Crosslinking Yield Research Articles

Photochemical and Collision-Induced Cross-Linking in Stereochemically Distinct Scaffolds of Peptides and Nitrile Imines in Gas-Phase Ions.

Intramolecular cross-linking between peptides and nitrile-imine intermediates was studied in stereochemically distinct conjugates in which the reacting components were mounted on cis-1,2-cyclohexane and trans-1,4-cyclohexane scaffolds that we call 1,2-s-peptides and 1,4-s-peptides, respectively. The nitrile-imine intermediates were generated by N2 loss from 2,5-diaryltetrazole tags upon UV-photodissociation at 213 and 250 nm or by collision-induced dissociation, and further interrogated by CID and UVPD-MS3. Peptide fragment ion series originating from linear structures and macrocyclic cross-links were distinguished and used to quantify the cross-linking yields. The yields in MS2 varied between 27% for AAAG conjugates to 78% for GAAAK conjugates, depending on the peptide sequence. The CID-MS3 yields were in a 57-97% range, depending on the peptide sequence. Structures of 1,2-s-peptide and 1,4-s-peptide ions as well as several of their nitrile-imine intermediates and cross-links were investigated by high-resolution cyclic ion mobility in combination with Born-Oppenheimer molecular dynamics and density functional theory calculations. Matches between the experimental and calculated collision cross sections and ion relative Gibbs energies were used to assign peptide structures. Peptide conjugates C-terminated with Gly and Lys residues underwent cross-linking by the carboxyl group, as established by MS3 sequencing and corroborated by carboxyl blocking experiments that lowered the cross-linking yields. Peptide conjugates C-terminated with Arg also cross-linked via the side-chain guanidine group. A notable feature of the 1,4-s-peptide ions was the participation of low-energy twist-boat cyclohexane conformers that was enforced by strong hydrogen bonds between the peptide and nitrile imine.

Tuning crosslinking of hybrid preceramic polymers in vat photopolymerization toward controlled ceramic yields

Control of preceramic polymer crosslinking for UV-curable processing is essential for fine 3D printing with high ceramic conversion for sustainable polymer-derived ceramics (PDC) engineering. While various factors influencing ceramic yield have been studied, the systematic exploration of the relationship between crosslinking and ceramic yield, especially when crosslinking increases volatile elements, remains open for further investigation. This study addresses this gap by utilizing vat photopolymerization (VP) additive manufacturing (AM) as a versatile platform for controlling preceramic crosslinking and ceramic yield. By rationally designing and tuning the photochemical crosslinking through digital light processing (DLP), we demonstrate that the ceramic yield can be enhanced from 64% to over 86%, even with added volatile elements. We reveal that the post-pyrolysis ceramic yield can be closely correlated with the pre-pyrolysis crosslinking of the preceramic network represented by its stiffness. This correlation thereby suggests a fast, energy-efficient, non-destructive methodology to predict and improve ceramic yield. Combined with these findings, our one-pot thiol-ene hybridization of polycarbosilane and polycarbosiloxane via DLP offers an exemplary method to generate hybrid preceramic polymers with tailored material properties toward target applications, and potentially even higher ceramic yields. This study thus contributes to achieving better resource- and energy-efficient preceramic polymer and PDC processing routes toward sustainable, advanced organic–inorganic materials manufacturing.

Cell fixation improves performance of in situ crosslinking mass spectrometry while preserving cellular ultrastructure

Crosslinking mass spectrometry (XL-MS) has the potential to map the interactome of the cell with high resolution and depth of coverage. However, current in vivo XL-MS methods are hampered by crosslinkers that demonstrate low cell permeability and require long reaction times. Consequently, interactome sampling is not high and long incubation times can distort the cell, bringing into question the validity any protein interactions identified by the method. We address these issues with a fast formaldehyde-based fixation method applied prior to the introduction of secondary crosslinkers. Using human A549 cells and a range of reagents, we show that 4% formaldehyde fixation with membrane permeabilization preserves cellular ultrastructure and simultaneously improves reaction conditions for in situ XL-MS. Protein labeling yields can be increased even for nominally membrane-permeable reagents, and surprisingly, high-concentration formaldehyde does not compete with conventional amine-reactive crosslinking reagents. Prefixation with permeabilization uncouples cellular dynamics from crosslinker dynamics, enhancing control over crosslinking yield and permitting the use of any chemical crosslinker.

Topology-selective photo-crosslinking of G-quadruplexes via dual G-quartet and groove recognition.

The novel photo-crosslinking ligand 6OTD-Bp, bearing an alkylamine benzophenone (Bp) with macrocyclic hexaoxazole (6OTD), was shown to preferentially ligate with hybrid G4s through recognizing both G-quartets and their characteristic wide groove. Higher crosslinking yield was observed for hybrid G4 with wider grooves.

Small molecule photocatalysis enables drug target identification via energy transfer

Over half of new therapeutic approaches fail in clinical trials due to a lack of target validation. As such, the development of new methods to improve and accelerate the identification of cellular targets, broadly known as target ID, remains a fundamental goal in drug discovery. While advances in sequencing and mass spectrometry technologies have revolutionized drug target ID in recent decades, the corresponding chemical-based approaches have not changed in over 50 y. Consigned to outdated stoichiometric activation modes, modern target ID campaigns are regularly confounded by poor signal-to-noise resulting from limited receptor occupancy and low crosslinking yields, especially when targeting low abundance membrane proteins or multiple protein target engagement. Here, we describe a broadly general platform for photocatalytic small molecule target ID, which is founded upon the catalytic amplification of target-tag crosslinking through the continuous generation of high-energy carbene intermediates via visible light-mediated Dexter energy transfer. By decoupling the reactive warhead tag from the small molecule ligand, catalytic signal amplification results in unprecedented levels of target enrichment, enabling the quantitative target and off target ID of several drugs including (+)-JQ1, paclitaxel (Taxol), dasatinib (Sprycel), as well as two G-protein-coupled receptors-ADORA2A and GPR40.

Azide- and diazirine-modified membrane lipids: Physicochemistry and applicability to study peptide/lipid interactions via cross-linking/mass spectrometry

Although the incorporation of photo-activatable lipids into membranes potentially opens new avenues for studying interactions with peptides and proteins, the question of whether azide- or diazirine-modified lipids are suitable for such studies remains controversial. We have recently shown that diazirine-modified lipids can indeed form cross-links to membrane peptides after UV activation and that these cross-links can be precisely determined in their position by mass spectrometry (MS). However, we also observed an unexpected backfolding of the lipid's diazirine-containing stearoyl chain to the membrane interface challenging the potential application of this modified lipid for future cross-linking (XL)-MS studies of protein/lipid interactions. In this work, we compared an azide- (AzidoPC) and a diazirine-modified (DiazPC) membrane lipid regarding their self-assembly properties, their mixing behavior with saturated bilayer-forming phospholipids, and their reactivity upon UV activation using differential scanning calorimetry (DSC), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), and MS. Mixtures of both modified lipids with DMPC were further used for photo-chemically induced XL experiments with a transmembrane model peptide (KLAW23) to elucidate similarities and differences between the azide and the diazirine moiety. We showed that both photo-reactive lipids can be used to study lipid/peptide and lipid/protein interactions. The AzidoPC proved easier to handle, whereas the DiazPC had fewer degradation products and a higher cross-linking yield. However, the problem of backfolding occurs in both lipids; thus, it seems to be a general phenomenon.

DEPENDENCE OF THE PROPERTIES OF SULFUR AND PEROXIDE VOLCANISES BASED ON A MIXTURE OF ISOPRENE WITH POLYVINYL ACETATE ON THE STRUCTURE OF THE VOLCANIZATION NETWORK

The effect on the crosslinking process of modified isoprene rubber (SKI-3-PVA) with the participation of sulfur and dicumyl peroxide (PD) in the presence of hexachloroparaxylene (HCPA) and 2,4-dichloro-6-dimethyl-amino sym triazine was studied. It was found that, depending on the length of time, the number of crosslinks in these systems (SKN-3-PVA + sulfur + GHPK + ZnO; SKI-3-PVA + PD + GHPK) is 2.5x105 and 1.9x105 mol / cm³, respectively, per Based on the observed changes in the parameter of the spatial grid of the crosslinked samples, the yield of effective crosslinks was calculated. It was shown that in the presence of dicumyl peroxide in filled systems, the number of crosslinks decreases, and a decrease in crosslinks is also observed. Comparative tests of elastomeric materials based on SKI-3 - PVA copolymers in liquid aggressive media were carried out. Sol-gel analysis found that with an increase in the content of the copolymer in isoprene, the concentration of the number of cross-linked molecules in the copolymer increases. It is shown that the physical and mechanical properties of filled vulcanizates are inferior to sulfuric ones in strength properties, but surpass peroxide ones in thermal aging and the action of solvents. It was found that an increase in aging with an increase in the heating time of the samples indicates the formation of additional cross-links. The role of zinc oxide in the peroxide crosslinking of the SKI-3 - PVA copolymer has been studied. It was found that the introduction of zinc oxide has a noticeable effect on the rate of sulfur and peroxide crosslinking of mixtures based on SKI-3 - PVA copolymers, an increase in the structuring time of mixtures, destructive processes are observed, and the number of crosslinks and crosslinks decreases. It was found that the physical and mechanical properties of peroxide vulcanizates are inferior to sulfuric ones in strength properties, but they are superior to peroxide ones in thermal aging. It is shown that an increase in the resistance to aging with an increase in the heating time of the samples indicates the formation of additional cross-links. As a result of the reaction with oxygen and the accumulation of interacting polar groups. A decrease in the degree of swelling is associated with the formation of C-C chemical bonds in elastomers, it effectively participates in the formation of cross-links during peroxide vulcanization. The nature of the interaction of sulfur PD, HCPC, and DCDEAST with elastomers was judged by the change in the conditional number of crosslinks (n´c) and the number of crosslinked molecules (1 \ Ms) in unfilled sulfur and peroxide vulcanizates. It is shown that in sulfur peroxide mixtures based on SKI-3-PVA with the proposed agents at room temperature it causes the formation of effective crosslinks, and with an increase in the duration of heating in systems 1, 2, the number of crosslinked molecules increases. An increase in resistance to aging with an increase in the heating time of the samples indicates the formation of additional cross-links as a result of the reaction with oxygen and the accumulation of interacting polar groups. A decrease in the degree of swelling is associated with the formation of C - C chemical bonds in elastomers, effectively participating in the formation of a network of crosslinks during peroxide vulcanization. Thus, the analysis of the results of the studies performed allows us to conclude that elastomers based on the isoprene copolymer SKI-3-PVA obtained with the participation of vulcanizing agents of sulfur and peroxide (PD) in the presence of hexachloroparaxylene (HCP) and triazine compounds are superior to those known in terms of thermal stability. aging in strength and accumulation of permanent deformation in aggressive environments. Keywords: isoprene, polyvinyl acetate, hexachloroparaxylene, peroxide dicumil, vulcanization, modification

Preparation of DNA interstrand cross-link repair intermediates induced by abasic sites

DNA interstrand cross-links (ICLs) are extremely deleterious DNA lesions, which can block different DNA transactions. A major step in ICL repair involves strand cleavage activities flanking the cross-linking site, also known as unhooking. The cleavage generates a single-stranded DNA remnant attached to the unbroken strand, often referred to as the unhooked ICL repair intermediates. The unhooked ICLs are substrates for specialized DNA polymerases, leading to the eventual restoration of the duplex DNA structure. Although these repair events have been outlined, the understanding of molecular details of the repair pathways has been hindered by the difficulty of preparing structurally defined ICL repair intermediates. Here, we present a straightforward method to prepare model ICL repair intermediates derived from a ubiquitous type of endogenous DNA modification, abasic (AP) sites. AP-derived ICLs have emerged as an important type of endogenous ICLs. We developed the method based on commercially available materials without the requirement of synthetic chemistry expertise. The method is expected to be accessible to any interested labs in the DNA repair community.• The method exploits the alkaline lability of ribonucleotides and uses designer oligonucleotides to create ICL repair intermediates with varying lengths of the unhooked strand.• Strand cleavage at ribonucleotides is achieved using NaOH, which avoids the potential for incomplete digestion during enzymatic workup due to specific substrate structures.• The method is grounded on the high cross-linking yield between an AP lesion and a nucleotide analog, 2-aminopurine, via reductive amination, developed by Gates and colleagues.

Gamma-Ray Modified Polymer/Clay Composites: Synthesis, Characterization, and Formulation Optimization Using Multivariate Calculus and Graph Theory

To enhance the oxidation strength and crosslinking yield of ultrahigh molecular weight polyethylene (UHMWPE), its composites were prepared by mixing 1%, 2%, and 3% (by wt.) of magnesium silicate hydrous (sepiolite) during this study. These composites were irradiated with 25 kGy and 50 kGy of irradiation doses in the open air. Subsequent to irradiation, the composites were characterized for updates in structure, oxidation strength, and degree of crosslinking while estimating the values of oxidation index (OI), crosslink density (Gx), and percent crystallinity (Xc), respectively. The reaction of modified sepiolite (SP) on irradiating UHMWPE/SP composites was evident from the origination of peaks at Si-O, O-Si-O, Si-O-Si, and Mg-OH and stretching vibration at 974 cm−1, 1014 cm−1, and 1080 cm−1, respectively. In addition, the moderate negative correlation of OI (−0.46) and strong positive correlation of Gx (0.87) with the absorbed dose, made obvious from correlation analysis, confirmed the effectiveness of SP in enhancing the oxidation strength and crosslink density of UHMWPE. Furthermore, cluster analysis (CA) grouped the composites as low, moderate, and best based on dissimilarities, i.e., oxidation strength and crosslink density, which was further confirmed by principal component analysis (PCA). PCA also revealed that the OI, Gx, and absorbed dose were the active variables for this best group. After confirming the effectiveness of SP (as an active filler for enhancing the oxidation strength and crosslink density of UHMWPE) and recognizing the OI, Gx, and absorbed dose as active variables, the next step was to determine the necessary minimum amount of SP concentration and radiation dose required for the composite that outperforms all others. For this, a parametric graph theory and matrix approach was employed to rank the composites of the best group, revealing that the UHMWPE/SP composite with 1% (by wt.) of Si12O30Mg8(OH)4(H2O)4·8H2O and irradiated with 50 kGy of irradiation dose was the most suitable choice of all existing alternatives tested in this study. Although this is the first attempt, to the best of our knowledge, where a smart approach using multivariate calculus and decision-making tools was utilized for figuring the best UHMWPE composite formulation along with appropriate treatment dose, the results and methodology could be extended for any polymer of industrial scale, such as those used in medical implants, defense armor, bulletproof jackets, etc.

Interstrand Cross-Link Formation Involving Reaction of a Mispaired Cytosine Residue with an Abasic Site in Duplex DNA.

The formation of interstrand cross-links in duplex DNA is important in biology, medicine, and biotechnology. Interstrand cross-links arising from the reaction of the aldehyde residue of an abasic (apurinic or AP) site with the exocyclic amino groups of guanine or adenine residues on the opposing strand of duplex DNA have previously been characterized. The canonical nucleobase cytosine has an exocyclic amino group but its ability to form interstrand cross-links by reaction with an AP site has not been characterized before now. Here it is shown that substantial yields of interstrand cross-links are generated in sequences having a mispaired cytosine residue located one nucleotide to the 3'-side of the AP site on the opposing strand (e.g., 5'XA/5'CA, where X = AP). Formation of the dC-AP cross-link is pH-dependent, with significantly higher yields at pH 5 than pH 7. Once formed, the dC-AP cross-link is quite stable, showing less than 5% dissociation over the course of 96 h at pH 7 and 37 °C. No significant yields of cross-link are observed when the cytosine residue is paired with its Watson-Crick partner guanine. It was also shown that a single AP site can engage with multiple nucleobase cross-linking partners in some sequences. Specifically, the dG-AP and dC-AP cross-links coexist in dynamic equilibrium in the sequence 5'CXA/5'CAG (X = AP). In this sequence, the dC-AP cross-link dominates. However, in the presence of NaBH3CN, irreversible reduction of small amounts of the dG-AP cross-link present in the mixture shifts the equilibria away from the dC-AP cross-link toward good yields of the dG-APred cross-link.

Crosslinking of human plasma C-reactive protein to human serum albumin via disulfide bond oxidation

Inter- and intra-molecular crosslinks can generate protein dysfunction, and are associated with protein aggregate accumulation in aged and diseased tissues. Crosslinks formed between multiple amino acid side chains can be reversible or irreversible. Disulfides formed either enzymatically, or as a result of oxidant-mediated reactions, are a major class of reversible crosslinks. Whilst these are commonly generated via oxidation of Cys thiol groups, they are also formed by ‘oxidant-mediated thiol-disulfide reactions’ via initial disulfide oxidation to a thiosulfinate or zwitterionic peroxide, and subsequent reaction with another thiol including those on other proteins. This generates new intermolecular protein-protein crosslinks. Here we demonstrate that photooxidation, or reaction with the biological oxidants HOCl and ONOOH, of the single disulfide present in the major human plasma inflammatory protein, C-reactive protein (CRP) can give rise to reversible disulfide bond formation with human serum albumin (HSA). This occurs in an oxidant dose-, or illumination-time-, dependent manner. These CRP-HSA crosslinks are formed both in isolated protein systems, and in fresh human plasma samples containing high, but not low, levels of CRP. The inter-protein crosslinks which involve Cys36 of CRP and Cys34 of HSA, have been detected by both immunoblotting and mass spectrometry (MS). The yield of protein-protein crosslinks depends on the nature and extent of oxidant exposure, and can be reversed by dithiothreitol and tris(2-carboxyethyl)phosphine hydrochloride. These data indicate that oxidation of disulfide bonds in proteins can be a source of novel inter-protein crosslinks, which may help rationalize the accumulation of crosslinked proteins in aged and diseased tissues.

Highly efficient and tunable miktoarm stars for HIPE stabilization and polyHIPE synthesis

PolyHIPEs, macroporous polymers synthesized within the external phases of high internal phase emulsions (HIPEs), have been developed for a wide range of applications (catalyst supports, tissue engineering, drug delivery) in which the leaching of traditional surfactants can become a significant liability. HIPEs can also be stabilized with relatively small amounts of amphiphilic solid nanoparticles, as well as with miktoarm stars, poly(divinylbenzene) (PDVB) cores bearing different types of polymer arms. In this paper, two types of stars bearing both poly(ethylene oxide) and polyacrylate arms were used to stabilize HIPEs, non-reactive stars and stars bearing reactive methacrylate groups. The 0.1 wt % of stars successfully used to stabilize HIPEs containing styrene and DVB were not enough, at low DVB contents in the HIPE, to provide the crosslinking support needed to resist the capillary stresses generated during drying. However, with sufficient DVB in the HIPE, star contents as low as 0.05 wt % were able to successfully stabilize a HIPE with 90% internal phase and produce a polyHIPE with a density of 0.06 g/cm3. As little as 0.07 wt % stars were enough to stabilize HIPEs containing long side-chain (meth)acrylates and ~0.7 wt % of a crosslinking polyhedral silsesquioxane (POSS) and yield polyHIPEs with densities of around 0.2 g/cm3. These polyHIPEs exhibited rapid shape recovery and recovery ratios of 100% when a temporary shape (a 70% compressive strain) was imposed above the melting point of the crystalline phase. Star stabilization combined with POSS crosslinking enable the generation of tunable shape memory polymer foams.

REINFORCEMENT OF RUBBER BY CARBON BLACK AND LIGNIN-COATED NANOCELLULOSE FIBRILS

ABSTRACT The reinforcement of rubber by a co-filler system of carbon black and lignin-coated nanocellulose fibrils (LCNF) is investigated. Natural rubber (NR)–polybutadiene (BR) blend compounds containing LCNF loadings of up to 20% of the total filler package are prepared, and the dispersion state of the LCNF is determined using interferometric and electron microscopy. The LCNF is found to be well dispersed on macro- and micro-dispersion length scales, with discrete fibrils tending to align in the milling/calendering grain direction. Cure properties—scorch, rate, and total yield of crosslinks—are unaffected by the presence of LCNF in the compounds. Tensile to break and cyclic tensile properties are found to be reasonably consistent with those of a conventional all carbon black control compound. Tear and laboratory abrasion resistance properties are maintained versus the control compound, while a systematic and substantial reduction in compound Payne Effect with increasing LCNF content is observed. Basic aging properties of the compounds are unaffected by the presence of LCNF. The potential benefits of LCNF as a lightweight, sustainable, and bio-derived reinforcing filler are outlined.

Cellulose Acetate of Rice Husk Blend Membranes: Preparation, Morphology and Application

Cellulose acetate blend membranes in this study synthesized from cellulose acetate (CA) of rice husk and NaA zeolites (Z) with N,N’-Methylene bis acrylamide (MBA) as cross-linker agent and gamma-rays from cobalt-60 source as a reaction initiator. Application of the membrane was carried out to increase the concentration of vetiverol in vetiver oil. The steps in this study were isolation the cellulose rice husk with alkali treatment, delignification, acetylation, preparation the cellulose acetate-NaA zeolites NaA-membrane by inverse phase technique, addition N,N’-Methylene bis acrylamide, and irradiation by gamma-rays. Swelling degree, crosslinking yield, tensile strength, membrane performance, FTIR, and SEM analysis were observed. The results obtained that the optimal irradiation dose for synthesis CA-Z-MBA membrane is 20 kGy. The CA-Z-MBA membrane has swelling degree of 4.44%, the tensile strength of 656.40 kg/cm2, and crosslinking yield of 6.61%. Performance of the CA-Z-MBA membrane reached the flux of 60.58 g/m2.h, and permeate concentration (CP) of 11.67%, the CP increase 5 times from 2.40% to 11.67%.

Preparation of nano-enzyme aggregates by crosslinking lipase with sodium tripolyphosphate

Cross-linked enzyme aggregates (CLEAs) are considered as an effective tool for the immobilization of enzyme. The ionic cross-linking agent-sodium tripolyphosphate (TPP) was first used in preparing CLEAs. Aspergillus niger lipase was precipitated with ammonium sulfate and further cross-linked by TPP. The factors including enzyme concentration, pH of cross-linking medium, TPP dosage and cross-linking time were optimized. Maximum recovery activity (99.5 ± 0.634 %) and cross-linking yield (88.4 ± 0.46 %) can be obtained under the optimal process conditions, which can illustrate TPP had little effect on enzyme activity. CLEAs showed improved activity over broad pH and temperature range compared to the free enzyme. The thermal stability was obviously improved compared to free enzyme under the optimal temperature (40℃) and the half-life was 7.5-fold higher than that of free enzyme. Moreover, scanning electron microscopy (SEM) revealed that CLEAs had a cavity with porous structure and the particle size was 249 ± 3.98 nm. X-ray diffraction (XRD) showed the crystallinity of the CLEAs decreased. The changes in secondary structures of CLEAs revealed the increment in conformational rigidity. Such results suggested that the CLEAs has ideal application prospects.

Directing Quinone Methide-Dependent Alkylation and Cross-Linking of Nucleic Acids with Quaternary Amines.

Polyamine and polyammonium ion conjugates are often used to direct reagents to nucleic acids based on their strong electrostatic attraction to the phosphoribose backbone. Such nonspecific interactions do not typically alter the specificity of the attached reagent, but polyammonium ions dramatically redirected the specificity of a series of quinone methide precursors. Replacement of a relatively nonspecific intercalator based on acridine with a series of polyammonium ions resulted in a surprising change of DNA products. Piperidine stable adducts were generated in duplex DNA that lacked the ability to support a dynamic cross-linking observed previously with acridine conjugates. Minor reaction at guanine N7, the site of reversible reaction, was retained by a monofunctional quinone methide-polyammonium ion conjugate, but a bisfunctional analogue designed for tandem quinone methide formation modified guanine N7 in only single-stranded DNA. The resulting intrastrand cross-links were sufficiently dynamic to rearrange to interstrand cross-links. However, no further transfer of adducts was observed in duplex DNA. An alternative design that spatially and temporally decoupled the two quinone methide equivalents neither restored the dynamic reaction nor cross-linked DNA efficiently. While di- and triammonium ion conjugates successfully enhanced the yields of cross-linking by a bisquinone methide relative to a monoammonium equivalent, alternative ligands will be necessary to facilitate the migration of cross-linking and its potential application to disrupt DNA repair.

Peroxide-initiated chemical modification of polyolefins: In search of a latent antioxidant

The ability of piperidine-based compounds to confer oxidative stability to polyolefin thermosets without compromising the yields of peroxide-initiated crosslinking and monomer grafting is demonstrated. Unlike phenolic, nitroxyl and phosphite antioxidants that lower the concentration of macroradical intermediates that support polyolefin modifications, additives based upon 2,2,6,6-tetramethylpiperidine (TEMPH) are shown to have little to no effect on the extent of LLDPE crosslinking or the conversion of vinyltriethoxysilane (VTEOS) to grafted hydrocarbon adducts. Notwithstanding this lack of interference in radical-mediated polymer modification, this class of hindered light stabilizer (HAS) compounds are shown to limit the extent of radical oxidation of linear low-density polyethylene (LLDPE) in an accelerated aging test. The origins of this paradox are discussed in terms of the current state of knowledge regarding HAS activation. The latent antioxidant concept is extended to an alternate approach, wherein 4-acryloyloxy-2,2,6,6-tetramethylpiperidine-N-oxyl (AOTEMPO) is used as an alkyl radical scavenger bearing an oligomerizable functional group. When added at a fraction of the peroxide loading used to produce an LLDPE thermoset, ATEMPO is shown to provide a predictable induction delay without impacting ultimate crosslink density, and produces polymer-bound alkoxyamine functionality that stabilizes the product against oxidation.

Photodissociative Cross-Linking of Diazirine-Tagged Peptides with DNA Dinucleotides in the Gas Phase.

Non-covalent complexes of DNA dinucleotides dAA, dAT, dGG, dGC, and dCG with diazirine-tagged Cys-Ala-Gln-Lys peptides were generated as singly charged ions in the gas phase. Laser photodissociation at 355nm of the diazirine ring in the gas-phase complexes created carbene intermediates that underwent covalent cross-linking to the dinucleotides. The dinucleotides differed in the cross-linking yields, ranging from 27 to 36% for dAA and dAT up to 90-98% for dGG, dGC, and dCG. Collision-induced dissociation tandem mass spectrometry (CID-MS3) of the cross-linked conjugates revealed that fragmentation occurred chiefly in the dinucleotide moieties, resulting in a loss of a nucleobase and backbone cleavages. The CID-MS3 spectra further revealed that cross-links were primarily formed in the 3'-nucleotides for the dAT, dGC, and dCG combinations. Gas-phase and solution structures of dGG complexes with S-tagged CAQK were investigated by Born-Oppenheimer molecular dynamics (BOMD) and density functional theory calculations. The low free-energy complexes had zwitterionic structures in which the peptide was protonated at the N-terminus and in the Lys residue whereas the carboxyl or dGG phosphate were deprotonated, corresponding to the respective (Cys+, Lys+, COO-)+ and (Cys+, Lys+, phosphate-)+ protomeric types. Both types preferred structures in which the peptide N-terminal cysteine carrying the S-photo-tag was aligned with the 3'-guanine moiety. BOMD trajectories at 310K were analyzed for close contacts of the incipient peptide carbene with the positions in dGG that pointed to frequent contacts with the N-1, NH2, and N-7 atoms of 3'-guanine, in agreement with the cross-linking results. Carbene insertion to the guanine N-1-H and NH2 bonds was calculated by density functional and Møller-Plesset perturbational theory to be 350-380kJmol-1 exothermic. Based on calculations, we proposed a mechanism for the carbene reaction with guanine starting with an exothermic attack at N-7 to form a dipolar intermediate that can close an aziridine ring in another exothermic reaction, forming a stable covalent cross link. Graphical Abstract.

PEDOT:PSS interfaces stabilised using a PEGylated crosslinker yield improved conductivity and biocompatibility.

The rapidly expanding fields of bioelectronics, and biological interfaces with electronic sensors and stimulators, are placing an increasing demand on candidate materials to serve as robust surfaces that are both biocompatible, stable and electroconductive. Amongst conductive polymers, poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) is a promising material in biomedical research due to its appropriate stability and high conductivity, however its intrinsic solubility requires a crosslinking process that can limit its conductivity and biocompatibility. Poly(ethylene glycol) is known to be a suitably anti-immunogenic moiety and its derivatives have been widely used for biomedical applications. In this study we investigate the application of poly(ethylene glycol)diglycidyl ether (PEGDE) as an effective crosslinker and conductive filler for PEDOT:PSS. From our interpretation of XPS analysis we hypothesise that the crosslinking reaction is occurring via the epoxy ring of PEGDE interacting with the sulfonic groups of excel PSS chains, which reaches a saturation at 3 w/v% PEGDE concentration. PEGDE crosslinked films did not disperse in aqueous environments, had enhanced electrical conductivity and imparted a significant degree of hydrophilicity to PEDOT:PSS films. This hydrophilicity and the presence of biocompatible PEGDE led to good cell viability and a significantly increased degree of cell spreading on PEDOT:PSS films. In comparison to widely reported chemical crosslinking via glycidoxy propyltrimethoxysilane (GOPS), this original crosslinking yields a highly hydrophilic 2D film substrate with increased electroconductive and biocompatibility properties, resulting in a next-generation formulation for bioengineering applications.

The human ribosome can interact with the abasic site in mRNA via a specific peptide of the uS3 protein located near the mRNA entry channel

The small subunit ribosomal protein uS3 is a critically important player in the ribosome-mRNA interactions during translation and has numerous functions not directly related to protein synthesis in eukaryotes. A peculiar feature of the human uS3 protein is the ability of its fragment 55–64 exposed on the 40S subunit surface near the mRNA entry channel to form cross-links with 3′-terminal dialdehyde derivatives of various unstructured RNAs and with abasic sites in single-stranded DNAs. Here we showed that the ability of the above uS3 fragment to cross-link to abasic sites in DNAs is inherent only in mature cytoplasmic 40S subunits, but not nuclear pre-40S particles, which implies that it may be relevant to the ribosome-mRNA interplay. To clarify this issue, we investigated interactions of human ribosomes with synthetic mRNA analogues bearing an abasic site protected by a photocleavable group at the 3′-termini. We found that these mRNA analogues can form specific complexes with 80S ribosomes and 40S subunits, where the undamaged upstream part of the analogue is fixed in the mRNA binding channel by interaction with the P-site tRNA, and the downstream part located outside the ribosome is cross-linked to the uS3 fragment 55–64. The yield of cross-links of the mRNA analogues was rather high when their undamaged parts were bound to the mRNA channel prior to deprotection of the abasic site enabling its covalent attachment to the 40S subunit via the uS3 protein, but not vice versa. Based on our findings, one can assume that abasic sites, which can occur in mRNAs due to oxidative stress and ageing, are able to interact directly with the uS3 fragment exposed on the 40S subunit surface near the mRNA entry channel during translation. Consequently, the 40S subunit can be considered as a potential mRNA quality controller.