Biotin Derivatives Research Articles

Biotinylation of Substituted Cysteines in the Nicotinic Acetylcholine Receptor Reveals Distinct Binding Modes for α-Bungarotoxin and Erabutoxin a

Although previous results indicate that alpha-subunit residues Trp(187), Val(188), Phe(189), Tyr(190), and Pro(194) of the mouse nicotinic acetylcholine receptor are solvent-accessible and are in a position to contribute to the alpha-bungarotoxin (alpha-Bgtx) binding site (Spura, A., Russin, T. S., Freedman, N. D., Grant, M., McLaughlin, J. T., and Hawrot, E. (1999) Biochemistry 38, 4912-4921), little is known about the accessibility of other residues within this region. By determining second-order rate constants for the reaction of cysteine mutants at alpha184-alpha197 with the thiol-specific biotin derivative (+)-biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctanediamine , we now show that only very subtle differences in reactivity (approximately 10-fold) are detectable, arguing that the entire region is solvent-exposed. Importantly, biotinylation in the presence of saturating concentrations of the long neurotoxin alpha-Bgtx is significantly retarded for positions alphaW187C, alphaF189C, and reduced wild-type receptors (alphaCys(192) and alphaCys(193)), further emphasizing their major contribution to the alpha-Bgtx binding site. Interestingly, although biotinylation of position alphaV188C is not affected by the presence of alpha-Bgtx, erabutoxin a, which is a member of the short neurotoxin family, inhibits biotinylation at position alphaV188C, but not at alphaW187C or alphaF189C. Taken together, these results indicate that short and long neurotoxins establish interactions with distinct amino acids on the nicotinic acetylcholine receptor.

Biotin reagents for antibody pretargeting. 4. Selection of biotin conjugates for in vivo application based on their dissociation rate from avidin and streptavidin.

An investigation was conducted to determine the affect of structural variation of biotin conjugates on their dissociation rates from Av and SAv. This information was sought to help identify optimal biotin derivatives for in vivo applications. Fifteen biotin derivatives were conjugated with a cyanocobalamin (CN-Cbl) derivative for evaluation of their "relative" dissociation rates by size exclusion HPLC analysis. Two biotin-CN-Cbl conjugates, one containing unaltered biotin and the other containing iminobiotin, were prepared as reference compounds for comparison purposes. The first structural variations studied involved modification of the biotinamide bond with a N-methyl moiety (i.e., sarcosine conjugate), lengthening the valeric acid side chain by a methylene unit (i.e., homobiotin), and replacing the biotinamide bond with thiourea bonds in two conjugates. The rate of dissociation of the biotin-CN-Cbl derivative from Av and SAv was significantly increased for biotin derivatives containing those structural features. Nine additional biotin conjugates were obtained by coupling amino acids or functional group protected amino acids to the biotin moiety. In the conjugates, the biotin moiety and biotinamide bond were not altered, but substituents of various sizes were introduced alpha to the biotinamide bond. The results obtained from HPLC analyses indicated that the rate of dissociation from Av or SAv was not affected by small substituents alpha to the biotinamide (e.g., methyl, hydroxymethyl, and carboxylate groups), but was significantly increased when larger functional groups were present. On the basis of the results obtained, it appears that biotin conjugates which retain an unmodified biotin moiety and have a linker molecule conjugated to it that has a small functional group (e.g., hydroxymethylene or carboxylate) alpha to the biotinamide bond are excellent candidates for in vivo applications. These structural features are obtained in the biotin amino acid conjugates: biotin-serine, biotin-aspartate, biotin-lysine, and biotin-cysteine. Importantly, these biotin derivatives can be readily conjugated with other molecules for specific in vivo applications. In our studies, these derivatives will be used in the design of new biotin conjugates to carry radionuclides for cancer therapy using the pretargeting approach.

Evaluation of biotin-dye conjugates for use in an HPLC assay to assess relative binding of biotin derivatives with avidin and streptavidin.

In this investigation, studies were conducted to determine if size exclusion HPLC could be used to assess relative association rates (on-rates) and dissociation rates (off-rates) of biotin derivatives from avidin (Av) and streptavidin (SAv). For easy detection and quantification of biotin derivatives, molecules that can be detected by UV absorbance were conjugated to biotin. Concern that conjugation of the chromophoric moieties (dyes) might affect biotin binding with Av and SAv or might interact with the HPLC column led to evaluation of 10 biotin-dye conjugates. The dyes conjugated with biotin included dansyl, cyanocobalamin (CN-Cbl), coumarin 343, Lissamine-rhodamine, fluorescein, Cascade Blue, Lucifer Yellow, Oregon Green, tetramethylrhodamine, and Alexa Fluor 594. The biotin-dye conjugates were initially evaluated to determine their peak characteristics on two different size exclusion HPLC columns. Measurement of the percent of biotin-dye conjugate bound with Av in the presence of an equal quantity of biotin provided an association rate relative to biotin. All of the biotin-dyes tested had association rates within a factor of 3x (slower) that of biotin. The relative dissociation rate of biotin-dye conjugates was assessed by challenging the biotin conjugate bound to Av or SAv with a large excess of biotin. All of the initial biotin-dye conjugates tested bound Av and SAv tightly resulting in very slow dissociation rates. From the biotin-dye conjugates studied, biotin-CN-Cbl, 6b, was selected as the best conjugate for the HPLC assay. To test the HPLC assay, an iminobiotin-CN-Cbl conjugate, 13a, and a biotin-sarcosine-CN-Cbl conjugate, 13b, were synthesized. The fact that the iminobiotin does not bind with Av at physiological pH was easily detected in the size exclusion HPLC assay. The biotin-sarcosine-CN-Cbl conjugate was expected to have a more rapid dissociation rate than the other biotin-dye conjugates. This was confirmed in that HPLC assay. Although 13b bound tightly with Av in the absence of added biotin, it was completely released within 1 h when challenged by an excess of biotin. A slower dissociation of 13b was noted with SAv. The results obtained indicate that CN-Cbl conjugates of biotin derivatives can be used to determine relative on-rates and off-rates of biotin derivatives with Av and SAv. The studies also demonstrated that the biotin-CN-Cbl conjugate, 6b, can be used as a reference compound to compare on-rates and off-rates of nonchromophoric biotin derivatives.

Use of microbial transglutaminase for the enzymatic biotinylation of antibodies

Nowadays many reagents are available for the biotinylation of proteins. As most of them bind to amino groups of the protein the degree of labelling differs from batch to batch and the possibility exists that the biological activity of the target protein may be affected by the labelling procedure. In the present study we have investigated an enzymatic approach to biotinylation using microbial transglutaminase (MTGase) from Streptoverticillium mobaraense. The proposed method is particularly suitable when only a few biotin molecules need to be attached to the target proteins. The enzyme catalyses the acyl transfer reaction between γ-carboxyamide groups and various primary amines. This was exploited for biotinylation using two amino-modified biotin derivatives, biotinamido-5-pentylamin (BIAPA) and biotinoyl-1,8-diamino-3,6-dioxaoctane (BIDADOO) as acyl acceptors and a monoclonal IgG against the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as the acyl donor. Kinetic studies revealed that the MTGase-mediated reaction proceeds with low velocity and is almost complete after 34 h. Conjugation ratios ranging from 1.1 to 1.9 biotins per IgG were found by mass spectrometry. To investigate the influence of antibody conjugation on antigen binding a competitive ELISA for the determination of 2,4-D employing MTGase-biotinoylated IgGs was developed. In this assay lower limits of detection of 0.3 and 1.0 μg/l of 2,4-D were achieved with BIDADOO- and BIAPA-modified antibodies, respectively.

Molecular imprinting of biotin derivatives and its application to competitive binding assay using nonisotopic labeled ligands.

Synthetic biotin-binding polymers were prepared by molecular imprinting. Methacrylic acid (MAA) was copolymerized with ethylene glycol dimethacrylate in the presence of biotin methyl ester (B-Me) in chloroform. Hydrogen-bonding-based complexation of B-Me with MAA generates the binding sites complementary to B-Me after extracting B-Me from the resulting copolymers. Data from NMR titration suggest a one-to-one prepolymerization complex formation of B-Me with MAA in chloroform. A possible complex structure was estimated by docking of the most stable conformers by intermolecular Monte Carlo conformational search under the assumption of a one-to-one association. The selectivity of the imprinted polymers was investigated and an imprinted polymer-based competitive binding assay for B-Me was demonstrated using biotin p-nitrophenyl ester as a nonisotopic-labeled ligand.

Antibody Conjugates for Cell Biology

Antibody conjugates are extremely useful reagents for probing many biologically and chemically important molecules in vitro or in vivo. This unit presents some basic protocols for conjugating antibodies with fluorescent dyes, with biotin, and with enzymes. These protocols have been selected because they are relatively easy to perform and have high rates of success. The basic approach for conjugating antibodies is to derivatize amino groups on the antibody with the dye or biotin derivative of choice. Conjugation at amine sites is simple to perform, and, with a fluorescent dye, it generally yields brightly fluorescent conjugates. The conjugates retain high levels of biological activity, notwithstanding the fact that the labels react with random amines on the antibody molecule, and they may even react near or at the antigen-binding site. Labeling of the amines is the most widely used commercial method to tag antibodies. In this unit, suggestions are presented for variations on these basic protocols that are useful when quantities of available antibody are limited. Methods for estimating antibody concentration are also included.

Reactivity of the Cysteine Residues in the Protein Splicing Active Center of the Mycobacterium tuberculosis RecA Intein

Protein splicing involves the self-catalyzed excision of an intervening polypeptide segment, an intein, from a precursor protein. The first two steps in the protein splicing process lead to the formation of ester intermediates through nucleophilic attacks by the side chains of cysteine, serine, or threonine residues adjacent to the splice junctions. Since both nucleophilic residues in the Mycobacterium tuberculosis RecA intein are cysteine, their reactivities could be compared by sulfhydryl group titration. This was accomplished by using fusion proteins containing a truncated RecA intein modified by mutation to prevent protein splicing, in which the cysteines at the splice junctions were the only sulfhydryl groups. The ability to undergo hydroxylamine-induced cleavage at the upstream splice junction showed that the modified intein was not impaired in the ability to form ester intermediates. Sulfhydryl titration with iodoacetamide, monitored by quantitating the residual thiols after reaction with a maleimide derivative of biotin, revealed a striking difference in the apparent pKa values of the cysteines at the two splice junctions. The apparent pKa of the cysteine at the upstream splice junction, which initiates the N–S acyl rearrangement leading to the linear ester intermediate, was approximately 8.2, whereas that of the cysteine residue at the downstream splice junction, which initiates the transesterification reaction converting the linear ester to the branched ester intermediate, was about 5.8. This suggests that the transesterification step is facilitated by an unusually low pKa of the attacking thiol group. Comparison of the rates of cleavage of the linear ester intermediates derived from the M. tuberculosis RecA and the Saccharomyces cerevisiae VMA inteins by dithiothreitol and hydroxylamine revealed that the former reacted relatively more slowly with dithiothreitol, suggesting that the RecA intein has diverged in the course of evolution to react preferentially with thiolate anions and thus lacks the basic groups that may facilitate nucleophilic attack by thiols in other inteins.

Concepts for the Syntheses of Biotinylated Steroids. Part I: Testosterone Derivatives as Immunochemical Probes

We describe synthetic strategies for the biotinylation of testosterone (T) at positions 3, 7alpha, 17alpha, and 19. These T probes are able to mimic ligand binding and may provide for a better understanding of the biospecific interaction with steroid-binding proteins such as the androgen receptor, anti-steroid antibodies, or steroid-binding serum globulins. For the 7alpha- and 17alpha-derivatives, biotinyl-N-hydroxy-succinimide esters with different types of spacer chains were used. The 3-biotin hydrazone derivative was produced using N-(epsilon-biotinyl)-caproyl hydrazide, whereas for the 19-biotinylation, a biotinyl-1-N-diamino-3, 6-dioxaoctane-amide was applied. Key reaction for the biotinylation at position 3 is the oximation of the 3-oxo function. The 17alpha-position is accessible by the reaction of the 3-protected 4-androsten-17-epoxide with oxygen in the beta-position, followed by nucleophilic ring opening with cyanide which provides the 17alpha-cyanomethyl derivative. The key step is the regioselective ketal protection of the 3-oxo function of androst-4-ene-3,17-dione using a stannoxane catalyst. An alternative pathway for the insertion of biotin at the 19-position was established by the synthesis of 17beta-hydroxy-androst-4-en-3-one-19-yl carboxymethyl ether. After activation by the carbodiimide method, the compound reacts with aminoterminal biotin derivatives. The copper(I)-catalyzed 1,6 Michael addition of 17-acetoxy-6,7-dehydro-T leads to 7alpha-derivatives by use of omega-silyl protected hydroxylalkyl-modified Grignard reagents. A functional group interconversion using the Staudinger reaction transforms the azide function into a primary omega-amino group. The absolute configurations of the different biotinylated derivatives were investigated by (1)H NMR studies. For the 7alpha-biotinylated T series, additionally, an X-ray analysis proved the axial position of the spacer group. This results in a vertical orientation of the biotin moiety toward the alpha-face of the planar tetracyclic backbone. Thus, a negligible alteration of the original structure of the upper beta-face offers the feasibility of applying the 7alpha-derivatives as optimal immunochemical tracers in competitive immunoassays. Biotinylated T derivatives should be also suitable for ligand-binding studies to the androgen receptor or to sex hormone-binding globulin.

Electrochemical analysis of immobilised chemical and genetic biotinylated alkaline phosphatase

Many approaches to enzyme immobilisation have been described but almost all are non-specific and result in a loss of biological activity or loss of enzyme due to weak or poor binding. In order to overcome these problems, we have investigated the fusion of biotin acceptor peptide sequences to the C-terminus of alkaline phosphatase (ALP) as a method to immobilise this enzyme through the avidin–biotin linkage. ALP from E coli. was expressed with the following sequences fused to its C-terminus: LEAIFEAQ KIEWR or LGGIFEAM KMEWR. These sequences have been reported to be biotinylated on the ε-amino group of the lysine residue (shown above in bold). Wild-type ALP was also biotinylated with a NHS derivative of a long chain biotin. Both genetically (GB) and chemically (CB) biotinylated enzymes were purified and their immobilisation to an avidin-coated electrode was investigated. The immobilised ALP was assayed electrochemically by following the conversion of p-aminophenylphosphate to p-aminophenol with amperometric determination of this product. The results showed that both biotinylated enzymes exhibited significantly higher binding to the electrode than the wild-type enzyme, which yields currents that are comparable to the background.

Interaction of biotin and biotinyl derivatives with avidin: conformational changes upon binding

We used FT-Raman and FT-IR spectroscopy to study avidin and its complexes with some biotin and biotinyl derivatives where the COOH group of the pentanoic acid chain is substituted by an ester or amidic group. To investigate the small structural changes due to the binding, the amide I Raman bands of Avidin and its complexes were analysed by two fairly comparable methods. The vibrational results indicate that the interaction with the ligands slightly modifies the secondary structure of the protein, decreasing the β-sheet content and increasing the α-helix percentage. To confirm the results obtained by Raman spectroscopy, we performed Gaussian curve-fitting procedure of the amide I and III infra-red regions of neat avidin and its complexes. The percentages obtained from the analysis of the Raman and IR spectra show the same trend. Thus, IR spectroscopy as an alternative or adjunct to Raman spectroscopy is a useful tool in evaluating small structural modifications produced by a bound ligand on a protein. The structural changes are mainly due to modifications in the hydrogen bonds with the pentanoic acid moiety of the ligand and, probably, are ascribed to conformational modifications in the surface loop which binds the strands of the β-barrel. In fact, biotin-N-succinimidyl ester, as well as biotin hydrazide, which have the same ability in the hydrogen-bond formation compared with biotin, induces avidine conformational changes similar to those observed in the avidin–biotin complex. The terminal group of the biotin derivatives seems to have a slight influence since the structural modifications of the protein observed upon the binding of the long-chain hydrazide-derivative ligands (biotinyl-aminocaproic hydrazide and biocitin hydrazide) are slightly greater than those obtained with the corresponding COOH containing ligands (biotinyl-aminocaproic acid and biocytin). Although the hydrogen bonds formed by the pentanoic acid chain of biotin are not essential to complex formation since they are not directly involved in the active site, the present study confirms that they can interact with the loops that lock biotin in the β-barrels of avidin and modulate the strength of the protein-ligand interaction. Copyright © 2000 John Wiley & Sons, Ltd.

Synthesis of New Biotin Derivatives

It is now established that the formation of C–S bonds during the conversion of dethiobiotin into biotin involves intermediate carbon radicals. To examine the behavior of dethiobiotin analogs that could lead to allylic and α-epoxyradicals, we have synthesized 4,5-dehydrodethiobiotins and the corresponding epoxides and 5,6-dehydrodethiobiotins. All these compounds have been labelled with 14C on the carboxyl group.

Development of new biotin/streptavidin reagents for pretargeting

The high affinity of biotin for streptavidin has made this pair of molecules very useful for in vivo applications. To optimize reagents for one potential in vivo application, antibody-based pretargeting of cancer, we have prepared a number of new biotin and streptavidin derivatives. The derivatives developed include new radiolabeled biotin reagents, new protein biotinylation reagents, and new biotin multimers for cross-linking and/or polymerization of streptavidin. We have also modified streptavidin by site-directed mutation and chemical modification to improve its in vivo characteristics, and have developed new reagents for cross-linking antibody fragments with streptavidin. A brief overview of these new reagents is provided.

Kinase Assay Based on Thiophosphorylation and Biotinylation

Protein kinases catalyze the transfer of the gamma-phosphate group from ATP to a serine, threonine or tyrosine residue of an acceptor protein. These enzymes play an important role in signal transduction. New inhibitors for these enzymes are actively being sought. In this article, we present a novel approach for detecting the activity of protein kinases, which could be useful for the high-throughput screening of chemical libraries. The method is based on the use of ATP gamma S instead of ATP in the phosphorylation reaction. This results in the transfer of a thiophosphate group onto a fluorescein-labeled acceptor peptide substrate. The mixture is then treated with a sulfur-reactive iodoacetyl derivative of biotin, which leads to the modification of the nucleophilic sulfur of the thiophosphate group and the generation of a fluorescently labeled, biotinylated molecule. Finally, streptavidin is added to the mixture and it binds to all biotinylated molecules present. The binding of streptavidin to the thiophosphorylated and biotinylated kinase substrate can be conveniently detected by measuring the change in fluorescence polarization of the fluorescent dye attached to the peptide. The detection of kinase inhibitors is demonstrated. The method is completely homogeneous and does not require any separation steps.

Enzyme amplification as detection tool in continuous-flow systems: I. Development of an enzyme-amplified biochemical detection system coupled on-line to flow-injection analysis

The on-line coupling of flow-injection analysis (FIA) to an enzyme-amplified biochemical detection (EA-BCD) system is described. The aim of this study is the development of a detection system able to detect biotin-containing compounds at low concentration levels. The detection system is based on the interaction of biotin with enzyme-labeled affinity proteins. Biotin possesses a high affinity to both streptavidin and anti-biotin Fab fragments, which are both tested. Several biotin derivatives are available with different reactive probes, which can be used to label analytes of interest. Therefore, biotin acts as a universal probe for the enzyme-amplified biochemical detection. Alkaline phosphatase (AP) was used as enzyme label. Several parameters, such as substrate type and concentration, concentration of enzyme-labeled affinity protein, reaction time and reaction temperature were examined. Biotin aminocaproic acid was used as a model compound. In addition to biotin aminocaproic hydrazide, other biotinylation reagents were also examined. With fluorescence detection of the enzyme-generated product, a mass detection limit of 1 fmol was achieved.

Functionalization of covalent DNA-streptavidin conjugates by means of biotinylated modulator components.

Covalent DNA-streptavidin conjugates are versatile biomolecular coupling reagents, since they have binding capacity for both a complementary nucleic acid and four molecules of biotin. The DNA-streptavidin hybrid molecules have been investigated for their capabilities to bind two different types of biotinylated components. Thus, (i) a functional biomolecule, e.g., a single-stranded DNA fragment or an enzyme and (ii) low-molecular weight biotin derivatives ("modulators") were coupled stepwise with the hybrid molecules. Modulators were D-biotin, aminobiotin, and biotin-fluorescein conjugate as well as a lysine-rich 10mer peptide, containing a biotin and a fluorescein substituent. These modulators were chosen to affected the hybridization properties of the DNA-streptavidin conjugates. As investigated by surface-plasmon resonance and microplate solid-phase hybridization measurements, D-biotin, biotin-fluorescein, and aminobiotin decreased the efficiency of hybridization with complementary, surface-bound oligonucleotides to a varying extent. The basic peptide increased the conjugate's hybridization efficiency. Moreover, it was demonstrated in two examples how modulators can be utilized as additional functional domains of streptavidin-based conjugates. First, fluorescein-containing modulators were used as hapten groups, allowing a sensitive detection by means of specific antibodies directed against the modulator. Second, the biotinylated peptide was used as a carrier molecule to attach multiple fluorogenic lanthanide-chelate groups to the streptavidin conjugate, enabling its sensitive detection by time-resolved fluorometry. The applicability of this kind of bioconjugation strategy to generate sensor-probes for gene detection assays was demonstrated.

A Raman study of the interactions of Streptavidin with Biotin and Biotinyl derivatives.

Raman spectra of Streptavidin and its complexes with some Biotin and Biotinyl derivatives were recorded. The β-sheet percentages of the secondary structure of Streptavidin, calculated by two different methods, decreased as a consequence of the interaction with Biotin derivatives. The Biotinyl derivatives did not significantly change the spectral features of the protein and, consequently, the secondary structure. The difference in behaviour between Biotin and Biotinyl derivatives, is owing to the absence of one hydrogen bond formed between the ligand and some aminoacid residues of the protein (Asn and Ser). The modification in the secondary structure should be ascribed to the surface loop which binds the strands of the β-barrel and not to the β-sheet strands which form the active site binding of the protein.

Accurate measurement of avidin and streptavidin in crude biofluids with a new, optimized biotin–fluorescein conjugate

A new biotin–fluorescein conjugate with an ethylene diamine spacer was found to be the first fluorescent biotin derivative which truly mimicked d-biotin in terms of high affinity, fast association, and non-cooperative binding to avidin and streptavidin tetramers. These exceptional properties were attributed to the small size/length of the new ligand since all larger/longer biotin derivatives are known for their mutual steric hindrance and anti-cooperative binding in 4:1 complexes with avidin and streptavidin tetramers. Specific binding of the new biotin–fluorescein conjugate towards avidin and streptavidin was accompanied by 84–88% quenching of ligand fluorescence. In the accompanying study this effect was used for rapid estimation of avidin and streptavidin in a new ‘single tube assay’. In the present study the strong quenching effect was utilized to accurately monitor stoichiometric titration of biotin-binding sites in samples with ≥200 pM avidin or streptavidin. The concentration was calculated from the consumption of fluorescent ligand up to the distinct breakpoint in the fluorescence titration profile which was marked by the abrupt appearance of strongly fluorescent ligands which were in excess. Due to this protocol the assay was not perturbed by background fluorescence or coloration in the unknown samples. The new fluorescence titration assay is particularly suited for quick checks on short notice because getting started only means to thaw an aliquot of a standardized stock solution of fluorescent ligand. No calibration is required for the individual assay and the ligand stock solution needs to be restandardized once per week (or once per year) when stored at −25°C (or at −70°C, respectively).

Comb-type prepolymers consisting of a polyacrylamide backbone and poly(L-lysine) graft chains for multivalent ligands.

The comb-type copolymers consisting of a polyacrylamide (PAAm) backbone and poly(L-lysine) (PLL) graft chains have been prepared as the "prepolymer" for designing multivalent ligands. To regulate the length and density of the clusters of primary amino groups, the Nalpha-carboxyanhydride of Nepsilon-carbobenzoxy (CBZ)-L-lysine was first polymerized using p-vinylbenzylamine as an initiator. The resulting poly(CBZ-L-lysine) macromonomer was then radically copolymerized with AAm, followed by the deprotection of amino groups. For the model study, the reactive clusters of primary amino groups were completely converted into anion clusters by the reaction with succinic anhydride. The model multivalent ligands having the biotin label on the PAAm backbone were prepared by the terpolymerization of the macromonomer, AAm, and the biotin derivative having a vinyl group. The enzyme-linked immunosorbent assay showed that the biotin with no spacer on the PAAm backbone was recognized by the avidin-peroxidase conjugate specifically. Therefore, the highly sensitive detection of the interaction between cells and various model multivalent ligands was possible. The selective labeling onto the PAAm backbone revealed that the converted anion clusters of graft chains interacted exclusively with the cell and that the backbone was inert to the interaction with the cell. These results indicate that the various PAAm-graft-PLL comb-type copolymers with the defined length and density of the PLL-grafts are the potential prepolymers to investigate and to optimize the affinity of the multivalent ligands for receptors.

Synthesis and evaluation of 99mTc/ 99Tc-MAG3-biotin conjugates for antibody pretargeting strategies

Four 99mTc-MAG3-biotin conjugates were synthesized to determine their potential use in antibody pretargeting strategies for radioimmunoscintigraphy (RIS). To use these 99mTc-MAG3-biotin conjugates as model compounds for 186Re-MAG3-biotin conjugates for radioimmunotherapy (RIT), nanomolar amounts of 99Tc were added as carrier to 99mTc. The biotin derivatives used for the preparation of the conjugates—biocytin, biotin hydrazide, biotinyl-piperazine, and biotinyl-diaminosuccinic acid—differed at the site that is regarded to be susceptible to hydrolysis by biotinidase present in human plasma. All four conjugates were produced with high radiochemical purity, were stable in PBS, and demonstrated full binding capacity to streptavidin. The 99mTc/ 99Tc-MAG3-labeled biotinyl-piperazine and biotinyl-diaminosuccinic acid conjugates were stable in mouse as well as human plasma, whereas the corresponding biocytin and biotin hydrazide conjugates were rapidly degraded. The biodistribution in nude mice at 30 min after injection was similar for all conjugates, and a rapid blood clearance and high intestinal excretion were both observed. It is concluded that the metabolic routing of a conjugate containing biotin and MAG3 is dominated by these two moieties. For this reason, MAG3-biotin conjugates do not seem suited for pretargeted RIT, for which quantitative and fast renal excretion is a prerequisite to minimize radiation toxicity. However, in a pretargeted RIS approach the 99mTc-MAG3-biotin conjugates might have potential.

Topological analysis of NHE1, the ubiquitous Na+/H+ exchanger using chymotryptic cleavage.

Proteases, glycosidases, and impermeant biotin derivatives were used in combination with antibodies to analyze the subcellular distribution and transmembrane disposition of the Na+/H+ exchanger NHE1. Both native human NHE1 in platelets and epitope-tagged rat NHE1 transfected into antiport-deficient cells were used for these studies. The results indicated that 1) the entire population of exchangers is present on the surface membrane of unstimulated platelets, ruling out regulation by recruitment of internal stores of NHE1; 2) the putative extracellular loops near the NH2 terminus are exposed to the medium and contain all the N- and O-linked carbohydrates; 3) by contrast, the putative extracellular loops between transmembrane domains 9-10 and 11-12 are not readily accessible from the outside and may be folded within the protein, perhaps contributing to an aqueous ion transport pathway; 4) the extreme COOH terminus of the protein was found to be inaccessible to extracellular proteases, antibodies, and other impermeant reagents, consistent with a cytosolic localization; and 5) detachment of approximately 150 amino acids from the NH2-terminal end of the protein had little effect on the transport activity of NHE1.