Preparation of surface molecularly imprinted polymeric microspheres and their recognition property for basic protein lysozyme

The surface imprinting of basic protein lysozyme (Lys) was carried out by designing a new route. The copolymerization of N-vinylpyrrolidone (NVP) and 2-hydroxyethyl methacrylate (HEMA) was first conducted in an inverse suspension polymerization system, and the crosslinked copolymeric microspheres HEMA/NVP were prepared. Subsequently, the esterification reaction of methacryloyl (MAO) chloride with the hydroxyl groups on the surfaces of HEMA/NVP microspheres was performed, and the modified microspheres MAO–HEMA/NVP, on which a mass of polymerisable double bonds were introduced, were obtained. In the presence of lysozyme, by initiating of K₂S₂O₈–NaHSO₃, the monomer methacrylic acid (MAA) in the solution was crosslink-polymerized on the surfaces of MAO–HEMA/NVP microspheres, resulting in the surface imprinting of lysozyme. After removing the template molecules, the lysozyme molecule-surface-imprinted material MIP-HEMA/NVP was obtained. Because there were strong interactions between lysozyme and monomer MAA, electrostatic interaction and hydrogen bonding, the lysozyme molecule-surface imprinting was successfully realized. The MIP-HEMA/NVP microspheres have very high binding affinity for lysozyme, and the binding capacity gets up to 216 mg/g. It is more important that MIP-HEMA/NVP microspheres have specific recognition selectivity for lysozyme, and the selectivity coefficient for lysozyme with respect to bovine hemoglobin (BHb), which was used as a contrast protein in the experiments, actually reaches 31.07. In the respect of protein imprinting, the imprinting material with such high performance is unwonted.     

Cleavable ester-linked magnetic nanoparticles for labeling of solvent-exposed primary amine groups of peptides/proteins

To study the solvent-exposed lysine residues of peptides/proteins, we previously reported disulfide-linked N-hydroxysuccinimide ester-modified silica-coated iron oxide magnetic nanoparticles (NHS–SS–SiO₂@Fe₃O₄ MNPs). The presence of a disulfide bond in the linker limits the use of disulfide reducing agent during protein digestion and allows unwanted disulfide formation between the MNPs and protein. In the current work, the disulfide bond was replaced with a cleavable ester group to synthesize NHS ester-modified SiO₂@Fe₃O₄ MNPs. Use of the cleavable ester group provides an improved method for protein labeling and allows the use of disulfide reducing agents during protein digestion.     

Cytotoxicity of mesoporous silica nanomaterials

We here measure the toxicity of MCM-41, a mesoporous silica nanomaterial, two of its functionalized analogs, AP-T, which has grafted aminopropyl groups and MP-T, which has grafted mercaptopropyl groups, and spherical silica nanoparticles (SiO₂), toward human neuroblastoma (SK-N-SH) cells. Since the particles studied are not soluble in aqueous media, the metric used to report the cytotoxicity of these materials is a new quantity, Q₅₀, which is the number of particles required to inhibit normal cell growth by 50%. Determining the number of particles per gram of material applied to the cells required both the calculated and experimentally determined surface areas of these nanomaterials. This study shows that Q₅₀ increases in the order, MCM-41 < MP-T < AP-T ≈ SiO₂, showing that on a per particle basis, MCM-41 is the most cytotoxic material studied. For the three mesoporous silica materials in this study, cytotoxicity appears related to the adsorptive surface area of the particle, although the nature of the functional group cannot be ruled out. Silica nanospheres have the lowest surface area of the particles studied but since they exhibit a Q₅₀ value similar to that of AP-T, shape may also be important in the cytotoxicity of these materials.     

E. coli tRNAPhe modified at the 3-(3-amino-3-carboxypropyl)uridine with a photoaffinity label is fully functional for aminoacylation and for ribosomal interaction

E. coli tRNA^Phe was modified at its 3-(3-amino-3-carboxypropyl)uridine residue with the N-hydroxysuccinimide ester of N-4-azido-2-nitrophenyl)glycine. Exclusive modification of this base was shown by two-dimensional TLC analysis of the T₁ oligonucleotide and nucleoside products of nuclease digestion. The fully modified tRNA could be aminoacylated to the same level as control tRNA. The aminoacylated tRNA was as active as control tRNA in non-enzymatic binding to the P site of ribosomes, and in EFTu-dependent binding to the rirobosomal A site. The functional activity of this photolabile modified tRNA allows it to be used to probe the A and P binding sites on ribosomes and on other proteins that interact with tRNA. Crosslinking to the ribosomal P site has been shown.     

Synthesis of N-hydroxysuccinimide esters of phosphatidylethanolamine and some properties of liposomes containing these derivatives

The N-hydroxysuccinimide (NHS) ester of N-suberyl-dimyristoylphosphatidylethanolamine (sub-DMPE) was synthesized by reaction of DMPE with disuccinimidyl suberate, and isolated by preparative plate chromatography. Liposomes, which contain NHS-sub-DMPE, can covalently bind compounds that possess a free amino group such as ε-dinitrophenyl-lysine. The extent of DNP-lysine binding is influenced by the time and temperature of incubation, the amount of NHS-sub-DMPE incorporated into the liposomes, and the initial concentration of DNP-lysine. Binding occurs as a consequence of the formation of a new dinitrophenylated compound which has been characterized. Although NHS-sub-DMPE is stable to storage in organic solvents, preformed liposomes rapidly lose their ability to bind DNP-lysine due to hydrolysis of the N-hydroxysuccinimide ester bond. These findings bear on the future applicability of liposomes, containing N-hydroxysuccinimide esters of PE, as illustrated by the preparation of immunogenic liposomes.     

Synthesis of 3-(3-hydroxyphenyl)pyrrolidine dopamine D3 receptor ligands with extended functionality for probing the secondary binding pocket

A series of 3-(3-hydroxyphenyl)pyrrolidine analogues which incorporate N-alkyl groups and N-butylamide-linked benzamide functionality have been synthesized and their in vitro binding affinities at human dopamine receptors have been evaluated. Our ligand design strategy was to take the 3-(3-hydroxyphenyl)pyrrolidine scaffold and extend functionality from the orthosteric binding site to the secondary binding pocket for enhancing affinity and selectivity for the D₃ receptor. The N-alkyl analogues constitute a homologous series from N-pentyl to N-decyl to probe the length/bulk tolerance of the secondary binding pocket of the D₃ receptor. Enantiomeric 3-(3-hydroxyphenyl)pyrrolidine analogues were also prepared in order to test the chirality preference of the orthosteric binding site for this scaffold. Benzamide analogues were prepared to enhance affinity and/or selectivity based upon the results of the homologous series.     

A nanoparticle-based immobilization assay for prion-kinetics study

Magnetic and gold coated magnetic nanoparticles were synthesized by co-precipitation of ferrous and ferric chlorides, and by the micromicelles method, respectively. Synthesized nanoparticles were functionalized to bear carboxyl and amino acid moieties and used as prion protein carriers after carbodiimide activation in the presence of N-hydroxysuccinimide. The binding of human recombinant prion protein (huPrPrec) to the surface of these nanoparticles was confirmed by FTIR and the size and structures of the particles were characterized by transmission electron microscopy. Findings indicate that the rate of prion binding increased only slightly when the concentration of prion in the reaction medium was increased. Rate constants of binding were very similar on Fe₃O₄@Au and Fe₃O₄-LAA when the concentrations of protein were 1, 2, 1.5, 2.25 and 3.57 μg/ml. For a 5 μg/ml concentration of huPrPrec the binding rate constant was higher for the Fe₃O₄-LAA particles. This study paves the way towards the formation of prion protein complexes onto a 3-dimensional structure that could reveal obscure physiological and pathological structure and prion protein kinetics.     

Isolation of isoaccepting tRNAs

The N-hydroxysuccinimide ester of succinated polyethylene oxide (polyethylene glycol 6000) has been prepared. The ester has been used to make the N-acyl derivatives of valyl-tRNA and phenylalanyl-tRNA from E. coli K-12. Because of the large molecular weight, high solubility in phenol, and the binding to Corning porous glass of polyethylene oxide, the acyl derivative, N-(succinated polyethylene oxide)-aminoacyl-tRNA, has been separated from unreacted tRNA. Since the reaction is reasonably specific for the amino group of the amino acid, large purifications have been obtained for tRNA_val and tRNA_phe. Evidence is presented to show that the ester can react with tRNA at a slow rate. The limitations on the purification due to this reaction are quantitatively evaluated. The highest ratios, pmoles aminoacyl-tRNA/ OD₂₆₀, obtained for valyl-tRNA and phenylalanyl-tRNA were 800 and 360.     

Structural insights into the targeting of mRNA GU-rich elements by the three RRMs of CELF1

The CUG-BP, Elav-like family (CELF) of RNA-binding proteins control gene expression at a number of different levels by regulating pre-mRNA splicing, deadenylation and mRNA stability. We present structural insights into the binding selectivity of CELF member 1 (CELF1) for GU-rich mRNA target sequences of the general form 5′-UGUN_xUGUN_yUGU and identify a high affinity interaction (K_d ∼ 100 nM for x = 2 and y = 4) with simultaneous binding of all three RNA recognition motifs within a single 15-nt binding element. RNA substrates spin-labelled at either the 3′ or 5′ terminus result in differential nuclear magnetic resonance paramagnetic relaxation enhancement effects, which are consistent with a non-sequential 2-1-3 arrangement of the three RNA recognition motifs on UGU sites in a 5′ to 3′ orientation along the RNA target. We further demonstrate that CELF1 binds to dispersed single-stranded UGU sites at the base of an RNA hairpin providing a structural rationale for recognition of CUG expansion repeats and splice site junctions in the regulation of alternative splicing.     

Molecularly imprinted polymers for RGD selective recognition and separation

Molecularly imprinted polymers that could recognize the tripeptide Arg-Gly-Asp have been produced with the use of two functional monomers and three different cross-linkers, respectively. Methacrylic acid and acrylamide were used as functional monomers and the role of the ethylene glycol dimethacrylate, trimethylpropane trimethacrylate and N,N′-methylene-bisacrylamide as crosslinking monomers, was investigated on their recognition capability. The % net rebinding and the imprinting factor values were obtained, giving for the methacrylic acid–trimethylpropane trimethacrylate polymer the highest values 12.3% and 2.44, respectively. In addition, this polymer presented lower dissociation constant (K _D) value and the higher B _max% of theoretical total binding sites than all the other polymers. Rebinding experiments with Lys-Gly-Asp, an analogue of Arg-Gly-Asp, and other different peptides, such as cholecystokinin C-terminal tri- and pentapeptide and gramicidin, further indicated the selectivity of methacrylic acid-trimethylpropane trimethacrylate copolymer for Arg-Gly-Asp giving specific selectivity factor values 1.27, 1.98, 1.31 and 1.67, respectively.     

Cobalt(II) Schiff base complex on multi-wall carbon nanotubes (MWNTs) by covalently grafted method: Synthesis,characterization and liquid phase epoxidation of cyclohexene by air

Cobalt [(OH)₂-salophen] (N,N′-bis(4-hydroxysalicylidene)phenylene-1,2-diamine) complex was covalently grafted on the chemical modification of multi-wall carbon nanotubes (MWNTs); [Co((OH)₂-salophen)]@MWNTs]. The as-products were characterized by spectroscopy (FT-IR, Raman, and UV–Vis), TGA, and TEM. The cobalt(II) Schiff-base complex covalently anchored on modified MWNTs was characterized by different techniques. The catalytic activity of the novel nanotubes based materials was tested in the epoxidation of cyclohexene in the iso-butyraldehyde/air system using acetonitrile as solvent and very high conversion was obtained. The experimental results indicated very good catalytic activity and selectivity in the epoxidation of cyclohexene. Repeated runs of the catalysts were carried out three times and the results indicated that the catalyst was stable for the epoxidation of cyclohexene.     

Heterocyclic aromatic amine content of selected beef flavors

Previous work has shown that meat extracts contain potent mutagenic and/or carcinogenic heterocyclic aromatic amines (HAAs). Because meat extracts and some beef flavors are produced from similar precursors and processing steps, the beef flavors may also contain HAAs. This study analyzed 24 commercial beef flavors and 2 food-grade beef extracts for creatine and creatinine concentrations, mutagenic activity and HAA concentrations (IQ, MeIQ, MeIQ_x, DiMeIQ_x, Glu-P-1, Glu-P-2 and PhIP). The creatine and creatinine levels of the flavors ranged from 0 to 73 and from 0 to 21 mg/g (dry wt.), respectively. The mutagenic activities of the flavors ranged from 0 to 3200 Salmonella typhimurium TA98 revertants/g (dry wt.). No direct relationship was found between creatine and/or creatinine concentrations and mutagenic activities. However, flavors with high creatine (> 1.5 mg/g) or creatinine (> 2 mg/g) levels exhibited higher mutagenic activities than did flavors with low levels of these compounds. Flavors with high mutagenic activities (> 1500 revertants/g) contained measurable amounts of HAAs. Three flavors contained MeIQ_x (7.2–21.2 ng/g [dry wt.]) and one contained DiMeIQ_x (4.2 ng/g [dry wt.]).     

Separating the isomers—Efficient synthesis of the N-hydroxysuccinimide esters of 5 and 6-carboxyfluorescein diacetate and 5 and 6-carboxyrhodamine B

Diacetate protection of 5 and 6-carboxyfluorescein followed by synthesis of the N-hydroxysuccinimide esters allowed ready separation of the two isomers on a multi-gram scale. The 5 and 6-carboxyrhodamine B N-hydroxysuccinimide esters were also readily synthesised and separated.     

Some molecular properties of an isolated acetylcholine receptor ion-translocation protein

An improved procedure for isolation and purification of acetylcholine receptor from Torpedo californica electroplax membranes is described. The purified material contains the neurotransmitter recognition site and a second binding subsite which complexes inorganic cations and bis-quaternary cholinergic analogs. In addition to the transmitter recognition site the isolated macromolecule contains the molecular features necessary for ion-translocation during postsynaptic depolarization, since a chemically excitable membrane can be formed from purified acetylcholine receptor and Torpedo phospholipids.     

Labeling proteins with fluorine-18 using N-succinimidyl 4-[18F]fluorobenzoate

Two methods were investigated for the no-carrier-added synthesis of N-succinimidyl 4-[¹⁸F]fluorobenzoate (S[¹⁸F]FB). The first, an attempted nucleophilic aromatic substitution by [¹⁸F]fluoride on N-succinimidyl 4-nitrobenzoate was unsuccessful. The second method involved three steps; [¹⁸F]fluoride for trimethylammonium substitution on 4-formyl-N,N,N-trimethylanilinium triflate, oxidation to 4-[¹⁸F]fluorobenzoic acid, followed by reaction with N-hydroxysuccinimide and dicyclohexylcarbodiimide to form S[¹⁸F]FB. Total synthesis and purification time was 100 min and the overall radiochemical yield was 25% (decay corrected). A monoclonal antibody F(ab′)₂ fragment could be labeled in 40–60% yield by reaction with S[¹⁸F]FB for 15–20 min. The tissue distribution in normal mice and in vitro tumor binding of the antibody F(ab′)₂ labeled by reaction with S[¹⁸F]FB were comparable to those observed for the fragment after radioiodination using N-succinimidyl 4-[¹²⁵I]iodobenzoate.     

Preparation of graft polypeptide binding bleomycin derivative

The esterification of carboxyl groups in poly-(l-glutamic acid) and atelo-collagen was carried out using N-hydroxysuccinimide and dicyclohexylcarbodiimide as coupling reagents in dimethylformamide (DMF) and/or dimethylsulphoxide (DMSO). The succinimide ester of polypeptide was allowed to react with the amino group of pepleomycin and then 2-aminoethanol in DMF and DMSO mixture. The graft polypeptide binding pepleomycin molecule is able to release the drug moiety during the biodegration of backbone polypeptides.     

Design,synthesis and biological evaluation of N-methyl-N-[(1,2,3-triazol-4-yl)alkyl]propargylamines as novel monoamine oxidase B inhibitors

Different azides and alkynes have been coupled via Cu-catalyzed 1,3-dipolar Huisgen cycloaddition to afford a novel family of N₁- and C₅-substituted 1,2,3-triazole derivatives that feature the propargylamine group typical of irreversible MAO-B inhibitors at the C4-side chain of the triazole ring. All the synthesized compounds were evaluated against human MAO-A and MAO-B. Structure–activity relationships and molecular modeling were utilized to gain insight into the structural and chemical features that enhance the binding affinity and selectivity between the two enzyme isoforms. Several lead compounds, in terms of potency (submicromolar to low micromolar range), MAO-B selective recognition, and brain permeability, were identified. One of these leads (MAO-B IC₅₀ of 3.54 μM, selectivity MAO-A/MAO-B index of 27.7) was further subjected to reversibility and time-dependence inhibition studies, which disclosed a slow and irreversible inhibition of human MAO-B. Overall, the results support the suitability of the 4-triazolylalkyl propargylamine scaffold for exploring the design of multipotent anti-Alzheimer compounds endowed with irreversible MAO-B inhibitory activity.     

A highly sensitive fluorescence sensor based on lucigenin/chitosan/SiO2 composite nanoparticles for microRNA detection using magnetic separation

In this paper, a convenient reverse‐phase microemulsion method for the synthesis of SiO₂ nanoparticles (NPs) by simply introducing the chitosan and fluorescent dye of lucigenin during the formation reaction of SiO₂ NPs was proposed. Addition of chitosan can make the SiO₂ NPs porous, and increases lucigenin molecule incorporation into chitosan/SiO₂ NPs nanopores based on electrostatic interaction and supermolecular forces. Therefore, fluorescence quantum yield of the lucigenin/chitosan/SiO₂ composite nanoparticles was increased by introduction of chitosan and compared with lucigenin/SiO₂ NPs without chitosan. Because the number of negative charges carried when using single‐stranded DNA (ssDNA) was different from that of double‐stranded DNA (dsDNA), the numbers of lucigenin/chitosan/SiO₂ composite nanoparticles with positive charge adsorbed using ssDNA or dsDNA were different. Consequently, fluorescence intensity caused using ssDNA or dsDNA/miRNA was clearly discriminative. With increase in target DNA/miRNA concentration, the difference in fluorescence intensity also increased, resulting in a good linear relationship between fluorescence intensity sensitizing value and target miRNA concentrations. Therefore, a new fluorescence analysis method for direct detection of let‐7a in human gastric cancer cell samples without enzyme, label free and no immobilization was established using lucigenin/chitosan/SiO₂ composite nanoparticles as a DNA hybrid indicator. The proposed method had high sensitivity and selectivity, low cost and the detection limit was 10 fM (S/N = 3).     

Structural manipulation of aporphines via C10 nitrogenation leads to the identification of new 5-HT7AR ligands

Aporphine alkaloids containing a C10 nitrogen motif were synthesized and evaluated for affinity at 5-HT_1AR, 5-HT_2AR, 5-HT₆R and 5-HT_7AR. Three series of racemic aporphines were investigated: 1,2,10-trisubstituted, C10 N-monosubstituted and compounds containing a C10 benzofused aminothiazole moiety. The 1,2,10-trisubstituted series of compounds as a group displayed modest selectivity for 5-HT_7AR and also had moderate 5-HT_7AR affinity. Compounds from the C10 N-monosubstituted series generally lacked affinity for 5-HT_2AR and 5-HT₆R and showed strong affinity for 5-HT_1A or 5-HT_7AR. Compounds in this series that contained an N6-methyl group were up to 27-fold selective for 5-HT_7AR over 5-HT_1AR, whereas compounds with an N6-propyl substituent showed a reversal in this selectivity. The C10 benzofused aminothiazole analogues showed a similar binding profile as the C10 N-monosubstituted series i.e. strong affinity for 5-HT_1AR or 5-HT_7AR, with selectivity between the two receptors being similarly influenced by N6-methyl or N6-propyl substituents. Compounds 29 and 34a exhibit high 5-HT_7AR affinity, excellent selectivity versus dopamine receptors and function as antagonists in 5-HT_7AR cAMP-based assays. Compounds 29 and 34a have been identified as new lead molecules for further tool and pharmaceutical optimization.