Highly sensitive and selective demethylase FTO detection using a DNAzyme-mediated CRISPR/Cas12a signal cascade amplification electrochemiluminescence biosensor with C-CN/PCNV heterojunction as emitter.

Fat mass and obesity-associated protein (FTO) has gained attention as the first RNA N6-methyladenosine (m6A) modification eraser due to its overexpression being associated with various cancers. In this study, an electrochemiluminescence (ECL) biosensor for the detection of demethylase FTO was developed based on DNAzyme-mediated CRISPR/Cas12a signal cascade amplification system and carboxylated carbon nitride nanosheets/phosphorus-doped nitrogen-vacancy modified carbon nitride nanosheets (C-CN/PCNV) heterojunction as the emitter. The biosensor was constructed by modifying the C-CN/PCNV heterojunction and a ferrocene-tagged probe (ssDNA-Fc) on a glassy carbon electrode. The presence of FTO removes the m6A modification on the catalytic core of DNAzyme, restoring its cleavage activity and generating activator DNA. This activator DNA further activates the trans-cleavage ability of Cas12a, leading to the cleavage of the ssDNA-Fc and the recovery of the ECL signal. The C-CN/PCNV heterojunction prevents electrode passivation and improves the electron-hole recombination, resulting in significantly enhanced ECL signal. The biosensor demonstrates high sensitivity with a low detection limit of 0.63 pM in the range from 1.0 pM to 100 nM. Furthermore, the biosensor was successfully applied to detect FTO in cancer cell lysate and screen FTO inhibitors, showing great potential in early clinical diagnosis and drug discovery.

Metabolism of toxic benzonitrile and hydroxybenzonitrile isomers via several distinct central pathway intermediates in a catabolically robust Burkholderia sp.

Aromatic nitriles are of considerable environmental concern, because of their hazardous impacts on the health of both humans and wildlife. In the present study, Burkholderia sp. strain BC1 was observed to be capable of utilizing toxic benzonitrile and hydroxybenzonitrile isomers singly, as sole carbon and energy sources. The results of chromatographic and spectrometric analyses in combination with oxygen uptake and enzyme activity studies, revealed the metabolism of benzonitrile as well as 2-, 3-, and 4-hydroxybenzonitriles by nitrile hydratase-amidase to the corresponding carboxylates. These carboxylates were further metabolized via central pathways, namely benzoate-catechol, salicylate-catechol, 3-hydroxybenzoate-gentisate and 4-hydroxybenzoate-protocatechute pathways in strain BC1, ultimately leading to the TCA cycle intermediates. Studies also evaluated substrate specificity profiles of both nitrile hydratase and amidase(s) involved in the denitrification of the nitriles. In addition, a few metabolic crosstalk events due to the induction of multiple operons by central metabolites were appraised in strain BC1. The present study illustrates the broad degradative potential of strain BC1, harboring diverse catabolic machinery of biotechnological importance, elucidating pathways for the assimilation of benzonitrile and that of hydroxybenzonitrile isomers for the first time.

Critical Evaluation of Polarizable and Nonpolarizable Force Fields for Proteins Using Experimentally Derived Nitrile Electric Fields.

Molecular dynamics (MD) simulations are frequently carried out for proteins to investigate the role of electrostatics in their biological function. The choice of force field (FF) can significantly alter the MD results, as the simulated local electrostatic interactions lack benchmarking in the absence of appropriate experimental methods. We recently reported that the transition dipole moment (TDM) of the popular nitrile vibrational probe varies linearly with the environmental electric field, overcoming well-known hydrogen bonding (H-bonding) issues for the nitrile frequency and, thus, enabling the unambiguous measurement of electric fields in proteins (J. Am. Chem. Soc. 2022, 144 (17), 7562-7567). Herein, we utilize this new strategy to enable comparisons of experimental and simulated electric fields in protein environments. Specifically, previously determined TDM electric fields exerted onto nitrile-containing o-cyanophenylalanine residues in photoactive yellow protein are compared with MD electric fields from the fixed-charge AMBER FF and the polarizable AMOEBA FF. We observe that the electric field distributions for H-bonding nitriles are substantially affected by the choice of FF. As such, AMBER underestimates electric fields for nitriles experiencing moderate field strengths; in contrast, AMOEBA robustly recapitulates the TDM electric fields. The FF dependence of the electric fields can be partly explained by the presence of additional negative charge density along the nitrile bond axis in AMOEBA, which is due to the inclusion of higher-order multipole parameters; this, in turn, begets more head-on nitrile H-bonds. We conclude by discussing the implications of the FF dependence for the simulation of nitriles and proteins in general.

Defect-Repaired g-C3N4 Nanosheets: Elevating the Efficacy of Sonodynamic Cancer Therapy Through Enhanced Charge Carrier Migration.

Sonodynamic therapy (SDT) has garnered growing interest owing to its high tissue penetration depth and minimal side effects. However, the lack of efficient sonosensitizers remains the primary limiting factor for the clinical application of this treatment method. Here, defect-repaired graphene phase carbon nitride (g-C3N4) nanosheets are prepared and utilized for enhanced SDT in anti-tumor treatment. After defect engineering optimization, the bulk defects of g-C3N4 are significantly reduced, resulting in higher crystallinity and exhibiting a polyheptazine imide (PHI) structure. Due to the more extended conjugated structure of PHI, facilitating faster charge transfer on the surface, it exhibits superior SDT performance for inducing apoptosis in tumor cells. This work focuses on introducing a novel carbon nitride nanomaterial as a sonosensitizer and a strategy for optimizing sonosensitizer performance by reducing bulk defects.

Nitrile-synthesizing enzymes and biocatalytic synthesis of volatile nitrile compounds: A review.

Nitriles (R-CN) comprise a broad group of chemicals industrially produced and used in fine chemicals, pharmaceuticals, and bulk applications, polymer chemistry, solvents, etc. Nitriles are important starting materials for producing carboxylic acids, amides, amines, and several other compounds. In addition, some volatile nitriles have been evaluated for their potential as ingredients in fragrance and flavor formulations. However, many nitrile synthesis methods have drawbacks, such as drastic reaction conditions, limited substrate scope, lack of readily available reagents, poor yields, and long reaction times. In contrast to chemical synthesis, biocatalytic approaches using enzymes can produce nitriles without harsh conditions, such as high temperatures and pressures, or toxic compounds. In this review, we summarize the nitrile-synthesizing enzymes from microorganisms, plants, and animals. Furthermore, we introduce several examples of biocatalytic synthesis of volatile nitrile compounds, particularly those using aldoxime dehydratase.

Prebiotically plausible chemoselective pantetheine synthesis in water.

Coenzyme A (CoA) is essential to all life on Earth, and its functional subunit, pantetheine, is important in many origin-of-life scenarios, but how pantetheine emerged on the early Earth remains a mystery. Earlier attempts to selectively synthesize pantetheine failed, leading to suggestions that "simpler" thiols must have preceded pantetheine at the origin of life. In this work, we report high-yielding and selective prebiotic syntheses of pantetheine in water. Chemoselective multicomponent aldol, iminolactone, and aminonitrile reactions delivered spontaneous differentiation of pantoic acid and proteinogenic amino acid syntheses, as well as the dihydroxyl, gem-dimethyl, and ß-alanine-amide moieties of pantetheine in dilute water. Our results are consistent with a role for canonical pantetheine at the outset of life on Earth.

Nitrile biosynthesis in nature: how and why?

Covering: up to the end of 2023Natural nitriles comprise a small set of secondary metabolites which however show intriguing chemical and functional diversity. Various patterns of nitrile biosynthesis can be seen in animals, plants, and microorganisms with the characteristics of both evolutionary divergence and convergence. These specialized compounds play important roles in nitrogen metabolism, chemical defense against herbivores, predators and pathogens, and inter- and/or intraspecies communications. Here we review the naturally occurring nitrile-forming pathways from a biochemical perspective and discuss the biological and ecological functions conferred by diversified nitrile biosyntheses in different organisms. Elucidation of the mechanisms and evolutionary trajectories of nitrile biosynthesis underpins better understandings of nitrile-related biology, chemistry, and ecology and will ultimately benefit the development of desirable nitrile-forming biocatalysts for practical applications.

Discovery of benzopyridone cyanoacetates as new type of potential broad-spectrum antibacterial candidates.

Unique benzopyridone cyanoacetates (BCs) as new type of promising broad-spectrum antibacterial candidates were discovered with large potential to combat the lethal multidrug-resistant bacterial infections. Many prepared BCs showed broad antibacterial spectrum with low MIC values against the tested strains. Some highly active BCs exhibited rapid sterilization capacity, low resistant trend and good predictive pharmacokinetic properties. Furthermore, the highly active sodium BCs (NaBCs) displayed low hemolysis and cytotoxicity, and especially octyl NaBC 5g also showed in vivo potent anti-infective potential and appreciable pharmacokinetic profiles. A series of preliminary mechanistic explorations indicated that these active BCs could effectively eliminate bacterial biofilm and destroy membrane integrity, thus resulting in the leakage of bacterial cytoplasm. Moreover, their unique structures might further bind to intracellular DNA, DNA gyrase and topoisomerase IV through various direct noncovalent interactions to hinder bacterial reproduction. Meanwhile, the active BCs also induced bacterial oxidative stress and metabolic disturbance, thereby accelerating bacterial apoptosis. These results provided a bright hope for benzopyridone cyanoacetates as potential novel multitargeting broad-spectrum antibacterial candidates to conquer drug resistance.

Identification and evolution of non-traditional nitrilase from Spirosoma linguale DSM 74 with high hydration activity.

Hydration, a secondary activity mediated by nitrilase, is a promising new pathway for amide production. However, low hydration activity of nitrilase or trade-off between hydration and catalytic activity hinders its application in the production of amides. Here, natural C-terminal-truncated wild-type nitrilase, mined from a public database, obtained a high-hydration activity nitrilase as a novel evolutionary starting point for further protein engineering. The nitrilase Nit-74 from Spirosoma linguale DSM 74 was successfully obtained and exhibited the highest hydration activity level, performing 50.7 % nicotinamide formation and 87.6 % conversion to 2 mM substrate 3-cyanopyridine. Steric hindrance of the catalytic activity center and the N-terminus of the catalytic cysteine residue helped us identify three key residues: I166, W168, and T191. Saturation mutations resulted in three single mutants that further improved the hydration activity of N-heterocyclic nitriles. Among them, the mutant T191S performed 72.7 % nicotinamide formation, which was much higher than the previously reported highest level of 18.7 %. Additionally, mutants I166N and W168Y exhibited a 97.5 % 2-picolinamide ratio and 97.7 % isonicotinamide ratio without any loss of catalytic activity, which did not indicate a trade-off effect. Our results expand the screening and evolution library of promiscuous nitrilases with high hydration activity for amide formation.

Introducing a protic ionic liquid from aminoethyl piperazine and triflic acid immobilized on pectin as a reusable nanocatalyst for the selective Schmidt synthesis of nitriles.

Preparation of reusable protic ionic liquid, triflic acid-immobilized aminoethyl piperazine-modified pectin (Pec-AEP-TfOH), with excellent activity and selectivity in modified Schmidt synthesis of nitriles from aldehydes and Si(CH3)3N3 has been described. The structure of the catalyst was characterized using FT-IR, XRD, FE-SEM, EDX-mapping, and TGA-DTA. The reaction demonstrated a broad substrate scope for a variety of benzaldehyde derivatives with electron withdrawing/donating substituents and heterocyclic aldehydes with yields between 85 and 96 % at room temperature. Also, the Pec-AEP-TfOH showed an excellent selectivity for the nitriles in which no formanilide was obtained. Furthermore, the Pec-AEP-TfOH revealed a remarkable chemoselectivity for aldehydes in the presence of acids or ketones. It is worth noting that TfOH as a precious superacid was immobilized for the first time in the selective Schmidt synthesis of nitriles to improve the eco-friendliness and economic efficiency of the process. Furthermore, the catalyst was cost-effective, metal-free, safe, scalable, and reusable (5 times) and its heterogeneity was confirmed by hot-filtration test.

Evaluation of structural and thermodynamic insight of ERß with DPN and derivatives through MMGBSA/MMPBSA methods.

Estrogen receptors (ERs) are nuclear factors that exist as two subtypes: ERα and ERß. Among the different selective ERß agonist ligands, the widely used ERß-selective agonist DPN (diarylpropionitrile) is highlighted. Recent experimental and thermodynamic information between R-DPN and S-DPN enantiomers with ERß is important for evaluating further the ability of MD simulations combined with end-point methods to reproduce experimental binding affinity and generate structural insight not provided through crystallographic data. In this research, starting from crystallographic data and experimental binding affinities, we explored the structural and thermodynamic basis of the molecular recognition of ERß with DPN and derivatives through triplicate MD simulations combined with end-point methods. Conformational analysis showed some regions with the highest mobility linked to ligand association that, at the time, impacted the total protein fluctuation. Binding free energy (ΔG) analysis revealed that the Molecular Mechanics Generalized-Born Surface Area (MMGBSA) approach was able to reproduce the experimental tendency with a strong correlation (R = 0.778), whereas per-residue decomposition analysis revealed that all the systems interacted strongly with eight residues (L298, E305, L339, M340, L343, F356, H475, and L476). The comparison between theoretical studies using the MMGBSA approach with experimental results provides new insights for drug designing of new DPN derivatives.

Structure-oriented engineering of nitrile hydratase: Reshaping of substrate access tunnel and binding pocket for efficient synthesis of cinnamamide.

Cinnamamide and its derivatives are the most common and important building blocks widely present in natural products. Currently, nitrile hydratase (NHase, EC 4.2.1.84) has been widely used in large-scale industrial production of nicotinamide and acrylamide, while its catalytic activity is extremely low or inactive for bulky nitrile substrates such as cinnamonitrile. Therefore, beneficial variant ßF37P/L48P/F51N were obtained from PtNHase of Pseudonocardia thermophila JCM3095 by reshaping of substrate access tunnel and binding pocket, which exhibited 14.88-fold improved catalytic efficiency compared to the wild-type PtNHase. Structure analysis, molecular dynamics simulations and dynamical cross-correlation matrix (DCCM) analysis revealed that the introduced mutations enlarged the substrate access tunnel and binding pocket, enhanced overall anti-correlated movements of enzymes, which would promote product release during the dynamic process of catalysis. In a hydration process, the complete conversion of 5 mM cinnamonitrile was achieved by ßF37P/L48P/F51N in a 50 mL reaction, with cinnamamide yield of almost 100 % and productivity of 0.736 g L-1 h-1. The study demonstrates the co-evolution of substrate access tunnel and binding pocket is an effective strategy, and provides a valuable reference for future research. Furthermore, NHases have huge potential for catalyzing bulky nitriles to form corresponding amides in large-scale industrial production.

Single-atom Ag-loaded carbon nitride photocatalysts for efficient degradation of acetaminophen: The role of Ag-atom and O2.

Carbon nitride has been extensively used as a visible-light photocatalyst, but it has the disadvantages of a low specific surface area, rapid electron-hole recombination, and relatively low light absorbance. In this study, single-atom Ag was successfully anchored on ultrathin carbon nitride (UTCN) via thermal polymerization, the catalyst obtained is called AgUTCN. The Ag hardly changed the carbon nitride's layered and porous physical structure. AgUTCN exhibited efficient visible-light photocatalytic performances in the degradation of various recalcitrant pollutants, eliminations of 85% were achieved by visible-light irradiation for 1 hr. Doping with Ag improved the photocatalytic performance of UTCN by narrowing the forbidden band gap from 2.49 to 2.36 eV and suppressing electron-hole pair recombination. In addition, Ag doping facilitated O2 adsorption on UTCN by decreasing the adsorption energy from -0.2 to -2.22 eV and favored the formation of O2·-. Electron spin resonance and radical-quenching experiments showed that O2·- was the major reactive species in the degradation of Acetaminophen (paracetamol, APAP).

The Nitrile Bis-Thiol Bioconjugation Reaction.

Electron-poor aryl nitriles are promising reagents for bioconjugation due to their high electrophilicity and selectivity for reaction with thiols, albeit generally in a reversible manner. A transient species has previously been observed in such reactions, involving the addition of two thiols to the nitrile functional group, forming a tetrahedral amino dithioacetal (ADTA). In this work, the reaction of heteroaryl nitriles with bis-thiols is explored in an attempt to generate stable ADTAs, which could facilitate new bioconjugation protocols. By use of a 1,2-dithiol, or the incorporation of an electrophilic trap into the aryl nitrile design, the formation of stable products is achieved. The resultant "nitrile bis-thiol" (NBT) reaction is then explored in the context of protein modification, specifically to carry out antibody conjugation. By addition of these nitriles to the reduced disulfide bond of an antibody fragment, it is shown that, depending on the reagent design, cysteine-to-lysine transfer or disulfide bridged NBT products can be generated. Both represent site-selective conjugates and are shown to be stable when challenged with glutathione under physiological conditions and upon incubation in serum. Furthermore, the NBT reaction is tested in the more challenging context of a full antibody, and all four disulfide bonds are effectively modified by these new one-carbon bridging reagents. Overall, this reaction of heteroaryl-nitriles with bis-thiols is shown to be highly efficient and versatile, of tunable reversibility, and offers enticing prospects as a new addition to the toolbox of biocompatible "click"-type reactions.

B, P, and S heteroatom doped, bio- and hemo-compatible 2D graphitic-carbon nitride (g-C3N4) with antioxidant, light-induced antibacterial, and bioimaging endeavors.

The synthesis of two-dimensional (2D) graphiticg-C3N4and heteroatom-doped graphitic H@g-C3N4(H: B, P, or S) particles were successfully done using melamine as source compounds and boric acid, phosphorous red, and sulfur as doping agents. The band gap values of 2Dg-C3N4, B50@g-C3N4, P50@g-C3N4, and S50@g-C3N4structures were determined as 2.90, 3.03, 2.89, and 2.93 eV, respectively. The fluorescent emission wavelengths of 2Dg-C3N4, B50@g-C3N4, P50@g-C3N4, and S50@g-C3N4structures were observed at 442, 430, 441, and 442 nm, respectively upon excitation atλEx= 325 nm. There is also one additional new emission wavelength was found at 345 nm for B50@g-C3N4structure. The blood compatibility test results ofg-C3N4, B50@g-C3N4, P50@g-C3N4, and S50@g-C3N4structures revealed that all materials are blood compatible with <2% hemolysis and >90% blood clotting indices at 100µg ml-1concentration. The cell toxicity of the prepared 2D graphitic structures were also tested on L929 fibroblast cells, and even the heteroatom doped hasg-C3N4structures induce no cytotoxicity was observed with >91% cell viability even at 250µg ml-1particle concentration with the exception of P50@g-C3N4which as >75 viability. Moreover, for 2Dg-C3N4, B50@g-C3N4, and S50@g-C3N4constructs, even at 500µg ml-1concentration, >90% cell viabilities was monitored. As a diagnostic material, B50@g-C3N4was found to have significantly high penetration and distribution abilities into L929 fibroblast cells granting a great potential in fluorescence imaging and bioimaging applications. Furthermore, the elemental doping with B, P, and S ofg-C3N4were found to significantly increase the photodynamic antibacterial activity e.g. more than half of bacterial elimination by heteroatom-doped forms ofg-C3N4under UVA treatment was achieved.

Residue dynamics and dietary risk assessment of new formulation of novaluron and lambda cyhalothrin on tomato.

Presence of residues on food commodities is major bottleneck of insecticide use under good agricultural practices (GAPs). The use of less persistent with two different mode of action insecticide is novel approach of getting maximum insect control without developing insecticide resistance. Novaluron, an insect growth disruptor and lambda cyhalothrin, a nerve poison has been used widely for the management of lepidopteran pests. Dissipation and consumer risk analysis studies were carried out on a new combination product of novaluron and lambda cyhalothrin are used for control insects of tomato at recommended standard dose of 71 + 14 g/ha and double dose of 142 + 28 g/ha of active ingredients. Extraction and cleanup of sample residues was done using QuEChERS technique and analyzed in GC-ECD. The residues of novaluron were dissipated within 10 to 15 days, and the residues of lambda cyhalothrin were at 7 to 10 days, both at the standard and double the standard dose, following a first order reaction kinetics. Analysis of risk and hazard quotient revealed that the test insecticides do not pose any dietary risk to consumer as TMDI < MPI and HQ < 1.

N-Terminal Cysteine Bioconjugation with (2-Cyanamidophenyl)boronic Acids Enables the Direct Formation of Benzodiazaborines on Peptides.

Benzodiazaborines (BDABs) have emerged as a valuable tool to produce stable and functional bioconjugates via a click-type transformation. However, the current available methods to install them on peptides lack bioorthogonality, limiting their applications. Here, we report a strategy to install BDABs directly on peptide chains using (2-cyanamidophenyl)boronic acids (2CyPBAs). The resulting BDAB is stabilized through the formation of a key intramolecular B-N bond. This technology was applied in the selective modification of N-terminal cysteine-containing functional peptides.

Cyclic Allene Approach to the Manzamine Alkaloid Keramaphidin B.

We report an approach to the core of the manzamine alkaloid keramaphidin B that relies on the strain-promoted cycloaddition of an azacyclic allene with a pyrone trapping partner. The cycloaddition is tolerant of nitrile and primary amide functional groups and can be complemented with a subsequent retro-Diels-Alder step. These efforts demonstrate that strained cyclic allenes can be used to build significant structural complexity and should encourage further studies of these fleeting intermediates.

Orthogonal translation with 5-cyanotryptophan as an infrared probe for local structural information, electrostatics, and hydrogen bonding.

Orthogonal translation is an efficient tool that provides many valuable spectral probes capable of covering different parts of the electromagnetic spectrum and thus enabling parameterization of various structural and dynamic phenomena in proteins. In this context, nitrile-containing tryptophan analogs are very useful probes to study local electrostatics and hydrogen bonding in both rigid and dynamic environments. Here, we report a semi-rational approach to engineer a tyrosyl-tRNA synthetase (TyrRS) variant of Methanocaldococcus jannaschii capable of incorporating 5-cyanotryptophan (5CNW) via orthogonal translation. We combined one round of the well-established positive selection system with saturation mutagenesis at preselected TyrRS positions, resulting in a novel 5CNW-specific enzyme that also exhibits high substrate tolerance to other aromatic noncanonical amino acids. We demonstrated the utility of our orthogonal pair by inserting 5CNW into the cyanobacteriochrome Slr1393g3, a bilin-binding photosensor of the phytochrome superfamily. The nitrile (CN) group of the inserted 5CNW provides non-invasive labeling in the local structural context while yielding information on local electrostatics and hydrogen bonding by IR spectroscopy. 5CNW is a versatile probe that can be used for both static and dynamic measurements.

AMOEBA Force Field Predicts Accurate Hydrogen Bond Counts of Nitriles in SNase by Revealing Water-Protein Interaction in Vibrational Absorption Frequencies.

Precisely quantifying the magnitude and direction of electric fields in proteins has long been an outstanding challenge in understanding biological functions. Nitrile vibrational Stark effect probes have been shown to be minimally disruptive to the protein structure and can be better direct reporters of local electrostatic field in the native state of a protein than other measures such as pKa shifts of titratable residues. However, interpretations of the connection between measured vibrational energy and electric field rely on the accurate molecular understanding of interactions of the nitrile group and its environment, particularly from hydrogen bonding. In this work, we compared the extent of hydrogen bonding calculated in two common force fields, the fixed charge force field Amber03 and polarizable force field AMOEBA, at 10 locations of cyanocysteine (CNC) in staphylococcal nuclease (SNase) against the experimental nitrile absorption frequency in terms of full width at half-maximum (FWHM) and frequency temperature line slope (FTLS). We observed that the number of hydrogen bonds correlated well in AMOEBA trajectories with respect to both the FWHM (r = 0.88) and the FTLS (r = -0.85), whereas the correlation of Amber03 trajectories was less reliable because the Amber03 force field predicted more hydrogen bonds in some mutants. Moreover, we demonstrated that contributions from the interactions between CNC and nearby water molecules were significant in AMOEBA trajectories but were not predicted by Amber03. We conclude that although the nitrile absorption peak shape could be qualitatively predicted by the fixed charge Amber03 force field, the detailed electrostatic environment measured by the nitrile probe in terms of the extent of hydrogen bonding could only be accurately observed in the AMOEBA trajectories, where the permanent dipole, quadrupole, and dipole-induced-dipole polarizable interactions were all taken into account. The significance of this finding to the goal of accurately predicting electric fields in complex biomolecular environments is discussed.