Maleimide Residues Research Articles

Radiotherapy-Triggered In Situ Tumor Vaccination Boosts Checkpoint Blockaded Immune Response via Antigen-Capturing Nanoadjuvants.

In situ vaccination (ISV) formed with the aid of intratumorally injected adjuvants has shed bright light on enhancing the abscopal therapeutic effects of radiotherapy. However, the limited availability of antigens resulting from the radiotherapy-induced immunogenic cell death largely hampers the clinical outcome of ISV. To maximally utilize the radiotherapy-induced antigen, we herein developed a strategy by capturing the radiotherapy-induced antigen in situ with a nanoadjuvant comprised of CpG-loaded Fe3O4 nanoparticles. The highly efficient click reaction between the maleimide residue on the nanoadjuvant and sulfhydryl group on the antigen maximized the bioavailability of autoantigens and CpG adjuvant in vivo. Importantly, combined immune checkpoint blockade can reverse T cell exhaustion after treatment with radiotherapy-induced ISV, thereby largely suppressing the treated and distant tumor. Mechanistically, metabolomics reveals the intratumorally injected nanoadjuvants disrupt redox homeostasis in the tumor microenvironment, further inducing tumor ferroptosis after radiotherapy. Overall, the current study highlights the immense potential of the innovative antigen-capturing nanoadjuvants for synergistically enhancing the antitumor effect.

Live-Cell Mitochondrial Targeted NIR Fluorescent Covalent Labeling of Specific Proteins Using a Dual Localization Effect.

Here, our designed water-soluble NIR fluorescent unsymmetrical Cy-5-Mal/TPP+ consists of a lipophilic cationic TPP+ subunit that can selectively target and accumulate in a live-cell inner mitochondrial matrix where a maleimide residue of the probe undergoes faster chemoselective and site-specific covalent attachment with the exposed Cys residue of mitochondrion-specific proteins. On the basis of this dual localization effect, Cy-5-Mal/TPP+ molecules remain for a longer time period even after membrane depolarization, enabling long-term live-cell mitochondrial imaging. Due to the adequate concentration of Cy-5-Mal/TPP+ reached in live-cell mitochondria, it facilitates site-selective NIR fluorescent covalent labeling with Cys-exposed proteins, which are identified by the in-gel fluorescence assay and LC-MS/MS-based proteomics and supported by a computational method. This dual targeting approach with admirable photostability, narrow NIR absorption/emission bands, bright emission, long fluorescence lifetime, and insignificant cytotoxicity has been shown to improve real-time live-cell mitochondrial tracking including dynamics and interorganelle crosstalk with multicolor imaging applications.

Human Serum Albumin Labelling with a New BODIPY Dye Having a Large Stokes Shift.

BODIPY dyes are photostable neutral derivatives of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene. These are widely used as chemosensors, laser materials, and molecular probes. At the same time, BODIPY dyes have small or moderate Stokes shifts like most other fluorophores. Large Stokes shifts are preferred for fluorophores because of higher sensitivity of such probes and sensors. The new boron containing BODIPY dye was designed and synthesized. We succeeded to perform an annulation of pyrrole ring with coumarin heterocyclic system and achieved a remarkable difference in absorption and emission maximum of obtained fluorophore up to 100 nm. This BODIPY dye was equipped with linker arm and was functionalized with a maleimide residue specifically reactive towards thiol groups of proteins. BODIPY residue equipped with a suitable targeting protein core can be used as a suitable imaging probe and agent for Boron Neutron Capture Therapy (BNCT). As the most abundant protein with a variety of physiological functions, human serum albumin (HSA) has been used extensively for the delivery and improvement of therapeutic molecules. Thiolactone chemistry provides a powerful tool to prepare albumin-based multimodal constructions. The released sulfhydryl groups of the homocysteine functional handle in thiolactone modified HSA were labeled with BODIPY dye to prepare a labeled albumin-BODIPY dye conjugate confirmed by MALDI-TOF-MS, UV-vis, and fluorescent emission spectra. Cytotoxicity of the resulting conjugate was investigated. This study is the basis for a novel BODIPY dye-albumin theranostic for BNCT. The results provide further impetus to develop derivatives of HSA for delivery of boron to cancer cells.

A genetically modified protein-based hydrogel for 3D culture of AD293 cells.

Hydrogels have strong application prospects for drug delivery, tissue engineering and cell therapy because of their excellent biocompatibility and abundant availability as scaffolds for drugs and cells. In this study, we created hybrid hydrogels based on a genetically modified tax interactive protein-1 (TIP1) by introducing two or four cysteine residues in the primary structure of TIP1. The introduced cysteine residues were crosslinked with a four-armed poly (ethylene glycol) having their arm ends capped with maleimide residues (4-armed-PEG-Mal) to form hydrogels. In one form of the genetically modification, we incorporated a peptide sequence ‘GRGDSP’ to introduce bioactivity to the protein, and the resultant hydrogel could provide an excellent environment for a three dimensional cell culture of AD293 cells. The AD293 cells continued to divide and displayed a polyhedron or spindle-shape during the 3-day culture period. Besides, AD293 cells could be easily separated from the cell-gel constructs for future large-scale culture after being cultured for 3 days and treating hydrogel with trypsinase. This work significantly expands the toolbox of recombinant proteins for hydrogel formation, and we believe that our hydrogel will be of considerable interest to those working in cell therapy and controlled drug delivery.

Characterization of metal-tagged antibodies used in ICP-MS-based immunoassays

A detailed characterization of metal-tagged antibodies is the prerequisite for the implementation of quantitative concepts in inductively coupled plasma-mass spectrometry (ICP-MS)-based bioanalysis or future medical diagnosis. In this paper, the common modification with bifunctional ligands containing maleimide residues as a reactive group was investigated in detail via size exclusion chromatography (SEC)-ICP-MS and liquid chromatography-time-of-flight (LC-TOF)-MS to determine the preservation of the antibody structure after tagging. Mouse monoclonal IgG modified with metal-coded tags (MeCATs) was used as a model system. Several antibody fragments were identified carrying different numbers of metal tags. In a second step, a functionality test was performed with isolated fragments where the antigen specificity was tested in a dot blot immunoassay.

NaGdF4 Nanoparticle-Based Molecular Probes for Magnetic Resonance Imaging of Intraperitoneal Tumor Xenografts in Vivo

Differently sized NaGdF(4) nanocrystals with narrow particle size distributions were synthesized by a high temperature approach. Upon ligand exchange, the as-prepared hydrophobic NaGdF(4) nanocrystals were transferred into water by using asymmetric PEGs simultaneously bearing phosphate and maleimide groups. Further investigations demonstrated that the water-soluble NaGdF(4) nanocrystals, coated by PEG bearing two phosphate groups on the same side, exhibit not only excellent colloidal stability in water and PBS buffer, but also higher T1 relaxivity than Gd-DTPA (Magnevist). Through "click" reaction between the maleimide residue on particle surface and thiol group from the partly reduced anti-EGFR monoclonal antibody (mAb), NaGdF(4)-PEG-mAb nanoprobes were constructed, and their biocompatibility and binding specificity were evaluated through in vitro experiments. A series of in vivo experiments were then carried out for detecting intraperitoneal tumor xenografts in nude mice by using magnetic resonance (MR) imaging technique. The results revealed that the NaGdF(4)-PEG-mAb probes possessed satisfying tumor-specific targeting ability and strong MR contrast enhancement effects.

Pyrazine-2,3-dicarbonitriles substituted with maleimide derivatives

Syntheses and spectroscopic characteristics are reported for eight pyrazine-2,3-dicarbonitriles substituted with maleimide residue in addition to phenyl, 2-thienyl, or 2-furyl substituent.

Metalization of polypropylene. I. Synthesis and melt free‐radical grafting of novel maleimides and methacrylates containing chelating moieties

AbstractSeveral chelating monomers with methacrylate and maleimide residues as polymerizable groups and diketone and aspartic acid residues as metal‐bonding groups were synthesized. A long‐chain aliphatic spacer was introduced to increase the solubility of the monomers in the polymer melt and to enhance the compatibility of grafted side chains and homopolymers with polypropylene (PP). PP was functionalized by melt free‐radical grafting, and some properties of the modified polymer were investigated. The grafting of a chelating methacrylic ester by injection molding afforded grafting yields of 30–50%. A comparison with products obtained by the grafting of commercially available acetoacetoxy methyl methacrylate showed the beneficial effect of the aliphatic spacer with respect to the grafting yield as well as the tensile strength and flexural modulus. The grafting of an aspartic acid based maleimide in an extruder afforded grafting yields of 80–95% and a significant increase in the surface wettability, as shown by contact‐angle measurements. A comparative study using samples obtained by the grafting of maleimidobenzoic acid indicated that the homogeneity‐increasing effect of the spacer was neutralized by the detrimental effect of the dicarboxylic chelating group. Nevertheless, the results showed that the maleimides were well suited for grafting by reactive extrusion because of their low susceptibility to homopolymerization under melt‐processing conditions. Preliminary metalization experiments showed the feasibility of plating without the use of corrosive etchants. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 3400–3413, 2003

Investigation of surface oligonucleotide-binding proteins of eucaryotic cells by affinity modification with reactive oligonucleotide derivatives

Interactions between oligodeoxyribonucleotides (ODN) with different sequences and cell proteins were examined using the affinity modification by [32P]-labeled reactive oligonucleotide derivatives. 3"-Terminal ribouridine oxidized with sodium periodate, 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine, and the maleimide residue were used as reactive groups. All the compounds used are specific reagents. The set of the discovered nucleic acid-binding (NA-binding) proteins depends on the chemical properties of the affinity reagent. The presence of the hydrophobic group at the 5"-terminus of the ODN molecule is the key factor determining the variety of the discovered NA-binding proteins. The cells of different origin (A431, HeLa, KB, MCF-7, Hap-2, K562, Cos-7, NIH/3T3, human-lung primary epithelial cells, and porcine kidney primary cells) are characterized by the same set of NA-binding proteins whose affinity modifications depends on the conditions of incubation of oligonucleotides with the cells. Treatments of cells disturbing the integrity of the cellular membrane (scrapping, treatment with trypsin, or cell permeabilization with streptolysin O or saponin), disrupt interactions between NA-binding proteins from native cells and ODN.

An immunoassay for the detection and quantitative determination of mycothiol

Mycothiol (MSH) is a glycosylated derivative of N-acetylcysteine that may have antioxidant functions in mycobacteria and other actinomycetes. To develop a highly specific assay for MSH, we capitalized on the selective binding of thiols to a maleimide residue linked to bovine serum albumin and employed affinity-purified polyclonal antibody and an enzyme-linked secondary antibody for detection. The assay was shown to be specific and to detect MSH at levels as low as 0.1 pmol when conducted in the form of a microtiter plate-based ELISA. A similar, nitrocellulose membrane-based immunoassay was shown to be useful for qualitative detection of MSH-producing bacterial colonies.

N-Polymethylenecarboxymaleimides—a new class of probes for membrane sulphydryl groups

Many membrane proteins contain essential sulphydry1 groups and are therefore susceptible to modification and subsequent inhibition by.electrophilic reagents. The availabilty of non-penetrant sulphydryl reagents such as p-chloromercuriphenylsulphonate and permeant reagents such as N-ethyl maleimide has allowed the localisation. of sulphydryl groups at either face of the membrane or within the membrane. Determination of the depth of sulphydryl groups within the membrane has not been possible until recently. Reagents consisting of a hydrophilic spacer arm and a reactive maleimide residue have been used to probe populations of sulphydryl groups on or inside the erythrocyte membrane [ 11. Similar reagents have been employed in investigations of the role of sulphydry1 groups in the transport of glucose into brush border membrane vesicles [2]. We have synthesised a series of N-polymethylenecarboxymaleimides for use as membrane-impermeant sulphydryl reagents. The positions of some of the essential sulphydryl groups of the phosphate-transporter protein of insect flight muscle and rat liver mitochondria have been determined using these compounds. tion coefficients were measured by vortexing 1 ml n-octanol with 1 ml potassium phosphate buffer @H 8 .O) containing 1 mM N-polymethylenecarboxymaleimide for 30 s. The concentration of the maleimide in each phase was determined by HPLC on a reversed phase-bonded silica column (Lichrosorb RF 2) eluted with methanol/SO mM potassium phosphate, pH 4 (3 :2) at 1 ml/min. Maleimides were detected by their absorption at 300 nm. Insect flight muscle mitochondria were prepared from&zrcophaga barbata asin [5]. Rat liver mitochondria were prepared as in [6]. Rat liver mitochondria (3 mg, 50 ~1) were diluted with 250 ~1 of a solution containing 200 mM sucrose, 50 mM KCl, 20 mM Tris-HCl, 1 mM EDTA (pH 8 .O) and incubated with the appropriate maleimide solution (20 nmol/mg protein) for 1 min at 22°C. The reaction was terminated by the addition of /3-mercaptoethanol to 0.5 mM. Aliquots of 1 ml were removed for the determination of phosphate transport as in [7]. Insect flight muscle mitochondria were incubated with maleimides (20 nmol/mg protein) in a medium containing 150 mM KCl, 10 mM sodium phosphate, 2 mM EGTA and 1 mM CaClz (PH 7.4). The reaction was terminated with @-mercaptoethanol and the ability to transport phosphate was assayed as above.

Improved Procedure for the Conjugation of Rabbit IgG and Fab' Antibodies with β-D-Galactosidase from <italic>Escherichia coil</italic> using <italic>N,N'-o-</italic>Phenylenedimaleimide<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

The procedures for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide were improved in several respects as compared with the previous methods (Eur. J. Biochem. 62, 285--292, 1976; J. Immunol. 116, 1554--1560, 1976). Maleimide residues were efficiently introduced into antibodies under an atmosphere of nitrogen; the average number of maleimide residues introduced into IgG and Fab' antibodies were 0.78 (0.65--0.86) and 0.86 (0.80--0.95) per molecule, respectively. The conjugation with the enzyme was performed at 4 degrees C at pH 6.5 for 15 or more hours. The conjugates were almost completely separated from unreacted IgG and Fab' by gel filtration. When the recoveries of IgG, Fab', and beta-D-galactosidase in the conjugates were 23-29, 35-44, and 99%, respectively, the average numbers of IgG and Fab' molecules conjugated with the enzyme were 1.5-1.7 and 2.1-2.8 per molecule, respectively. There was no significant impairment of beta-D-galactosidase activity or the activity of anti-human IgG antibody to bind to human IgG upon conjugation. However, the conjugate preparation was heterogeneous, and one-third of each preparation consisted of aggregated conjugates less useful in sandwich enzymoimmunoassay than the remaining material. The conjugate with Fab' antibody gave lower control values in sandwich enzymoimmunoassay with silicone rubber as a solid phase than that with IgG antibody.

A New Method for Preparation of an Antiserum to Penicillin and Its Application for Novel Enzyme Immunoassay of Penicillin1

A new method for the preparation of ampicillin-BSA conjugate by a three step procedure was developed. The first step is the introduction of a maleimide residue to ampicillin by a cross-linking reagent, MBS. The second step is reductive cleavage of disulfide bonds in BSA. The third step is thioether formation between the introduced maleimide residues and the reduced thiol groups. The obtained ampicillin-BSA conjugated raised an anti-ampicillin serum in rabbits. A new reagent, MPGS, was used for enzyme labelling of ampicillin to avoid immunological cross reaction. Using the enzyme labelled ampicillin and anti-ampicillin serum, enzyme immunoassay of ampicillin was successful in detecting 4 ng to 1 mug. Cross reactivities of anti-ampicillin to ampicillin analogs were studied by the enzyme immunoassay to find that the antiserum is specific to penicillin especially to ampicillin but hardly reacts with cephalosporins or the penicilloic acid derivative of ampicillin.

Enzyme Immunoassay of Viomycin1

A new cross-linking reagent of the hetero-bisfunctional type, a N-(maleimidobenzoyloxy)-succinimide (MBS) was prepared and used for enzyme labelling of viomycin under mild aqueous conditions by a two-step process. In the first step a maleimide residue was selectively introduced onto the N1-amino group of viomycin with a limited amount of MBS. The second step consisted of thioether formation between the maleimide residue and free thiol groups of beta-D-galactosidase. An antiserum to viomycin was raised in rabbit by immunization with a viomycin-BSA conjugate. The conjugate was prepared by protecting N6-amino group of viomycin with an acetyl group and succinylating the N1-amino group, activating the carboxyl group by a mixed anhydride method and coupling it with the amino groups of bovine serum albumin (BSA). The specificity of the antiserum was proved by an enzyme immunoassay based on the competition between viomycin and its enzyme conjugate toward diluted solutions of the antiserum. By use of the viomycin-enzyme conjugate and the antiserum to viomycin, enzyme immunoassay of viomycin was successfully performed by the competitive binding procedure with the double-antibody method, and 0.1 to 4 ng of the antibiotic could be detected.

Thermal degradation of poly(bismaleimides)

AbstractThermal decomposition of poly(bismaleimides) has been investigated by using programmed and isothermal thermogravimetric analysis (TGA) in nitrogen. Reaction rates and overall activation energies were calculated from isothermal weight less studies. For five aliphatic poly(bismaleimides) a linear correlation between the activation energies and the number of methylene groups in the sequence between the maleimide residues was found. Aliphatic poly(bismaleimides) follow first‐order kinetic law up to a conversion of 60‐70% having activation energies between 196 and 256 kj/mole. poly(2,4‐bismaleimidotoluene) which was found to have in highest polymer decomposition temperature (PDT) did follow the first‐order kinetics up to a conversion of 60% in contrast to other aromatic poly(bismaleimides). In addition to the TGA, the pyro‐field ion mass spectra of the polymers were recorded and are discussed.

Enzyme-Linked Immunoassay: Conjugation of the Fab′ Fragment of Rabbit IgG with β-D-Galactosidase from E. Coli and its Use for Immunoassay

Abstract A method for the conjugation of the Fab′ fragment of rabbit IgG with β-D-galactosidase from Escherichia coli is described. The method consists of two main steps: treatment of the Fab′ fragments containing sulfhydryl groups with excess N,N′-o-phenylenedimaleimide, to introduce maleimide residues into the fragments, and then incubation of the dimaleimide-treated Fab′ fragments with β-D-galactosidase, which also contains sulfhydryl groups, to form the rabbit Fab′-β-D-galactosidase complex. More than 90% of the enzyme used can be converted to the Fab′-enzyme complex, and the complex is readily separated from free Fab′ fragments by chromatography on a Sepharose 6B column. The application of the rabbit Fab′-β-D-galactosidase complex for immunoassay of macromolecular antigens is shown by measuring human IgG by the sandwich method. The rabbit (anti-human IgG) IgG-coupled Sepharose 4B is incubated with human IgG and then with the rabbit (anti-human IgG) Fab′-enzyme complex, and the enzyme activity bound to the Sepharose is measured. In this way it is possible to determine as little as 0.3 fmoles of human IgG.

Enzyme-linked immunoassay. Conjugation of rabbit anti-(human immunoglobulin G) antibody with beta-D-galactosidase from Escherichia coli and its use for human immunoglobulin G assay.

Rabbit immunoglobulin G (IgG) was reduced by incubating with 10 mM 2-mercaptoethylamine and then treated with excess amounts of N,N'-o-phenylenedimaleimide. As a result, maleimide residues were introduced into rabbit IgG molecules. Rabbit IgG containing maleimide residues could be coupled to beta-D-galactosidase from Escherichia coli which has sulfhydryl groups in the molecule. The resulting rabbit IgG (antibody)-enzyme complex may be useful for immunoassay of antigens. As an example, human IgG was assayed by the sandwich method using the rabbit anti-(human IgG) IgG-beta-D-galactosidase complex and amounts of human IgG as small as 3 fmoles were measurable.

Infrared spectroscopic determination of the kinetic data of the polymerization of aliphatic bismaleimides

Bismaleimides polymerize at elevated temperatures, forming highly crosslinked, high temperature-resistant polymers. From the decrease of the ν(HC) band at 3100 cm−1, rate constants and activation energies of the polymerization of four homologous aliphatic bismaleimides were determined. Up to a 20–30% conversion, the polymerization is pseudofirst order. An almost linear correlation between the activation energy and the number of methylene groups in the sequence between the maleimide residues was found.