Reaction of the cytosine nucleus with hydrazine in the presence of bisulphite

Abstract

1. Introduction The presence of a reactive hydrazine group in ti- amino cytosine provides ample opportunities for the specific introduction of labels (fluorescent or radio- active), combinations with carbonyl-containing carriers (cellulose or Sephadex) and for other specific secondary reactions. Also, p-amino cytidine is an effective mutagen [ 11, this effect being evidently caused by the double functional specificity of this compound in the systems of matrix biosynthesis similar to p-hydroxy- and fl-methoxy-derivatives of cytidine and adenosine [2]. By hydrazine action in aqueous neutral solutions the conversion of cytosine into the p-amino cytosine is slow even in rather vigorous conditions [ 3] . As was shown earlier [4-81 , the nucleophilic substitution of the exocyclic amino group of cytosine is greatly facilitated by the presence of bisulphite. In this paper it is shown that p-amino cytosine derivatives can be obtained in mild conditions when cytidine is treated with a bisulphite-hydrazine mix- ture and some kinetic characteristics of this reaction are adduced. 2. Materials and methods Sodium metabisulphite NazSzOs was prepared as described earlier [8] . Hydrazine hydrate was an analytically pure preparation. The reaction was followed by the changes of u.v.- spectra in lOO-fold water dilutions of portions of the reaction mixture. 212 7 mg of 2’-desoxycytidine-5’-phosphate were dissolved in 2 ml of 1 M sodium metabisulphite solution containing hydrazine (hydrazine concentration, pH values and temperature are given in table 1). Immedi- ately after nucleotide dissolution and then after fixed intervals of 0.1 ml portions were taken and diluted in 10 ml of wafer. The spectra were measured lo- 15 minutes after dilution. To study the velocity of the reverse reaction, the mixtures were incubated until no further spectral changes occurred and then portions were diluted SO-fold with 0.5 M buffer solutions (pH 5.0 - sodium acetate, pH 6.0 and 7.0 - sodium phosphate, pH 7.9 - TrisHCl, pH 9.0 - glycine-NaOH). The diluted mixtures were incubated at the required temperature and their spectra measured periodically. The constants of reaction velocity were calculated according to the formula: +? lgDDOI;m t 00