Diazonium with tyrosine

Figure 11.17 The diazonium group of p-diazobenzoylbiocytin can react with tyrosine or histidine residues in proteins to form diazo bonds.

The catalytically essential nature of tyrosine 85 and its proximity to the substrate binding site and to tyrosine 115 were demonstrated from studies of modification with tetranitromethane (71) and from studies of intramolecular cross-linking of aminotyrosyl residues (72). The bro-moacetamidophenyl (69) and diazonium (70) reagents obtained from aminophenyl-pdT both react selectively and exclusively with tyrosine 85. This residue is situated, stereochemically, such that its hydroxyl group can interact with the 3 -phosphate of pdTp. 

Aromatic amine-containing haptens are converted to diazonium salts with ice-cold nitrous acid. Diazonium salts can then react with a protein at alkaline pH (around 9) through electrophilic attack of the diazonium salt at histidine, tyrosine and(or) tryptophan residues of the carrier protein (Table 7). 

The synthesized CPMV-alkyne 42 was subjected to the CuAAC reaction with 38. Due to the strong fluorescence of the cycloaddition product 43 as low as 0.5 nM, it could be detected without the interference of starting materials. TMV was initially subjected to an electrophilic substitution reaction at the ortho-position of the phenol ring of tyrosine-139 residues with diazonium salts to insert the alkyne functionality, giving derivative 44 [100]. The sequential CuAAC reaction was achieved with greatest efficiency yielding compound 45, and it was found that the TMV remained intact and stable throughout the reaction. 

Figure 1.11 Tyrosine residues are subject to nucleophilic and electrophilic reactions. The unprotonated phe-nolate ion may be alkylated or acylated using a variety of bioconjugate reagents. Its aromatic ring also may undergo electrophilic addition using diazonium chemistry or Mannich condensation, or be halogenated with radioactive isotopes such as 12iI.

The phenolic group of tyrosine undergoes iodina-tion (Eq. 3-44), acylation, coupling with diazonium compounds, and other reactions. 

Diazotization of proteins with diazonium-l/f-tetrazole in previous work has been studied in terms of reaction with histidine and tyrosine residues (50, 51). When E. coli pyrophosphatase was incubated with this reagent, there was pseudo-first-order inactivation, but analysis of the... 

Oxidation of two out of 13 tryptophan residues in a cellulase from Penicillium notatum resulted in a complete loss of enzymic activity (59). There was an interaction between cellobiose and tryptophan residues in the enzyme. Participation of histidine residues is also suspected in the catalytic mechanism since diazonium-l-H-tetrazole inactivated the enzyme. A xylanase from Trametes hirsuta was inactivated by N-bromosuc-cinimide and partially inactivated by N-acetylimidazole (60), indicating the possible involvement of tryptophan and tyrosine residues in the active site. As with many chemical modification experiments, it is not possible to state definitively that certain residues are involved in the active site since inactivation might be caused by conformational changes in the enzyme molecule produced by the change in properties of residues distant from the active site. However, from a summary of the available evidence it appears that, for many / -(l- 4) glycoside hydrolases, acidic and aromatic amino acid residues are involved in the catalytic site, probably at the active and binding sites, respectively. 

Aromatic amines may be converted to their diazonium salts with nitrous acid. The hapten may then be bound via azo linkages to the tyrosine (shown), histidine, lysine, and possibly arginine and tryptophane residues of the carrier protein by mixing the... 

Another problem was the number of chelating groups attached to each protein. Diazonium reagents react with many different amino acid side chains including those of lysine, tyrosine, and histidine (24). There-... 

Much of Landsteiner s pioneer work was carried out with haptens that were aromatic amines. The compounds were converted to diazonium salts with nitrous acid and aUowed to react with proteins at alkaline pH (approximately 9). Reaction occurred primarily with histidine, tyrosine, and tryptophan residues of the protein carrier. For a representative procedure, see Kabat (p. 799 seq.). An interesting application of this procedure was the preparation of a chloramphenicol-protein conjugate which was used to elicit antibodies specific for chloramphenicol. In this case, a prior reduction of the nitro group of chloramphenicol to an amino group was required. As early as 1937, carcinogenic compounds were conjugated to protein carriers by means of their isocyanate derivatives which were prepared from amines. Immune sera were raised, and their properties were studied. - ... 

Reactive properties of aryl diazonium salts Aryl diazonium cations are electrophilic reagents and appear to react predominantly with four types of amino acids - lysine, tyrosine, histidine and cysteine. Although the precise structure of the cysteine derivatives is unclear, aryl diazonium salts can react with lysine, histidine and tyrosine as indicated in eq. (5.3) below to give the various substituted derivatives. 

As standards for spectrophotometric measurements, Traylor and Singer (1967) prepared the monoazo derivatives of histidine and tyrosine with the various diazonium salts. The spectra of the products of reaction of N-acetyl histidine with the diazonium salt were obtained after reaction of a 100-fold excess of N-acetyl histidine with the diazonium salt in 0.1 M borate at pH 9.0. The spectral constants for the histidine and tyrosine derivatives are recorded in Table 5.1. 

The diazonium salt is readily prepared from the aminophenyl group, but does not result from treatment of an aliphatic amine (such as the APTES-derivatized surface) with nitrous acid. The activated surface is now ready for enzyme coupling, since diazonium salts are very reactive toward protein tyrosine residues (Eq. 4.4) ... 

Some effective cross-linking agents are less suitable for the conjugation of enzyme to antibody. Diazonium salts of aromatic compounds react preferentially with histidine and tyrosine which are very often located in or near the active site of the enzyme. Protecting the active sites with inhibitors (Jansen et al., 1971) is often cumbersome and, consequently, not very popular. 

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