Biotin Derivatives Research Articles

Immobilization of biomolecules on polyurethane membrane surfaces.

Lipophilic polymer membranes incorporating binding sites are widely used in various potentiometric, amperometric, and optical sensors. Here, we report on the biofunctional modification of the surface of a Ca(2+)-selective membrane. A photoactivatable biotin derivative was synthesized and covalently immobilized on a soft polyurethane membrane. The modified polymer was characterized by X-ray photoelectron spectroscopy (XPS) as well as by potentiometric measurements. The selective binding of streptavidin by the photo-cross-linked biotin derivative was demonstrated. The surface coverage obtained with different experimental protocols was analyzed by autoradiography using [(35)S]-streptavidin. The new approach may significantly extend the scope of applicability of potentiometric sensors.

Directional degradation of β-chitin by chitinase A1 revealed by a novel reducing end labelling technique

A novel procedure for labelling the molecular ends of β-chitin crystals has been established. By introducing a hydrazide derivative of biotin at the reducing end of a chitin chain, followed by a specific interaction between biotin and streptavidin coupled with a colloidal gold particle, the chain directionality of β-chitin microcrystals could be directly visualized by transmission electron microscopy. This method allowed to certify the parallelism of the chitin chains in the β-chitin microcrystals, and also to label the reducing tips of β-chitin microcrystals degraded by Bacillus circulans chitinase A1. With these substrates, the labelling occurred only at their tapered tip, which indicates that the digestion of these crystals proceeded from their reducing end. The generalization of this new labelling method to other polysaccharide crystals is discussed.

Synthesis of novel biotin anchors

Details are reported of the synthesis of biotinylated thiol and cholesteryl derivatives. The biotin derivative has been used to form multilayer films on gold through interaction with streptavidin.

Biotin Derivatives of [formula omitted]-Phe-Pro-Arg-CH2Cl for Active-Site-Specific Labeling of Thrombin and Other Serine Proteinases

Biotin derivatives of peptide chloromethyl ketones have ideal properties for specific labeling of the catalytic sites of serine proteinases but have not been widely used as probes because of the difficulty of synthesis and their instability. To make the reagents more accessible, a simple, economical method was developed for preparation of three biotin derivatives of the thrombin-specific inhibitor d-Phe-Pro-Arg-CH2Cl containing increasing lengths of the spacer connecting biotin. Reaction of the peptide with biotin-succinimidyl esters and purification by conventional chromatography yielded the compounds in 91–96% purity. The biotin-labeled inhibitors bound avidin with stoichiometries of 0.88–1.02 mol biotin compound/mol avidin subunits and irreversibly inactivated human thrombin with stoichiometries of 0.89–1.10 mol inhibitor/mol thrombin. Comparison of the three inhibitors by Western blotting indicated that a ≥7- to 14-atom spacer was needed for sensitive (∼10 ng) detection of thrombin, with the derivative lacking a spacer only weakly detected because of its greatly reduced affinity for avidin. Application of the compounds to identify catalytically active products of factor Xa-catalyzed human prethrombin 1 activation in the absence of the protein cofactor, factor Va, allowed the direct observation of transient, low levels of the active intermediate, meizothrombin des-fragment 1, in addition to thrombin. Formation of this intermediate is concluded to reflect an intrinsic property of factor Xa activation of prethrombin 1 that is modulated by factor Va. The methods developed for preparation and characterization of the biotin-labeled inhibitors may be applicable to other tripeptide chloromethyl ketones, and the reagents can be employed for labeling of serine proteinases of diverse substrate specificity.

Intramolecular hydrogen bonding of (+)-biotin and biotin derivatives in organic solvents

The intramolecular hydrogen bonding of (+)-biotin and biotin derivatives in mixtures of CDCl3 and DMSO-d6 was investigated by H NMR spectroscopy. The dynamic changes observed in the chemic al shifts for the 1-NH and 3-NH protons with changes in solvent composition and temperature confirmed the presence of an intramolecular hydrogen bond between the valeryl side chain hydrogen bond acceptor and the 3-NH proton in a range of biotin derivatives.

Evaluation of a tertiary amine labeling protocol for peptides and proteins using Ru(bpy) 33+-based chemiluminescence detection

The properties of four ruthenium complexes and their efficiency as probes for chemiluminescent (CL) detection were evaluated, and a protocol for CL detection of a protein labeled with a tertiary amine was proposed. Of the four ruthenium complexes studied, Ru(bpy) 3 3+ proved to exhibit the characteristics necessary for optimum CL detection. Two biotin derivatives were synthesized containing a tertiary amine functionality and then incubated with the protein, avidin. The addition of the tertiary amine moiety to biotin made the derivative more sensitive to Ru(bpy) 3 3+-based CL. Quantitative data were obtained as a model of a labeling protocol by varying the number of biotins per avidin. Limit of detection (LOD) for either derivative were determined to be ∼50 nM, for the lowest concentration studied. The relative luminescence response increased with increasing number of tertiary amine labels per molecule of avidin. The results showed the potential of this approach for the detection of proteins using this tertiary amine labeling methodology.

Aspartyl protease inhibitor pepstatin binds to the presenilins of Alzheimer's disease.

Mutations in the presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease by altering gamma-secretase cleavage of the amyloid precursor protein, the last step in the generation of Abeta peptide. Ablation of presenilin (PS) genes, or mutation of two critical aspartates, abolishes gamma-secretase cleavage, suggesting that PS may be the gamma-secretases. Independently, inhibition experiments indicate that gamma-secretase is an aspartyl protease. To characterize the putative gamma-secretase activity associated with presenilins, lysates from human neuroblastoma SH-SY5Y and human brain homogenates were incubated with biotin derivatives of pepstatin, followed by immunoprecipitation of PS and associated proteins, and biotin detection by Western blotting. Precipitation with PS1 antibodies, directed to either N-terminal or loop regions, yielded the same 43 kDa band, of apparent molecular mass consistent with that of full-length PS1, although it may represent an aspartyl protease complexed with PS1. Incubation of cell lysates with pepstatin-biotin, followed by streptavidin precipitation and PS1 Western blotting, revealed PS1 fragments and full-length protein, indicating that pepstatin-biotin bound to both cleaved and uncleaved PS1. Binding could be competed by gamma-secretase inhibitor L-685,458 and could not be achieved with a PS1 mutant lacking the two transmembrane aspartates. Pepstatin-biotin was also shown to bind to PS2. PS1 was specifically absorbed to pepstatin-agarose, with an optimal pH of 6. Binding of pepstatin-biotin to PS1 from lymphocytes of a heterozygous carrier of pathologic exon 9 deletion was markedly decreased as compared to control lymphocytes, suggesting that this PS1 mutation altered the pepstatin binding site.

Biotin labelling of amines by polymer-assisted solution-phase synthesis

An efficient and simple polymer-assisted approach has been developed to biotinylate multifunctional compounds bearing an amino functionality. Biotin was immobilized on aminomethylated polystyrene via the Kenner safety catch linker, activated and subsequently transferred to the amino function of the target compounds chemoselectively, thus avoiding protecting group operations. This approach holds potential for the introduction of spacer-modified biotin derivatives.

Biotin reagents for antibody pretargeting. 5. Additional studies of biotin conjugate design to provide biotinidase stability.

An investigation was conducted in which the stabilities of four structurally different biotin derivatives were assessed with regard to biotinamide bond hydrolysis by the enzyme biotinidase. The biotin derivatives studied contained an extra methylene in the valeric acid chain of biotin (i.e., homobiotin), or contained conjugated amino acids having hydroxymethylene, carboxylate, or acetate functionalities on a methylene alpha to the biotinamide bond. The biotinidase hydrolysis assay was conducted on biotin derivatives that were radioiodinated at high specific activity, and then subjected to diluted human serum at 37 degrees C for 2 h. After incubation, assessment of biotinamide bond hydrolysis by biotinidase was readily achieved by measuring the percentage of radioactivity that did not bind with avidin. As controls, an unsubstituted biotin derivative which is rapidly cleaved by biotinidase and an N-methyl-substituted biotin derivative which is stable to biotinidase cleavage were included in the study. The results indicate that increasing the distance from the biotin ring structure to the biotinamide bond by one methylene only decreases the rate of biotinidase cleavage, but does not block it. The data obtained also indicate that placing a hydroxymethylene, carboxylate, or acetate alpha to the biotinamide bond is effective in blocking the biotinamide hydrolysis reaction. These data, in combination with data previously obtained, which indicate that biotin derivatives containing hydroxymethylene or carboxylate moieties retain the slow dissociation rate of biotin from avidin and streptavidin [Wilbur, D. S., et al. (2000) Bioconjugate Chem. 11, 569-583], strongly support incorporation of these structural features into biotin derivatives being used for in vivo targeting applications.

Determination of biotin in foodstuffs and pharmaceutical preparations using a biosensing system based on the streptavidin–biotin interaction

An affinity biosensor system for the determination of biotin and its derivatives based on the specific non-covalent interaction between biotin and streptavidin is described. The permanent immobilisation of streptavidin on controlled-pore-glass (CPG) provides an adequate active support in which the specific and competitive reaction between biotin derivatives and biotin labelled with peroxidase (POD) takes place. Biotin was used as a model. The development of this competitive assay was revealed by determining the amount of POD which is not retained in the active support by using a derivatising reaction with fluorimetric detection. The method exhibits a linear range from 0.1 to 8.0 μg l −1 of biotin ( r 2=0.9986, n=9) with a precision, expressed as R.S.D., lower than 3.8% and a sampling frequency of 10 h −1. The approach has been applied to the determination of biotin in natural juices and pharmaceutical preparations with recoveries ranging between 88.1 and 104.8%.

Negatively Charged Polymers Protect Antinuclear Antibody against Inactivation by Acylating Agents

For many practical applications, monoclonal antibodies must be chemically modified without any significant loss in their immunoreactivity. In some situations, however, the amino acid residue crucial for antibody activity may be highly reactive toward the modifying agent, which results in antibody inactivation. The method to prevent inactivation of a modification-sensitive antinuclear monoclonal antibody by acylating agents was developed. The method is based on the hypothesis that a highly reactive amino group exists within, or in the vicinity of, the binding site of the antibody, providing crucial interaction with negatively charged moieties of DNA. It has been shown that negatively charged polymers, such as dextran sulfate or heparin, may provide temporary protection, presumably interacting noncovalently with this amino group and thus masking it. The protecting molecule can be removed later by chromatography on a protein A column, thus regenerating modified but not inactivated antibody in the free form for use in subsequent applications. In particular, we have modified antibody 2C5 with a chelating agent, diethylenetriaminepentaacetic acid (DTPA) without the loss of activity. Modified antibody was labeled with radioactive isotope, 111In, via chelation by antibody-attached DTPA. The labeled antibody was shown to demonstrate the same specificity of binding to nucleosomes as the nonmodified antibody, so it may be used in immunoscintigraphy or biodistribution studies. The method might be useful for the modification of other modification-sensitive antibodies with other acylating chemicals, such as crosslinking agents or biotin derivatives.

Preparation and evaluation of radioiodinated biotin derivatives which contain ionic charges

Preparation and evaluation of radioiodinated biotin derivatives which contain ionic charges

Incorporation of an aldehyde function in oligonucleotides.

A nucleotide-like phosphoramidite building block that has the nucleic base replaced by the tert-butyldimethylsilyl-protected styrene glycol was synthesized. After the automatic synthesis of an oligonucleotide incorporating this synthon, the benzaldehyde function was generated by fluoride deprotection and oxidation by sodium periodate. In a similar manner, an oligonucleotide where a nucleic base was replaced by the (CH2)8CH=O chain was synthesized and conjugated with biotin derivatives.

Binding of Biotin to Gold Surfaces Functionalized by Self-Assembled Monolayers of Cystamine and Cysteamine: Combined FT-IRRAS and XPS Characterization

As part of our project of developing a new IR-based immunosensor, we investigated the functionalization of gold substrates with thin organic films containing biotin ligands. A two-step procedure was developed consisting of the chemisorption of short amine-terminated organosulfur compounds, followed by their reaction at the solid liquid interface with an activated ester derivative of biotin. Covalent binding of biotin to these attachment layers was assessed by Fourier transform infrared reflection–absorption spectroscopy (FT-IRRAS) and X-ray photoelectron spectroscopy (XPS). The interaction of activated biotin with alcohol- and carboxylic acid-terminated monolayers was also investigated, and, as expected, no binding occurred. Moreover, mixed layers of short alcohol- and amine-terminated thiolates were successfully constructed at the gold surfaces and were shown to be the most efficient for the covalent binding of biotin thanks to the blocking effect of the thioalcohol, which prevented direct adsorption of biotin to the gold surface.

Investigation of the spontaneous transferability of a phospholipid-poly(ethylene glycol)-biotin derivative from small unilamellar phospholipid vesicles to magnetoliposomes

High-gradient magnetophoresis was used to monitor the spontaneous transfer between phospholipid vesicles and magnetoliposomes of dipentadecanoylphosphatidylethanolamine (DC 15PE) and of its conjugate with a biotinylated PEG molecule (DC 15-PEG-B). For the latter molecule, a half-time of 70 min was found, whereas DC 15PE did not move. The observed transferability of PEGylated phospholipids offers a unique method for generating Stealth ®-magnetoliposomes.

Identification of surface proteins and antigens from larval stages of Ascaris suum by two-dimensional electrophoresis.

An understanding of the biology of the cuticle in the larval stages of Ascariodea is of importance since the cuticle molecules not only possess a variety of functions related to survival but also have a potential role as a target for immunoprophylaxis. Thus, we made a preliminary characterization of surface proteins and antigens from 3rd-stage larvae (L3) and lung-stage larvae of Ascaris suum using two biotin-derivatives and two-dimen sional polyacrylamide gel electrophoresis (2D-PAGE). The proteins labelled with biotin comprised a total of 37 and 32 spots, with molecular weights (Air) ranging from 15 to 101 kDa and isoelectric points (pI) from 3.8 to 7.6, in L3 and lung-stage larvae, respectively. The profiles revealed that the individual spots bound to one or both biotin derivatives. In addition, stage-common and stage-specific spots were found in L3 and lung-stage larvae. 2D-PAGE/immunoblotting analysis was performed with antisera from rabbits repeatedly inoculated with A. suum L3. Nineteen spots were recognized as surface antigens, with Mr ranging from 32 to 66 kDa and pI from 4.9 to 7.6, from L3 and lung-stage larvae after alignment of the immunoblots with the profile of the surface proteins. These spots were found to include stage-common and stage-specific antigens. Identification of surface proteins by biotin labelling combined with 2D-PAGE allows a substantial shortening of sample preparation time for the target proteins, and will be a viable method for protein analysis of surface proteins and antigens of A. suum L3 and lung-stage larvae.

Modulation of the diffusion coefficient of a hapten-modified redox species as a basis for an amplified electrochemical affinity assay

An affinity assay was developed that is based on the modulation of the diffusion coefficient of an electroactive label upon complementary recognition leading to an increase of the molecular weight. Using an electroanalytical technique, which is correlated to the diffusion coefficient of the redox species, e.g., cyclic voltammetry, the decrease of the diffusion coefficient can be monitored as a decrease of the diffusion-limited current. Signal amplification was achieved by redox recycling using a microelectrode that is positioned in close distance to a conducting surface. The amplification rate can be adapted by varying the distance between the microelectrode and the conducting surface. As a model system for molecular recognition, the biotin–streptavidin system has been chosen using a ferrocene-labeled biotin derivative as electroactive species. The generality of the approach was proved by extension of the basic assay to related sandwich assays and by miniaturization using a wall-free droplet cell.

Binding of a diverse set of ligands to avidin and streptavidin: an accurate quantitative prediction of their relative affinities by a combination of molecular mechanics and continuum solvent models.

We report calculations of free energies of binding, DeltaG(bind), between a diverse set of nine ligands and avidin as well as between a peptide and streptavidin using the recently developed MM/PBSA approach. This method makes use of a molecular dynamics simulation of the ligand-protein complex to generate a thermally averaged ensemble of conformations of the molecules that are involved in the complex formation. Based on this set of structures, a free energy of binding is calculated using molecular mechanical and continuum solvent energies as well as including estimates of the nonpolar solvation free energy and solute entropy. We compare in our simulations different classes of ligands, involving biotin derivatives, the dye 2-(4'-hydroxyazobenzene)benzoic acid (HABA), and a cyclic hexapeptide, which cover a large range of binding free energies from -5 to -20 kcal/mol. Our calculations are able to reproduce experimental DeltaG(bind) values with a very good correlation coefficient of r(2) = 0.92. This agreement is considerably better than the results obtained with an alternate approach, the linear interaction energy approximation, for this system (r(2) = 0.55).

Sequence similarities between a novel putative G protein-coupled receptor and Na+/Ca2+ exchangers define a cation binding domain.

cDNA clones encoding a novel putative G protein-coupled receptor have been characterized. The receptor is widely expressed in normal solid tissues. Consisting of 1967 amino acid residues, this receptor is one of the largest known and is therefore referred to as a very large G protein-coupled receptor, or VLGR1. It is most closely related to the secretin family of G protein-coupled receptors based on similarity of the sequences of its transmembrane segments. As demonstrated by cell surface labeling with a biotin derivative, the recombinant protein is expressed on the surface of transfected mammalian cells. Whereas several other recently described receptors in this family also have large extracellular domains, the large extracellular domain of VLGR1 has a unique structure. It has nine imperfectly repeated units that are rich in acidic residues and are spaced at intervals of approximately 120 amino acid residues. These repeats resemble the regulatory domains of Na+/Ca2+ exchangers as well as a component of an extracellular aggregation factor of marine sponges. Bacterial fusion proteins containing two or four repeats specifically bind 45Ca in overlay experiments; binding is competed poorly by Mg2+ but competed well by neomycin, Al3+, and Gd3+. These results define a consensus cation binding motif employed in several widely divergent types of proteins. The ligand for VLGR1, its function, and the signaling pathway(s) it employs remain to be defined.

Benzyl 5-{2-[2-(diethoxyphosphinoyl)acetyl]-3-oxo-7-thia-2,4-diazabicyclo[3.3.0]oct-6-yl}pentanoate, a novel biotin derivative

The title compound, C23H33N2O7PS, has its phospho­no­acetate carbonyl group rotated slightly out of the plane of the ureido ring, with a C—N—C—O torsion angle of −6.9 (4)°. The sulfur-containing ring has an envelope conformation, while the ureido ring is nearly planar.