, designs new proteins and sends designs to a fully automated robotic system that experimentally tests the designed proteins and provides feedback to improve the agent’s understanding of the system. We deploy four SAMPLE agents with the goal of engineering glycoside hydrolase enzymes with enhanced thermal tolerance. Despite showing individual differences in their search behavior, all four agents quickly converge on thermostable enzymes. Self-driving laboratories automate and accelerate the scientific discovery process and hold great potential for the fields of protein engineering and synthetic biology.

Tinzl M., Diedrich J.V., Mittl P.R., Clémancey M., Reiher M., Proppe J., Latour J., Hilvert D.

Wackelin D.J., Mao R., Sicinski K.M., Zhao Y., Das A., Chen K., Arnold F.H.

Xu Y., Chen H., Yu L., Peng X., Zhang J., Xing Z., Bao Y., Liu A., Zhao Y., Tian C., Liang Y., Huang X.

Enzymes are recognized as exceptional catalysts for achieving high stereoselectivities1–3, but their ability to control the reactivity and stereoinduction of free radicals lags behind that of chemical catalysts4. Thiamine diphosphate (ThDP)-dependent enzymes5 are well-characterized systems that inspired the development of N-heterocyclic carbenes (NHCs)6–8 but have not yet been proved viable in asymmetric radical transformations. There is a lack of a biocompatible and general radical-generation mechanism, as nature prefers to avoid radicals that may be harmful to biological systems9. Here we repurpose a ThDP-dependent lyase as a stereoselective radical acyl transferase (RAT) through protein engineering and combination with organophotoredox catalysis10. Enzyme-bound ThDP-derived ketyl radicals are selectively generated through single-electron oxidation by a photoexcited organic dye and then cross-coupled with prochiral alkyl radicals with high enantioselectivity. Diverse chiral ketones are prepared from aldehydes and redox-active esters (35 examples, up to 97% enantiomeric excess (e.e.)) by this method. Mechanistic studies reveal that this previously elusive dual-enzyme catalysis/photocatalysis directs radicals with the unique ThDP cofactor and evolvable active site. This work not only expands the repertoire of biocatalysis but also provides a unique strategy for controlling radicals with enzymes, complementing existing chemical tools. Enzyme-bound ketyl radicals derived from thiamine diphosphate are selectively generated through single-electron oxidation by a photoexcited organic dye and shown to lead to enantioselective radical acylation reactions.

Li J., Kumar A., Lewis J.C.

AbstractDespite the unique reactivity of vitamin B12 and its derivatives, B12‐dependent enzymes remain underutilized in biocatalysis. In this study, we repurposed the B12‐dependent transcription factor CarH to enable non‐native radical cyclization reactions. An engineered variant of this enzyme, CarH*, catalyzes the formation γ‐ and δ‐lactams through either redox‐neutral or reductive ring closure with marked enhancement of reactivity and selectivity relative to the free B12 cofactor. CarH* also catalyzes an unusual spirocyclization by dearomatization of pendant arenes to produce bicyclic 1,3‐diene products instead of 1,4‐dienes provided by existing methods. These results and associated mechanistic studies highlight the importance of protein scaffolds for controlling the reactivity of B12 and expanding the synthetic utility of B12‐dependent enzymes.

Gao S., Das A., Alfonzo E., Sicinski K.M., Rieger D., Arnold F.H.

Lukowski A.L., Hubert F.M., Ngo T., Avalon N.E., Gerwick W.H., Moore B.S.

Meng Z., Yang Z., Xu C., Gao X., Liu Z.

Ettenhuber P., Hansen M.B., Poier P.P., Shaik I., Rasmussen S.E., Madsen N.K., Majland M., Jensen F., Olsen L., Zinner N.T.

Wang B., Zhou T., Shen Y., Hu J., Zhou J., Tang J., Han R., Xu G., Schwaneberg U., Wang B., Ni Y.

Xu Z., Liu J., Hu H., Ma J., Yang H., Chen J., Xu H., Hu H., Luo H., Chen G.

Osteoarthritis is one of the most common degenerative joint diseases, which seriously affects the life of middle-aged and elderly people. Traditional treatments such as surgical treatment and systemic medication, often do not achieve the expected or optimal results, which leads to severe trauma and a variety of side effects. Therefore, there is an urgent need to develop novel therapeutic options to overcome these problems. Hydrogels are widely used in biomedical tissue repairing as a platform for loading drugs, proteins and stem cells. In recent years, smart-responsive hydrogels have achieved excellent results as novel drug delivery systems in the treatment of osteoarthritis. This review focuses on the recent advances of endogenous stimuli (including enzymes, pH, reactive oxygen species and temperature, etc.) responsive hydrogels and exogenous stimuli (including light, shear, ultrasound and magnetism, etc.) responsive hydrogels in osteoarthritis treatment. Finally, the current limitations of application and future prospects of smart responsive hydrogels are summarized.

Singh N., Lane S., Yu T., Lu J., Ramos A., Cui H., Zhao H.

Proteins are the molecular machines of life with numerous applications in energy, health, and sustainability. However, engineering proteins with desired functions for practical applications remains slow, expensive, and specialist-dependent1–3. Here we report a generally applicable platform for autonomous protein engineering that integrates machine learning and large language models with biofoundry automation to eliminate the need for human intervention, judgement, and domain expertise. Requiring only an input protein sequence and a quantifiable way to measure fitness, this autonomous platform can be applied to engineer virtually any protein. As a proof of concept, we engineeredArabidopsis thalianahalide methyltransferase (AtHMT)4for a 90-fold improvement in substrate preference and 16-fold improvement in ethyltransferase activity, along with developing aYersinia mollaretiiphytase (YmPhytase)5,6variant with 26-fold improvement in activity at neutral pH. This was accomplished in four rounds over four weeks, while requiring construction and characterization of fewer than a total of 500 variants for each enzyme. This platform for autonomous experimentation paves the way for rapid advancements across diverse industries, from medicine and biotechnology to renewable energy and sustainable chemistry.

Bellou M.G., Skonta A., Chatzikonstantinou A.V., Polydera A.C., Katapodis P., Voutsas E., Stamatis H.

Biocatalytic processes for the formation of bioactive compounds and biopolymer preparations that can be applied in pharmaceuticals and cosmetics are gaining increasing interest due to their safety and sustainability, relying on environmentally friendly approaches and biocompatible compounds. In this work, we investigate the implementation of various Deep Eutectic Solvents (DES) in the laccase-catalyzed oxidation of hydroxytyrosol (HT), aiming to produce its oligomer derivatives such as HT dimer and trimer. The composition of the reaction mixture in which the oligomers’ yield was the highest was 70% v/v Bet:PG (1:4 molar ratio). The oligomers formed were subsequently used for the non-enzymatic grafting of chitosan (CS) and the development of bioactive chitosan-based nanogels (NG). Grafted chitosan nanogels were prepared by ionic gelation using sodium tripolyphosphate (TPP) as a cross-linking agent. The functionalized chitosan was characterized using Fourier-Transform Infrared (FTIR) and Nuclear Magnetic Resonance (NMR) spectroscopy, while Scanning Electron Microscopy (SEM) was employed for nanogel characterization. Compared to unmodified chitosan nanogels, grafted chitosan nanogels exhibited almost ten-fold higher antioxidant activity and approximately 20% greater antibacterial activity.

Mansoor S., Frasnetti E., Cucchi I., Magni A., Bonollo G., Serapian S.A., Pavarino L.F., Colombo G.

Cheng L., Bo Z., Krohn-Hansen B., Yang Y.

Fu W., Liu A., Yang Y.

Yang J., Lal R.G., Bowden J.C., Astudillo R., Hameedi M.A., Kaur S., Hill M., Yue Y., Arnold F.H.

Abstract Directed evolution (DE) is a powerful tool to optimize protein fitness for a specific application. However, DE can be inefficient when mutations exhibit non-additive, or epistatic, behavior. Here, we present Active Learning-assisted Directed Evolution (ALDE), an iterative machine learning-assisted DE workflow that leverages uncertainty quantification to explore the search space of proteins more efficiently than current DE methods. We apply ALDE to an engineering landscape that is challenging for DE: optimization of five epistatic residues in the active site of an enzyme. In three rounds of wet-lab experimentation, we improve the yield of a desired product of a non-native cyclopropanation reaction from 12% to 93%. We also perform computational simulations on existing protein sequence-fitness datasets to support our argument that ALDE can be more effective than DE. Overall, ALDE is a practical and broadly applicable strategy to unlock improved protein engineering outcomes.

Bronstein L.M., Matveeva V.G.

Multifunctional catalysts have received considerable attention in the cascade reactions of biomass processing. A cascade (or tandem) reaction is realized when multiple reaction steps that require different catalysts are performed in a one-step process. These reactions require bi- or multifunctional catalysts or catalyst mixtures to serve successfully at each reaction step. In this review article, we discuss the major factors of the catalyst design influencing the structure–property relationships, which could differ depending on the catalyst type. The major factors include the amounts and strengths of acidic and basic sites, interactions between those and metal sites, synergetic effects, nanoparticle sizes and morphology, nanostructures, porosity, etc. The catalysts described in this review are based on zeolites, mesoporous solids, MOFs, and enzymes. The importance of continuous cascade processes is also examined.