N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)-cyclohexane-1-carboxamide (TPICC): a heterobifunctional reagent for immobilization of biomolecules on glass surface

An efficient heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)cyclohexane-1-carboxamide (TPICC) has been developed by a simple 'two step reaction' for the preparation of bioconjugates and immobilization of biomolecules such as oligonucleotides, peptides and proteins on the glass surface. The isothiocyanate functionality at one end of the reagent, TPICC was found specific for the ligands having either aminoalkyl (RNH(2)) or mercaptoalkyl (R-SH) functionality. The synthesis of bioconjugates with the reagent was achieved through its isothiocyanate functionality at one end via the formation of stable thiourea linkage with aminoalkyl and dithiocarbamate linkage with mercaptoalkyl derivatives. The triethoxysilyl functionality of the reagent has been utilized for specific coupling with the virgin glass surface by a very simple methodology.     

A new heterobifunctional reagent for immobilization of biomolecules on glass surface

Synthesis of a new heterobifunctional reagent, [N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine] (NTMTA) is described for the immobilization of a variety of biomolecules on glass surface. Its triethoxysilyl group reacts with glass surface and trifluoroethanesulfonate ester structure reacts selectively with aminoalkyl/mercaptoalkyl function in biomolecules. The immobilization can be achieved by two ways involving two steps. The first route involves the reaction of NTMTA with glass beads followed by attachment of aminoalkyl- or mercaptoalkylated biomolecules. The second one involves the reaction of biomolecules, viz., oligonucleotides, proteins, etc., with NTMTA via their aminoalkyl or mercaptoalkyl functions to form a biomolecule conjugate, which is then reacted with glass beads (unmodified) to complete immobilization process. This has been demonstrated by successful immobilization of 5'-mercaptoalkyl- or aminoalkylated oligonucleotides and some commonly used enzymes on glass beads using NTMTA reagent.     

N-(3-Triethoxysilylpropyl)-4-(N'-maleimidylmethyl)cyclohexanamide (TPMC): a heterobifunctional reagent for immobilization of oligonucleotides on glass surface

A new heterobifunctional reagent, namely, N-(3-triethoxysilylpropyl)-4-(N'-maleimidylmethyl)cyclohexanamide (TPMC) was developed and its potentiality for fixing of thiol (-SH) modified oligonucleotides were tested. The covalent attachment of oligonucleotides with the reagent was achieved through its maleimide functionality at one end via stable thioether linkage while the other end bearing triethoxysilyl functionality has been utilized for coupling with the virgin glass surface with simplified methodologies. Immobilization of oligonucleotides was achieved by two alternating ways. The PATH-1 involves formation of conjugate of reagent and SH-modified oligonucleotides through thioether linkage and was subsequently immobilized on unmodified glass surface through triethoxysilyl group and alternatively, PATH-2 involves reaction of reagent first with unmodified glass surface to get maleimide functionality on the surface and then the SH-modified oligonucleotides were immobilized via thioether linkage. The specificity of immobilization was tested by hybridization study with complementary fluorescein labeled oligonucleotide strand.     

N-(Iodoacetyl)-N'-(anthraquinon-2-oyl)-ethylenediamine (IAED): a new heterobifunctional reagent for the preparation of biochips

Design and synthesis of a new heterobifunctional reagent, N-(iodoacetyl)-N'-(anthraquinon-2-oyl)-ethylenediamine (IAED), have been described for the preparation of oligonucleotide-based biochips. The performance of the featured reagent is probed by the immobilization of thiolated and thiophosphorylated oligonucleotides on modified glass microslides via two routes (routes A and B). The immobilization procedure was accelerated by performing a chemical reaction between thiolated oligomers and the iodoacetyl moiety of the reagent under microwaves (MW), where it is completed in just 10 min. The quality of the constructed oligonucleotide microarrays was tested by performing a hybridization assay with a complementary target and subsequently used for the detection of base mismatches. The immobilized probes were found to be thermally stable.     

Construction of oligonucleotide arrays on a glass surface using a heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA)

A rapid method for construction of oligonucleotide arrays on a glass surface, using a novel heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA), has been described. The heterobifunctional reagent, NTMTA, carries two different thermoreactive groups. The triethoxysilyl group on one end is specific towards silanol functions on the virgin glass surface, while the trifluoroethanesulfonyl (tresyl) group on the other end of the reagent reacts specifically with aminoalkyl- or mercaptoalkyl- functionalized oligonucleotides. Immobilization of oligonucleotides on a glass surface has been realized via two routes. In the first one (A), 5′- aminoalkyl- or mercaptoalkyl-functionalized oligonucleotides were allowed to react with NTMTA to form a oligonucleotide-triethoxysilyl conjugate which, in a subsequent reaction with unmodified (virgin) glass microslide, results in surface-bound oligonucleotides. In the second route (B), the NTMTA reagent reacts first with a glass microslide whereby it generates trifluoroethanesulfonate ester functions on it, which in a subsequent step react with 5′-aminoalkyl or mercaptoalkyl oligonucleotides to generate support-bound oligonucleotides. Subsequently, the oligonucleotide arrays prepared by both routes were analyzed by hybridization experiments with complementary oligonucleotides. The constructed microarrays were successfully used in single and multiple nucleotide mismatch detection by hybridizing these with fluorescein-labeled complementary oligonucleotides. Further more, the proposed method was compared with the existing methods with respect to immobilization efficiency of oligonucleotides.     

Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent.

A heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate, was synthesized. Its N-hydroxysuccinimide ester group reacts with amino groups and the 2-pyridyl disulphide structure reacts with aliphatic thiols. A new thiolation procedure for proteins is based on this reagent. The procedure involves two steps. First, 2-pyridyl disulphide structures are introduced into the protein by the reaction of some of its amino groups with the N-hydroxysuccinimide ester sie of the reagent. The protein-bound 2-pyridyl disulphide structures are then reduced with dithiothreitol. This reaction can be carried out without concomitant reduction of native disulphide bonds. The technique has been used for the introduction of thiol groups de novo into ribonuclease, gamma-globulin, alpha-amylase and horseradish peroxidase. N-Succinimidyl 3-(2-pyridyldithio)propionate can also be used for the preparation of protein-protein conjugates. This application is based on the fact that protein-2-pyridyl disulphide derivatives (formed from the reaction of non-thiol proteins with the reagent) react with thiol-containing proteins (with native thiols or thiolated by, for example, the method described above) via thiol-disulphide exchange to form disulphide-linked protein-protein conjugates. This conjugation technique has been used for the preparation of an alpha-amylase-urease, a ribonuclease-albumin and a peroxidase-rabbit anti-(human transferrin) antibody conjugate. The disulphide bridges between the protein molecules can easily be split by reduction or by thiol-disulphide exchange. Thus conjugation is reversible. This has been demonstrated by scission of the ribonuclease-albumin and the alpha-amylase-urease conjugate into their components with dithiothreitol. N-Succinimidyl 3-(2-pyridyldithio)propionate has been prepared in crystalline form, in which state (if protected against humidity) it is stable on storage at room temperature (23 degrees C).     

Photoreactive 111In-cyclodextrin inclusion complex: a new heterobifunctional reagent for antibody labeling

The compound of interest, N-5-azido-2-nitrobenzoylaminomethyl-¹¹¹In-acetylacetone-α-cyclodextrin (CD) (V) was synthesized by the selective tosylation of α-CD to form 6-tosyl-6-deoxy-CD, which was then reacted with NaN₃ to form 6-azido-6-deoxy-CD (II). This was followed by catalytic hydrogenation to yield III. Compound III and ¹¹¹In-acetylacetone were mixed to form an inclusion complex, which was then reacted with N-5-azido-2-nitrobenzoyloxysuccinimide to yield compound V. Anti-melanoma MAbTP41.2 was added to compound V, followed by immediate photoreactivation labeling by u.v. light at 320 nm. The final product VI was purified from a Sephadex G-50 column. ¹¹¹In-DTPA-MAbTP41.2 was also prepared as a control.Immunoreactivity via the cell-binding assay of VI was 87%, compared with 57% by the BADTPA method. Biodistribution in non-tumor rats yielded a liver concentration in %ID/g of 3.5, 1.7 and 1.0 for compound VI, compared to the 5.5, 5.2 and 3.1 for the BADTPA compound, at 4, 24 and 48 h post-injection, respectively.     

Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces

A procedure for covalent immobilization of functional proteins on silica substrates was developed using thiol-terminal silanes and heterobifunctional cross-linkers. Using this procedure, a high density of functional antibodies was bound to glass cover slips and silica fibers. The amount of anti-IgG antibody immobilized was determined to be in the range of 0.66 to 0.96 ng/mm2 using radiolabeled antibody. The relative amount of IgG antigen bound by the immobilized antibody (0.37 to 0.55 mol antigen/mol antibody) was three to five times greater than other investigators have reported. In addition, the amount of protein nonspecifically adsorbed to the antibody-coated surface was further reduced by the addition of blocking agents so that nonspecific adsorption of protein antigens represented only 2-6% of the total antigen binding. With this low background, IgG antigen binding could be measured at levels as low as 150 fmol when an antigen concentration of 3 pmol/ml was applied. The process for antibody immobilization is straightforward, easy to perform, and adaptable for modifying mass quantities of biosensor components.     

3-(Trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine: Synthesis and chemical characterization of a heterobifunctional carbene-generating crosslinking reagent

A new hydrophobic heterobifunctional photocrosslinking reagent 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine (TRIMID), a carbene precursor, and its radioiodinated analogue [¹²⁵I]TRIMID, have been synthesized and chemically characterized. The reagents were applied for membrane protein modification in human erythrocyte membranes and purple membranes fromHalobacterium halobium. Covalent labeling of the anion transport protein (band 3) via the isothiocyanate function was confirmed. Radiolabeled TRIMID was detected in at least two thermolysin-generated transmembrane fragments of the anion transport protein, and half-maximal inhibition of the erythrocyte anion transport activity was attained with 2.2 mM reagent. In bacteriorhodopsin (BR), a common binding site for the monofunctional phenylisothiocyanate and the bifunctional crosslinking reagent was identified: preincubation of purple membranes with TRIMID suppressed phenylisothio-[¹⁴C]-cyanate binding to BR. [¹²⁵I]TRIMID was recovered in V-1, the N-terminal segment of BR, which includes the phenylisothiocyanate binding site Lys-41. Light-induced intramolecular crosslinking of band 3-derived thermolytic fragments was not observed, although the carbene was generatedin situ and photocrosslinking of the protease V8 fragments of BR was not detected. Chemical and physicochemical characteristics of the new reagent are discussed with regard to limitations imposed for photoinduced site-directed crosslink formation.     

A high-density poly(ethylene glycol) polymer brush for immobilization on glass-type surfaces.

Label-free heterogeneous phase detection critically depends on the properties of the interfacial layer. We have obtained high-density monomolecular poly(ethylene glycol) (PEG) layers by solvent-free coupling of homo-bifunctional PEGs (2,000 g/mol) at 75 degrees C to silica surfaces silanized with glycidyloxipropyltrimethoxysilane (GOPTS). Characterization by ellipsometry and contact angles revealed that PEG layers up to 3.4 ng/mm2 with low roughness and flexibility were obtained. Specific and non-specific binding at these PEG surfaces was monitored by reflectometric interference spectroscopy (RIfS). No significant non-specific adsorption upon incubation of 1 mg/ml ovalbumin was detectable (< 10 pg/mm2), and 150 pg/mm2 upon incubation of 10% calf serum, less than 10% of the amount adsorbed to the solely silanized surfaces. The terminal functional groups of the PEG layers were utilized to couple ligands and a protein. Specific protein interaction with these immobilized compounds was detected with saturation loadings in the range of protein monolayers (2-4 ng/mm2). The excellent functional properties, the high stability of the layers, the generic and practical coupling procedure and the versatility for immobilizing compounds of very different functionality make these PEG layers very attractive for application in label-free detection with silica or metal-oxide based transducers.     

N-(3-(p-azido-m-[125I]iodophenyl)propionyl)-succinimide--a heterobifunctional reagent for the synthesis of radioactive photoaffinity ligands: synthesis of a carrier-free 125I-labeled cardiac glycoside photoaffinity label

A new heterobifunctional reagent, N-(3-(p-azido-m-iodophenyl)propionyl)-succinimide (AIPPS), was synthesized and chemically characterized. The radiochemical form of the reagent, [125I]AIPPS, should be of general use as a photoactive reagent for the derivatization of free amino groups on a large variety of biologically active compounds, including many hormones. Amino-containing ligands can be derivatized with [125I]AIPPS in a method which is similar to that used for the 125I-labeled Bolton-Hunter reagent (N-(3-(p-hydroxyphenyl)propionyl)-succinimide). The added advantage with [125I]AIPPS, however, is that the ligand derivative is made both photoactive and radioactive in a single step. As an example of how this reagent can be used, we have prepared carrier-free [125I]AIPPS and reacted it with the amino-containing cardiac glycoside, 4-amino-4,6-dideoxyglucosyl digitoxigenin (GluD). The radioiodinated cardiac glycoside, [125I]AIPP-GluD, was purified by thin-layer chromatography and was carrier-free with a specific radioactivity of 2175 Ci/mmol. [125I]AIPP-GluD was an effective photoaffinity label for Na,K-ATPase as shown by specific photoaffinity labeling of purified canine kidney enzyme and human erythrocyte enzyme.     

Polyketone polymer: a new support for direct enzyme immobilization

Polyketone polymer -[-CO-CH(2)-CH(2)-](n)-, obtained by copolymerization of ethene and carbon monoxide, is utilized for immobilization of three different enzymes, one peroxidase from horseradish (HRP) and two amine oxidases, from bovine serum (BSAO) and lentil seedlings (LSAO). The easy immobilization procedure is carried out in diluted buffer, at pH 7.0 and 3 degrees C, gently mixing the proteins with the polymer. No bifunctional reagents and spacer arms are required for the immobilization, which occurs exclusively via a large number of hydrogen bonds between the carbonyl groups of the polymer and the -NH groups of the polypeptidic chain. Experiments demonstrate a high linking capacity of polymer for BSAO and an extraordinary strong linkage for LSAO. Moreover, activity measurements demonstrate that immobilized LSAO totally retains the catalytic characteristics of the free enzyme, where only a limited increase of K(M) value is observed. Finally, the HRP-activated polymer is successfully used as active packed bed of an enzymatic reactor for continuous flow conversion and flow injection analysis of hydrogen peroxide containing solutions.     

2-(3-Amino-3-deoxy-beta-D-xylofuranosyl)thiazole-4-carboxamide: a new tiazofurin analogue with potent antitumour activity

A new tiazofurin analogue, 2-(3-amino-3-deoxy-beta-d-xylofuranosyl)thiazole-4-carboxamide (3), was synthesized starting from d-glucose and evaluated for its in vitro antiproliferative activity against a panel of human tumour cell lines. Compound 3 exhibited the most powerful cytotoxicity against K562 cells, being approximately 100-fold more potent than tiazofurin. This analogue was also active against Jurkat, HT-29 and HeLa malignant cells, with respective IC(50) values being ca. 2-, 27- and 17-fold lower than those observed for tiazofurin. Remarkably, compound 3 did not exhibit any significant cytotoxicity towards normal foetal lung MRC-5 cell line.     

N-(1-pyrene)maleimide: a fluorescent cross-linking reagent.

N-(1-Pyrene)maleimide is nonfluorescent in aqueous solution but forms strongly fluorescent adducts with sulfhydryl groups of organic compounds or proteins. The conjugation reactions of N-(1-pyrene)maleimide are relatively fast and can be monitored by the increase in fluorescence intensity of the pyrene chromophore. In cases where primary amino groups are also present in the system, we have observed a red shift of the emission spectra of the fluorescent adducts subsequent to the initial conjugation, as characterized by the disappearance of three emission peaks at 376, 396, and 416 nm, and the appearance of two new peaks at 386 and 405 nm. Model studies with N-(1-pyrene)maleimide adducts of L-cysteine and cysteamine indicate that the spectral shift is the result of an intramolecular aminolysis of the succinimido ring in the adducts. Evidence from both chemical analysis and nuclear magnetic resonance studies of the addition products supports this reaction scheme. N-(1-Pyrene)maleimide adducts of N-acetyl-L-cysteine and beta-mercaptoethanol, which have no free amino group, do not exhibit a spectral shift. Among several protein conjugates only the N-(1-pyrene)maleimide adduct of bovine serum albumin (PM-BSA) shows the spectral shift resembling that of PM-cysteine. N-(1-Pyrene)maleimide reacts with the sulfhydryl group of the single cysteine residue at position 34 in BSA. The finding that the alpha-amino group of the N-terminus in PM-BSA is blocked after the spectral shift is completed strongly suggests that N-(1-pyrene)maleimide cross-links the N-terminus and the cysteine residue in BSA. The relative proximity of the sulfhydryl and amino groups is very critical in the cross-linking as demonstrated by the observation that the spectral shift observed with PM-BSA can be prevented by addition of denaturing reagents such as 1% sodium dodecyl sulfate immediately after labeling, and by the failure of PM-glutathione to undergo the intramolecular aminolysis. Since the intramolecular rearrangement of PM adducts is associated with characteristic fluorescence changes, N-(1-pyrene)maleimide can serve as a fluorescent cross-linking reagent which provides information about the spatial proximity of sulfhydryl and amino groups in proteins.     

2,3,5,6-Tetrafluorophenyl N-(S-benzoylthioacetyl)glycylglycyl- p-aminobenzoate, a heterobifunctional (99m)Tc ligand for precomplexed antibody labeling

Heterobifunctional (99m)Tc ligands are useful for antibody labeling using the precomplexation route. The aim of this work was to synthesize a ligand, which has sufficient chemical stability to be complexed with (99m)Tc without inactivating the reactive conjugation group. Using 2,3,5,6-tetrafluorophenyl N-(S-benzoylthioacetyl)glycylglycyl-p-aminobenzoate (OC2) >60% of the (99m)Tc complex was obtained at 80 degrees C in 20 min, which was separated from the free ligand and impurities by HPLC. After solvent evaporation, (99m)Tc-OC2 was conjugated with the monoclonal antibody mAb425 in 50% radiochemical yield. In all, the labeling method required about 1 h preparation time. The immunoreactive fraction of the (99m)Tc-OC2 mAb425 conjugate was 81%, indicating preserved binding capability after conjugation. Compared to recently described methods, which need in situ activation of the (99m)Tc complex, the application of OC2 saved time and reduced the number of manipulations with radioactive material.     

Plasma modified surfaces for covalent immobilization of functional biomolecules in the absence of chemical linkers: towards better biosensors and a new generation of medical implants

Plasma modification and plasma polymer deposition are valuable technologies for the preparation of surfaces for the covalent binding of biomolecules for applications such as biosensors, medical prostheses, and diagnostic devices as well as surfaces for enzyme-mediated reactions. Covalency is conveniently tested by the ability of the surface to retain the attached molecules after vigorous washing with sodium dodecyl sulphate (SDS). Covalency is indicated if the fraction of protein retained lies above the curve characteristic of physisorption. Confidence in covalency is strengthened when the washing protocol is aggressive enough to remove all adsorbed protein from a control significantly more hydrophobic than the test surface. The use of linker chemistry to space the molecules from the surface is in some cases beneficial. However, the use of linker chemistry is not necessary to retain molecular function for long periods when the polymer surface is modified by energetic bombardment. The energetic bombardment retains hydrophilicity of the surface by crosslinking the subsurface, and this appears to facilitate retention of protein function. Energetic bombardment also increases the functional life of molecules immobilized and then freeze dried on plasma-modified surfaces. Analysis of the surfaces shows that the covalent binding mechanism is related to the presence of free radicals on the surface and in the subsurface regions. The unpaired electrons associated with the radicals appear to be mobile within the modified region and can diffuse to the surface to take part in binding interactions. Proactive implantable devices can make use of these principles of covalent attachment by seeding the surface of an implant with a biomolecule that elicits the desired interaction with cells and prevents undesirable responses.     

Synthesis of a new heterobifunctional linker, N-[4-(aminooxy)butyl]maleimide, for facile access to a thiol-reactive 18F-labeling agent

A new heterobifunctional linker containing an aldehyde-reactive aminooxy group and a thiol-reactive maleimide group, namely N-[4-(aminooxy)butyl]maleimide, was synthesized as a stable HCl salt by O-alkylation of either N-hydroxyphthalimide or N-(4-monomethoxytrityl)hydroxylamine, followed by N-alkylation of maleimide, in an overall yield of 18% (seven steps) or 29% (five steps), respectively. This heterobifunctional linker allowed a simple and efficient synthesis of a maleimide-containing thiol-reactive (18)F-labeling agent. Thus, N-[4-[(4-[(18)F]fluorobenzylidene)aminooxy]butyl]maleimide (specific activity: approximately 3000 Ci/mmol at end of synthesis) was synthesized in two steps involving the preparation of 4-[(18)F]fluorobenzaldehyde, followed by its aminooxy-aldehyde coupling reaction to the heterobifunctional linker, with an overall radiochemical yield of approximately 35% (decay corrected) within approximately 60 min from end of bombardment. Initial (18)F-labeling experiments were carried out using a thiol-containing tripeptide glutathione (GSH) and a 5'-thiol-functionalized oligodeoxynucleotide (5'-S-ODN) in phosphate-buffered saline (PBS, pH 7.5). After standing at room temperature for 10 min, the (18)F-labeled GSH and 5'-S-ODN were obtained in (18)F-labeling yields of approximately 70% and approximately 5% (decay-corrected), respectively. The heterobifunctional linker is easy to synthesize and provides a facile access to the maleimide-containing thiol-reactive (18)F-labeling agent, which could be advantageously employed in the development of (18)F-labeled biomomolecules for use with positron emission tomography.     

Preparation and use of synthetic cell culture surfaces. A new reagent for the covalent immobilization of proteins and glycoproteins on a nonionic inert matrix

To study cell interactions with external molecules immobilized on a chemically defined nonionic, inert matrix, we have prepared flat polyacrylamide matrices containing covalently attached carbohydrate or protein. A new acrylamide derivative, containing a terminal 1,2-dihydroxy group, was synthesized and then copolymerized with acrylamide and bisacrylamide to make 20% polyacrylamide matrices, which could be oxidized with NaIO4 to generate reactive aldehyde groups. Molecules containing a free amine (e.g. proteins or glycopeptides) can be coupled to the aldehyde-activated matrix by formation of a Schiff base and reduction with NaCNBH3 to form a stable -CH2-NH-bond. Unreacted aldehyde groups are reduced to hydroxyl groups with NaBH4. In order to immobilize polysaccharides on the activated surfaces, these molecules are first modified to contain a free amine. We have described a procedure to convert purified hyaluronic acid oligosaccharides to a reactive alkylamine derivative uniquely modified at the reducing end (Raja, R. H., LeBoeuf, R. D., Stone, G. W., and Weigel, P. H. (1984) Anal. Biochem. 139, 168-177). The covalent attachment of [3H]hyaluronate-amine, [14C]ethanolamine, or 125I-bovine serum albumin, to activated surfaces was complete within 5-24 h. The amount immobilized was directly proportional to the amine concentration and to the aldehyde content of the matrix and inversely proportional to the molecular weight of the amine. About 90% of the available aldehyde groups reacted with ethanolamine, whereas less than 0.01% reacted with albumin. Molecules larger than 3300 Da were excluded from the interior of the matrix and could therefore only be attached to the surface of the matrix. These synthetic surfaces can be used in long term culture experiments to study cellular interactions with virtually any type of immobilized molecule.