Abstract
The acidity constants of the twofold protonated acyclic nucleotide analogue 9-[2-(phosphonomethoxy)- ethyl]-8-azaadenine, H2(9,8aPMEA)±, as well as the stability constants of the M(H;9,8aPMEA)+ and M(9,8aPMEA) complexes with the metal ions M2+ =Ni2+, Cu2+ or Zn2+, have been determined by potentiometric pH titrations in aqueous solution at I=0.1 M (NaNO3) and 25℃. The result for the release of the first proton from H2(9,8aPMEA)+ (pKa= 2.73), which originates from the (N1)H+ site, was confirmed by UV-spectrophotometric measurements. Application of previously determined straight-line plots of log KMM(R-PO3) versus PKH3(R-HPO3)' for simple phosph(on)ate ligands, R- PO-, where R represents a residue without an affinity for metal ions, proves that the primary binding site of 9,8aPMEA2- is the phosphonate group for all three metal ions studied. By stability constant comparisons with related ligands it is shown, in agreement with conclusions reached earlier for the Cu(PMEA) system [PMEA2-=dianion of 9-[2- (phosphonomethoxy)ethyl]adenine], that in total four different isomers are in equilibrium with each other, i.e. (i) an open isomer with a sole phosphonate coordination, M(PA)op, where PA2-=PMEA2-or 9,8aPMEA2-, (ii) an isomer with a 5-membered chelate involving the ether oxygen, M(PA)cl/o, (iii) an isomer which contains 5- and 7-membered chelates formed by coordination of the phosphonate group, the ether oxygen and the N3 site of the adenine residue, M(PA)cl/O/N3, and finally (iv) a macrochelated isomer involving N7, M(PA)cl/]N7. The Cu2+ systems of PMEA2- and 9,8aPMEA2- behave quite alike; the formation degrees for Cu(PA)op, CuM(PA)cl/O, Cu(PA)cl/O/N3 and Cu(PA)cl/N3 are approximately 16, 32, 45 and 7%, respectively, which shows that Cu(PA)cl/N7 is a minority species. In the Ni2+ and Zn2+ systems the open isomer is the dominating one followed by M(PA)cl/O, but there are indications that the other two isomers also occur to some extent.
Highlights
The acyclic nucleoside phosphonate, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), known as Adefovir [1], can be considered as an analogue of (2’-deoxy)adenosine 5’-monophosphate ((d)AMP2-) [2].PMEA has excellent antiviral properties [1] and in the form of its bis(pivaloyloxymethyl)ester, Adefovir dipivoxil, it has recently been approved by the US Food and Drug Administration (FDA) for the treatment [3]of hepatitis B patients; these people suffer from an infection of a DNA virus
Since polymerases depend on the presence of metal ions [4], we have studied over the past few years the metal ion-binding properties of PMEA in detail [2,5,6], and suggested a mechanism [7] which explains why diphosphorylated PMEA is initially an excellent substrate for nucleic acid polymerases [8,9]
The stability determining binding site of PMEA2- is the phosphonate group; biologically important metal ions like Mg2+, Ca2+, Mn2+ and Zn2+ are able to interact with the ether oxygen atom and this gives rise to the following intramolecular equilibrium (1) [2,5,6]: HO
Summary
The acyclic nucleoside phosphonate, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), known as Adefovir [1], can be considered as an analogue of (2’-deoxy)adenosine 5’-monophosphate ((d)AMP2-) [2].PMEA has excellent antiviral properties [1] and in the form of its bis(pivaloyloxymethyl)ester, Adefovir dipivoxil, it has recently been approved by the US Food and Drug Administration (FDA) for the treatment [3]of hepatitis B patients; these people suffer from an infection of a DNA virus. The stability determining binding site of PMEA2- is the phosphonate group; biologically important metal ions like Mg2+, Ca2+, Mn2+ and Zn2+ are able to interact with the ether oxygen atom and this gives rise to the following intramolecular equilibrium (1) [2,5,6]: HO %"M2+ R-- O,,, ,,,0 () ",M2+ This proposed metal ion-ether oxygen interaction is crucial for the suggested polymerase mechanism [7] which agrees with the observation that deletion of this ether oxygen or a change in its position in the aliphatic chain leads to compounds which are biologically inactive [8,9,10].
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