Tolidine, Diazotized

Benzidine is a known carcinogen and should be handled with extreme caution (Fourth Annual Report on Carcinogens NTP 85-002, 1985, p. 37). The solid and its vapors may be rapidly absorbed through skin. Protective clothing and the use of a fume hood are 

B/s-diazotized benzidine has been used to create active-hydrogen-reactive spacer arms on chromatographic matrices (Silman et al., 1966 Lowe and Dean, 1971). The compound may be used similarly to o-tolidine for the conjugation of active-hydrogen-containing molecules (see Section 9.1, this chapter, and Chapter 2, Section 6.1). 

Reactive halogen crosslinkers are mainly specific for sulfhydryl groups at physiological pH, however at more alkaline pH values they can readily cross-react with amines and the imidazole nitrogens of histidine residues. Some reactivity with hydroxyl-containing compounds also may be realized, particularly with dichloro-s-triazine derivatives under alkaline conditions. 

Most of the bis-alkyl halides referenced in the literature are unavailable commercially, and therefore must be synthesized. Some key references to the preparation and use of these compounds for the crosslinking of sulfhydryl-containing proteins and other molecules include Goodlad (1957), Segal and Hurwitz (1976), Ewig and Kohn (1977), Wilchek and Givol (1977), Prestayko et al. (1981), Luduena et al. (1982), Hiratsuka (1988), and Aliosman et al. (1989). 

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Figure 19.23 The conjugation of a tyrosine-containing carrier protein and a tyrosine-containing peptide may be done using bis-diazotized tolidine to form diazo crosslinks.

Mix 0.5 ml of the peptide solution with 0.5 ml of the carrier protein solution. Chill on ice. Add 0.4 ml of the fois-diazotized tolidine solution. There should be a color change from orange to red almost immediately. Continue the reaction for 2 hours on ice in the dark. 

Cell walls in the necrotic tissue of these wounds were browned. Staining with diazotized Q-tolidine and toluidine blue confirmed the polypheno-lic nature of these brown depositions, which may have resulted from the polymerization of the stilbenes present in large quantities in spruce bark. Phenolic residues were deposited on the walls of certain cells internal to the necrotic tissues by 10 days after wounding. By 36 days these cells had become thick-walled. The precise nature of substances responsible for this thickening has not been determined, variable responses being obtained with histochemical tests for lignin (cf. Table I). Suberin was detectable in cells immediately underlying the thick walled cells, which corresponded to the... 

Figure 151 Bis-diazotized tolidine can form cross-links with proteins through available tyrosine, histidine, or lysine residues.

If the hydrochloric acid is added all at once instead of in two portions as described, a solid will be obtained consisting of o-tolidine coated with its dihydrochloride and the diazotization will occur slowly. 

Benzo purpurin.— Tolidine is di-methyl benzidine, that is it bears the same relation to toluene and ortho-toluidine that benzidine does to benzene and aniline. When this is diazotized and the tetrazonium salt coupled with sodium naphthionate the disazo compound resulting is a dye known as benzopmpurin 4B, which is also a substantive dye of a red color. 

In a i-l. beaker 21.2 g. (o.i mole) of d-tolidine is made into a thin paste with 300 cc. of water and dissolved by the addition of 20 cc. (23.6 g., 0.23 mole) of concentrated hydrochloric acid (sp. gr. 1.18), warming a little if necessary. The solution is cooled to 10° with ice, mechanical agitation is started, and 21 cc. (35 g., 0.24 mole) more of concentrated hydrochloric acid added (Note i). This causes partial separation of o-tolidine dihydrochloride. Keeping the temperature at 10-15°, a solution of 14.5 g. (0.2 mole) of gs per cent sodium nitrite in 40 cc. of water is run in rapidly, holding back the last 10 per cent which is added slowly and only as needed to give a distinct positive test for nitrous acid on starch-iodide paper. This excess is maintained for one-half hour. Throughout the diazotization an excess of hydrochloric acid should be present as shown by a strong test... 

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