Optimizing the conversion of phosphoenolpyruvate to lactate by enzymatic channeling with mixed nanoparticle display.

Co-assembling enzymes with nanoparticles (NPs) into nanoclusters allows them to access channeling, a highly efficient form of multienzyme catalysis. Using pyruvate kinase (PykA) and lactate dehydrogenase (LDH) to convert phosphoenolpyruvic acid to lactic acid with semiconductor quantum dots (QDs) confirms how enzyme cluster formation dictates the rate of coupled catalytic flux (kflux) across a series of differentially sized/shaped QDs and 2D nanoplatelets (NPLs). Enzyme kinetics and coupled flux were used to demonstrate that by mixing different NP systems into clusters, a >10× improvement in kflux is observed relative to free enzymes, which is also ≥2× greater than enhancement on individual NPs. Cluster formation was characterized with gel electrophoresis and transmission electron microscopy (TEM) imaging. The generalizability of this mixed-NP approach to improving flux is confirmed by application to a seven-enzyme system. This represents a powerful approach for accessing channeling with almost any choice of enzymes constituting a multienzyme cascade.

Exploration of the In Vitro Violacein Synthetic Pathway with Substrate Analogues.

Evolution has gifted enzymes with the ability to synthesize an abundance of small molecules with incredible control over efficiency and selectivity. Central to an enzyme's role is the ability to selectively catalyze reactions in the milieu of chemicals within a cell. However, for chemists it is often desirable to extend the substrate scope of reactions to produce analogue(s) of a desired product and therefore some degree of enzyme promiscuity is often desired. Herein, we examine this dichotomy in the context of the violacein biosynthetic pathway. Importantly, we chose to interrogate this pathway with tryptophan analogues in vitro, to mitigate possible interference from cellular components and endogenous tryptophan. A total of nine tryptophan analogues were screened for by analyzing the substrate promiscuity of the initial enzyme, VioA, and compared to the substrate tryptophan. These results suggested that for VioA, substitutions at either the 2- or 4-position of tryptophan were not viable. The seven analogues that showed successful substrate conversion by VioA were then applied to the five enzyme cascade (VioABEDC) for the production of violacein, where l-tryptophan and 6-fluoro-l-tryptophan were the only substrates which were successfully converted to the corresponding violacein derivative(s). However, many of the other tryptophan analogues did convert to various substituted intermediaries. Overall, our results show substrate promiscuity with the initial enzyme, VioA, but much less for the full pathway. This work demonstrates the complexity involved when attempting to analyze substrate analogues within multienzymatic cascades, where each enzyme involved within the cascade possesses its own inherent promiscuity, which must be compatible with the remaining enzymes in the cascade for successful formation of a desired product.

Multienzymatic Cascades and Nanomaterial Scaffolding-A Potential Way Forward for the Efficient Biosynthesis of Novel Chemical Products.

Synthetic biology is touted as the next industrial revolution as it promises access to greener biocatalytic syntheses to replace many industrial organic chemistries. Here, it is shown to what synthetic biology can offer in the form of multienzyme cascades for the synthesis of the most basic of new materials-chemicals, including especially designer chemical products and their analogs. Since achieving this is predicated on dramatically expanding the chemical space that enzymes access, such chemistry will probably be undertaken in cell-free or minimalist formats to overcome the inherent toxicity of non-natural substrates to living cells. Laying out relevant aspects that need to be considered in the design of multi-enzymatic cascades for these purposes is begun. Representative multienzymatic cascades are critically reviewed, which have been specifically developed for the synthesis of compounds that have either been made only by traditional organic synthesis along with those cascades utilized for novel compound syntheses. Lastly, an overview of strategies that look toward exploiting bio/nanomaterials for accessing channeling and other nanoscale materials phenomena in vitro to direct novel enzymatic biosynthesis and improve catalytic efficiency is provided. Finally, a perspective on what is needed for this field to develop in the short and long term is presented.

Multiplexed DNA and Protease Detection with Orthogonal Energy Transfer on a Single Quantum Dot Scaffolded Biosensor.

Almost all pathogens, whether viral or bacterial, utilize key proteolytic steps in their pathogenesis. The ability to detect a pathogen's genomic material along with its proteolytic activity represents one approach to identifying the pathogen and providing initial evidence of its viability. Here, we report on a prototype biosensor design assembled around a single semiconductor quantum dot (QD) scaffold that is capable of detecting both nucleic acid sequences and proteolytic activity by using orthogonal energy transfer (ET) processes. The sensor consists of a central QD assembled via peptidyl-PNA linkers with multiple DNA sequences that encode complements to genomic sequences originating from the Ebola, Influenza, and COVID-19 viruses, which we use as surrogate targets. These are hybridized to complement strands labeled with a terbium (Tb) chelate, AlexaFluor647 (AF647), and Cy5.5 dyes, giving rise to two potential FRET cascades: the first includes Tb → QD → AF647 → Cy5.5 (→ = ET step), which is detected in a time-gated modality, and QD → AF647 → Cy5.5, which is detected from direct excitation. The labeled DNA-displaying QD construct is then further assembled with a RuII-modified peptide, which quenches QD photoluminescence by charge transfer and is recognized by a protease to yield the full biosensor. Each of the labeled DNAs and peptides can be ratiometrically assembled to the QD in a controllable manner to tune each of the ET pathways. Addition of a given target DNA displaces its labeled complement on the QD, disrupting that FRET channel, while protease addition disrupts charge transfer quenching of the central QD scaffold and boosts its photoluminescence and FRET relay capabilities. Along with characterizing the ET pathways and verifying biosensing in both individual and multiplexed formats, we also demonstrate the ability of this construct to function in molecular logic and perform Boolean operations; this highlights the construct's ability to discriminate and transduce signals between different inputs or pathogens. The potential application space for such a sensor device is discussed.

Enzyme assembly on nanoparticle scaffolds enhances cofactor recycling and improves coupled reaction kinetics.

Enzyme activity can be many times enhanced in configurations where they are displayed on a nanoparticle (NP) and this same format sometimes even provides access to channeling phenomena within multienzyme cascades. Here, we demonstrate that such enhancement phenomena can be expanded to enzymatic cofactor recycling along with the coupled enzymatic processes that they are associated with. We begin by showing that the efficiency of glucose driven reduction of nicotinamide adenine dinucleotide (NAD+ → NADH) by glucose dehydrogenase (GDH) is enhanced ca. 5-fold when the enzyme is displayed on nanocrystalline semiconductor quantum dots (QDs) which are utilized as prototypical NP materials in our experimental assays. Coupling this enzymatic step with NADH-dependent lactate dehydrogenase (LDH) conversion of lactate to pyruvate also increases the latter's rate by a similar amount when both enzymes were jointly incorporated into self-assembled QD-based nanoclusters. Detailed agarose gel mobility assays and transmission electron microscopy imaging studies confirm that both tetrameric enzymes assemble to and crosslink the QDs into structured nanoclusters via their multiple-pendant terminal (His)6 sequences. Unexpectedly, control experiments utilizing blocking peptides to prevent enzyme-crosslinking of QDs resulted in even further enhancement of individual enzyme on-QD kinetic activity. This activity was also probed revealing that 200-fold excess peptide/QD addition enhanced individual GDH and LDH on-QD kcat a further 2- and 1.5×, respectively, above that seen just by QD display to a maximum of ∼10-fold GDH enhancement. The potential implications for how these enzyme kinetics-enhancing phenomena can be applied to single and multi-enzyme cascaded reactions in the context of cofactor recycling and cell-free synthetic biology are discussed.

Self assembling nanoparticle enzyme clusters provide access to substrate channeling in multienzymatic cascades.

Access to efficient enzymatic channeling is desired for improving all manner of designer biocatalysis. We demonstrate that enzymes constituting a multistep cascade can self-assemble with nanoparticle scaffolds into nanoclusters that access substrate channeling and improve catalytic flux by orders of magnitude. Utilizing saccharification and glycolytic enzymes with quantum dots (QDs) as a model system, nanoclustered-cascades incorporating from 4 to 10 enzymatic steps are prototyped. Along with confirming channeling using classical experiments, its efficiency is enhanced several fold more by optimizing enzymatic stoichiometry with numerical simulations, switching from spherical QDs to 2-D planar nanoplatelets, and by ordering the enzyme assembly. Detailed analyses characterize assembly formation and clarify structure-function properties. For extended cascades with unfavorable kinetics, channeled activity is maintained by splitting at a critical step, purifying end-product from the upstream sub-cascade, and feeding it as a concentrated substrate to the downstream sub-cascade. Generalized applicability is verified by extending to assemblies incorporating other hard and soft nanoparticles. Such self-assembled biocatalytic nanoclusters offer many benefits towards enabling minimalist cell-free synthetic biology.

Alternative design strategies to help build the enzymatic retrosynthesis toolbox.

Most of the complex molecules found in nature still cannot be synthesized by current organic chemistry methods. Given the number of enzymes that exist in nature and the incredible potential of directed evolution, the field of synthetic biology contains perhaps all the necessary building blocks to bring about the realization of applied enzymatic retrosynthesis. Current thinking anticipates that enzymatic retrosynthesis will be implemented using conventional cell-based synthetic biology approaches where requisite native, heterologous, designer, and evolved enzymes making up a given multi-enzyme pathway are hosted by chassis organisms to carry out designer synthesis. In this perspective, we suggest that such an effort should not be limited by solely exploiting living cells and enzyme evolution and describe some useful yet less intensive complementary approaches that may prove especially productive in this grand scheme. By decoupling reactions from the environment of a living cell, a significantly larger portion of potential synthetic chemical space becomes available for exploration; most of this area is currently unavailable to cell-based approaches due to toxicity issues. In contrast, in a cell-free reaction a variety of classical enzymatic approaches can be exploited to improve performance and explore and understand a given enzyme's substrate specificity and catalytic profile towards non-natural substrates. We expect these studies will reveal unique enzymatic capabilities that are not accessible in living cells.

Homogeneous Electrocatalytic Reduction of CO2 by a CrN3O Complex: Electronic Coupling with a Redox-Active Terpyridine Fragment Favors Selectivity for CO.

Electrocatalyst design and optimization strategies continue to be an active area of research interest for the applied use of renewable energy resources. The electrocatalytic conversion of carbon dioxide (CO2) is an attractive approach in this context because of the added potential benefit of addressing its rising atmospheric concentrations. In previous experimental and computational studies, we have described the mechanism of the first molecular Cr complex capable of electrocatalytically reducing CO2 to carbon monoxide (CO) in the presence of an added proton donor, which contained a redox-active 2,2'-bipyridine (bpy) fragment, CrN2O2. The high selectivity for CO in the bpy-based system was dependent on a delocalized CrII(bpy•-) active state. Subsequently, we became interested in exploring how expanding the polypyridyl ligand core would impact the selectivity and activity during electrocatalytic CO2 reduction. Here, we report a new CrN3O catalyst, Cr(tpytbupho)Cl2 (1), where 2-(2,2':6',2″-terpyridin-6-yl)-4,6-di-tert-butylphenolate = [tpytbupho]-, which reduces CO2 to CO with almost quantitative selectivity via a different mechanism than our previously reported Cr(tbudhbpy)Cl(H2O) catalyst. Computational analyses indicate that, although the stoichiometry of both reactions is identical, changes in the observed rate law are the combined result of a decrease in the intrinsic ligand charge (L3X vs L2X2) and an increase in the ligand redox activity, which result in increased electronic coupling between the doubly reduced tpy fragment of the ligand and the CrII center. The strong electronic coupling enhances the rate of protonation and subsequent C-OH bond cleavage, resulting in CO2 binding becoming the rate-determining step, which is an uncommon mechanism during protic CO2 reduction.

Inverse potential scaling in co-electrocatalytic activity for CO2 reduction through redox mediator tuning and catalyst design.

Electrocatalytic CO2 reduction is an attractive strategy to mitigate the continuous rise in atmospheric CO2 concentrations and generate value-added chemical products. A possible strategy to increase the activity of molecular systems for these reactions is the co-catalytic use of redox mediators (RMs), which direct reducing equivalents from the electrode surface to the active site. Recently, we demonstrated that a sulfone-based RM could trigger co-electrocatalytic CO2 reduction via an inner-sphere mechanism under aprotic conditions. Here, we provide support for inner-sphere cooperativity under protic conditions by synthetically modulating the mediator to increase activity at lower overpotentials (inverse potential scaling). Furthermore, we show that both the intrinsic and co-catalytic performance of the Cr-centered catalyst can be enhanced by ligand design. By tuning both the Cr-centered catalyst and RM appropriately, an optimized co-electrocatalytic system with quantitative selectivity for CO at an overpotential (η) of 280 mV and turnover frequency (TOF) of 194 s-1 is obtained, representing a three-fold increase in co-catalytic activity at 130 mV lower overpotential than our original report. Importantly, this work lays the foundation of a powerful tool for developing co-catalytic systems for homogeneous electrochemical reactions.

Enzymatic Laser-Induced Graphene Biosensor for Electrochemical Sensing of the Herbicide Glyphosate.

Glyphosate is a globally applied herbicide yet it has been relatively undetectable in-field samples outside of gold-standard techniques. Its presumed nontoxicity toward humans has been contested by the International Agency for Research on Cancer, while it has been detected in farmers' urine, surface waters and crop residues. Rapid, on-site detection of glyphosate is hindered by lack of field-deployable and easy-to-use sensors that circumvent sample transportation to limited laboratories that possess the equipment needed for detection. Herein, the flavoenzyme, glycine oxidase, immobilized on platinum-decorated laser-induced graphene (LIG) is used for selective detection of glyphosate as it is a substrate for GlyOx. The LIG platform provides a scaffold for enzyme attachment while maintaining the electronic and surface properties of graphene. The sensor exhibits a linear range of 10-260 µ m, detection limit of 3.03 µ m, and sensitivity of 0.991 nA µ m -1. The sensor shows minimal interference from the commonly used herbicides and insecticides: atrazine, 2,4-dichlorophenoxyacetic acid, dicamba, parathion-methyl, paraoxon-methyl, malathion, chlorpyrifos, thiamethoxam, clothianidin, and imidacloprid. Sensor function is further tested in complex river water and crop residue fluids, which validate this platform as a scalable, direct-write, and selective method of glyphosate detection for herbicide mapping and food analysis.

Homogeneous Catalytic Reduction of O2 to H2O by a Terpyridine-Based FeN3O Complex.

We report a new terpyridine-based FeN3O catalyst, Fe(tpytbupho)Cl2, which reduces O2 to H2O. Variable concentration and variable temperature spectrochemical studies with decamethylferrocene as a chemical reductant in acetonitrile solution enabled the elucidation of key reaction parameters for the catalytic reduction of O2 to H2O by Fe(tpytbupho)Cl2. These mechanistic studies suggest that a 2 + 2 mechanism is operative, where hydrogen peroxide is produced as a discrete intermediate, prior to further reduction to H2O. Consistent with this proposal, the spectrochemically measured first-order rate constant k (s-1) value for H2O2 reduction is larger than that for O2 reduction. Further, significant H2O2 production is observed under hydrodynamic conditions in rotating ring-disk electrode measurements, where the product can be swept away from the cathode surface before further reduction occurs.

Mediated Inner-Sphere Electron Transfer Induces Homogeneous Reduction of CO2 via Through-Space Electronic Conjugation.

The electrocatalytic reduction of CO2 is an appealing method for converting renewable energy sources into value-added chemical feedstocks. We report a co-electrocatalytic system for the reduction of CO2 to CO comprised of a molecular Cr complex and dibenzothiophene-5,5-dioxide (DBTD) as a redox mediator, which achieves high activity (TOF=1.51-2.84×105  s-1 ) and quantitative selectivity. Under aprotic or protic conditions, DBTD produces a co-electrocatalytic response with 1 by coordinating trans to the site of CO2 binding and mediating electron transfer from the electrode with quantitative efficiency for CO. This assembly is reliant on through-space electronic conjugation between the π frameworks of DBTD and the bpy fragment of the catalyst ligand, with contributions from dispersive interactions and weak sulfone coordination.

Non-covalent assembly of proton donors and p-benzoquinone anions for co-electrocatalytic reduction of dioxygen.

The two-electron and two-proton p-hydroquinone/p-benzoquinone (H2Q/BQ) redox couple has mechanistic parallels to the function of ubiquinone in the electron transport chain. This proton-dependent redox behavior has shown applicability in catalytic aerobic oxidation reactions, redox flow batteries, and co-electrocatalytic oxygen reduction. Under nominally aprotic conditions in non-aqueous solvents, BQ can be reduced by up to two electrons in separate electrochemically reversible reactions. With weak acids (AH) at high concentrations, potential inversion can occur due to favorable hydrogen-bonding interactions with the intermediate monoanion [BQ(AH) m ]˙-. The solvation shell created by these interactions can mediate a second one-electron reduction coupled to proton transfer at more positive potentials ([BQ(AH) m ]˙- + nAH + e- ⇌ [HQ(AH)(m+n)-1(A)]2-), resulting in an overall two electron reduction at a single potential at intermediate acid concentrations. Here we show that hydrogen-bonded adducts of reduced quinones and the proton donor 2,2,2-trifluoroethanol (TFEOH) can mediate the transfer of electrons to a Mn-based complex during the electrocatalytic reduction of dioxygen (O2). The Mn electrocatalyst is selective for H2O2 with only TFEOH and O2 present, however, with BQ present under sufficient concentrations of TFEOH, an electrogenerated [H2Q(AH)3(A)2]2- adduct (where AH = TFEOH) alters product selectivity to 96(±0.5)% H2O in a co-electrocatalytic fashion. These results suggest that hydrogen-bonded quinone anions can function in an analogous co-electrocatalytic manner to H2Q.

DFT Study on the Electrocatalytic Reduction of CO2 to CO by a Molecular Chromium Complex.

A variety of molecular transition metal-based electrocatalysts for the reduction of carbon dioxide (CO2) have been developed to explore the viability of utilization strategies for addressing its rising atmospheric concentrations and the corresponding effects of global warming. Concomitantly, this approach could also meet steadily increasing global energy demands for value-added carbon-based chemical feedstocks as nonrenewable petrochemical resources are consumed. Reports on the molecular electrocatalytic reduction of CO2 mediated by chromium (Cr) complexes are scarce relative to other earth-abundant transition metals. Recently, our group reported a Cr complex that can efficiently catalyze the reduction of CO2 to carbon monoxide (CO) at low overpotentials. Here, we present new mechanistic insight through a computational (density functional theory) study, exploring the origin of kinetic selectivity, relative energetic positioning of the intermediates, speciation with respect to solvent coordination and spin state, as well as the role of the redox-active bipyridine moiety. Importantly, these studies suggest that under certain reducing conditions, the formation of bicarbonate could become a competitive reaction pathway, informing new areas of interest for future experimental studies.

Mechanistic insight into initiation and regioselectivity in the copolymerization of epoxides and anhydrides by Al complexes.

Pentacoordinate Al catalysts comprising bipyridine (bpy) and phenanthroline (phen) backbones were synthesized and their catalytic activity in epoxide/anhydride copolymerization was investigated and compared to (t-Busalph)AlCl. Stoichiometric reactions of tricyclic anhydrides with Al alkoxide complexes produced ring-opened products that were characterized by NMR spectroscopy, mass spectrometry, and X-ray crystallography, revealing key regio- and stereochemical aspects.

Electrocatalytic CO2 Reduction to Formate with Molecular Fe(III) Complexes Containing Pendent Proton Relays.

Previously, we reported an iron(III) complex with 6,6'-([2,2'-bipyridine]-6,6'-diyl)bis(2,4-ditertbutyl-phenol) as a ligand (Fe(tbudhbpy)Cl, 1) as catalytically competent for the electrochemical reduction of CO2 to formate (Faradaic efficiency FEHCO2- = 68 ± 4%). In mechanistic experiments, an essential component was found to be a pre-equilibrium reaction involving the association of the proton donor with the catalyst, which preceded proton transfer to the Fe-bound O atoms upon reduction of the Fe center. Here, we report the synthesis, structural characterization, and reactivity of two iron(III) compounds with 6,6'-([2,2'-bipyridine]-6,6'-diyl)bis(2-methoxy-4-methylphenol) (mecrebpy[H]2, Fe(mecrebpy)Cl, 2) and 6,6'-([2,2'-bipyridine]-6,6'-diyl)bis(4-(tert-butyl)benzene-1,2-diol) (tbucatbpy[H]4, Fe(tbucatbpy), 3) as ligands, where pendent -OMe and -OH groups are poised to modify the protonation reaction involving the Fe-bound O atoms. Differences in selectivity and activity for the electrocatalytic reduction of carbon dioxide (CO2) to formate (HCO2-) between complexes 1-3 were assessed via cyclic voltammetry and controlled potential electrolysis (CPE) experiments in N,N-dimethylformamide. Mechanistic studies suggest that the O atoms in the secondary coordination sphere are important for relaying the exogenous proton donor to the active site through a preconcentration effect, which leads to the JHCO2- (partial catalytic current density for formate) increasing by 3.3-fold for 2 and 1.2-fold for 3 in comparison to the JHCO2- of 1. These results also suggest that there is a difference in the strength of the interaction between the pendent functional groups and the sacrificial proton donor between 2 and 3, resulting in quantifiable differences in catalytic activity and efficiency. CPE experiments demonstrate an increased FEHCO2- = 85 ± 2% for 2, whereas 3 had a lower FEHCO2- = 71 ± 3%, with CO and H2 generated as co-products in each case to reach mass balance. These results indicate that using secondary sphere moieties to modulate metal-ligand interactions and multisite electron and proton transfer reactivity in the primary coordination sphere through reactant preconcentration can be a powerful strategy for enhancing electrocatalytic activity and selectivity.

Electrocatalytic reduction of dioxygen by Mn(iii) meso-tetra(N-methylpyridinium-4-yl)porphyrin in universal buffer.

The electrochemical characterization of manganese(iii) meso-tetra(N-methylpyridinium-4-yl)porphyrin pentachloride ([Mn(TMPyP)Cl][Cl]4) via cyclic voltammetry (CV) and UV-vis spectroelectrochemistry (UV-vis SEC) was performed across the entire pH domain in aqueous buffered conditions. Assessment of the homogeneous electrocatalytic efficiency for the oxygen reduction reaction (ORR) from pH 3 to 6 using rotating-ring disk electrode experiments (RRDE) found it to be selective for water (82 to 93%). The observed efficiency for water is in contrast to previous reports on electrocatalytic ORR activity by Mn porphyrins in aqueous systems, which identified H2O2 as the primary product using indirect RDE methods only. The results described here are consistent with recent reports on the electrocatalytic behavior of Mn porphyrins under nonaqueous conditions, where the similar selectivity for water was also determined by RRDE methods. At pH 1, UV-vis SEC experiments also revealed that decomposition was occurring; free-base porphyrin was observed after the application of reducing potentials.