Buffer catalysis of amino proton exchange in compounds of adenosine, cytidine and their endocyclic N-methylated derivatives.

Abstract

The use of buffer catalysts having a wide range of pK (dissociation) values (4-12) provides the first estimates of two generally useful empirical parameters of amino proton exchange in compounds of adenine and cytosine. These are a nucleobase amino group dissociation constant (pKD) and the 'encounter frequency' for proton transfer (kD), which can be used to predict amino proton exchange rates. Values of amino pKD fall in the range 8.6-9.4 for the unsubstituted nucleobases and their endocyclic N-methylated derivatives. Similar values of kD are obtained for all nucleobases (1 X 10(8) M-1 s-1). These constants were obtained from a statistical fit of second-order catalytic rate constants for amino proton exchange, measured by amino 1H-NMR lineshape at varying field frequencies (100, 300 and 360 MHz). These results confirm the requirement for buffer conjugate base formation and nucleobase protonation, but point to a different mechanism of exchange at low pH; most probably direct amino protonation for adenine, but not for cytosine compounds. Anionic buffer conjugate bases (phosphate and acetate) show a greater catalytic effect than neutral (nitrogen) bases, especially with cytosine compounds. The use of high concentrations of sodium perchlorate to sharpen amino 1H resonances of 1-methyladenosine is examined, with respect to chemical and rotational exchange and NMR line broadening.