Crosslinking Efficiency Research Articles

Digital light processing of customized elastic scaffolds by efficient thiol-yne crosslinking

Digital light processing (DLP) based 3D printing technology has the advantages of fast printing rate and high spatial resolution in the manufacture of tissue engineering scaffolds. However, the limited availability of printable materials for DLP technology, especially the lack of biodegradable elastomeric materials hinders the development of customized soft tissue scaffolds. Herein, liquid poly(4-methyl-ε-caprolactone) dialkynylate (PMCLDY) was prepared as a DLP printable ink based on efficient thiol-yne photocrosslinking. With the aid of photorheology and in situ FT-IR and tensile tests, an ink formulation for DLP printing was optimized as the P50 ink (PMCLDY to diluent PEGDY mass ratio of 1), which possessed fast cure rates (<15 s), low viscosity (<2 Pa·s), moderate mechanical properties (failure strain > 40 %) and maximum toughness (0.153 MJ·m−3). A commercial DLP printer equipped with the P50 ink realized 3D scaffolds models with microstructure and macroscopic shape precisely. It was demonstrated that the 3D printed scaffold with biodegradability could withstand dynamic compression cycles. Furthermore, the cytocompatibility experiments showed that the scaffold could effectively support the growth and proliferation of rat bone marrow stromal cells (BMSCs). These results demonstrate that the P50 ink is a promising material for manufacturing elastic scaffolds by DLP printing for tissue engineering applications.

Rheological behaviors and foaming behaviors of crosslinked ethylene–vinyl acetate with polyfunctional monomers

AbstractTo produce ethylene–vinyl acetate (EVA) foams with a higher foaming ratio and stable cell size, the efficiency of the crosslinking reaction is required to conduct simultaneously with the blowing reaction. Hence, the multifunctional monomer trimethylolpropane trimethacrylate (TMP) as a crosslinking promoter and a crosslinking agent dicumyl peroxide are used together as a crosslinking system to EVA foaming, when chemical blowing agent azodicarbonamide (ADC) is used. The TMP concentration of approximately 1.0 phr (per hundred resin) is most effective for balancing the expansion ratio and fine cell size. In the SEM diagram and expansion ratio results, it can be found that when the concentration of TMP ranges from 0 to 1.0 phr, the cell mean size is reduced from 360.3 to 140.5 μm and the expansion ratio is reduced from 13.6 to 9.1 at 170°C. The results of time‐sweep experiment of thermogravimetric analysis (TGA) show that the foaming time of ADC in the melt increases with the addition of TMP. Capillary rheometer and rotational rheometer tests show that the viscosity and the crosslinking efficiency of the melt are improved with the TMP addition. The curing time has been reduced from 2.93 to 2.22 min for 170°C.

Visible light-mediated cross-linking of injectable gellan gum hydrogels embedding human chondrocytes

Gellan gum, a polysaccharide with structural similarities to glycosaminoglycans, is gaining attention in cartilage tissue engineering. It can be chemically modified with methacrylic moieties to produce a photo-crosslinkable formulation, called methacrylated gellan gum (GGMA), envisaging the injection and the in situ cross-linking through light on the cartilage surface.This study aimed to investigate the visible light cross-linking of GGMA, underlying the impact of steam sterilization on its chemical and rheological properties and cross-linking efficiency. After an initial assessment of the sterilization effects, we evaluated a combination of hydrogel concentrations (1 and 2 % w/v), ruthenium/sodium persulfate concentrations (0.1/1, 0.2/2 and 0.5/5 mM ratios), and light exposure times (30, 60 and, 120 s). The formulations with the lowest sol fraction were characterized for mechanical and showed self-healing properties. Biological characterization on encapsulated chondrocytes in hydrogels showed high cell viability with increased metabolic activity over two weeks. The expression of genes encoding collagen type II and cartilage oligomeric matrix protein was upregulated in the softer formulation (E = 0.95 ± 0.48 kPa) composed of 2 % w/v GGMA, 0.1/1 mM Ru/SPS, and photo-crosslinked for 60 s. These findings represent an interesting overview of the potential application of visible-light crosslinked GGMA in the clinical scenario to treat cartilage defects.

Highly efficient cross-linking of reactive dyes in the amorphous zones of cotton fabric to simultaneously improve color and wrinkle properties

Dyeing and wrinkle-finishing of cotton fabric is generally achieved through two steps, which consumes a lot of energy and generates a lot of colored wastewaters. In this study, we report an energy-saving and cost-reducing dyeing process that enhanced the cross-linking degree of double reactive group dye in the amorphous zone of cotton fabric, which in turn improved the wrinkle resistance of cotton fabric as well as the utilization of the dye. Compared with the traditional dyeing processes, the method could improve K/S values by 69.7%, dye fixation by 22.4%, and wrinkle recovery angle by 21.5% for dyed cotton fabric. The wrinkle recovery angle of the dyed cotton fabric increased with the increase of cross-linking degree. The cross-section of cotton fibers changed from a flattened girdle shape to an elliptical or rounded shape, and the mid-cavity structure became smaller or even disappeared. In addition, the cross-linked structures could hinder the degradation of fiber macromolecular chains by cellulases. This meant that the better the wrinkle resistance of the dyed cotton fabric, the lower the glucose content in the degradation solution. Compared with the conventional dyeing processes, this dyeing method could be saved 36.5% of dyes and 28.4% of alkali for per 1 kg of cotton fabric, and the cost could be saved by 56.1%. The dye concentration in the waste water was reduced by 43.2%. This method had certain advantages in terms of economic and environmental.

Multi-functional epoxides cross-linked collagen sponges for tissue engineering scaffolds

With the efficient cross-linking abilities and the flexible regulation abilities to the performances of cross-linked products, the multi-functional aliphatic epoxides were once widely used to cross-link the collagen-based materials in the last century. In present work, the multi-functional epoxides were used to construct and cross-link collagen sponges for tissue engineering scaffolds, which was hoped to board the theoretical system of epoxides and explore their potentials for modern applications. The bi- to tetra-functional epoxides were used to cross-link collagen solutions and establish the gel-like precursors, then using freeze-drying to form the final sponges. The SEM observed that the sponges had shown regular porous structures with a wide range of pore sizes from 160 to 440 μm. The sponges had presented the resistance to enzymatic degradation, shape-remaining ability, and reversible compressibility in aqueous environments, which all could be regulated through the functionalities of epoxides. The regulation abilities of multi-functional epoxides on the performances of sponges had been mainly achieved through the cross-linking degrees that the higher functionality of epoxides would bring higher cross-linking degree. Such higher cross-linking degrees could enhance the elastic behaviors of gel-like precursors, and improve the compressive strengths and thermal stabilities of sponges. Nevertheless, the multi-functional epoxides had barely affected the safety of collagen sponges at the cellular level according to the results of CCK8 assay and the SEM and CLSM images of L929 fibroblasts cultured on the cross-sections of sponges.Graphical abstract

Covalent Capture and Selection of DNA-Encoded Chemical Libraries via Photo-Activated Lysine-Selective Crosslinkers.

Covalent crosslinking probes have arisen as efficient toolkits to capture and elucidate biomolecular interaction networks. Exploiting the potential of crosslinking in DNA-encoded chemical library (DEL) selection methods significantly boosted bioactive ligand discovery in complex physiological contexts. Herein, we incorporated o-nitrobenzyl alcohol (o-NBA) as a photo-activated lysine-selective crosslinker into divergent DEL formats and achieved covalent capture of ligand-target interactions featuring improved crosslinking efficiency and site specificity. In addition, covalent DEL selection was realized with the modularly designed o-NBA-functionalized mock libraries.

Orthogonally Crosslinked Gelatin-Norbornene Hydrogels for Biomedical Applications.

The thiol-norbornene photo-click reaction has exceptionally fast crosslinking efficiency compared with chain-growth polymerization at equivalent macromer contents. The orthogonal reactivity between norbornene and thiol/tetrazine permits crosslinking of synthetic and naturally derived macromolecules with modularity, including poly(ethylene glycol) (PEG)-norbornene (PEGNB), gelatin-norbornene (GelNB), among others. For example, collagen-derived gelatin contains both cell adhesive motifs (e.g., Arg-Gly-Asp or RGD) and protease-labile sequences, making it an ideal macromer for forming cell-laden hydrogels. First reported in 2014, GelNB is increasingly used in orthogonal crosslinking of biomimetic matrices in various applications. GelNB can be crosslinked into hydrogels using multi-functional thiol linkers (e.g., dithiothreitol (DTT) or PEG-tetra-thiol (PEG4SH) via visible light or longwave ultraviolet (UV) light step-growth thiol-norbornene reaction or through an enzyme-mediated crosslinking (i.e., horseradish peroxidase, HRP). GelNB-based hydrogels can also be modularly crosslinked with tetrazine-bearing macromers via inverse electron-demand Diels-Alder (iEDDA) click reaction. This review surveys the various methods for preparing GelNB macromers, the crosslinking mechanisms of GelNB-based hydrogels, and their applications in cell and tissue engineering, including crosslinking of dynamic matrices, disease modeling, and tissue regeneration, delivery of therapeutics, as well as bioprinting and biofabrication.

Furan-modified PNA probes for covalent targeting and ligation of nucleic acids.

While natural oligonucleotides (ONs) are increasingly used as therapeutic and diagnostic tools, they still face certain challenges such as low resistance to enzymatic degradation, potential immunogenicity, and delivery issues, which can limit their applications. Peptide Nucleic Acids (PNAs) are promising alternatives due to their high affinity for DNA and RNA, the high resistance to enzymatic degradation, and the easy introduction of a wide range of potential modifications. Chemical modifications that enable the covalent targeting of specific DNA and RNA strands offer additional advantages, including enhanced potency. The current study focuses on the utilization of furan-PNAs as pro-reactive probe systems and their applications to DNA and RNA targeting. Specifically, in this methodological paper, we provide practical insights into the design, synthesis, and application of furan-containing PNA probes for achieving efficient PNA-DNA and PNA-RNA interstrand crosslinking (ICL), as well as ON-templated PNA-PNA ligation systems. Furthermore, we discuss the applications of these probes in targeting DNA secondary structures, such as G-quadruplexes and i-motifs, target pull-down assays, and on-surface detection.

Site-specific crosslinking and assembly of tetrameric β-glucuronidase improve glycyrrhizin hydrolysis.

In this study, eight nonconserved residues with exposed surfaces and flexible conformations of the homotetrameric PGUS (β-glucuronidase from Aspergillus oryzae Li-3) were identified. Single-point mutation into cysteine enabled the thiol-maleimide reaction and site-specific protein assembly using a two-arm polyethylene glycol (PEG)-maleimide crosslinker (Mal2 ). The Mal2 (1k) (with 1 kDa PEG spacer)-crosslinked PGUS assemblies showed low crosslinking efficiency and unimproved thermostability except for G194C-Mal2 (1k). To improve the crosslinking efficiency, a lengthened crosslinker Mal2 (2k) (with 2 kDa PEG spacer) was used to produce PGUS assembly and a highly improved thermostability was achieved with a half-life of 47.2-169.2 min at 70°C, which is 1.04-3.74 times that of wild type PGUS. It is found that the thermostability of PGUS assembly was closely associated with the formation of inter-tetramer assembly and intratetramer crosslinking, rather than the PEGylation of the enzyme. Therefore, the four-arm PEG-maleimide crosslinker Mal4 (2k) (with 2 kDa PEG spacer) was employed to simultaneously increase the inter-tetramer assembly and intratetramer crosslinking, and the resulting PGUS assemblies showed further improved thermostabilities compared with Mal2 (2k)-crosslinked assemblies. Finally, the application of PGUS assemblies with significantly improved thermostability to the bioconversion of GL proved that the PGUS assembly is a strong catalyst for glycyrrhizin (GL) hydrolysis in industrial applications.

ОЦЕНКА ГЕНОТОКСИЧЕСКОГО ВОЗДЕЙСТВИЯ МИТОМИЦИНА С НА ПРИМЕРЕ APOE НОКАУТНЫХ МЫШЕЙ

BACKGROUND Mitomycin C (MMC) has a wide spectrum of genotoxicity, including inhibition of DNA synthesis, clastogenesis and mutagenesis. As an immediate-action clastogen requiring exclusively intracellular reductive activation, MMC initiates efficient DNA cross-linking. Most studies are aimed at studying single-stage acute effects, which are caused by high doses of mutagen. In turn, there are no or very few studies aimed at studying the chronic effects of MMC. Polychrome red blood cells have been accepted as a suitable target for the evaluation of micronuclei in both acute and cumulative injury. The in vivo micronucleus test is well established as a standard assay for assessing genotoxicity at the chromosomal level of mouse erythrocytes. THE AIM OF THE STUDY To create a chronic genotoxic effect of mitomycin C without lethal outcome in ApoE knockout mice while selecting the optimal dose of MMC. MATERIALS AND METHODS The study design included 6 groups of ApoE-/- mice, two doses of MMC with a concentration of 0.1 and 0.5 mg/kg, one-time and three-time administration. To assess genotoxicity, 1000 polychrome erythrocytes (PCEs) extracted from the bone marrow of a mousefemur were counted on each sample, and PCEs with micronuclei were identified. RESULTS This study aimed to find the optimal dose of MMC that has a clear genotoxic effect and does not lead to death in an ApoE knockout mouse model. With a single injection, PCEs with chromosomal damage were more common (more than 2 times; p &lt; 0.05) in groups of mice with MMC administration (0.1 and 0.5 mg/kg; 0.39 % and 0.26 %, respectively) compared to the control group (0.15 %). We also found that the frequency of occurrence of PCEs with micronuclei in groups of mice with MMC dose of 0.1 and 0.5 mg/kg (0.36 % and 0.47 %, respectively) and three-time administration exceeded this indicator in mice from the control group (0.2 %). CONCLUSION The present study on the determination of the optimal dose of mitomycin C provides further evidence that 0.1 mg/kg is the threshold value for genotoxic effects caused by MMC. An increase in the frequency of micronucleated immature red blood cells in animals exposed to a mutagen is an indicator of induced structural or numerical chromosomal aberrations. Our results further suggest that careful selection of MMC dose is critical. Dose-response studies in rodents can provide useful information on mechanisms and dose selection for long-term toxicity studies.

Dose-Response of the Mitomycin C Genotoxic Effect on the ApoE Knockout Mice

Polychromic erythrocytes have been accepted as a suitable target for micronucleus (MN) evaluation in both acute and cumulative injury. Mitomycin C (MMC) also has a wide range of genotoxicity, including inhibition of DNA synthesis, clastogenesis and mutagenesis. As an immediate clastogen requiring exclusively intracellular reductive activation, MMS initiates efficient DNA crosslinking. The in vivo micronucleus assay has established itself as a standard assay for evaluating chromosomal genotoxicity in mouse erythrocytes. Most of the studies are focused on the study of acute acute effects, which is caused by high doses of the mutagen. In turn, there are no or very few studies aimed at studying the chronic effects of MMS. The aim of the study is to create a chronic genotoxic effect of MMS without lethal outcome in ApoE–/– mice when selecting the optimal dose of MMS. The design of the study included 6 groups of ApoE–/– mice, two doses of MMC at a concentration of 0.1 and 0.5 mg/kg, single and three doses. Each group consisted of four females and one male. To assess genotoxicity, 1000 polychromic erythrocytes (PChE) extracted from the femoral bone marrow were counted on each sample, PChE with micronuclei were detected, and the proportion of reticulocytes was counted. A dose of 0.5 mg/kg showed a clear cytotoxic effect, expressed in a violation of erythropoiesis, and more precisely in a decrease in the proportion of reticulocytes. In our study, the concentration of the mutagen, namely 0.1 mg/kg, was shown to cause a clear genotoxic effect without reaching the threshold of cytotoxicity. Dose-response studies in rodents can provide useful information on the mechanisms of toxicity and dose selection for long-term toxicity studies.

Thermal/Blue Light Induced Cross-Linking of Acrylic Coatings with Diazo Compounds.

The thermal curing of industrial coatings (e.g., car painting and metal coil coatings) is accompanied by a substantial energy consumption due to the intrinsically high temperatures required during the curing process. Therefore, the development of new photochemical curing processes-preferably using visible light-is in high demand. This workdescribes new diazo-based cross-linkers that can be used to photocure acrylic coatings using blue light.This workdemonstrates that the structure of the tethered diazo compounds influences the cross-linking efficiency, finding that side reactions are suppressed upon engineering greater molecular flexibility. Importantly,this work shows that these diazo compounds can be employed as either thermal or photochemical cross-linkers, exhibiting identical crosslinking performances. The performance of diazo-cross-linked coatingsis evaluated to reveal excellent water resistance and demonstrably similar material properties to UV-cured acrylates. These studies pave the way for further usage of diazo-functionalized cross-linkers in the curing of paints and coatings.

Use of covalent dynamic networks as binders on epoxy‐based carbon fiber composites: Effect on properties, processing, and recyclability

AbstractComposites are gaining widespread market interest due to their outstanding mechanical properties and light weight. However, this growth is hindered by the lack of sustainable recycling solutions. This research aims to integrate reversible chemistries to advanced carbon fiber‐reinforced composite materials for recycling in a so‐called eco‐design, circular approach. Diene and dienophile reactions are generated at the surface of the carbon fiber allowing a bond–debond interaction with the epoxy matrix. The application of the Diels–Alder adduct on the fiber modifies its polarity and structure. This research assesses a biobased and synthetic epoxy equivalent and demonstrates that this newly developed interphase chemistry has a direct impact on the crosslinking efficiency of the resin, as well as on their interlaminar properties. Furthermore, it has been observed that depending on the type of resin, these changes can have differing impacts on final properties of the composite, reducing the crosslinking density up to 50% in some modifications while in others the Tg remains constant.

A novel efficient wet process for preparing cross-linked starch: Impact of urea on cross-linking performance

Although wet processes are promising for preparing cross-linked starch, they are currently challenged by lower cross-linking efficiency and the requirement of large amounts of salts. Herein, an efficient and greener wet process was proposed, in which the cross-linking performance between sodium hexametaphosphate (SHMP) and starch was enhanced with the aid of urea. The maximum degree of substitution (DS) of the urea-phosphorylated cross-linked starch (UPCS) was 0.040 at 35 °C, while that of the conventional phosphorylated cross-linked starch (CPCS) was 0.031 at 45 °C. Compared with CPCS, the maximum DS of UPCS was elevated by 29.03 %, but its optimum cross-linking temperature was reduced by 10 °C, indicating that the cross-linking efficiency of this novel wet process was greatly improved by urea. The structural difference between UPCS and CPCS was confirmed by using a series of techniques including 31P NMR and 13C NMR. Zeta potential results suggested that urea may promote starch cross-linking by preventing the closure of active sites through hydrophobic interactions. Due to the structural reinforcement of starch by urea, UPCS showed better thermal stability, water resistance, acid and alkali resistance, and steady shear tolerance properties. This study provides a facile wet process for the fabrication and application of cross-linked starch materials.

The DEAD-Box RNA Helicase Ded1 Is Associated with Translating Ribosomes.

DEAD-box RNA helicases are ATP-dependent RNA binding proteins and RNA-dependent ATPases that possess weak, nonprocessive unwinding activity in vitro, but they can form long-lived complexes on RNAs when the ATPase activity is inhibited. Ded1 is a yeast DEAD-box protein, the functional ortholog of mammalian DDX3, that is considered important for the scanning efficiency of the 48S pre-initiation complex ribosomes to the AUG start codon. We used a modified PAR-CLIP technique, which we call quicktime PAR-CLIP (qtPAR-CLIP), to crosslink Ded1 to 4-thiouridine-incorporated RNAs in vivo using UV light centered at 365 nm. The irradiation conditions are largely benign to the yeast cells and to Ded1, and we are able to obtain a high efficiency of crosslinking under physiological conditions. We find that Ded1 forms crosslinks on the open reading frames of many different mRNAs, but it forms the most extensive interactions on relatively few mRNAs, and particularly on mRNAs encoding certain ribosomal proteins and translation factors. Under glucose-depletion conditions, the crosslinking pattern shifts to mRNAs encoding metabolic and stress-related proteins, which reflects the altered translation. These data are consistent with Ded1 functioning in the regulation of translation elongation, perhaps by pausing or stabilizing the ribosomes through its ATP-dependent binding.

H2O2-Inducible DNA Cross-linking Agents Capable of Releasing Multiple DNA Alkylators as Potent Anticancer Prodrugs.

Three compounds with arylboronate esters conjugated with two equivalent nitrogen mustards [bis(2-chloroethyl)methylamine, HN2] have been synthesized and characterized. These inactive small molecules selectively react with H2O2 to produce multiple DNA cross-linkers, such as two HN2 molecules alongside a bisquinone methide (bisQM), leading to efficient DNA ICL formation. In comparison to other amine functional groups, using HN2 as a leaving group greatly improves the DNA cross-linking efficiency of these arylboronate esters as well as cellular activity. The introduction of HN2 in these arylboronate ester analogues favored the generation of bisQM that can directly cross-link DNA. Two equivalents of HN2 are also generated from these compounds upon treatment with H2O2, which directly produces DNA ICL products. The cumulative effects of HN2 and bisQM on DNA cross-linking makes these molecules highly effective H2O2-inducible DNA ICL agents. The three compounds with HN2 as a leaving group showed greatly enhanced cytotoxicity towards cancer cells in comparison to those containing trimethyl amine as a leaving group. This provides an effective strategy for further design of novel potential ROS-activated anticancer prodrugs.

Adducts of Carbon Black with a Biosourced Janus Molecule for Elastomeric Composites with Lower Dissipation of Energy.

Elastomer composites with low hysteresis are of great importance for sustainable development, as they find application in billions of tires. For these composites, a filler such as silica, able to establish a chemical bond with the elastomer chains, is used, in spite of its technical drawbacks. In this work, a furnace carbon black (CB) functionalized with polar groups was used in replacement of silica, obtaining lower hysteresis. CBN326 was functionalized with 2-(2,5-dimethyl-1H-pyrrol-1-yl)-1,3-propanediol (serinol pyrrole, SP), and samples of CB/SP adducts were prepared with different SP content, ranging from four to seven parts per hundred carbon (phc). The entire process, from the synthesis of SP to the preparation of the CB/SP adduct, was characterized by a yield close to 80%. The functionalization did not alter the bulk structure of CB. Composites were prepared, based on diene rubbers-poly(1,4-cis-isoprene) from Hevea Brasiliensis and poly(1,4-cis-butadiene) in a first study and synthetic poly(1,4-cis-isoprene) in a second study-and were crosslinked with a sulfur-based system. A CB/silica hybrid filler system (30/35 parts) was used and the partial replacement (66% by volume) of silica with CB/SP was performed. The composites with CB/SP exhibited more efficient crosslinking, a lower Payne effect and higher dynamic rigidity, for all the SP content, with the effect of the functionalized CB consistently increasing the amount of SP. Lower hysteresis was obtained for the composites with CB/SP. A CB/SP adduct with approximately 6 phc of SP, used in place of silica, resulted in a reduction in ΔG'/G' of more than 10% and an increase in E' at 70 °C and in σ300 in tensile measurements of about 35% and 30%, respectively. The results of this work increase the degrees of freedom for preparing elastomer composites with low hysteresis, allowing for the use of either silica or CB as filler, with a potentially great impact on an industrial scale.

In vivo PAR-CLIP (viP-CLIP) of liver TIAL1 unveils targets regulating cholesterol synthesis and secretion

System-wide cross-linking and immunoprecipitation (CLIP) approaches have unveiled regulatory mechanisms of RNA-binding proteins (RBPs) mainly in cultured cells due to limitations in the cross-linking efficiency of tissues. Here, we describe viP-CLIP (in vivo PAR-CLIP), a method capable of identifying RBP targets in mammalian tissues, thereby facilitating the functional analysis of RBP-regulatory networks in vivo. We applied viP-CLIP to mouse livers and identified Insig2 and ApoB as prominent TIAL1 target transcripts, indicating an important role of TIAL1 in cholesterol synthesis and secretion. The functional relevance of these targets was confirmed by showing that TIAL1 influences their translation in hepatocytes. Mutant Tial1 mice exhibit altered cholesterol synthesis, APOB secretion and plasma cholesterol levels. Our results demonstrate that viP-CLIP can identify physiologically relevant RBP targets by finding a factor implicated in the negative feedback regulation of cholesterol biosynthesis.

Synthesis of Highly Stable and Reactive Nitrile N-Oxides and Their Application as Catalyst-Free Crosslinkers

Nitrile N-oxide-based click ligation is a very promising technique for polymer crosslinking due to the unique advantages of catalyst-free and byproduct-free reactions. For this technique, nitrile N-oxides that are sufficiently stable at room temperature and have high reactivity under mild conditions are very desirable. Usually, neighboring bulky substituents are introduced to enhance the stability of nitrile N-oxides, which however bring the reactivity down due to the high steric hindrance. Herein, methyl groups, small-sized substituents, were introduced into the o,o′-positions of phenyl nitrile N-oxides to enable high stability and high reactivity. Thus, several novel aromatic nitrile N-oxides were synthesized. Among them, ditopic aromatic nitrile N-oxides were prepared through three simple steps including etherification, oximation, and oxidative dehydrogenation, whereas stable tritopic aromatic nitrile N-oxides were synthesized by directly introducing the nitrile N-oxide functionality into the corresponding trifunctional compounds to avoid undesirable side reactions. These nitrile N-oxides as ligation reagents exhibited very high reactivity, and efficient catalyst-free crosslinking of natural rubber was achieved at room temperature to give elastomers with good mechanical properties, revealing the potential of such nitrile N-oxides as useful crosslinkers for unsaturated bond-containing polymers. This work provides reliable synthesis of stable ditopic/tritopic aromatic nitrile N-oxides, broadening the application of the nitrile N-oxide-based click ligation technique toward catalyst-free and byproduct-free polymer crosslinking.

Identification of photocrosslinking peptide ligands by mRNA display

Photoaffinity labelling is a promising method for studying protein-ligand interactions. However, obtaining a specific, efficient crosslinker can require significant optimisation. We report a modified mRNA display strategy, photocrosslinking-RaPID (XL-RaPID), and exploit its ability to accelerate the discovery of cyclic peptides that photocrosslink to a target of interest. As a proof of concept, we generated a benzophenone-containing library and applied XL-RaPID screening against a model target, the second bromodomain of BRD3. This crosslinking screening gave two optimal candidates that selectively labelled the target protein in cell lysate. Overall, this work introduces direct photocrosslinking screening as a versatile technique for identifying covalent peptide ligands from mRNA display libraries incorporating reactive warheads.