Novel diadenosine polyphosphate analogs with oxymethylene bridges replacing oxygen in the polyphosphate chain

Dinucleoside polyphosphates (Np n N′s; where N and N′ are nucleosides and n  = 3–6 phosphate residues) are naturally occurring compounds that may act as signaling molecules. One of the most successful approaches to understand their biological functions has been through the use of Np n N′ analogs. Here, we present the results of studies using novel diadenosine polyphosphate analogs, with an oxymethylene group replacing one or two bridging oxygen(s) in the polyphosphate chain. These have been tested as potential substrates and/or inhibitors of the symmetrically acting Ap 4 A hydrolase [bis(5′‐nucleosyl)‐tetraphosphatase (symmetrical); EC 3.6.1.41] from E. coli and of two asymmetrically acting Ap 4 A hydrolases [bis(5′‐nucleosyl)‐tetraphosphatase (asymmetrical); EC 3.6.1.17] from humans and narrow‐leaved lupin. The six chemically synthesized analogs were: ApCH 2 OpOCH 2 pA ( 1 ), ApOCH 2 pCH 2 OpA ( 2 ), ApOpCH 2 OpOpA ( 3 ), ApCH 2 OpOpOCH 2 pA ( 4 ), ApOCH 2 pOpCH 2 OpA ( 5 ) and ApOpOCH 2 pCH 2 OpOpA ( 6 ). The eukaryotic asymmetrical Ap 4 A hydrolases degrade two compounds, 3 and 5 , as anticipated in their design. Analog  3 was cleaved to AMP (pA) and β,γ‐methyleneoxy‐ATP (pOCH 2 pOpA), whereas hydrolysis of analog  5 gave two molecules of α,β‐oxymethylene ADP (pCH 2 OpA). The relative rates of hydrolysis of these analogs were estimated. Some of the novel nucleotides were moderately good inhibitors of the asymmetrical hydrolases, having K i values within the range of the K m for Ap 4 A. By contrast, none of the six analogs were good substrates or inhibitors of the bacterial symmetrical Ap 4 A hydrolase.