Derivatization of phosphopeptides with mercapto- and amino-functionalized conjugate groups by phosphate elimination and subsequent Michael addition.

Kinetics of the beta-elimination of the phosphate group from H-Tyr-Ser(PO3H2)-Phe-OH and H-Tyr-Thr(PO3H2)-Phe-OH and subsequent addition of thiols and amines to the dehydroalaninyl and beta-methyldehydroalaninyl residues formed, were followed by RP HPLC under alkaline conditions in the absence and presence of Ba2+ ions. By this reaction sequence, the phosphoserinyl peptide was conjugated with mono-N-(2-mercaptoethyl)amide of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (4), a mercapto-functionalized pentapeptide, H-His-Gly-Gly-His-Gly-NH(CH2)4SH, and an amino-functionalized fluorescent dye, 5-dimethylaminonaphthalene-1-[N-(5-aminopentyl)]sulfonamide (dansyl cadaverine). The beta-methyldehydroalanine residue was, in turn, observed to be a poor Michael acceptor.

Hydrolytic reactions of diribonucleoside 3',5'-(3'-N-phosphoramidates): kinetics and mechanisms for the P-O and P-N bond cleavage of 3'-amino-3'-deoxyuridylyl-3',5'-uridine.

Hydrolytic reactions of 3'-amino-3'-deoxyuridylyl-3',5'-uridine (2a), an analogue of uridylyl-3',5'-uridine having the 3'-bridging oxygen replaced with nitrogen, have been followed by RP HPLC over a wide pH range. The only reaction taking place under alkaline conditions (pH > 9) is hydroxide ion-catalyzed hydrolysis (first-order in [OH(-)]) to a mixture of 3'-amino-3'-deoxyuridine 3'-phosphoramidate (7) and uridine (4). The reaction proceeds without detectable accumulation of any intermediates. At pH 6-8, a pH-independent formation of 3'-amino-3'-deoxyuridine 2'-phosphate (3) competes with the base-catalyzed cleavage. Both 3 and in particular 7 are, however, rather rapidly dephosphorylated under these conditions to 3'-amino-3'-deoxyuridine (5). In all likelihood, both 3 and 7 are formed by an intramolecular nucleophilic attack of the 2'-hydroxy function on the phosphorus atom, giving a phosphorane-like intermediate or transition state. Under moderately acidic conditions (pH 2-6), the predominant reaction is acid-catalyzed cleavage of the P-N3' bond (first-order in [H(+)]) that yields an equimolar mixture of 5 and uridine 5'-phosphate (6). The reaction is proposed to proceed without intramolecular participation of the neighboring 2'-hydroxyl group. Under more acidic conditions (pH < 2), hydrolysis to 3 and 4 starts to compete with the cleavage of the P-N bond, and this reaction is even the fastest one at pH < 1. Formation of 2'-O,3'-N-cyclic phosphoramidate as an intermediate appears probable, although its appearance cannot be experimentally verified. The rate constants for various partial reactions have been determined. The reaction mechanisms and the effect that replacing the 3'-oxygen with nitrogen has on the behavior of the phosphorane intermediate are discussed.