300 P. Alexander, S. F. Cousens and K. A. Stagey 



Reaction with nucleic acids 



Our attention has so far been confined almost entirely to 

 DNA*; in this substance one might expect reaction with the 

 phosphate and the amino imino groups and ring nitrogen of 

 the purine and pyrimidines, all of which are in the electro- 

 philic (or reactive) form at pH 7. With mustard gas — present 

 in very large excess — Elmore and co-workers (1948) obtained 

 extensive esterification of the phosphate groups of DNA and 

 indication for reactions with amino groups based on changes in 

 the titration curve, but more recent work on the titration 

 behaviour of DNA invalidates their interpretation. Alexander 

 (1952) found that all the other types of radiomimetic alkylat- 

 ing agents readily esterified the phosphate groups of DNA 

 under mild conditions. 



With the biologically highly active mustard from /-phenyl- 

 alanine (Bergel and Stock, 1954), i.e. CB 3025. 



/CM 9 • CH 9 * CI 



COOHCHCH2 



0~K 



Cxl2 ' Cxl2 ' ^1 



NH2 



we were able to show, when the mustard was not present in 

 excess, that the only reaction which could be detected with 

 native DNA is esterification of the phosphate groups. By 

 using an automatic titrator the rate of release of acid due to 

 hydrolysis of the mustard group could be followed while 

 maintaining the pH throughout at 7. In the presence of DNA 

 the amount of acid produced is decreased (Fig. 2) and this 

 provides a direct measure of the number of phosphate groups 

 which have been esterified. Acid is liberated to the same 

 extent when the mustard group is hydrolysed by the water 

 or when it alkylates an amino group : 



>NCH2CH2C1 + H2O — > >NCH2CH2 0H + HCl 



>NCH2CH2C1 + RNH2 — > >NCH2CH2NHR + HCl. 



* The DNA we used was obtained from the sperm heads of herring by a 

 modification of the method of Kay, Simmons and Bounce (1952). The protein 

 content is less than • 1 per cent and the molecular weight of different batches 

 as determined by light-scattering ranged from 6 to 6 • 8 X 10^. 



