Reactive Amino Research Articles

Insight into the distribution of amino groups along the chain of chemically deacetylated hyaluronan

Deacetylated hyaluronan (daHA) containing reactive free amino groups is an important intermediate for further modification. Comparing direct and indirect NMR and HPLC to characterize the degree of HA deacetylation (DD), direct NMR approach using area ratio of anomeric CH and CH–NH2 groups was the most precise one. To describe the substitution pattern, daHA was selectively cleaved by nitrous acid generated in situ or hyaluronan lyase from Streptococcus pneumoniae. The resulting oligomers were identified by LC-ESI-MS. The experimental distribution of these oligomers was compared with theoretically expected random oligomer distribution. Independently on the starting HA molecular weight and deacetylation conditions, the experimental data differed from the random distribution model and suggested that deacetylation of certain N-acetyl-d-glucosamine had reduced the probability of deacetylation at the neighbouring disaccharide. This phenomenon was explained by conformational changes of HA caused by intra- and intermolecular interactions between positively charged amino and negatively charged carboxylic groups.

Construction of an immunotoxin via site-specific conjugation of anti-Her2 IgG and engineered Pseudomonas exotoxin A

BackgroundImmunotoxins consisting of a toxin from bacteria or plants and a targeting module have been developed as potent anti-cancer therapeutics. The majority of them, especially those in preclinical or clinical testing stages, are fusion proteins of a toxin and antibody fragment. Immunotoxins based on full-length antibodies are less studied, even though the fragment crystallizable (Fc) domain plays an important role in regulating the concentration of immunoglobulin G (IgG) in the serum and in antibody-mediated immune responses against pathogens.ResultsWe devised a method to site-specifically conjugate IgG and another protein using a cysteine residue introduced into the IgG and a bio-orthogonally reactive unnatural amino acid incorporated into the other protein. The human epidermal growth factor receptor 2 (Her2)-targeting IgG, trastuzumab, was engineered to have an unpaired cysteine in the heavy chain, and an unnatural amino acid with the azido group was incorporated into an engineered Pseudomonas exotoxin A (PE24). The two protein molecules were conjugated site-specifically using a bifunctional linker having dibenzocyclooctyne and maleimide groups. Binding to Her2 and interaction with various Fc receptors of trastuzumab were not affected by the conjugation with PE24. The trastuzumab-PE24 conjugate was cytotoxic to Her2-overexpressing cell lines, which involved the inhibition of cellular protein synthesis due to the modification of elongation factor-2.ConclusionsWe constructed the site-specifically conjugated immunotoxin based on IgG and PE24, which induced target-specific cytotoxicity. To evaluate the molecule as a cancer therapeutic, animal studies are planned to assess tumor regression, half-life in blood, and in vivo immunogenicity. In addition, we expect that the site-specific conjugation method can be used to develop other antibody-protein conjugates for applications in therapeutics and diagnostics.

Enhanced elastomer toughness and fracture properties imparted by chemically reactive flat nanoparticles

A remarkable enhancement in the mechanical and fracture properties of elastomers, imparted by flat predominantly 2-dimensional nanoparticles, is reported. Elastomers of hydrogenated nitrile butadiene rubber (HNBR), incorporating surface-functionalized montmorillonite clay, nanographite, carbon fiber, or carbon black, were thermally crosslinked in the presence of a bismaleimide coagent using a peroxide free-radical generator. Clay platelets, side-functionalized with octadecyl amine and edge-functionalized with reactive amino groups, were used to achieve good dispersion in the elastomer matrix. The clay-filled elastomer exhibited multiple glass transitions in their dynamic mechanical analysis (DMA) and approx. 6 times higher tear resistance, 4× higher rubbery plateau modulus, 2× higher modulus of toughness, and 2× higher crack initiation resistance than an elastomer containing equivalent carbon black concentration. Nanographite also resulted in property enhancement, albeit to a lower extent than nanoclay. Flat fillers are evidently able to arrest crack growth better than fillers with spherical or elongated shapes. DMA loss tangent data, and dissipated energy fraction calculated from the cyclic tensile test data, show that the flat fillers are better able to release strain energy in the form of viscous dissipation, rather than use it for increasing the crack surface area. SEM images of fractured surfaces are reported and discussed.

Rapid and Quantitative Profiling of Substrate Specificity of ω‐Transaminases for Ketones

Abstractω‐Transaminases (ω‐TAs) have gained growing attention owing to their capability for asymmetric synthesis of chiral amines from ketones. Reliable high‐throughput activity assay of ω‐TAs is essential in carrying out extensive substrate profiling and establishing a robust screening platform. Here we report spectrophotometric and colorimetric methods enabling rapid quantitation of ω‐TA activities toward ketones in a 96‐well microplate format. The assay methods employ benzylamine, a reactive amino donor for ω‐TAs, as a cosubstrate and exploit aldehyde dehydrogenase (ALDH) as a reporter enzyme, leading to formation of benzaldehyde detectable by ALDH owing to concomitant NADH generation. Spectrophotometric substrate profiling of two wild‐type ω‐TAs of opposite stereoselectivity was carried out at 340 nm with 22 ketones, revealing subtle differences in substrate specificities that were consistent with docking simulation results obtained with cognate amines. Colorimetric readout for naked eye detection of the ω‐TA activity was also demonstrated by supplementing the assay mixture with color‐developing reagents whose color reaction could be quantified at 580 nm. The colorimetric assay was applied to substrate profiling of an engineered ω‐TA for 24 ketones, leading to rapid identification of reactive ketones. The ALDH‐based assay is expected to be promising for high‐throughput screening of enzyme collections and mutant libraries to fish out the best ω‐TA candidate as well as to tailor enzyme properties for efficient amination of a target ketone.

A Single Reactive Noncanonical Amino Acid Is Able to Dramatically Stabilize Protein Structure.

A p-isothiocyanate phenylalanine mutant of the homodimeric protein homoserine o-succinyltransferase (MetA) was isolated in a temperature dependent selection from a library of metA mutants containing noncanonical amino acids. This mutant protein has a dramatic increase of 24 °C in thermal stability compared to the wild type protein. Peptide mapping experiments revealed that the isothiocyanate group forms a thiourea cross-link to the N terminal proline of the other monomer, despite the two positions being >30 Å apart in the X-ray crystal structure of the wild type protein. These results show that an expanded set of building blocks beyond the canonical 20 amino acids can lead to significant changes in the properties of proteins.

Effect of heat treatment on the binding of selected flavor compounds to myofibrillar proteins.

The influence of heat-induced structural modifications of grass carp myofibrillar protein (MP) on its ability to bind to selected aldehydes (hexanal, heptanal, octanal and nonanal) was investigated. The interactions of MP and flavor compounds were investigated using HS-GC-MS, intrinsic fluorescence spectra, Raman spectra, SDS-PAGE, turbidity, total sulfhydryl content and surface hydrophobicity. The ability to bind to aldehydes was strongly influenced by changes in the structure and surface of proteins during the heating process (0-30 min). During the first 0-10min of heating, the flavor-binding ability increased, which is likely attributable to increased surface hydrophobicity and total sulfhydryl content, and to the unfolding of secondary structures of MP by exposure to reactive amino acids, sulfhydryl groups and hydrophobic bonding sites. Nevertheless, lengthy heating (>10min) caused protein refolding and accelerated aggregation of protein, thus reducing hydrophobic interactions and weakening the resultant capacity of MP to bind to flavor compounds. The results suggested that hydrophobic interactions were enhanced upon short-term heating, whereas long-term heating weakend them. The results provide information concerning improvement of the flavor profile of freshwater fish surimi products. © 2019 Society of Chemical Industry.

Bifunctionality of Carbon Nanotubes – Chitosan Film Based Biosensor for Selective Determination of Atropine in Biological Fluids and Leaf Extract of Datura Stramonium

Atropine is a tropane alkaloid, which is most commonly found in Solanaceae family and serves as a drug with wide variety of effects. It is a racemic mixture of d-hyoscyamine and l-hyoscyamine, with most of its physiological effects due to l-hyoscyamine. The drug has extensive clinical applications because of its strong parasympatolytic, antispasmodic and anticholinergic actions. The anticholinergic activity results in blurred vision, vasodilation, increased heart rate and delirium. It is used to cure organic phosphorous pesticides poisoning. Atropine acts pharmacologically via blocking acetylcholine receptors of the muscarine subtype, which causes not only peripheral effect symptoms such as tachycardia, dilated pupils, and decreased gastrointestinal motility, but also affects the central nervous system and causes agitation, disorientation and hallucinations. It has been established as a very efficacious antidote to poisoning with parasympathomimetic nerve agents. Atropine has several medicinal applications like reducing rigidity in Parkinsonism, bronchial asthma, treating bradycardia in emergency settings, alleviating the viscera angina, peptic ulcers, renal colic and biliary colic. In spite of its wide clinical applications, the use of the highly toxic atropine in suicides and homicides has also been described in case reports. Since, the dosage level of atropine in pharmaceutical preparation is key to the effective treatment of diseases; its quantitative determination is of considerable significance in medicinal applications. Carbon nanotubes aroused attention in the burgeoning field of electrochemical sensors due to their unique properties including improved electrochemical activity. The single walled carbon nanotubes (SWCNTs) based biosensor is one of the immense frontiers in analytical chemistry, which is because of the high electrical conductivity, large surface area, ability to alleviate surface fouling and remarkable biochemical stability of SWCNTs. Chitosan is a cellulose – based biopolymer with large number of reactive amino and hydroxyl functional groups rendering the properties of excellent film-forming ability, high water permeability, good adhesion, and susceptibility to chemical modifications. SWCNTs have the potential to form stable and uniform nanocomposite dispersion in chitosan solution via covalent attachment that provide a platform for the development of biosensor. The integration of nanomaterials with biopolymer in the field of sensors and biosensors have allowed improving analytical performance. The main objective of this work is to develop an electrochemical sensor with enhanced sensitivity and augmented selectivity in the desired environment. In the present investigation, we have explored a non-destroyable surface decoration of single walled carbon nanotubes with biopolymer chitosan composite film coated on a glassy carbon electrode for the first time for the selective and ultrasensitive determination of atropine. The biosensor exhibited notable stability, exemplary reproducibility and uniformity in the composite film, short response time and an excellent catalytic activity towards the electro oxidation of atropine leading to a significant improvement in sensitivity. The developed biosensor displayed an electrocatalytic effect towards the anodic oxidation of atropine with selective recognition. Under the optimized conditions, the calibration curve for atropine concentration was linear in the range from 100 nM – 150 µM with the limits of quantification and detection as 54.9 and 16.5 nM, respectively. The high sensitivity was estimated to be 2.7 µA µM-1 for the proposed method. The selectivity of the developed sensor was substantiated by performing interference study in the presence of commonly existing biological metabolites. The developed biosensor was efficaciously applied to determine atropine in pharmaceutical samples available in the market. The biological relevance of the present method has been demonstrated by the analysis of atropine in media of human plasma and urine samples that correlate best with the pharmacological or toxicological effects. In addition, the versatility of the fabricated sensor was corroborated by the selective detection of atropine in the leaf extract of Datura stramonium.

Genetically Introducing Biochemically Reactive Amino Acids Dehydroalanine and Dehydrobutyrine in Proteins.

Expansion of the genetic code with unnatural amino acids (Uaas) has significantly increased the chemical space available to proteins for exploitation. Due to the inherent limitation of translational machinery and the required compatibility with biological settings, function groups introduced via Uaas to date are restricted to chemically inert, bioorthogonal, or latent bioreactive groups. To break this barrier, here we report a new strategy enabling the specific incorporation of biochemically reactive amino acids into proteins. A latent bioreactive amino acid is genetically encoded at a position proximal to the target natural amino acid; they react via proximity-enabled reactivity, selectively converting the latter into a reactive residue in situ. Using this Genetically Encoded Chemical COnversion (GECCO) strategy and harnessing the sulfur-fluoride exchange (SuFEx) reaction between fluorosulfate-l-tyrosine and serine or threonine, we site-specifically generated the reactive dehydroalanine and dehydrobutyrine into proteins. GECCO works both inter- and intramolecularly, and is compatible with various proteins. We further labeled the resultant dehydroalanine-containing protein with thiol-saccharide to generate glycoprotein mimetics. GECCO represents a new solution for selectively introducing biochemically reactive amino acids into proteins and is expected to open new avenues for exploiting chemistry in live systems for biological research and engineering.

A carbon dot and gold nanoparticle-based fluorometric immunoassay for 8-hydroxy-2'-deoxyguanosine in oxidatively damaged DNA.

A method is described for the fluorometric determination of DNA containing oxidatively damaged product 8-hydroxy-2'-deoxyguanosine (DNA-8-OHdG). Carbon dots (CDs) were modified with glutaraldehyde for DNA conjugation, and antibody against 8-OHdG was immobilized on gold nanoparticles (AuNPs). The presence of DNA-8-OHdG can be linked to CDs by reaction of amino groups on DNA with glutaraldehyde. AuNPs were brought closely to CDs by specific immune reaction between 8-OHdG and antibody on AuNPs. Under 350nm photoexcitation, the emission of CDs with a peak at 440nm is quenched by the AuNPs and not restored. In the presence of DNA-8-OHdG, the measured fluorescence intensity decreases and quenching efficiency increases. The limit of detection is 700 pM, and the assay works in the 0.01nM to 25μM DNA-8-OHdG concentration range. The method is perceived to possess a good potential as a tool for detecting biomarkers for DNA damage due to oxidative stress. Graphical abstract A fluorometric immunoassay for detecting 8-hydroxy-2'-deoxyguanosine (8-OHdG) in oxidatively damaged DNAis reported. It isbased on the use of carbon dots (CDs) and gold nanoparticles (AuNPs). Black wavy lines represent DNA. Yellow polygonal sharps represent 8-OHdG. Blue and pink balls represent CDs and AuNPs, respectively.

Hydrolysis of homocysteine thiolactone results in the formation of Protein-Cys-S-S-homocysteinylation.

An increased level of homocysteine, a reactive thiol amino acid, is associated with several complex disorders and is an independent risk factor for cardiovascular disease. A majority (>80%) of circulating homocysteine is protein bound. Homocysteine exclusively binds to protein cysteine residues via thiol disulfide exchange reaction, the mechanism of which has been reported. In contrast, homocysteine thiolactone, the cyclic thioester of homocysteine, is believed to exclusively bind to the primary amine group of lysine residue leading to N-homocysteinylation of proteins and hence studies on binding of homocysteine thiolactone to proteins thus far have only focused on N-homocysteinylation. Although it is known that homocysteine thiolactone can hydrolyze to homocysteine at physiological pH, surprisingly the extent of S-homocysteinylation during the exposure of homocysteine thiolactone with proteins has never been looked into. In this study, we clearly show that the hydrolysis of homocysteine thiolactone is pH dependent, and at physiological pH, 1 mM homocysteine thiolactone is hydrolysed to ~0.71 mM homocysteine within 24 h. Using albumin, we also show that incubation of HTL with albumin leads to a greater proportion of S-homocysteinylation (0.41 mol/mol of albumin) than N-homocysteinylation (0.14 mol/mol of albumin). S-homocysteinylation at Cys34 of HSA on treatment with homocysteine thiolactone was confirmed using LC-MS. Further, contrary to earlier reports, our results indicate that there is no cross talk between the cysteine attached to Cys34 of albumin and homocysteine attached to lysine residues.

The Changes of Mitochondria in Substantia Nigra and Anterior Cerebral Cortex of Hepatic Encephalopathy Induced by Thioacetamide.

ABSTRACTHepatic encephalopathy (HE) is a neuropsychiatric syndrome resulting from chronic or acute liver failure. Under the condition of HE, various factors such as reactive oxygen species, inflammatory factors, ammonia poisoning and amino acids alteration lead to changes of mitochondria. Selective depletion of damaged mitochondrion is essential for maintaining the morphology and function of mitochondria and cells. In this study, molecular biology analysis was used to analyze the mitochondrial morphology in the substantia nigra (SN) and anterior cerebral cortex (ACC) of the HE mice. The results revealed that the drp1, mfn1 and mfn2 increased in mRNA level of SN, which indicated the changes of mitochondrial morphology in HE mice. The drp1 and mfn2 genes were up‐regulated, then, the Opa1 exhibited no significant change in the ACC of HE mice. Further study demonstrated that the mitochondrial autophagy related genes, pink1 and parkin, increased in SN, while the parkin reduced in ACC of HE mice. In addition, uncoupling protein (ucp2) increased in mRNA level of SN and ACC, and the ucp4 had no change or reduced in SN and ACC, respectively. These findings suggested that the mitochondrial dynamics is different in the SN and ACC of HE mice. Therefore, our results indicated that mitochondrial dynamics provided a potential treatment strategy for HE through the fission, fusion and autophagy of genes. Anat Rec, 302:1169–1177, 2019. © 2018 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Synthesis of poly(1,5-diaminonaphthalene) microparticles with abundant amino and imino groups as strong adsorbers for heavy metal ions.

Poly(1,5-diaminonaphthalene) microparticles with abundant reactive amino and imino groups on their surface were synthesized by one-step oxidative polymerization of 1,5-diaminonaphthalene using ammoniumpersulfate as the oxidant. The molecular, supramolecular, and morphological structures of the microparticles were systematically characterized by IR and UV-vis spectroscopies, elementary analysis, wide-angle X-ray diffractometry, and transmission electron microscopy. The microparticles demonstrate electrical semiconductivity and high resistance to strong acid and alkali, and strong adsorption capability for lead(II), mercury(II), and silver(I) ions. The experimental conditions for adsorption of Pb(II) were optimized by varying the persulfate/monomer ratio, adsorption time, sorbent concentration, and pH value of the Pb(II) solution. The maximum adsorption capacity is 241mg·g-1 for particles after a 24h-exposure to a solution at an initial Pb(II) concentration of 29mM. The adsorption data fit a Langmuir isotherm and follow a pseudo-second-order reaction kinetics. This indicates a chemical adsorption that is typical for a chelation interaction between Pb(II) and amino/imino groups on the sorbent. Graphical abstract Poly(1,5-diaminonaphthalene) microparticles with abundant functional amino and imino groups have been synthesized by one-step direct polymerization of non-volatile 1,5-diaminonaphthalene in aqueous medium for sustainable preparation of high-performance adsorbents to strongly adsorb lead(II), mercury(II), and silver(I)ions.

New orthogonally trifunctionalized morpholine nucleosides

Three novel uracil-based morpholine nucleosides each containing three different orthogonally reactive functional groups, namely, amino, carboxy and acetylene ones, were synthesized. The obtained monomers are intended for the synthesis of labeled nucleotide, nucleic acid or peptide mimics.

A review on applications of chitosan-based Schiff bases.

Biopolymers have become very attractive as they are degradable, biocompatible, non-toxic and renewable. Due to the intrinsic reactive amino groups, chitosan is vibrant in the midst of other biopolymers. Using the versatility of these amino groups, various structural modifications have been accomplished on chitosan through certain chemical reactions. Chemical modification of chitosan via imine functionalization (RR'CNR″; R: alkyl/aryl, R': H/alkyl/aryl and R″: chitosan ring) is significant as it recommends the resultant chitosan-based Schiff bases (CSBs) for the important applications in the fields like biology, catalysis, sensors, water treatment, etc. CSBs are usually synthesized by the Schiff condensation reaction between chitosan's amino groups and carbonyl compounds with the removal of water molecules. In this review, we first introduce the available synthetic approaches for the preparation of CSBs. Then, we discuss the biological applications of CSBs including antimicrobial activity, anticancer activity, drug carrier ability, antioxidant activity and tissue engineering capacity. Successively, the applications of CSBs in other fields such as catalysis, adsorption and sensors are demonstrated.

The effect of high UV radiation exposure environment on the novel PVC polymers.

Although plastic induces environmental damages, almost the consumption of poly(vinyl chloride) never stops increasing. Therefore, this work abstracted by two parts, first, synthesis of Schiff bases 1-4 compounds through the reaction of amino group with appropriate aromatic aldehyde, reaction of PVC with Schiff bases compounds 1-4 in THF to form a new modified PVC-1, PVC-2, PVC-3, and PVC-4. The structures of Schiff bases 1-4 and the modified PVC-1, PVC-2, PVC-3, and PVC-4 have been characterized by different spectroscopic analyses. Second, the influence of introducing 4-amino-1,2,4-triazole as a pendent groups into PVC chain investigated on photostability rules of tests. The modified polymers photostability investigated by observing indices (ICO, Ipo, and IOH), weight loss, UV and morphological studies, and all results obtained indicated that PVC-1, PVC-2, PVC-3 and PVC-4 gave lower growth rate of ICO, IPO, and IOH through UV exposure time. The photostability are given as PVC-4 < PVC-3 < PVC-2 < PVC-1 from different mechanisms which suggested building on existence of 4-amino-1,2,4-triazole moieties in the polymer chain.

Identification of [6-Hydroxy-2-(hydroxymethyl)-5-oxo-5,6-dihydro-2 H-pyran-3-yl]-cysteine (HHPC) as a Cysteine-specific Modification Formed from 3,4-Dideoxyglucosone-3-ene (3,4-DGE).

Glucose degradation products (GDPs) are formed from glucose and other reducing sugars during heat treatment, for example, in heat-sterilized peritoneal dialysis fluids or foods. Because of their reactive mono- and dicarbonyl structure, they react readily with proteins, resulting in the formation of advanced glycation end products (AGEs), loss of protein functionality, and cytotoxicity. Among the GDPs, 3,4-dideoxyglucosone-3-ene (3,4-DGE) exerts the strongest effects despite its relatively low concentration levels. The goal of the present study was therefore to identify the structure of specific protein modifications deriving from 3,4-DGE. A nonapeptide containing the reactive amino acids lysine, arginine, and cysteine was incubated with 3,4-DGE and the dominant GDPs 3-deoxyglucosone (3-DG) and 3-deoxygalactosone (3-DGal) in concentrations as present in peritoneal dialysis fluids (235 μM 3-DG, 100 μM 3-Gal, and 11 μM 3,4-DGE). Glycation rate and product formation were determined by ultra-HPLC-MS/MS (UHPLC-MS/MS). 3,4-DGE showed the strongest glycation activity. After 2 h of incubation, 3,4-DGE had modified 57% of the nonapeptide, whereas 3-DG had modified only 2% and 3-DGal had modified 29% of the peptide. A stable 3,4-DGE-derived cysteine modification was isolated. Its structure was determined by comprehensive NMR and MS experiments to be [6-hydroxy-2-(hydroxymethyl)-5-oxo-5,6-dihydro-2 H-pyran-3-yl]-cysteine (HHPC), which represents a novel cysteine-AGE derived from 3,4-DGE. The results indicate that 3,4-DGE might contribute to a severe loss of protein functionality by forming cysteine-specific AGEs, such as HHPC.

High temperature phthalonitrile nanocomposites with silicon based nanoparticles of different nature and surface modification: Structure, dynamics, properties

A series of phthalonitrile nanocomposites, obtained from bisphenol A based bisphthalonitrile (BAPhN) with 0.5 wt.% of different reactive amino- or epoxy-functionalized polyhedral oligomeric silsesquioxane (POSS) nanoparticles or montmorillonite (MMT) nanolayers, were synthesized and studied in detail. Their structure, dynamics and properties were characterized by TEM, mid-IR, far-IR and EDX spectroscopies, DSC, and by DMA and TGA measurements performed in air and N2 atmosphere at temperatures from 200 to 600–700 °C. Predominance of phthalocyanine macrocycles in the nodes of the matrix network is suggested; a satisfactory dispersion and a quasi-regular distribution of POSS nanoparticles have been revealed. For post-cured nanocomposites, DMA demonstrated an anomalous (“quasi-phase”) glass transition, dynamic heterogeneity in this transition, and “constrained dynamics” effects. Tg (DMA) was found to vary from 4600 to 560°С. After a high-temperature treatment in N2 atmosphere, disappearance of relaxation spectrum and glass transition as well as the constancy of the nanocomposite modulus E’≈ 3.2 GPa at 20–600°С were observed. A relatively satisfactory thermal stability of the nanocomposites was registered at temperatures up to ∼500 °C in air and 700 °C in N2.

Facile synthesis and characterization of covalently colored polyurethane latex based on the chain extension of water-soluble dye monomer

In this work, a new method for facile synthesis of covalently colored polyurethane latex was developed. A series of blue dye monomers were synthesized via one-step reaction of a reactive dye and amino compounds, and then employed as chain extenders to react with polyurethane prepolymer in the emulsification process to prepare the covalently colored polyurethane latexes. The chemical structures and the properties of dye monomers and the corresponding polymers were investigated, and the UV–Vis spectra of covalently colored polyurethane featured fairly consistent with dye monomers with maximum absorption wavelength at 620 nm and 580 nm, since the chemical structure of chromophore maintained unchanged. The conversion of dye monomer reached more than 92% with 1.0 wt% dye content, whereas the conversion decreased with the increase of the dye amount and the adding time. Compared with the noncovalently latex film, the light fastness of the covalently colored latex film was greatly promoted due to the chemical bonding of chromophores with polymer matrix.

Selective Derivatization of Hexahistidine-Tagged Recombinant Proteins.

Covalent modification of proteins is extensively used in research and industry for biosensing, medical diagnostics, targeted drug delivery, and many other practical applications. The conventional method for production of protein conjugates has changed little in the last 20years mostly relying on reactions of side chains of cysteine and lysine residues. Due to the presence of large numbers of similar reactive amino acid residues in proteins, common synthetic methods generally produce complex mixtures of products, which are difficult to separate. An emerging alternative technology for covalent modification of proteins involves formation of a covalent bond with a hexahistidine affinity tag present in a majority of recombinant proteins without interfering with other amino acid residues. The approach is based on formation of a ternary complex of the hexahistidine sequence with a bivalent metal cation chelated by ligand bearing an electrophilic Baylis-Hillman ester group capable of subsequent formation of a covalent bond with one of the histidine residues of the tag. The reaction proceeds under mild reaction conditions in neutral aqueous solutions under high dilutions (10-5 to 10-4M) providing a stable covalent bond between the label and an imidazole residue in a hexahistidine tag at either C- or N-terminus. Because hexahistidine affinity tag methodology is a de-facto standard for preparation of recombinant proteins our approach can be easily implemented for selective derivatization of these proteins with fluorescent groups, alkynyl groups for "click" reactions, or biotinylation.

Preparation of poly(2,4,6‐triaminopyrimidine‐TMC)/P84 composite nanofiltration membrane with enhanced chlorine resistance and solvent resistance

AbstractBACKGROUNDNanofiltration membrane has attracted great study interest because of its high permselectivity and low operating cost. However, most commercial nanofiltration membranes are still not applicable for chemical separation processes due to their low resistance to organic solvent and chlorination.RESULTS2,4,6‐triaminopyrimidine (TAPI) was employed as an aqueous phase monomer to prepare poly (2,4,6‐triaminopyrimidine‐TMC) composite nanofiltration membrane with interfacial polymerization. Polyimide (P84) ultrafiltration (UF) membrane was crosslinked with TAPI and used as the supporting membrane. The composite membrane showed high rejection (91.4%) of Na2SO4 and a high flux (7.3 L m−2 h−1) at 0.3 MPa and ambient temperature. The membrane also showed high stability during 120 h immersion in N,N‐dimethylacetamide (DMAc) and acetone. The performance of membrane immersed in 200 ppm NaClO solution did not decline over 96 h.CONCLUSIONTAPI with reactive amino group can be used as aqueous phase monomer to prepare poly (2,4,6‐triaminopyrimidine‐TMC) composite nanofiltration membrane with high permselectivity. The pyrimidine ring of TAPI endows the functional layer with excellent chlorine resistance while the crosslinking of P84 membrane with TAPI significantly improves the solvent resistance. © 2018 Society of Chemical Industry