Fei Zhao

Enzymes that catalyze non-natural C–C bond-forming reactions are powerful tools in asymmetric synthesis, yet reprogramming their active sites to invert stereochemical outcome remains challenging. Building on our recently engineered S _N Arase, S _N Ar1.3, which performs enantioselective nucleophilic aromatic substitutions with carbon nucleophiles, we now report the evolution of an enantiocomplementary biocatalyst (eS _N Ar1.3) that displays enhanced activity and expanded substrate scope. Structural and computational analyses uncover both conserved and divergent features between S _N Ar1.3 and eS _N Ar1.3. Despite retaining similar electrophile binding poses and a conserved catalytic arginine, the halide-binding pocket of S _N Ar1.3 has been abandoned in eS _N Ar1.3. Instead, His23 has emerged as a key motif that works with Arg124 to accurately position the nucleophilic substrate. Calculations reveal that Arg124 also plays a crucial role in facilitating halide release during catalysis. Our study demonstrates how evolution can reshape enzyme mechanisms in unforeseen ways, highlighting the importance of exploring diverse trajectories to access new functions.

Developing enzymatic strategies to selectively construct C–C and C–heteroatom bonds is a major objective in modern biocatalysis. Here we combine genetic code reprogramming and laboratory evolution to establish a family of allylic transferase enzymes that can achieve a remarkable breadth of chemistry, enabling the generation of valuable motifs including γ‑butenolides, chiral amines and all-carbon quaternary centres. Our enzymes operate through the formation of electrophilic imidazolium intermediates that can be generated from reagents equipped with a para -nitrophenol leaving group to facilitate high-throughput evolution. These intermediates can be intercepted with diverse carbon and nitrogen nucleophiles to generate densely functionalized products featuring Cβ or Cγ stereocentres. Interestingly, structural analysis suggests that catalysis proceeds through an unanticipated ternary complex involving para -nitrophenol and the incoming substrate nucleophile. This study adds a family of C–C and C–N bond-forming enzymes to the biocatalytic repertoire and illustrates how artificial enzymes can achieve precise control over challenging chemical conversions.

Arginine side chains play important roles in the catalytic sites of many natural and engineered enzymes. This versatility is further exemplified by the recent development of artificial enzymes for S _N Ar processes, which rely on a key catalytic arginine embedded within a haloacid dehalogenase scaffold. Although powerful, the catalytic proficiency of these first generation S _N Arases remains limited, meaning their activity is restricted to highly activated electrophile and nucleophile coupling partners. Here we address these limitations by developing miniature de novo S _N Arases that offer far greater catalytic potency and synthetic scope. To this end, a computationally designed NTF2-like protein featuring an activated arginine embedded within its hydrophobic core was optimized over successive rounds of directed evolution. The enhanced catalytic proficiency offered by these optimized designs allows them to operate efficiently on poorly activated substrates and has enabled the development of unprecedented regio- and enantioselective S _N Ar transformations. Structural and biochemical analysis of the most active enzyme reveals a remarkably sophisticated active site, whereby a key catalytic Arg17 embedded in an extended polar network works in synergy with an aromatic-rich substrate binding pocket to accurately position and activate the S _N Ar coupling partners. This study provides new insights to guide the design of arginine-containing catalytic sites that will enable the future development of de novo enzymes for diverse chemical transformations.

The enantioselective construction of C(sp ³ )─S stereocentres remains a major challenge in catalysis due to the distinct electronic and steric features of sulfur, compared to oxygen or nitrogen atoms, which complicate both stereocontrol and configurational stability at the C─S bond. Here, we report that the ene‐reductase biocatalyst ENE‐101 catalyses the highly enantioselective reduction of β‐vinyl sulfones, enabling the direct formation of C(sp ³ )–S(VI) stereocentres in excellent yields and enantiomeric excesses (up to >99% ee). Whereas the corresponding β‐vinyl sulfides are unreactive towards ENE‐101, the S(II) to S(VI) oxidation activates the C═C bond toward enzymatic reduction. Computational studies reveal that the sulfone moiety enhances alkene electrophilicity and promotes favourable substrate orientation and binding within the ENE‐101 active site. The biocatalyst exhibits broad substrate scope, tolerating diverse β‐vinyl sulfones. This work establishes sulfur(VI) activation as an effective strategy to expand the reactivity landscape of ene‐reductase biocatalysts for the asymmetric C─S bond formation.

Herein, we report the regio‐ and stereoselective synthesis of 5‐alkylidene‐pyrrolidin‐2‐one derivatives through an Ag (I) or Au (I)‐catalyzed cascade reaction between linear alkynoic acids and primary amines. This protocol features excellent regio‐ and stereoselectivity, good functional group tolerance, broad substrate scope, good to high yields, and high bond‐forming efficiency, thus providing an efficient and convenient route to a variety of 5‐alkylidene‐pyrrolidin‐2‐ones.

A Rh( iii )-catalyzed redox-neutral [4 + 1] annulation of N -aryl phthalazinediones with alkynes is reported. This method features a broad substrate scope, excellent chemo-/regio-/stereoselectivity, and moderate to high yields.

Nucleophilic aromatic substitutions (S_NAr) are among the most widely used processes in the pharmaceutical and agrochemical industries^1–4, allowing convergent assembly of complex molecules through C–C and C–X (X = O, N, S) bond formation. S_NAr reactions are typically carried out using forcing conditions, involving polar aprotic solvents, stoichiometric bases and elevated temperatures, which do not allow for control over reaction selectivity. Despite the importance of S_NAr chemistry, there are only a handful of selective catalytic methods reported that rely on small organic hydrogen-bonding or phase-transfer catalysts^5–11. Here we establish a biocatalytic approach to stereoselective S_NAr chemistry by uncovering promiscuous S_NAr activity in a designed enzyme featuring an activated arginine¹². This activity was optimized over successive rounds of directed evolution to afford an engineered biocatalyst, S_NAr1.3, that is 160-fold more efficient than the parent and promotes the coupling of electron-deficient arenes with carbon nucleophiles with near-perfect stereocontrol (>99% enantiomeric excess (e.e.)). S_NAr1.3 can operate at a rate of 0.15 s⁻¹, perform more than 4,000 turnovers and can accept a broad range of electrophilic and nucleophilic coupling partners, including those that allow construction of challenging 1,1-diaryl quaternary stereocentres. Biochemical, structural and computational studies provide insights into the catalytic mechanism of S_NAr1.3, including the emergence of a halide binding pocket shaped by key catalytic residues Arg124 and Asp125. This study brings a landmark synthetic reaction into the realm of biocatalysis to provide an efficient and versatile platform for catalytic S_NAr chemistry.

Biocatalysis has been widely employed for the generation of carbon-carbon/heteroatom stereocentres, yet its application in chiral C(sp³)–S bond construction is rare and limited to enzymatic kinetic resolutions. Herein, we describe the enantioselective construction of chiral C(sp³)–S bonds through ene-reductase biocatalyzed conjugate reduction of prochiral vinyl sulfides. A series of cooperative sequential/concurrent chemoenzymatic and biocatalytic cascades have been developed to access a broad range of chiral sulfides, including valuable β-hydroxysulfides bearing two adjacent C(sp³)–S and C(sp³)–O stereocentres, in a stereoconvergent manner with good to excellent yields (up to 96%) and enantioselectivities (up to >99% ee). Notably, this biocatalytic strategy allows to overcome the long-standing shortcomings of catalyst poisoning and C(sp²)/C(sp³)–S bond cleavage faced in transition-metal-catalyzed hydrogenation of vinyl sulfides. Finally, the potential of this methodology is also exemplified by its broader application in the stereoconvergent assembly of chiral C(sp³)–N/O/Se bonds with good to excellent enantioselctivities.

Herein, we report the rhodium-catalyzed tunable divergent synthesis of tetrasubstituted 1,3-enynes and alkynylated benzofurans via C−H alkenylation/DG migration and [3 + 2] annulation, respectively.

Chiral β‐hydroxysulfides are an important class of organic compounds which find broad application in organic and pharmaceutical chemistry. Herein we describe the development of novel biocatalytic and chemoenzymatic methods for the enantioselective synthesis of β‐hydroxysulfides by exploiting ketoreductase (KRED) enzymes. Four KREDs were discovered from a pool of 384 enzymes identified and isolated through a metagenomic approach. KRED311 and KRED349 catalysed the synthesis of β‐hydroxysulfides bearing a stereocentre at the C−O bond with opposite absolute configurations and excellent ee values by novel chemoenzymatic and biocatalytic‐chemical‐biocatalytic (bio‐chem‐bio) cascades starting from commercially available thiophenols/thiols and α‐haloketones/alcohols. KRED253 and KRED384 catalysed the enantioselective synthesis of β‐hydroxysulfides bearing a stereocentre at the C−S bond with opposite enantioselectivities by dynamic kinetic resolution (DKR) of racemic α‐thioaldehydes.