Reactive Amino Research Articles

Site-Specific Immobilization Boosts the Performance of a Galectin-1 Biosensor.

The analysis of protein-bound glycans has gained significant attention due to their pivotal roles in physiological and pathological processes like cell-cell recognition, immune response, and disease progression. Routine methods for glycan analysis are challenged by the very similar physicochemical properties of their carbohydrate components. As an alternative, lectins, which are proteins that specifically bind to glycans, have been integrated into biosensors for glycan detection. However, the effectiveness of protein-based biosensors depends heavily on the immobilization of proteins on the sensor surface. To enhance the sensitivity and/or selectivity of lectin biosensors, it is crucial to immobilize the lectin in an optimal orientation for ligand binding without compromising its function. Random immobilization methods often result in arbitrary orientation and reduced sensitivity. To address this, we explored a directed immobilization strategy relying on a reactive noncanonical amino acid (ncAA) and bioorthogonal chemistry. In this study, we site-specifically incorporated the reactive noncanonical lysine derivative, Nε-((2-azidoethoxy)carbonyl)-l-lysine, into a cysteine-less single-chain variant of human galectin-1 (scCSGal-1). The reactive bioorthogonal azide group allowed the directed immobilization of the lectin on a biosensor surface using strain-promoted azide-alkyne cycloaddition. Biolayer interferometry data demonstrated that the controlled, directed attachment of scCSGal-1 to the biosensor surface enhanced the binding sensitivity to glycosylated von Willebrand factor by about 12-fold compared to random immobilization. These findings emphasize the importance of controlled protein orientation in biosensor design. They also highlight the power of single site-specific genetic encoding of reactive ncAAs and bioorthogonal chemistry to improve the performance of lectin-based diagnostic tools.

Fully aminated p‐polyaramid effectively strengthen and toughen Bisphenol A epoxy resin and its warp‐knitted carbon fiber composites

AbstractStemming from the cost‐effectiveness and broad applicability, many modification researches on Bisphenol A epoxy resin (DGEBA) and its carbon fiber composites have been done extensively. Here, following the concept of molecular composite, we prepared fully aminated p‐polyaramid with reactive amino side groups by polycondensation and reduction reaction, aiming to construct a molecular distribution of the reinforcement within the resin matrix and achieve a satisfactory enhancement effect. Among them, after adding 1 wt% fully aminated p‐polyaramid, Young's modulus and tensile toughness of the DGEBA epoxy resin (EP) increased significantly to 2.34 ± 0.03 GPa (+68.3%) and 4402 ± 213 kJ/m3 (+30.5%), the flexural modulus and toughness reached 4.14 ± 0.04 GPa (+42.8%) and 0.44 ± 0.01 J (+83.3%). Moreover, the Tg of EP was only decreased by 2.7°C. In addition, their warp‐knitted bidirectional carbon fiber composites also have enhancements in both flexural properties and interlaminar shear strength (all improved by more than 20%). What is noticeable is that the preparation and blending of fully aminated p‐polyaramid are simpler and more effective than most other micro‐ or nanofillers, and the dispersion is more uniform. Thus, it can act as a highly effective reinforcement and strengthen the properties of DGEBA and its carbon fiber composite materials significantly.Highlights Fully aminated p‐polyaramid is uniformly dispersed in EP by chemical bonds. DGEBA is strengthened and toughened without a significant decrease in Tg. The tensile and flexural modulus of EP is increased by 68.3% and 42.8%. Fully aminated p‐polyaramid can reinforce warp‐knitted carbon fiber composite.

LuCl3/B(C6F5)3 Cocatalyzed Reductive Deoxygenation of Ketones, Aldehydes, Alcohols, and Ethers to Alkanes with Pinacolborane.

This report describes the LuCl3/B(C6F5)3 cocatalyzed reductive deoxygenation of 67 ketones, aldehydes, alcohols, and ethers to alkanes under mild conditions. The strategy tolerates reactive amino, hydroxyl, nitro, halogen, vinyl, and ester functional groups, and the results demonstrate rare chemoselective deoxygenation of α,β-unsaturated ketones. Isotopic labeling experiments, control experiments, and derivatization studies are used to elucidate the reaction mechanism.

Top-down Proteomics for the Characterization and Quantification of Calreticulin Arginylation.

Arginylation installed by arginyltransferase 1 (ATE1) features an addition of arginine (Arg) to the reactive amino acids (e.g., Glu and Asp) at the protein N-terminus or side chain. Systemic removal of arginylation after ATE1 knockout (KO) in mouse models resulted in heart defects leading to embryonic lethality. The biological importance of arginylation has motivated the discovery of arginylation sites on proteins using bottom-up approaches. While bottom-up proteomics is powerful in localizing peptide arginylation, it lacks the ability to quantify proteoforms at the protein level. Here we developed a top-down proteomics workflow for characterizing and quantifying calreticulin (CALR) arginylation. To generate fully arginylated CALR (R-CALR), we have inserted an R residue after the signaling peptide (AA1-17). Upon overexpression in ATE1 KO cells, CALR and R-CALR were purified by affinity purification and analyzed by LCMS in positive mode. Both proteoforms showed charge states ranging from 27-68 with charge 58 as the most intense charge state. Their MS2 spectra from electron-activated dissociation (EAD) showed preferential fragmentation at the protein N-terminals which yielded sufficient c ions facilitating precise localization of the arginylation sites. The calcium-binding domain (CBD) gave minimum characteristic ions possibly due to the abundant presence of >100 D and E residues. Ultraviolet photodissociation (UVPD) compared with EAD and ETD significantly improved the sequence coverage of CBD. This method can identify and quantify CALR arginylation at absence, endogenous (low), and high levels. To our knowledge, our work is the first application of top-down proteomics in characterizing post-translational arginylation in vitro and in vivo.

Coordination chemistry of 4-aminopyrazole: Structure and physicochemical properties of cobalt(II) chlorido complexes, and amino group reactivity towards a ketone to yield an imine bond

An outline of the in situ construction of a coordination polymer by reacting a mononuclear cobalt(II) complex, functionalised with an amine substituent, with acetone is given. The methodology involved 4-amino-3,5-diisopropyl-1-pyrazole (denoted as L1pzHNH2) as the precursor, putative linker molecule. Reaction of the ligand with anhydrous cobalt(II) chloride, yielded a four-coordinated cobalt(II) chlorido complex, [CoCl2(L1pzHNH2)2]. When the complex was reacted with acetone, a four-coordinated cobalt(II) chlorido coordination polymer, [CoCl2{L1pzHN=C(CH3)2}]n, with an imine bond, was obtained. The complexes were characterised by various spectroscopic techniques. X-ray crystallography shows [CoCl2(L1pzHNH2)2]·2(thf) to comprise a tetrahedrally coordinated cobalt(II) centre with the donors being two chlorido and two pyrazolyl-N atoms. Similarly, [CoCl2{L1pzHN=C(CH3)2}]n features a Cl2N2 donor set defining an approximately tetrahedral geometry with the imine-N donors being derived from a pyrazolyl ring and a symmetry related exocyclic imine-N atom with the result that a one-dimensional coordination polymer with a helical topology is formed. When the uncoordinated precursor molecule, L1pzHNH2, was reacted with acetone, the formation of the imine was confirmed by 1H NMR measured in (CD3)2CO solution. However, from the 1H NMR measured for the imine in CDCl3 solution, a 70:30 equilibrium between the imine and amine was established. It is concluded that the nucleophilicity of the amine group is enhanced by the binding of cobalt(II) to the pyrazole nitrogen in [CoCl2(L1pzHNH2)2]·2(thf), thereby favouring irreversible imine formation. The facile formation of [CoCl2{L1pzHN=C(CH3)2}]n in the solid-state also correlates with the reduced solubility of the coordination polymer structure in organic solvents.

Accelerated Degradation of DiXyl-α,ε-Lys-ARP via Interaction between Extra-Added Xylose and Monosubstituted Lys-ARPs during Maillard Reaction.

Lysine (Lys) is capable of forming a di-substituted Amadori rearrangement product (ARP) with xylose (Xyl), designated as diXyl-α,ε-Lys-ARP. DiXyl-α,ε-Lys-ARP degradation was characterized by two steps: Initially, Xyl-α- and Xyl-ε-Lys-ARP were formed through elimination or hydrolysis at specific Nα/Nε positions of the corresponding enol and imine intermediates, which were then further degraded to dicarbonyl compounds and regenerated Lys. Xyl-α- or Xyl-ε-Lys-ARP had a reactive free amino group (ε-NH2 or α-NH2), both of which were still highly reactive and able to undergo further reactions with Xyl. Therefore, the diXyl-α,ε-Lys-ARP/Xyl model system was established to explore the impact of extra-added Xyl on diXyl-α,ε-Lys-ARP degradation behavior. Extra-added Xyl remarkably affected the degradation pathway of diXyl-α,ε-Lys-ARP by capturing the Xyl-α- and Xyl-ε-Lys-ARP to regenerate diXyl-α,ε-Lys-ARP. This interaction between Xyl and mono-substituted Lys-ARPs promoted the shift of chemical equilibrium toward the degradation of diXyl-α,ε-Lys-ARP, thereby accelerating its degradation rate. This degradation was markedly facilitated by the elevated temperature and pH values. Interestingly, the yield of Xyl-α- and Xyl-ε-Lys-ARP was particularly dependent on the pH during diXyl-α,ε-Lys-ARP degradation. Xyl-ε-Lys-ARP was the dominant product at pH 5.5-7.5 while Xyl-α-Lys-ARP possessed a relatively higher content under weak alkaline conditions, which was related to the reactivities of the Nα/Nε positions under various reaction conditions.

Liposome-Papain Conjugates for Catalytic Digestion of Antibody Producing Fab Fragments.

Papain is useful for the enzymatic digestion of various proteins to produce functional peptides or protein fragments. Immobilized papain being reactive toward proteins and easily removable from a reaction mixture is worth developed. In the present work, liposomes were applied as colloidal carriers of papain for the catalytic digestion of polyclonal immunoglobulin G (IgG). Papain was covalently conjugated at pH = 7.0 via tris-succinimidyl aminotriacetate (TSAT) to liposomes incorporated with 5 mol % poly(ethylene glycol)-tethered lipid with a reactive amino group. The papain-conjugated liposome (liposome-papain) catalyzed the hydrolysis of Nα-benzoyl-l-arginine 4-nitroanilide hydrochloride (BAPNA) at pH = 5.0-7.0. The activity of liposome-papain significantly increased with increasing temperature from 25 to 50 °C. The Michaelis constant Km was determined with respect to the liposome-papain- and free papain-catalyzed reactions with BAPNA at 37 °C as Km = 1.11 ± 0.13 and 11.6 ± 2.9 mM, respectively. Liposome-papain was applied to the catalytic digestion of 10 mg·mL-1 IgG at 37 °C for 24 h at pH = 5.0-7.0. The reaction mixture could be analyzed without pretreatment by using the affinity columns immobilized with the protein A or protein L ligand because colloidal liposome-papain quickly flowed through the chromatographic stationary phase, exhibiting little proteolytic effect on the proteinaceous ligands. The analysis clearly demonstrated the catalytic production of antigen-binding fragments (Fab) from IgG in an enzyme concentration- and pH-dependent manner. Liposome-papain with 15 or 50 mol % anionic lipids also catalyzed the formation of Fab from IgG. The above results demonstrated that liposome-papain was useful to digest IgG and to purify Fab formed with the affinity chromatography.

Mathematical modelling of genipin-bovine serum albumin interaction using fluorescence intensity measurements

The interaction between genipin and a model protein bovine serum albumin (BSA), with and without the addition of acetic acid, has been studied experimentally and by modelling. The number of amino groups available to react was determined to be 5.6 % of the total number of amino acid building blocks on BSA. Fluorescence intensity was used to record the progress of the reaction over the 24 h, while the modelling study focused on capturing the kinetic profiles of the reaction. The experiments revealed a slow start to the BSA and genipin interaction, that subsequently accelerated in an S-shaped curve which the modelling study linked with the existence of the feedback cycle for both reactive amino groups and genipin. At BSA concentrations ≥30 mg/mL the reaction was accelerated in the presence of acid, while below 30 mg/mL the acidified conditions delayed the onset of the reaction. Contrary to the reaction mechanisms previously proposed, a degree of breakdown of the fluorescent links in the products formed was denoted both experimentally and in a modelling study. This indicated the reversibility of the processes forming fluorescent product/s and suggested feasibility of the successful release of the protein following prospective encapsulation within the genipin-crosslinked hydrogel structure.

Parylene Double-Layer Coated Screen-Printed Carbon Electrode for Label-Free and Reagentless Capacitive Aptasensing of Gliadin.

Celiac patients are required to strictly adhere to a gluten-free diet because even trace amounts of gluten can damage their small intestine and leading to serious complications. Despite increased awareness, gluten can still be present in products due to cross-contamination or hidden ingredients, making regular monitoring essential. With the goal of guaranteeing food safety for consuming labeled gluten-free products, a capacitive aptasensor was constructed to target gliadin, the main allergic gluten protein for celiac disease. The success of capacitive aptasensing was primarily realized by coating a Parylene double-layer (1000 nm Parylene C at the bottom with 400 nm Parylene AM on top) on the electrode surface to ensure both high insulation quality and abundant reactive amino functionalities. Under the optimal concentration of aptamer (5 μM) used for immobilization, a strong linear relationship exists between the amount of gliadin (0.01-1.0 mg/mL) and the corresponding ΔC response (total capacitance decrease during a 20 min monitoring period after sample introduction), with an R2 of 0.9843. The detection limit is 0.007 mg/mL (S/N > 5), equivalent to 0.014 mg/mL (14 ppm) of gluten content. Spike recovery tests identified this system is free from interferences in corn and cassava flour matrices. The analytical results of 24 commercial wheat flour samples correlated well with a gliadin ELISA assay (R2 = 0.9754). The proposed label-free and reagentless capacitive aptasensor offers advantages of simplicity, cost-effectiveness, ease of production, and speediness, making it a promising tool for verifying products labeled as gluten-free (gluten content <20 ppm).

Progresses and Developments in Chitosan-based Materials and its Derivatives for Biomedical and Biosensing Applications

Abstract: This extensive review highlights the different techniques used to extract chitosan, its antibacterial characteristics, and its wide range of applications across various industries. Thanks to its exceptional solubility and notable chemical and biological properties, it has been used in several applications. It is biodegradable, and biocompatible, and has numerous reactive amino side groups that enable chemical modification and the development of various practical derivatives. Several academic publications have extensively covered many characteristics of chitosan and its applications in different industries, such as medicine. Chitosan can impart different functions in multiple fields, such as antibacterial, antiviral, and other biological features. Chitosan has various applications across various industries. Due to its exceptional solubility and significant chemical and biological properties, it has been utilized in numerous applications. Chitosan is biodegradable and biocompatible, and its multiple reactive amino side groups enable chemical modification and the development of various practical derivatives. Chitosan can provide different functions in numerous fields, such as antibacterial, antiviral, and other biological features. This review primarily focuses on chitosan's sources and extraction methods, providing an up-to-date overview of its properties. Additionally, the review highlights the diverse applications of chitosan, whether in its original state or modified and incorporated into nanocomposites, in various fields, making it a highly versatile and commonly used material in the biomedical industry.

Stereocontrolled Synthesis of Aryl C-Nucleosides under Ambient Conditions.

A stereocontrolled synthesis of an aryl C-nucleoside has been developed using D-ribals and arylboronic acids catalyzed by palladium without additional ligands in common solvents under an open-air atmosphere at room temperature. This protocol features very mild conditions, simplicity in operation, exclusive β-stereoselectivity, broad substrate scopes, and good compatibility with reactive amino and hydroxyl groups. The functionalization of unsaturated C-nucleosides and the late-stage glycosylation of natural products/drugs demonstrated the high practicality of this strategy.

Robust mechanical strength and high colorfastness of amorphous photonic crystals on fabrics with durable antibacterial and anti-static properties

The technique of utilizing structural color dyeing presents a sustainable and energy-conserving approach to textile dyeing. However, the coating with structural coloration always exhibited cracks and inadequate colorfastness attributed to the presence of capillary tension. In this study, we employed a hydrophilic silane called 3-aminopropyltriethoxysilane (KH550) to chemically modify the fabric. This method endowed the fabric with reactive amino groups, thereby augmenting the interfacial bonding strength between polydopamine (PDA)@SiO2@Ag+/Ag NPs and the fabric substrate through metal coordination and covalent bonds. Additionally, by utilizing catechol complexation, we successfully loaded Ag ions onto the PDA coating of composite microspheres. These Ag ions acted as linkers during self-assembly of microspheres. The active amino groups on the surface of chemically modified cotton fabric reacted with PDA at a specific temperature, resulting in the formation of robust covalent bonds simultaneously. The resulting fabric demonstrated excellent water resistance as well as efficient antibacterial and anti-static properties, which were confirmed through various mechanical tests such as washing, folding, and rubbing experiments. This research presents an innovative approach for creating durable structural colored fabrics with multifunctional properties based on metal coordination and covalent bonds that can be effectively applied in textile printing and dyeing.

Cysteine S-acetylation is a post-translational modification involved in metabolic regulation.

Cysteine is a reactive amino acid central to the catalytic activities of many enzymes. It is also a common target of post-translational modifications (PTMs), such as palmitoylation. This longchain acyl PTM can modify cysteine residues and induce changes in protein subcellular localization. We hypothesized that cysteine could also be modified by short-chain acyl groups, such as cysteine S-acetylation. To test this, we developed sample preparation and non-targeted mass spectrometry protocols to analyze the mouse liver proteome for cysteine acetylation. Our findings revealed hundreds of sites of cysteine acetylation across multiple tissue types, revealing a previously uncharacterized cysteine acetylome. Cysteine acetylation shows a marked cytoplasmic subcellular localization signature, with tissue-specific acetylome patterns and specific changes upon metabolic stress. This study uncovers a novel aspect of cysteine biochemistry, highlighting short-chain modifications alongside known long-chain acyl PTMs. These findings enrich our understanding of the landscape of acyl modifications and suggest new research directions in enzyme activity regulation and cellular signaling in metabolism.

Investigating of epoxy nanocomposites structure and properties that contain both pristine and aminosilane-treated silicon carbide (SiC) particles

This article presents a methodology for functionalization of silicon carbide (SiC) through chemical modification using γ-aminopropyltriethoxysilane (APTES) and its subsequent dispersion in an epoxy composition. The research revealed that functionalizing SiC particles with γ-aminopropyltriethoxysilane (SiC(APTES)) enhanced their chemical compatibility with the epoxy composition, facilitating the dispersion of SiC particles. Furthermore, it was observed that the functionalization of the filler had a profound impact on the structure, curing kinetics, and physical and mechanical properties of epoxy nanocomposites. The addition of SiC(APTES) into the epoxy composition resulted in a significant reinforcement of the material. Specifically, the flexural stress and flexural modulus of elasticity increased by 179% and 74%, respectively, while the impact strength experienced a remarkable improvement of 462%. Additionally, the tensile strength and tensile modulus of elasticity increased by 83% and 70%, respectively, compared to the epoxy composite without SiC. The application of SiC(APTES) also played a crucial role in initiating the polymerization process through the involvement of reactive amino groups, leading to a reduction in the initial curing temperature and an amplification of the thermal effects of the polymerization reaction. Moreover, the presence of functionalized SiC significantly influenced the structure of the epoxy composite, thereby contributing to its enhanced strength. In summary, the inclusion of SiC in the epoxy composition not only bolstered the material but also improved its thermal stability.

Catalytic effect of N-phenylaminopropyl polyheadral oligomeric silsesquioxane in the synthesis of hybrid nanocomposites based on polycyanurate

Organic-inorganic nanocomposites based on heat-resistant crosslinked polycyanurate (PCN) and N-phenylaminopropyl polyhedral oligomeric silsesquioxane (NPAP-POSS), containing eight reactive secondary amino groups, were synthesized using the in situ reactive formation method. Fourier transmission infrared (FTIR) spectroscopy and dynamic differential scanning calorimetry (DSC) methods were used to study the effect of NPAP-POSS on the kinetics of bisphenol E dicyanate ester (DCBE) polycyclotrimerization during the formation of PCN in PCN/NPAP-POSS nanocomposites. The content of the nanofiller was varied from 0.05 to 1.00 wt.%. Based on the results of FTIR spectral studies, the main kinetic peculiarities of PCN formation were found and their changes under the action of NPAP-POSS nanofiller were determined. A significant catalytic effect of NPAP-POSS on the polycyclotrimerization of DCBE was found, which is confirmed by a decrease in the time of the onset of auto-acceleration, an acceleration of the conversion of cyanate groups of DCBE and the formation of triazine cycles of PCN, an increase in the values of the maximum reaction rate, a decrease in the duration of the reaction, etc. The dynamic DSC method also confirmed the catalytic effect of NPAP-POSS on the formation of PCN in the nanocomposites and established the main kinetic characteristics depending on the content of the nanofiller: a significant decrease in the temperature of the exothermic maximum, an increase in the reaction enthalpy, non-monotonic changes in the induction period and reaction rate, etc. From the analysis of the experimental data, it was concluded that the detected changes in the kinetics of the in situ reaction formation of PCN/NPAP-POSS nanocomposites and the recorded catalytic effect of the nanofiller are due to the fact that two chemical processes occur during the synthesis of the nanocomposites: chemical interaction of –O–C≡N groups of DCBE with secondary –NH groups of NPAP-POSS, which led to further embedding of nanoparticles into the resulting polymer matrix and the direct polycyclotrimerization of DCBE with formation of hybrid polycyanurate network. Schemes of the sequential reactions explaining the catalytic effect of the nanofiller in the synthesis of hybrid PCN/NPAP-POSS nanocomposites are proposed. It was concluded that under the selected conditions of the synthesis, the greatest catalytic effect of the nanofiller is manifested at its content of 0.10 wt.%, since for this sample the maximum shift of the reaction exothermic peak towards lower temperatures, the maximum reaction rate, and the minimum induction period and reaction start temperature were recorded. The results of the research make it possible to optimize the synthesis of heat-resistant materials promising for use in special-purpose structures.

Studying the Structure and Properties of Epoxy Composites Modified by Original and Functionalized with Hexamethylenediamine by Electrochemically Synthesized Graphene Oxide.

In this study, we used multilayer graphene oxide (GO) obtained by anodic oxidation of graphite powder in 83% sulfuric acid. The modification of GO was carried out by its interaction with hexamethylenediamine (HMDA) according to the mechanism of nucleophilic substitution between the amino group of HMDA (HMDA) and the epoxy groups of GO, accompanied by partial reduction of multilayer GO and an increase in the deformation of the carbon layers. The structure and properties of modified HMDA-GO were characterized using research methods such as scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction spectroscopy and Raman spectroscopy. The conducted studies show the effectiveness of using HMDA-OG for modifying epoxy composites. Functionalizing treatment of GO particles helps reduce the free surface energy at the polymer-nanofiller interface and increase adhesion, which leads to the improvement in physical and mechanical characteristics of the composite material. The results demonstrate an increase in the strength and elastic modulus in bending by 48% and 102%, respectively, an increase in the impact strength by 122%, and an increase in the strength and elastic modulus in tension by 82% and 47%, respectively, as compared to the pristine epoxy composite which did not contain GO-HMDA. It has been found that the addition of GO-HMDA into the epoxy composition initiates the polymerization process due to the participation of reactive amino groups in the polymerization reaction, and also provides an increase in the thermal stability of epoxy nanocomposites.

Abstract 3315: Preclinical studies of TSN1611, a potent, selective, and orally bioavailable KRASG12D inhibitor

Abstract Background: KRAS mutations are the most frequently encountered driver oncogene, involved in ~25% of all human cancers [1,2]. KRASG12D is the predominant KRAS mutation isoform, detected in approximately 35% of pancreatic cancer, 13% of colorectal cancer, and 5% of NSCLC [3]. Compared to KRASG12C, targeting KRASG12D has proven to be more challenging since the target protein lacks a reactive amino acid residue for irreversible inhibitory modification by a ligand. Herein, we disclose TSN1611, a potent and selective KRASG12D inhibitor, which possesses favorable oral PK profiles and demonstrates significant in vitro and in vivo anti-tumor activity in various KRASG12D-mutant models. Method: Biochemical HTRF assay was used to measure the inhibition of TSN1611 to both GDP-bound and GTP-bound state of KRASG12D. Biophysical SPR method was used to directly measure the binding of TSN1611 to GDP-bound KRASG12D and KRASWT. Cell based activities were evaluated in a series of in vitro cell proliferation assay utilizing Ba/F3 cells engineered with KRASG12D or non-KRASG12D mutations and tumor cell lines harboring KRASG12D mutation. Human cancer cell-derived xenograft models of HPAC (pancreatic) and GP2D (colorectal) were used to evaluate its in vivo antitumor effect. in vitro and in vivo PK studies were performed in mouse, rat, and dog. Systematic nonclinical safety evaluations, including safety panel screen testing, safety pharmacology studies, and repeat-dose toxicity studies were carried out to assess its preliminary toxicity profile. Results: TSN1611 inhibited both active (GTP-bound) and inactive (GDP-bound) forms of KRASG12D protein at IC50 1.23 and 1.49 nM, respectively; the KD value of its direct binding to KRASG12D protein is 1.93 pM in SPR assay. TSN1611 demonstrated potent anti-proliferation activity against several tumor cell lines harboring KRASG12D mutation, and excellent selectivity over cells of NRAS, HRAS, and other KRAS isoforms. It also showed dose-dependent anti-tumor efficacy in GP2D and HPAC models. Mechanism of action studies concluded that the antitumor effect of TSN1611 is resulted from its effective inhibition of KRAS signaling pathway. Oral bioavailability and safety profile across multiple species supported its further development. Conclusion: TSN1611 is a selective KRASG12D inhibitor. It exhibited excellent selectivity and activity both in vitro and in vivo; it demonstrated favorable physicochemical properties, oral PK profiles, and brain penetration potential; it also showed acceptable margins of safety. The preclinical data supports further development. Pending regulatory submission and review, the phase I/II study is planned to start in H1 of 2024.

Increased reactive carboxyl and free alfa-amino groups from fish type I collagen peptides by Alcalase® hydrolysis exhibit higher antibacterial and antioxidant activities

Abstract This study aimed to generate low molecular weight peptides (LMWP) from fish collagen type I hydrolysed by increasing activity of Alcalase® from 0.0 to 12.0 (AU-A per 100 g) at 55 °C and 7.4 of pH for 3 h 40 min. The results showed that all enzyme activity levels caused 34–55 % reductions in protein recovery, 1.0–3.0 folds’ increase in free α-amino groups and 1.7–3.2 folds’ increase in carboxyl groups. Degree of hydrolysis ranged from 20 to 30 % with increasing enzyme activity. The number average molecular weight significantly reduced from 3200 g/mol in 0.0 AU-A per 100 g enzyme activity to 1151, 1398, 1175, 1040 and 1246 g/mol in 2.4, 4.8, 7.2, 9.6 and 12.0 AU-A per 100 g enzyme activities, respectively. Depending upon enzyme activity level, the produced LMWP with reactive carboxyl and amino end-groups exhibited 2.5- to 4.0-fold increases in antioxidant capacity and 1.0–3.5 log cfu/ml inhibition of four pathogen bacteria. Highest inhibition of 2.5 log cfu/ml in Escherichia coli was obtained from 2.4 AU-A per 100 g enzyme activity and 3.5 log cfu/ml in Listeria monocytogenes from 9.6 and 12.0 AU-A per 100 g enzyme activity levels. Infrared spectroscopy clearly identified reactive end-groups and showed remarkably differences in molar absorptivity of various molecular regions between non-enzyme and enzyme treated collagen type I molecule. A 9.6 and 12.0 AU-A per 100 g enzyme activity levels were found optimally effective to generate LMWP. In conclusion, LMWP exhibited high antioxidant and antibacterial activity due to increased functional reactive end-groups, and these bio-active peptides may have greater potentialities in various food and pharmaceutical applications.

Superoxide versus peroxide activation of dye decolorizing peroxidases for bioelectrocatalysis

Here we show that dye decolorizing peroxidases can be electrochemically activated for substrate oxidation without addition of exogenous H2O2, yielding activities similar or better than in their conventional operation. The method is based on the in situ generation of O2•−, •OH and H2O2. These species bind the heme to form catalytically competent oxoiron intermediates. Activation of the Pseudomonas putida enzyme is mainly mediated by O2•−, while those from Thermobifida fusca and Saccharomonospora viridis show preferential activation by H2O2. This differential reactivity is explained by the dynamics of a conserved distal aspartate residue. In contrast to previous reports on the Bacillus subtilis enzyme, none of the peroxidases studied here show significant •OH mediated activation, as this radical is quenched by reactive amino acids abundant in these proteins. These results pave the way for the development of bioelectrocatalytic reactors based on dye decolorizing peroxidases for water remediation and biomass processing.

Self-healing properties of GO nanofiltration membranes based on dialdehyde cellulose nanocrystals and ethylenediamine facilitate efficient separation of dyes

Graphene oxide (GO) membranes can purify wastewater due to their unique two-dimensional channels, but they are susceptible to mechanical damage during use, which reduces membrane performance. In this paper, EGO nanosheets were prepared by grafting ethylenediamine (EDA) small molecules onto GO at high temperatures and introducing aldehyde groups on the molecular chain of double aldehyde cellulose nanocrystals (DACNC), which reacted with reactive amino groups on the surface of the EGO nanosheets via a Schiff base reaction to create interlayer nanochannels in the composite membrane. The best performing EGO-DACNC24 composite membranes were prepared by adding different aldehyde substitution degrees and different amounts of DACNC to mediate the crosslinked interlayer nanochannels and network structure of the composite membranes. The water flux is more than 10 times that of pure GO membrane, and the rejection rate of dye molecules larger than 400 Da is always more than 99%. Excellent separation results are achieved after 120 h of operation due to the structural stability of the membrane resulting from chemical cross-linking. Damaged in practical applications, small amounts of added DACNC can achieve self-healing through the synergistic effect of dynamic Schiff base and hydrogen bonding interactions.