Synthesis of typical diazonium salts

Traylor and Singer (1967) have provided a detailed synthesis of the diazonium salts XIX and XX. Compound XIX has been used as an affinity label for acetylcholinesterase on erythrocyte membrane (Wofsy and Michaeli 1967) and for the cholinergic receptor from eel electroplax (Changeux et al. 1967). XX has been used for the modifica- 

These same workers accomplished the synthesis of the radioactive XX as follows. Sodium nitrite (38.2 mg, 0.553 mmole) in 0.17 ml of water was added dropwise to a solution of 34.4 mg of ( H)-m-nitro-aniline and 30.9 mg of carrier m-nitroaniline in 0.5 ml of 50 % fluoroboric acid and 0.25 ml of water. The reaction mixture was stirred magnetically during the addition and for an additional 1 hr. After the product precipitated as a cream solid, it was filtered and washed successively with ice cold 25 % fluoroboric acid, anhydrous ethanol and anhydrous ether. The product could be recrystallized as white needles from acetone-ether if the temperature did not exceed 50°C. The yield was 39.3 mg specific activity was 7.72 x 10 dpm//imole. The product can be stored at — 20°C in 0.01 M hydrochloric acid at a concentration of 2x10 M. 

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. 

Spectral properties of monoazo coupling products with histidine and tyrosine  

The monoazo derivative of tyrosine formed with XX was prepared in the following way. A solution of 237 mg (1.0 mmole) of XX in 30 ml of 0.01 N HCl was added dropwise to N-chloroacetyltyrosine (309 mg, 1.2 mmoles) in 25 ml of 0.2 M sodium phosphate buffer, pH 6.2, with continuous stirring at 0°C. Aqueous sodium hydroxide was added constantly to maintain the reaction mixture at pH 6.2. Three hr after the completion of the addition of N-chloroacetyl tyrosine, the stirring was discontinued and the reaction mixture was left at room temperature overnight. The reaction mixture formed a gel and then was acidified to pH 2 with 12 N HCl. The product precipitated out and could be recrystallized from methanol and then aqueous acetone. The crystals thus obtained melted at 214-215° with decomposition. 

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