Histidine 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). 

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

The most important applications of the in situ photochemical fluorodediazoniation start from heterocyclic amines for which the diazonium tetrafluoroborates are impossible to isolate. In this way, fluorinated imidazoles,123-253,254-257 261 benzimidazoles,262 pyrazoles,254-256,262 indazoles,158 thiazoles,259 and triazoles259-262 have been prepared (Table 4). Some of these are the precursors of biologically active compounds such as fluoro-substituted histidines or histamines,123,253-257,258 or 5-fluoro-l/ -D-ribofuranosylimidazole-4-carboxamide.261... 

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. 

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. 

The interesting diazonium sulphate betaine (495) has been prepared by diazotisation of the corresponding amino-sulphate and shown to couple under physiological conditions with cysteine, tryptophan, and histidine. 

H.G. Higgins, D. Fraser, The reaction of amino acids and proteins with diazonium compounds. 1. A spectrophotometric study of azo-derivatives of histidine and tyrosine, Australian J., Sci. Res. Ser. A Phys. Sciences 1952, 5, 736-753. 

It is interesting that when tyrosine is acylated with benzoyl chloride under this condition, it forms a dibenzoyl derivative that cannot react with the diazotized sulfanilic acid whereas histidine yields only a monobenzoyl derivative that can further react with diazonium acid. However, it has been found that when pyridine is added to scavenge the generated HCl, the reaction yield might be reduced." ... 

---