A novel biotinylated heterobifunctional cross-linking reagent bearing an aromatic diazirine

The synthesis of a p-[(3-trifluoromethyl)diazirine-3-yl]benzoic acid derivative is described as a new carbene generating heterobifunctional cross-linking reagent. The cross-linker carries a biotin moiety in order to make use of avidin—biotin technology for specific manipulation of cross-linked components. To evaluate the ability of this reagent, the inter-subunit cross-linking of egg-white avidin tetramer was investigated. As a typical application of avidin—biotin technology for cross-linking experiments, a chemiluminescent detection method was examined to identify photobiotinylated components. A cross-linked dimeric product with an apparent molecular mass of 38 kDa was clearly visualized by the combined use of a horseradish peroxidase—streptavidin conjugate and a luminol-based chemiluminescent system.     

A new radioactive diazofluorene based heterobifunctional reagent

Synthesis of a radioactive photoactivable heterobifunctional reagent, N-oxysuccinimide ester of 2-[¹⁴C]glycyl carboxy-9-diazofluorene is described. This reagent on photolysis gives rise to a reactive carbene which rapidly inserts into solvents like methanol. The probe can be easily linked to aldolase which on photolysis gives rise to aldolase dimer, trimer and tetramer depending on the density of linked probe. This probe has also been linked to concanavalin A. The radioactive concanavalin A so obtained was incubated with erythrocyte ghosts and photolysed. The membrane protein analysis by gel electrophoresis indicated that concanavalin A has been covalently crosslinked to band 3.     

Conjugation of soluble CD4 without loss of biological activity via a novel carbohydrate-directed cross-linking reagent.

Chemical conjugates of recombinant soluble CD4 (sCD4) with toxins, or with antibodies that activate cytotoxic T cells, can be used to direct selective killing of human immunodeficiency virus (HIV)-infected cells. This approach takes advantage of the ability of sCD4 to bind with high affinity to gp120, the envelope protein of HIV-1, which is expressed on actively infected cells. However, conjugation of sCD4 via reagents that target amino groups may reduce its affinity for gp120, since at least one such group is important for gp120 binding. Here, we describe a novel cross-linking reagent which enables the conjugation of sCD4 via its carbohydrate moieties rather than its free amino groups. This heterobifunctional reagent, 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), combines a nucleophilic hydrazide with an electrophilic maleimide, thereby allowing coupling of carbohydrate-derived aldehydes to free thiols. We describe conditions by which MPBH is coupled selectively to the sialic acid residues of sCD4, and exemplify the use of MPBH by conjugating sCD4 to hemoglobin and to beta-galactosidase. We show that, whereas conjugation of sCD4 via amino groups markedly reduces its gp120 binding affinity, conjugation via the carbohydrate chains using MPBH does not affect binding. Moreover, we demonstrate the ability of a sCD4-MPBH-fluorescein conjugate to label HIV-infected human CEM cells selectively. These results indicate that, by targeting its carbohydrate moieties, sCD4 can be cross-linked to other molecules without compromising its function. The approach described here can be useful for glycoproteins in which amino groups, but not carbohydrates, are important for function. More generally, this approach can be considered for use in cross-linking glycoconjugates to compounds which either contain thiols, or to which thiols can be added.     

Didecyl squarate--a practical amino-reactive cross-linking reagent for neoglycoconjugate synthesis

The present paper describes the synthesis and use of the hydrophobic squaric decyl ester glycosides in neoglycoconjugate chemistry. The 2-aminoethyl glycosides of -D-mannopyranose, lactose, globotriose, globotetraose, GM3, and sialyl Lewis^x, as well as the 2-(2-aminoethylthio)ethyl glycoside of -D-mannopyranose, -D-glucopyranose, and galabiose were reacted with squaric acid didecyl ester to afford the hydrophobic squaric decyl ester glycosides. These glycosides were efficient reagents for the conjugation to amino-functional microtiter plates, BSA and aminated Sepharose EAH 4B. The decyl ester moiety of the squaric decyl ester glycosides constitutes a traceless hydrophobic tag, which has the major advantage, as compared to the corresponding ethyl esters, that it enables easy purification of the glycosides with silica chromatography and that unreacted excesses glycosides from conjugation reaction mixtures can easily be recovered by means of C18 solid phase extraction.     

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.     

Radioiodination of a photoactivatable heterobifunctional reagent

The N-hydroxysuccinimide ester of 4-azidosalicylic acid, a photoactivable heterobifunctional reagent, can be radioiodinated. The low efficiency (3%) of the radioiodination by a previously published method (I. Ji and T. H. Ji, 1982, Anal. Biochem. 121, 286-289) has been increased to 63% by substituting the solvent, acetone, with others such as aqueous acetonitrile, dimethylformamide, or dimethyl sulfoxide. The resulting 125I reagent was used for derivatizing human choriogonadotropin. The radioactive hormone derivative was crosslinked to the alpha beta dimer upon photolysis.     

A novel cross-linking reagent for bovine hemoglobin modification

A novel cross-linking reagent, methoxypolyethelene glycol-glutamic acid, was synthesized and used to modify bovine hemoglobin. Bis-tetrameric hemoglobin with moderate affinity for O₂ was obtained under the controlled reaction conditions.     

A new nucleic acid-protein cross-linking reagent

Summary A new photoactivable reagent is described, which allows the formation of RNA-protein crosslinks via disulfide bridges in combination with mercaptobutyrimidate.The reconstituted L24 protein-23S RNA complex from the large subunit of E. coli ribosomes has been used as a model system for the cross-linking. The main advantages of the reagent are the absence of U.V. generated cross-links, since photoactivation is carried out at 360 nm, on one hand and the ease of cleavage of the cross-link by mild reduction (-mercaptoethanol) on the other.     

A new carbene based heterobifunctional reagent. Photochemical crosslinking of aldolase

The synthesis of a new photoactivatable heterobifunctional crosslinking reagent, the N-oxysuccinimide ester of 2-carboxy-9-diazofluorene, is described. The ability of the parent chromophore 2-carbomethoxy-9-diazofluorene to insert into cyclohexane and methanol has been established. The reagent has been linked to aldolase and the stoichiometry determined. Photolysis of the probe-linked aldolase indicated that photolysis was very rapid and that the photolysed product was constituted of crosslinked dimer, trimer and tetramer. Increase in concentration of probe linked to aldolase followed by photolysis gave rise to largely tetramer and higher oligomers of aldolase. The use of this carbene-based reagent vis a vis arylazide-based reagent for studying protein crosslinking is discussed.     

Efficient coupling of glycopeptides to proteins with a heterobifunctional reagent

A heterobifunctional linking reagent containing a masked aldehydo group and acyl hydrazide was synthesized for coupling of glycopeptides and other amino-containing compounds to proteins. After conversion to acyl azide, the reagent reacts with the amino group of a glycopeptide, and the modified glycopeptide is deacetalized with a weak acid to unmask the aldehydo group, which is then conjugated to bovine serum albumin (BSA) by reductive alkylation with pyridine-borane. The overall reaction scheme proceeds under relatively mild conditions. When the protein amino group was in a large excess (greater than 6-fold) of the aldehyde reagent, the efficiency of conjugation was as high as 88% even at submicromole levels. As a test case for application of this reagent, 6-aminohexyl beta-D-galactopyranoside (Gal-AH) was attached to the linking reagent and conjugated to BSA at various aldehyde-to-protein molar ratios ranging from 25 to 200. The level of O-galactosyl residue incorporated into BSA by this reagent far exceeded that observed in a similar reductive alkylation involving S-galactoside reagents [Lee, R. T., & Lee, Y. C. (1980) Biochemistry 19, 156-163]. By use of the present conjugating procedure, as many as 112 mol of Gal-AH residues were incorporated per mole of BSA, which represents near total modification of the amino groups. Some binding characteristics of the new BSA derivatives were studied in the mammalian hepatic galactose/N-acetylgalactosamine specific lectin system along with other types of BSA derivatives (containing S-galactosyl residues). In general, the behavior of the new derivatives was similar to that of other types. For instance, the affinity increased exponentially at low sugar substitution levels (up to 30 mol of galactosyl residues/mol of BSA), and the slope of exponential increase and affinity at a given sugar substitution level was similar to those of other types.     

A new cleavable reagent for cross-linking and reversible immobilization of proteins.

We have prepared a new bifunctional reagent for the cross-linking and reversible immobilization of proteins through their amine groups. This compound, ethylene glycolyl bis(succinimidyl succinate), reacts rapidly with proteins, at pH 7 and at high dilution. The resulting protein cross-links are readily cleaved at pH 8.5 using hydroxylamine for 3–6 hr. at 37°C. Substantial enzymatic activity was observed with lactic dehydrogenase after such reversible cross-linking. Trypsin immobilized on agarose using this reagent retains full specific activity, is stable for weeks in the cold, and may be released with hydroxylamine at 25°C. This compound appears suitable for studies involving proteins with essential disulfide linkages.     

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.     

A novel tetrafunctional reagent for simultaneous intramolecular and intermolecular cross-linking of bovine hemoglobin

A novel tetrafunctional reagent, alpha,gamma,alpha',gamma'-tetra-succinimidyl-hexanediamide-di-glutamate ester (HDG(OSu)(4)), was successfully synthesized, and a well-defined cross-linked bovine hemoglobin (mainly 128 kDa) was prepared with this reagent. Due to the spatial structure of this cross-linking reagent, the intramolecular and intermolecular cross-linking of bovine hemoglobin was formed simultaneously in one reaction. Although the cross-linked bovine hemoglobin showed a slight decrease in half-saturated O(2) pressure value (P(50), from 28.1 mm Hg to 21.7 mm Hg) and Hill coefficient (from 2.5 to 2), due to the cross-linkage, it still performed well for O(2) delivery.     

Divinyl sulfone as a cross-linking reagent for oligomeric proteins

The kinetics of the nucleophilic addition reactions of divinyl sulfone to amino groups of glycine and model proteins was studied in aqueous solution at 30 degrees C. The rate constants for glycine, bovine serum albumin, and alpha 1-casein were (4.84 +/- 0.58) x 10(-1), (2.97 +/- 0.31) x 10(-2), and (2.38 +/- 0.49) x 10(-2) M-1s-1, respectively. Divinyl sulfone was proposed as a cross-linking reagent for the qualitative detection of protein association in solution. The cross-linking capacity of divinyl sulfone was compared to that of 1,3,5-triacryloylhexahydro-s-triazine.     

A disulfide-bridge bifunctional imidoester as a reversible cross-linking reagent.

A disulfide-bridged bifunctional imidoester, dimethyl 3, 3′ dithio-bispropionimidate (DTP) has been prepared and investigated as a reagent to introduce covalent cross-links in proteins that can subsequently be broken by mild reduction. Such reversible cross-links were shown to be introduced by DTP in the soluble subunit proteins aldolase and Concanavalin A. DTP was also used to modify human intact erythrocytes. Such modification rendered the erythrocytes resistant to hypotonic lysis; subsequent treatment with mercaptoethanol lysed the cells. After DTP-modification of the cells, the hemoglobin contained in them could still be reversibly oxygenated and deoxygenated.     

Synthesis of a water-soluble protein cross-linking reagent

2,4-Difluoro-5-nitrobenzenesulfonic acid has been synthesized by the sulfonation of 2,4-difluoronitrobenzene, and precipitated with KCl as the potassium sulfonate. The structure was confirmed by chemical and spectroscopic methods (IR, 1H-NMR, 13C-NMR, 19F-NMR, UV, MS and ultimate organic analysis). Lysozyme was cross-linked with the potassium sulfonate and with 1,5-difluoro-2,4-dinitrobenzene. The products were analysed by SDS-PAGE and compared. The cross-linking conditions were optimized.     

A novel method for modification of tumor cells with bacterial superantigen with a heterobifunctional cross-linking agent in immunotherapy of cancer

Bacterial superantigens (SAGs) bind to cognate Vβ elements of T-cell receptors on T-cells and to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells to activate T-cell subsets expressing the Vβ elements. We examined the possibility that the direct binding of SAGs (staphylococcal enterotoxins B [SEB] and A [SEA]) to tumor cells decreases the toxicity of SAGs, and that antitumor immunity can be induced with the aid of T-helper-1 (Th1)-type cytokines and monokines released from T-cells and monocytes, respectively, by activation with SAGs. In this context, we have developed a general method for conjugating SEB and SEA directly to tumor cells with a heterobifunctional cross linking agent, N-(γ-maleimidobutyryloxy) sulfosuccinimide sodium salt. Using this method, we have succeeded in conjugating SEB to a sufficient extent as to induce strong tumor immunity. Both in vitro T-cell culture with SEB-bearing Meth A cells and in vivo immunization with SEB-bearing Meth A cells induce strong antitumor activity. These results suggest that the direct conjugation of SAGs including SEB and SEA to tumor cells is a powerful and useful method for immunotherapy of cancer.     

Controlled coupling of mildly reduced proteins to Sepharose gelatin by heterobifunctional reagent

The heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate was utilized for controlled coupling of mildly reduced BSA and lysozyme to Sepharose gelatin to prepare immunoabsorbents. Each reaction step was examined and quantitated. The free amino groups on gelatin after coupling gelatin to cyanogen bromide-activated Sepharose as well as the number of 3-(2-pyridyldithio) propionyl groups on Sepharose gelatin were quantitated. Coupling of mildly reduced BSA as well as lysozyme to PDP-Sepharose gelatin occurred through sulfhydryl-disulfide exchange and permitted the formation of an immunoabsorbent. The immunoabsorbents were capable of binding the respective antibody and the eluted antibodies were pure and free of plasma proteins.     

Preparation of peptide-protein immunogens using N-succinimidyl bromoacetate as a heterobifunctional crosslinking reagent

Synthetic peptides derived from human fibrin were unidirectionally conjugated to three carrier proteins (bovine serum albumin, bovine alpha-lactalbumin, and keyhole limpet hemocyanin) by a method that employs N-succinimidyl bromoacetate. This heterobifunctional crosslinking reagent was prepared with a 79% yield in gram quantities from inexpensive starting materials. With this reagent, carrier proteins were first bromoacetylated, then reacted with the thiol groups of cysteine-containing peptides. The extent of peptide conjugation was assessed by amino acid analysis after acid hydrolysis, which liberated 1 mol of S-carboxymethylcysteine for each mole of thioether linkage between peptide and protein. The results of several conjugation experiments indicated that the efficiency of peptide incorporation ranged between 22 and 37% based on the recovery of S-carboxymethylcysteine relative to lysine. When the conjugates were used as immunogens, the S-carboxymethyl linkage was not antigenic in comparison with the S-maleimidobenzoyl linkage, even though their antipeptide immunoreactivities were similar.     

An efficient synthesis of a heterobifunctional coupling agent

An efficient synthesis of 4-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl]-N-(6-[[6-([6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl]amino)-6-oxohexyl]amino]-6-oxohexyl)cyclohexanecarboxamide (12), a heterobifunctional coupling agent, was developed, which is critical for chemoselective conjugation of proteins and enzymes. The synthesis involved seven steps starting from 6-{[(benzyloxy)carbonyl]amino}hexanoic acid (1), and multigram quantities of coupling agent (12) were prepared using this protocol in excellent overall yield and 99.6% purity by reversed phase HPLC. The new method is suitable for the synthesis of coupling agent 12, consistently with purity >99%, and is useful for the preparation of other analogous coupling agents.