A New Method of Chemical Modification of N6‐Amino Group in Adenine Nucleotides with Formaldehyde and a Thiol and Its Application to Preparing Immobilized ADP and ATP

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Abstract
Reaction of AMP with formaldehyde and 3-mercaptopropionic acid at pH 11.7 gave a new AMP derivative, N6-[(2-carboxyethyl)thiomethyl]-AMP (I) in 91% yield and reaction at pH 3.1 gave another new derivative, N6,N6-bis[(2-carboxyethyl) thiomethyl]-AMP (II) in 57% yield. The structures were determined by their 13C and 1H NMR spectra coupled with those of the simple analogs, N6-[(2-carboxyethyl)thiomethyl]-9-methyladenine and N6,N6-bis[(2-carboxyethyl)thiomethyl]-9-methyladenine which were synthesized from 9-methyladenine in the same way as for derivatives I and II. ADP and ATP were treated in the same way as AMP to afford the corresponding carboxyl derivatives, N6-[(2-carboxyethyl)thiomethyl]-ADP (V), N6-[(2-carboxyethyl)thiomethyl]-ATP (VI), N6,N6-bis[(2-carboxyethyl)thiomethyl]-ADP (X) and N6,N6-bis[(2-carboxyethyl)thiomethyl]-ATP (XI) in 71%, 75%, 53% and 40% yield, respectively. These compounds were coupled to 1,3-diaminopropane with a water-soluble carbodiimide to give the corresponding amino derivatives, N6-{[N-(3-aminopropyl)carbamoylethyl]thiomethyl}-ADP (VIII), N6-{[N-(3-aminopropyl)carbamoylethyl]thiomethyl}-ATP (XI), N6,N6-bis{[N-(3-aminopropyl)carbamoylethyl]thiomethyl}-ADP (XIII) and N6,N6-bis{[N-(3-aminopropyl)carbamoylethyl]thiomethyl}-ATP (XIV), which were further bound to CNBr-activated dextran to give new polymer-bound derivatives of ADP and ATP. These free and bound derivatives were tested for their coenzymic activities against several kinases [glycerol-3-phosphate dehydrogenase (EC 1.1.1.8); lactate dehydrogenase (EC 1.1.1.27); glucose-6-phosphate dehydrogenase (EC 1.1.1.49); glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12); hexokinase (EC 2.7.1.1); glycerol kinase (EC 2.7.1.30); pyruvate kinase (EC 2.7.1.40); acetate kinase (EC 2.7.2.1.); phosphoglycerate kinase (EC 2.7.2.3); and apyrase (EC 3.6.1.5).]. The activities of the ADP derivatives, V, VIII, X, XIII dextran-bound VIII, and dextran-bound XIII against acetate kinase were 82%, 81%, 68%, 55%, 35% and 15%, respectively, relative to ADP and those of the ATP derivatives, VI, IX, XI, XIV, dextran-bound IX and dextran-bound XIV against hexokinase were 88%, 94%, 60%, 81%, 58% and 49%, respectively, relative to ATP.