Tyrosine diazonium

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. 

Tyrosine may be targeted specifically for modification through its phenolate anion by acylation, through electrophilic reactions such as the addition of iodine or diazonium ions, and by Mannich condensation reactions. The electrophilic substitution reactions on tyrosine s ring all occur at the ortho position to the —OH group (Figure 1.11). Most of these reactions proceed effectively only when tyrosine s ring is ionized to the phenolate anion form. 

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.

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

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

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. 

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-... 

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