A new procedure for the determination of the N-terminal groups in peptides and proteins is described. The free amino-groups are allowed to react with 2-fluoro-3-nitropyridine or 2-fluoro-5-nitropyridine in slightly alkaline solution ; the resulting derivatives are easily hydrolyzed in dilute hydrochloric acid in sealed tubes to give the corresponding nitropyridyl amino acids. Most of these compounds are quantitatively recovered under these conditions : in particular it is possible to estimate proline and glycine as N-terminal residues.After isolation the nitropyridyl amino acids can be estimated spectrophotometrically ; in some cases it is possible to regenerate the parent amino acid by treating the derivatives with strongly alkaline solutions. The adequacy of the present techniques in relation to those of other workers was tested on insulin, lysozyme and aldolase ; the advantages and peculiarities in comparison with these procedures are discussed.It is well known that the most satisfactory procedure for the determination of the N-terminal residues in peptides and proteins is that of substituting under mild conditions the a-NH, group with a radical stable enough to survive hydrolysis and so constituted as to permit identification and estimation of the N-terminal amino acids after hydrolysis [1-31. The possibility of regenerating the parent amino acids from the derivatives so obtained, offers further advantages since amino acids can be identified and estimated accurately [4,5]. These techniques are of the greatest importance in determining the yield of particular end groups set free by enzymatic or chemical hydrolysis. For this purpose and in order to recover the N-terminal amino acids without hydrolytic losses, it is clearly desirable to use the least drastic conditions of hydrolysis which will allow cleavage of the adjacent peptide bonds.Recently we have established that the acid hydrolysis of 3-and 5-nitro-2-pyridyl peptides is anchimerically assisted and the reaction proceeds quickly in dilute hydrochloric acid [6] ; the pH-rate profiles parallel the ionisation curves of the heterocyclic nitrogen atom. Because of this neighboring group participation highly preferential cleavage of the peptide bond a t the end of the chain is possible. The current difficulties in the determination of the N-terminal groups prompted us to extend the method which employs 2-chloro-3,5-dinitropyridine as reagent [7-91 to the analogous reactions involving 2-fluoro-3-nitropyridine and 2-fluoro-5-nitropyridine.I n earlier studies on the reactivity of some haloheteroaromatic derivatives [lo], we have observed that 2-chloro-3,5-dinitropyridine condenses readily with amino acids and peptides ; mononitro-chloropyridines, however, are not very reactive and failed to condense under the mild conditions required. Although not as reactive as 2-chloro-3,5-dinitropyridine mononitro-fluoropyridines couple with peptides and proteins smoothly in slightly alkaline solution to give practically quantitative yields of the derivatives within 24 hours at...
