Methylene analogues of adenosine 5′‐tetraphosphate

Adenosine 5′‐polyphosphates have been identified in vitro , as products of certain enzymatic reactions, and in vivo . Although the biological role of these compounds is not known, there exist highly specific hydrolases that degrade nucleoside 5′‐polyphosphates into the corresponding nucleoside 5′‐triphosphates. One approach to understanding the mechanism and function of these enzymes is through the use of specifically designed phosphonate analogues. We synthesized novel nucleotides: α,β‐methylene‐adenosine 5′‐tetraphosphate (pppCH 2 pA), β,γ‐methylene‐adenosine 5′‐tetraphosphate (ppCH 2 ppA), γ,δ‐methylene‐adenosine 5′‐tetraphosphate (pCH 2 pppA), αβ,γδ‐bismethylene‐adenosine 5′‐tetraphosphate (pCH 2 ppCH 2 pA), αβ, βγ‐bismethylene‐adenosine 5′‐tetraphosphate (ppCH 2 pCH 2 pA) and βγ, γδ‐bis(dichloro)methylene‐adenosine 5′‐tetraphosphate (pCCl 2 pCCl 2 ppA), and tested them as potential substrates and/or inhibitors of three specific nucleoside tetraphosphatases. In addition, we employed these p 4 A analogues with two asymmetrically and one symmetrically acting dinucleoside tetraphosphatases. Of the six analogues, only pppCH 2 pA is a substrate of the two nucleoside tetraphosphatases (EC 3.6.1.14), from yellow lupin seeds and human placenta, and also of the yeast exopolyphosphatase (EC 3.6.1.11). Surprisingly, none of the six analogues inhibited these p 4 A‐hydrolysing enzymes. By contrast, the analogues strongly inhibit the ( asymmetrical ) dinucleoside tetraphosphatases (EC 3.6.1.17) from human and the narrow‐leafed lupin. ppCH 2 ppA and pCH 2 pppA, inhibited the human enzyme with K i values of 1.6 and 2.3 n m , respectively, and the lupin enzyme with K i values of 30 and 34 n m , respectively. They are thereby identified as being the strongest inhibitors ever reported for the ( asymmetrical ) dinucleoside tetraphosphatases. The three analogues having two halo/methylene bridges are much less potent inhibitors for these enzymes. These novel nucleotides should prove valuable tools for further studies on the cellular functions of mono‐ and dinucleoside polyphosphates and on the enzymes involved in their metabolism.