Catalytic Turnover Research Articles

Proton transfer in cytochrome bd-I from E. coli involves Asp-105 in CydB

Cytochrome bds are bacterial terminal oxidases expressed under low oxygen conditions, and they are important for the survival of many pathogens and hence potential drug targets. The largest subunit CydA contains the three redox-active cofactors heme b558, heme b595 and the active site heme d. One suggested proton transfer pathway is found at the interface between the CydA and the other major subunit CydB. Here we have studied the O2 reduction mechanism in E. coli cyt. bd-I using the flow-flash technique and focused on the mechanism, kinetics and pathway for proton transfer.Our results show that the peroxy (P) to ferryl (F) transition, coupled to the oxidation of the low-spin heme b558 is pH dependent, with a maximum rate constant (~104 s−1) that is slowed down at higher pH. We assign this behavior to rate-limitation by internal proton transfer from a titratable residue with pKa ~ 9.7. Proton uptake from solution occurs with the same P➔F rate constant. Site-directed mutagenesis shows significant effects on catalytic turnover in the CydB variants Asp58B➔Asn and Asp105B➔Asn variants consistent with them playing a role in proton transfer. Furthermore, in the Asp105B➔Asn variant, the reactions up to P formation occur essentially as in the wildtype bd-I, but the P➔F transition is specifically inhibited, supporting a direct and specific role for Asp105B in the functional proton transfer pathway in bd-I. We further discuss the possible identity of the high pKa proton donor, and the conservation pattern of the Asp-105B in the cyt. bd superfamily.

Crystal structure and mechanistic studies of the PPM1D serine/threonine phosphatase catalytic domain

Protein phosphatase 1D (PPM1D, Wip1) is induced by the tumor suppressor p53 during DNA damage response signaling and acts as an oncoprotein in several human cancers. Although PPM1D is a potential therapeutic target, insights into its atomic structure were challenging due to flexible regions unique to this family member. Here we report the first crystal structure of the PPM1D catalytic domain to 1.8 Å resolution. The structure reveals the active site with two Mg2+ ions bound, similar to other structures. The flap subdomain and B-loop, which are crucial for substrate recognition and catalysis, were also resolved, with the flap forming two short helices and three short β-strands that are followed by an irregular loop. Unexpectedly, a nitrogen–oxygen–sulfur bridge was identified in the catalytic domain. Molecular dynamics simulations and kinetic studies provided further mechanistic insights into the regulation of PPM1D catalytic activity. In particular, the kinetic experiments demonstrated a magnesium concentration-dependent lag in PPM1D attaining steady-state velocity, a feature of hysteretic enzymes that show slow transitions compared with catalytic turnover. All combined, these results advance the understanding of PPM1D function and will support the development of PPM1D-targeted therapeutics.

Two-Dimensional Cu(II)-MOF with Lewis Acid-Base Bifunctional Sites for Chemical Fixation of CO2 and Bioactive 1,4-DHP Synthesis via Hantzsch Condensation.

Five- and six-membered heterocycles containing nitrogen or oxygen have been considered as privileged scaffolds in organic chemistry and the chemical industry because of their usage in high-value commodities. Herein, we report a two-dimensional (2D) Cu(II)-based MOF catalyst, IITKGP-40, via the strategic employment of ample Lewis acid-base bifunctional sites (open metal nodes and free pyrazine moieties) along the pore wall. IITKGP-40 could convert toxic CO2 to cyclic carbonates in an atom-economical manner under solvent-free conditions and aromatic aldehyde to bioactive 1,4-DHPs via Hantzsch condensation. Exceptional catalytic performance (99%) and turnover number under mild reaction conditions for CO2 fixation using sterically hindered styrene oxide, and good-to-excellent yields for a wide range of aromatic aldehydes toward 1,4-dihydropyridines (1,4-DHPs) make IITKGP-40 promising as a multipurpose heterogeneous catalyst. Moreover, to demonstrate the practical utility of the catalyst, two biologically important drug molecules, diludine and nitrendipine analogue, have also been synthesized. IITKGP-40 is recyclable for at least three consecutive runs without significant loss of activity, making it promising for real-time applications.

Methane Formation Induced via Face-to-Face Orientation of Cyclic Fe Porphyrin Dimer in Photocatalytic CO2 Reduction.

Iron porphyrins are known to provide CH4 as an eight-electron reduction product of CO2 in a photochemical reaction. However, there are still some aspects of the reaction mechanism that remain unclear. In this study, we synthesized iron porphyrin dimers and carried out the photochemical CO2 reduction reactions in N,N-dimethylacetamide (DMA) containing a photosensitizer in the presence of 1,3-dimethyl-2-phenyl-2,3-dihydro-1H-benzo[d]imidazole (BIH) as an electron donor. We found that, despite a low catalytic turnover number, CH4 was produced only when these porphyrins were facing each other. The close proximity of the cyclic dimers, distinguishing them from a linear Fe porphyrin dimer and monomers, induced multi-electron CO2 reduction, emphasizing the unique role of their structural arrangement in CH4 formation.

Engineering supramolecular dynamics of self-assembly and turnover of oncogenic microRNAs to drive their synergistic destruction in tumor models

Rationally-engineered functional biomaterials offer the opportunity to interface with complex biology in a predictive, precise, yet dynamic way to reprogram their behaviour and correct shortcomings. Success here may lead to a desired therapeutic effect against life-threatening diseases, such as cancer. Here, we engineered “Crab”-like artificial ribonucleases through coupling of peptide and nucleic acid building blocks, capable of operating alongside and synergistically with intracellular enzymes (RNase H and AGO2) for potent destruction of oncogenic microRNAs. “Crab”-like configuration of two catalytic peptides (“pincers”) flanking the recognition oligonucleotide was instrumental here in providing increased catalytic turnover, leading to ≈30-fold decrease in miRNA half-life as compared with that for “single-pincer” conjugates. Dynamic modeling of miRNA cleavage illustrated how such design enabled “Crabs” to drive catalytic turnover through simultaneous attacks at different locations of the RNA-DNA heteroduplex, presumably by producing smaller cleavage products and by providing toeholds for competitive displacement by intact miRNA strands. miRNA cleavage at the 5′-site, spreading further into double-stranded region, likely provided a synergy for RNase H1 through demolition of its loading region, thus facilitating enzyme turnover. Such synergy was critical for sustaining persistent disposal of continually-emerging oncogenic miRNAs. A single exposure to the best structural variant (Crab-p-21) prior to transplantation into mice suppressed their malignant properties and reduced primary tumor volume (by 85 %) in MCF-7 murine xenograft models.

A role for conformational changes in enzyme catalysis

The role played by conformational changes in enzyme catalysis is controversial. In addition to examining specific enzymes, studying formal models can help identify the conditions under which conformational changes promote catalysis. Here, we present a model demonstrating how conformational changes can break a generic trade-off due to the conflicting requirements of successive steps in catalytic cycles, namely high specificity for the transition state to accelerate the chemical transformation and low affinity for the products to favor their release. The mechanism by which the trade-off is broken is a transition between conformations with different affinities for the substrate. The role of the effector that induces the transition is played by a substrate “handle,” a part of the substrate that is not chemically transformed but whose interaction with the enzyme is nevertheless essential to rapidly complete the catalytic cycle. A key element of the model is the formalization of the constraints causing the trade-off that the presence of multiple states breaks, which we attribute to the strong chemical similarity between successive reaction states—substrates, transition states, and products. For the sake of clarity, we present our model for irreversible one-step unimolecular reactions. In this context, we demonstrate how the different forms that chemical similarities between reaction states can take impose limits on the overall catalytic turnover. We first analyze catalysts without internal degrees of freedom and then show how two-state catalysts can overcome their limitations. Our results recapitulate previous proposals concerning the role of conformational changes and substrate handles in a formalism that makes explicit the constraints that elicit these features. In addition, our approach establishes links with studies in the field of heterogeneous catalysis, where the same trade-offs are observed and where overcoming them is a well-recognized challenge.

Weak Interaction Activates Esters: Reconciling Catalytic Activity and Turnover Contradiction by Tailored Chalcogen Bonding.

The activation of esters by strong Lewis acids via the formation of covalent adducts is a classic strategy to give reactivity; however, this approach frequently incurs limited turnover due to the low efficiency in the dissociation of catalyst from a stable catalyst-product complex. While the use of some weak interaction catalysts that can easily dissociate from any bonding complexes in the reaction system would solve this catalyst turnover problem, the poor catalytic activity in the ester activation that can be provided by these noncovalent forces in turn sets up a formidable challenge. Herein, we describe the activation and catalytic transformation of esters by weak interactions, which provides a promising platform to reconcile the catalytic activity and turnover problems. Several tailored chalcogen-bonding catalysts were developed for the activation of esters, enabling achieving several inherently low reactive Diels-Alder reactions as well as the ring-opening polymerization of lactones through weak chalcogen bonding interactions. This supramolecular catalysis approach is particularly highlighted by its capability to promote some uncommon Diels-Alder reactions involving using dienes bearing electron-withdrawing groups coupled by α,β-unsaturated ester as dienophiles and substrate incorporating competitive Lewis basic sites, in which typical strong Lewis acids showed low catalytic efficiency, while representative hydrogen and halogen bonding catalysts were inactive.

Can the Rate of a Catalytic Turnover Be Altered by Ligands in the Absence of Direct Binding Interactions?

Second sphere coordination effects ubiquitous in enzymatic catalysis occur through direct interactions, either covalent or non-covalent, with reaction intermediates and transition states. We present herein evidence of indirect second sphere coordination effects in which ligation of water/alkanols far removed from the primary coordination sphere of the active site nevertheless alter energetic landscapes within catalytic redox cycles in the absence of direct physicochemical interactions with surface species mediating catalytic turnovers. Density functional theory, in situ X-ray absorption and infrared spectroscopy, and a wide array of steady-state and transient CO oxidation rate data suggest that the presence of peripheral water renders oxidation half-cycles within two-electron redox cycles over μ3-oxo-bridged trimers in MIL-100(M) more kinetically demanding. Communication between ligated water and the active site appears to occur through the Fe-O-Fe backbone, as inferred from spin density variations on the central μ3-oxygen 'junction'. Evidence is provided for the generality of these second sphere effects in that they influence different types of redox half-cycles or metals, and can be amplified or attenuated through choice of coordinating ligand. Specifically in the case of MIL-100(M) materials, the Cr isostructure can be made to kinetically mimic the Fe variant by disproportionately hindering oxidation half-cycles relative to the reduction half-cycles. Kinetic and spectroscopic inferences presented here significantly expand both the conceptual definition of second sphere effects as well as the palette of synthetic levers available for tuning catalytic redox performance through chemical ligation.

Breaking a Molecular Scaling Relationship Using an Iron-Iron Fused Porphyrin Electrocatalyst for Oxygen Reduction.

The design of efficient electrocatalysts is limited by scaling relationships governing trade-offs between thermodynamic and kinetic performance metrics. This ″iron law″ of electrocatalysis arises from synthetic design strategies, where structural alterations to a catalyst must balance nucleophilic versus electrophilic character. Efforts to circumvent this fundamental impasse have focused on bioinspired applications of extended coordination spheres and charged sites proximal to a catalytic center. Herein, we report evidence for breaking a molecular scaling relationship involving electrocatalysis of the oxygen reduction reaction (ORR) by leveraging ligand design. We achieve this using a binuclear catalyst (a diiron porphyrin), featuring a macrocyclic ligand with extended electronic conjugation. This ligand motif delocalizes electrons across the molecular scaffold, improving the catalyst's nucleophilic and electrophilic character. As a result, our binuclear catalyst exhibits low overpotential and high catalytic turnover frequency, breaking the traditional trade-off between these two metrics.

Ligand Field Sensitive Spin Acceleration in the Iron-Catalyzed [2 + 2] Cycloaddition of Unactivated Alkenes and Dienes.

Redox-active pyridine(diimine) (PDI) iron catalysts promote the reversible [2 + 2] cycloaddition of alkenes and dienes to cyclobutane derivatives that have applications ranging from fuels to chemically recyclable polymers. Metallacycles were identified as key intermediates, and spin crossover from the singlet to the triplet surface was calculated to facilitate the reductive coupling step responsible for the formation of the four-membered ring. In this work, a series of sterically and electronically differentiated PDI ligands was studied for the [2 + 2] cycloaddition of ethylene and butadiene to vinylcyclobutane. Kinetic studies revealed that the fastest and slowest turnover were observed with equally electron-deficient supporting ligands that either feature phenyl-substituted imine carbon atoms (MeBPDI) or a pyrazine core (MePZDI). While the oxidative cyclization was comparatively slow for both catalysts, the rate of reductive coupling─determined by stoichiometric 13C2H4 labeling studies─correlated with the turnover frequencies. Two-state density functional theory studies and the distinct electronic structures of related (iPrBPDI) and (iPrPZDI) iron methyl complexes revealed significantly different ligand field strengths due to either diminished ligand σ-donation (MeBPDI) or promoted metal π-backbonding (MePZDI). Spin acceleration, leading to fast reductive coupling and catalytic turnover, was promoted in the case of the weaker ligand field and depends on both the nature and position of the electron-withdrawing group. This study provides strong evidence for the role of two-state reactivity in C(sp3)-C(sp3) bond formation and insights on how ligand design either promotes or inhibits spin acceleration in earth-abundant metal catalysis.

Methane-H2S Reforming Catalyzed by Carbon and Metal Sulfide Stabilized Sulfur Dimers.

H2S reforming of methane (HRM) provides a potential strategy to directly utilize sour natural gas for the production of COx-free H2 and sulfur chemicals. Several carbon allotropes were found to be active and selective for HRM, while the additional presence of transition metals led to further rate enhancements and outstanding stability (e.g., Ru supported on carbon black). Most metals are transformed to sulfides, but the carbon supports prevent sintering under the harsh reaction conditions. Supported by theoretical calculations, kinetic and isotopic investigations with representative catalysts showed that H2S decomposition and the recombination of surface H atoms are quasi-equilibrated, while the first C-H bond scission is the kinetically relevant step. Theory and experiments jointly establish that dynamically formed surface sulfur dimers are responsible for methane activation and catalytic turnovers on sulfide and carbon surfaces that are otherwise inert without reaction-derived active sites.

Effects of Xylanase A double mutation on substrate specificity and structural dynamics

While protein activity is traditionally studied with a major focus on the active site, the activity of enzymes has been hypothesized to be linked to the flexibility of adjacent regions, warranting more exploration into how the dynamics in these regions affects catalytic turnover. One such enzyme is Xylanase A (XylA), which cleaves hemicellulose xylan polymers by hydrolysis at internal β-1,4-xylosidic linkages. It contains a “thumb” region whose flexibility has been suggested to affect the activity. The double mutation D11F/R122D was previously found to affect activity and potentially bias the thumb region to a more open conformation. We find that the D11F/R122D double mutation shows substrate-dependent effects, increasing activity on the non-native substrate ONPX2 but decreasing activity on its native xylan substrate. To characterize how the double mutant causes these kinetics changes, nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations were used to probe structural and flexibility changes. NMR chemical shift perturbations revealed structural changes in the double mutant relative to the wild-type, specifically in the thumb and fingers regions. Increased slow-timescale dynamics in the fingers region was observed as intermediate-exchange line broadening. Lipari-Szabo order parameters show negligible changes in flexibility in the thumb region in the presence of the double mutation. To help understand if there is increased energetic accessibility to the open state upon mutation, alchemical free energy simulations were employed that indicated thumb opening is more favorable in the double mutant. These studies aid in further characterizing how flexibility in adjacent regions affects the function of XylA.

Analysis of the electron transfer pathway in small laccase by EPR and UV–vis spectroscopy coupled with redox titration

Bacterial small laccases (SLAC) are promising industrial biocatalysts due to their ability to oxidize a broad range of substrates with exceptional thermostability and tolerance for alkaline pH. Electron transfer between substrate, copper centers, and O2 is one of the key steps in the catalytic turnover of SLAC. However, limited research has been conducted on the electron transfer pathway of SLAC and SLAC-catalyzed reactions, hindering further engineering of SLAC to produce tunable biocatalysts for novel applications. Herein, the combinational use of electron paramagnetic resonance (EPR) and ultraviolet–visible (UV–vis) spectroscopic methods coupled with redox titration were employed to monitor the electron transfer processes and obtain further insights into the electron transfer pathway in SLAC. The reduction potentials for type 1 copper (T1Cu), type 2 copper (T2Cu) and type 3 copper (T3Cu) were determined to be 367 ± 2 mV, 378 ± 5 mV and 403 ± 2 mV, respectively. Moreover, the reduction potential of a selected substrate of SLAC, hydroquinone (HQ), was determined to be 288 mV using cyclic voltammetry (CV). In this way, an electron transfer pathway was identified based on the reduction potentials. Specifically, electrons are transferred from HQ to T1Cu, then to T2Cu and T3Cu, and finally to O2. Furthermore, superhyperfine splitting observed via EPR during redox titration indicated a modification in the covalency of T2Cu upon electron uptake, suggesting a conformational alteration in the protein environment surrounding the copper sites, which could potentially influence the reduction potential of the copper sites during catalytic processes. The results presented here not only provide a comprehensive method for analyzing the electron transfer pathway in metalloenzymes through reduction potential measurements, but also offer valuable insights for further engineering and directed evolution studies of SLAC in the aim for biotechnological and industrial applications.

The Structural Role of N170 in Substrate-Assisted Deacylation in KPC-2 β-Lactamase.

The amino acid substitutions in Klebsiella pneumoniae carbapenemase 2 (KPC-2) that have arisen in the clinic are observed to lead to the development of resistance to ceftazidime-avibactam, a preferred treatment for KPC bearing Gram-negative bacteria. Specific substitutions in the omega loop (R164-D179) result in changes in the structure and function of the enzyme, leading to alterations in substrate specificity, decreased stability, and more recently observed, increased resistance to ceftazidime/avibactam. Using accelerated rare-event sampling well-tempered metadynamics simulations, we explored in detail the structural role of R164 and D179 variants that are described to confer ceftazidime/avibactam resistance. The buried conformation of D179 substitutions produce a pronounced structural disorder in the omega loop - more than R164 mutants, where the crystallographic omega loop structure remains mostly intact. Our findings also reveal that the conformation of N170 plays an underappreciated role impacting drug binding and restricting deacylation. The results further support the hypothesis that KPC-2 D179 variants employ substrate-assisted catalysis for ceftazidime hydrolysis, involving the ring amine of the aminothiazole group to promote deacylation and catalytic turnover. Moreover, the shift in the WT conformation of N170 contributes to reduced deacylation and an altered spectrum of enzymatic activity.

The Structural Role of N170 in Substrate‐Assisted Deacylation in KPC‐2 β‐Lactamase

AbstractThe amino acid substitutions in Klebsiella pneumoniae carbapenemase 2 (KPC‐2) that have arisen in the clinic are observed to lead to the development of resistance to ceftazidime‐avibactam, a preferred treatment for KPC bearing Gram‐negative bacteria. Specific substitutions in the omega loop (R164‐D179) result in changes in the structure and function of the enzyme, leading to alterations in substrate specificity, decreased stability, and more recently observed, increased resistance to ceftazidime/avibactam. Using accelerated rare‐event sampling well‐tempered metadynamics simulations, we explored in detail the structural role of R164 and D179 variants that are described to confer ceftazidime/avibactam resistance. The buried conformation of D179 substitutions produce a pronounced structural disorder in the omega loop ‐ more than R164 mutants, where the crystallographic omega loop structure remains mostly intact. Our findings also reveal that the conformation of N170 plays an underappreciated role impacting drug binding and restricting deacylation. The results further support the hypothesis that KPC‐2 D179 variants employ substrate‐assisted catalysis for ceftazidime hydrolysis, involving the ring amine of the aminothiazole group to promote deacylation and catalytic turnover. Moreover, the shift in the WT conformation of N170 contributes to reduced deacylation and an altered spectrum of enzymatic activity.

Dicationic Acridinium/Carbene Hybrids as Strongly Oxidizing Photocatalysts.

A new design concept for organic, strongly oxidizing photocatalysts is described based upon dicationic acridinium/carbene hybrids. A highly modular synthesis of such hybrids is presented, and the dications are utilized as novel, tailor-made photoredox catalysts in the direct oxidative C-N coupling. Under optimized conditions, benzene and even electron-deficient arenes can be oxidized and coupled with a range of N-heterocycles in high to excellent yields with a single low-energy photon per catalytic turnover, while commonly used acridinium photocatalysts are not able to perform the challenging oxidation step. In contrast to traditional photocatalysts, the hybrid photocatalysts reported here feature a reversible two-electron redox system with regular or inverted redox potentials for the two-electron transfer. The different oxidation states could be isolated and structurally characterized supported by NMR, EPR, and X-ray analysis. Mechanistic experiments employing time-resolved emission and transient absorption spectroscopy unambiguously reveal the outstanding excited-state potential of our best-performing catalyst (+2.5 V vs SCE), and they provide evidence for mechanistic key steps and intermediates.

Modulating Transition Metal Reactivity with Force

AbstractThe reactivity and selectivity of a transition metal catalyst is intimately related to its ligand‐sphere geometry, and, in many cases, the ideal ligand geometry for one step of a catalytic cycle is poorly matched to the ideal ligand geometry for another. For this reason, methods for reversibly modulating ligand geometry on the time scale of catalytic turnover or monomer enchainment are highly desirable. Mechanical force represents a heretofore untapped approach to modulate catalyst geometry and/or reactivity, with the potential to do so on the timescale of catalytic turnover or monomer enchainment. Macroscopic mechanical forces are large, directional and localized to an extent that differentiates them from other forms of energy input such as heat or light. In this Concept, we describe our efforts to address the fundamental challenges associated with force‐modulated transition metal catalysis by employing molecular force probe ligands comprising a stiff stilbene photoswitch tethered to rotationally flexible biaryl bisphosphine ligand. Our efforts to date include the modulation of catalytic activity through force‐mediated ligand perturbations, quantification of the force‐coupled ligand effects on the energetics of elementary organometallic transformations, and evaluation of the mechanisms of force transduction in these systems.

Bridging the Catalytic Turnover Gap Between Single-Atom Iron Nanozymes and Natural Enzymes by Engineering the First and Second Shell Coordination.

Single-atom nanozymes (SAzymes) constitute a promising category of enzyme-mimicking materials with outstanding catalytic performance. The performance of SAzymes improves through modification of the coordination environments around the metal center. However, the catalytic turnover rates of SAzymes, which are key measures of the effectiveness of active site modifications, remain lower than those of natural enzymes, especially in peroxidase-reactions. Here, the first and second shell coordination tuning strategy that yields SAzymes with structures and activities analogous to those of natural enzymes is reported. The optimized SAzyme exhibits a turnover rate of 52.7 s-1 and a catalytic efficiency of 6.97 × 105 M-1 s-1. A computational study reveals that axial S-ligands induce an alternative reaction mechanism, and SO2- functional groups provide hydrogen bonds to reduce the activation energy. In addition, SAzyme shows superior anti-tumor ability in vitro and in vivo. These results demonstrate the validity of coordination engineering strategies and the carcinostatic potential of SAzymes.

Tuning the peroxidase activity of artificial P450 peroxygenase by engineering redox-sensitive residues.

Cytochrome P450 monooxygenases (P450s) are well recognized as versatile bio-oxidation catalysts. However, the catalytic functions of P450s are highly dependent on NAD(P)H and redox partner proteins. Our group has recently reported the use of a dual-functional small molecule (DFSM) for generating peroxygenase activity of P450BM3, a long-chain fatty acid hydroxylase from Bacillus megaterium. The DFSM-facilitated P450BM3 peroxygenase system exhibited excellent peroxygenation activity and regio-/enantioselectivity for various organic substrates, such as styrenes, thioanisole, small alkanes, and alkylbenzenes. Very recently, we demonstrated that the DFSM-facilitated P450BM3 peroxygenase could be switched to a peroxidase by engineering the redox-sensitive tyrosine residues in P450BM3. Given the great potential of P450 peroxidase for C-H oxyfunctionalization, we herein report scrutiny of the effect of mutating redox-sensitive residues on peroxidase activity by deeply screening all redox-sensitive residues of P450BM3, namely methionines, tryptophans, cysteines, and phenylalanines. As a result, six beneficial mutations at positions M212, F81, M112, F173, M177, and F77 were screened out from 78 constructed mutants, and significantly enhanced the peroxidase activity of P450BM3 in the presence of Im-C6-Phe, a typical DFSM molecule. Further combination of the beneficial mutations resulted in a more than 100-fold improvement in peroxidase activity compared with that of the combined parent enzyme and DFSM, comparable to or better than most natural peroxidases. In addition, mutations of redox-sensitive residues even dramatically increased, by more than 300-fold, the peroxidase activity of the starting F87A enzyme in the absence of the DFSM, despite the far lower apparent catalytic turnover number compared with the DFSM-P450 system. This study provides new insights and a potential strategy for regulating the catalytic promiscuity of P450 enzymes for multiple functional oxidations.

Borane-catalysed C2-selective indole reductive functionalisation.

Indolines are common motifs within pharamceuticals and natural products. Boron catalysis enables the chemoselective allylation of indoles to give allylic indolines in excellent diastereoselectivity. Mechanistic studies revealed in situ formation of the allylic borane, allylation of the imine tautomer of the indole and B-N/B-H transborylation for catalytic turnover.