Metal Ion-Binding Properties of the Diphosphate Ester Analogue, Methylphosphonylphosphate, in Aqueous Solution

The stability constants of the 1:1 complexes formed between methylphosphonylphosphate (MePP3-), CH3P(O)-2-O-PO32-, and Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, or Cd2+ (M2+) were determined by potentiometric pH titration in aqueous solution (25 °C; l = 0.1 M, NaNO3). Monoprotonated M(H;MePP) complexes play only a minor role. Based on previously established correlations for M2+-diphosphate monoester complex-stabilities and diphosphate monoester β-group. basicities, it is shown that the M(Mepp)- complexes for Mg2+ and the ions of the second half of the 3d series, including Zn2+ and Cd2+, are on average by about 0.15 log unit more stable than is expected based on the basicity of the terminal phosphate group in MePP3-. In contrast, Ba(Mepp)- and Sr(Mepp)- are slightly less stable, whereas the stability for Ca(Mepp)- is as expected, based on the mentioned correlation. The indicated increased stabilities are explained by an increased basicity of the phosphonyl group compared to that of a phosphoryl one. For the complexes of the alkaline earth ions, especially for Ba2+, it is suggested that outersphere complexation occurs to some extent. However, overall the M(Mepp)- complexes behave rather as expected for a diphosphate monoester ligand.


INTRODUCTION
Phosphonate derivatives like 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) or (S)-9[3hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) belong to a promising class of nucleotide analogues with antiviral properties [ 1] and indeed, there is hope that therapeutic agents agains, HIV will result from this class. [2,3]PMEA 2and HPMPA 2are evidently analogues of adenosine 5monophosphate (AMP2-) and 2'-deoxyadenosine 5'-monophosphate (dAMp2-). [ 4,5] After twofold phosphorylation by cellular nucleotide kinases, [ 6] the resulting triphosphate analogues can serve as substrates for viral DNA polymerase or reverse transcriptase which subsequently terminate the growing nucleic acid chain. [ 2,7] The fact that most nucleotide-relevant enzymes, like DNA and RNA polymerases, kinases and ATP synthases, are metal ion-(often Zn2+)-dependent [ 8,9] and that they use nucleotides as substrates only in the form of (mostly Mg2+) complexes [ 9] has led to intensive studies of the metal [5] ion-binding properties of HPMPA and especially of PMEA and its derivatives. [ 2,1,11] However, at this time the coordinating properties of the phosphorylated compounds are unknown. Since replacement of an O-P bond by a C-P bond makes a compound more basic, as is known for example from the properties of methyl phosphate [ 12] and methylphosphonate, [ 4] it is desirable to learn how the metal ion-binding properties change if an ester bond of a diphosphate is replaced by a C-P bond. The most simple of the latter mentioned compounds is evidently methylphosphonylphosphate, CH3P(O)-O-PO -(Mepp3-), which can be considered as a diphosphate monoester analogue. [ 13] Very recently we have established the correlation between metal ion complex stability and ligand basicity for a series of structurally related diphosphate monoester ligands by constructing log K4M/R DP) versus PKH(R DP) P Its. [14] Based on these results we are now in the position to study and to va]uate the rrielal "ion-binding properties of methylphosphonylphosphate. We are reporting here the stability constants of the M(MePP)complexes with Ba2+, Sr2+, Ca2+, Mg2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, and Cd2+. As we shall see, in most instances a slight stability enhancement is observed which we attribute to the increased basicity of the phosphonyl group. Vol. 6, No. 6, 1999 Metal [13].
All the other reagents were the same as used previously. [ 12,4]   Under the above given conditions, for the stability constants of the protonated M(H;MePP) complexes (the formation degree of which is small) only estimates could be obtained, which are in part also based on our previous experience with related ligands (for details see ref. [14] and also footnote [22]  The first proton of this species will be released with pK a < 1.5 (cf., e.g., [2]), which means outside of the pH range of this study. For the present case the following two deprotonation equilibria have to be considered:  (1) and (2)) and of Some Related Protonated Phosphonate and Phosphate Ligands (L) as Determined by Potentiometric pH Titrations in Aqueous Solutions at 25 C and 0.1 M (NaNO3) a Acid: H2L or H3L As one might expect, replacement of an O-P bond by a C-P bond is most pronounced if it concerns the P atom in the vicinity of which also the acid-base reaction takes place. Indeed, from the first two entries in Table 1, which refer to the protonated forms of methylphosphonate and methyl phosphate, it is evident that both acidity constants are strongly affected: Replacement of the electron-withdrawing CH30 group by a CH3 group leads to an increase of the PKa values by about to 1.2 pK units. The same replacement in methyl diphosphate leads to a similar but much In H2(MePP)-one proton is certainly bound at the terminal I-phosphate group, whereas the other proton could in principle either be located at the phosphonyl or also at the terminal phosphate group. Since, as we have seen above, replacement of an O-P bond by a C-P bond increases mainly the basicity of the corresponding group, the dominating tautomer of H2(MePP)is expected to be CH3P(O)(OH)-O-P(O)2(OH)-. 3

.2. Stability Constants of M(H;MePP) and M(MePP)-Complexes
The experimental data of the potentiometric pH titrations may be completely described by considering equilibria (1) and (2) as well as (3) (6) The constants for eqs (3b), (4b), and (Sb) are listed in columns 2, 3, and 4 of Table 2, respectively. To the best of our knowledge none of these constants has been determined before. [ 19] Since all the acidity constants, pKHM(HMePP= 3.3 to 5.5 (Table .2., column 4), of the M(H;MePP) complexes are lower than those o{ the FI(MeP,P) 2species, P KH(MePPi 6.57 (Table  1), but also significantly larger than those of H2(MePP)-, PKH2(MePP 1.85, it i cleai" that the metal ions must reside at the phosphonyl phosphate residue and the pi'oton at the terminal phosphate group. As far as the structure of the M(MePP)complexes is concerned, there can be no doubt that they exist as chelates, a formal structure of which is shown at the left (see also the comment regarding Ba(MePP)and outersphere complexation in Section 3.3). It is interesting to note that the order of the stabilities of the M(MePP)complexes does not strictly follow the common Irving-Williams sequence, but instead they follow the order now repeatedly observed [ 12,2] for the stabilities of phosphate metal ion complexes, i.e., Ba 2+ < Sr 2+ < Ca 2+ < Mg 2+ < Ni 2+ < Co 2+ < Mn 2+ < Cu 2+ > Zn 2+ < Cd2+.  (3)) and M(MePP)-Complexes (eq.(4)) as Estimated a and Determined, b Respectively, by Potentiometric pH Titrations in Aqueous Solutions, Together with the Negative Logarithms of the Acidity Constants (eqs (5) and (6) [22] regarding Ba(MePP)-in Section 3.3).
b The errors given are three times the standard errors of the mean value or the sum of the probable systematic errors whichever is larger. The error limits of the derived data, in the present case for column 4, were calculated according to the error propagation after Gauss. c The values listed for the Cu2+/MePP system appear also in table of [13]. d These three values are also given in footnote 131a of [21]. Methylphosphonylphosphate, in Aqueous Solution expected on the basis of the basicity of MePP3-, i.e., on PKfH-IH(,MePP) 6.57, whereas the data point for the Ca 2+ system fits on the reference line and the one for the Ba z+ system is below.
A more rigorous evaluation of this observation is possible because the straight-line equations for the log MIR-DP) versus pKlH(,R.DP) plots for all ten metal ions considered in this study have been defined (seetabl 4 in [14]). Consequently, with a known PKa value of a monoprotonated diphosphate monoester one can calculate the stability of its corresponding M(R-DP)complex. For the present case this means that we are in the position to compare the measured (exptl) stability constants with those calculated (calcd) according to the indicated procedure. This comparison is done best by defining the stability difference expressed in equation (7): The corresponding results are summarized in Table 3. [141. c Regarding the error limits see footnote b of Table 2.
It is evident that the M(MePP)complexes for Mg 2+ and especially for the metal ions of the second half of the 3d series, including Zn 2+ and Cd2+, are on average by about 0.15 log unit more stable than is expected on the basis of the basicity of the terminal phosphate group in MePP3-. It appears to be logic to attribute this increased stability to the higher basicity of the phosphonyl group, compared to that of a phosphoryl group as commonly present in a diphosphate monoester ligand.
Based on this explanation one wonders why the stability "increase" for the alkaline earth ions is in part reversed and becomes even negative following the order Mg 2+ (log zl 0.08+0.04) > Ca 2+ (0.00,-0.04) > Sr 2+ (-0.08+0.05) > Ba 2+ (-0.14+0.06). [ 22] This order is reverse to that of the ionic radii, but it parallels the hydrated radii [ 23] of the alkaline earth ions. Therefore, it is our belief that for Mg 2+ and the divalent ions of the 3d series, including Zn 2+ and Cd2+, complex formation with MePP 3occurs overwhelmingly innersphere (cf. also [14]), whereas for Ca2+, Sr2+, and Ba 2+ outersphere complex formation plays an increasing role. Maybe the larger a metal ion is, the more the methyl group at the phosphorus atom affects solvation and metal ion binding. 4. CONCLUSIONS The present results obtained with MePP 3show that replacement of the ester-phosphoryl group by a phosphonyl residue slightly affects the complex forming properties. Indeed, for the biologically most significant metal ions, i.e., Mg 2+ and Zn2+, a small stability increase is observed. However, since this stability increase is small compared to the overall stability constants of these complexes, one may conclude that, for example, the metal ion-binding properties of monophosphorylated and diphosphorylated PMEA, i.e., the resulting chains then correspond to diphosphate and triphosphate residues, closely resemble of the parent nucleotides as far as metal ion binding at the phosphate residues in a 1:1 ratio is concerned. In accord herewith, in mixed ligand complexes MePP 3behaves as expected for a simple diphosphate monoester. [ 13,24] However, if a 2:1 metal ion ratio is considered, the metal ion binding properties of (2'-deoxy)adenosine 5'-triphosphate (dATp4-/ATP4-) are different from those of diphoshorylated PMEA, i.e. PMEApp4-. From studies of the metal ion promoted hydrolysis of ATP it was concluded[ 2527] that for a facilitated phosphoryl transfer one metal ion should be , coordinated and the other should be located at the terminal ,-phosphate group of the triphosphate chain. Indeed, X-ray structural studies of a kinase have confirmed this. [ 28] For a nucleotidyl transfer as it is catalyzed by nucleic acid polymerases the two metal ions should be in a M(o)-M(13,,) coordination to facilitate the bond break between theand -phosphate groups. [ 25,26] X-ray studies confirmed also in this case that two metal ions are involved.[ 29,30] As far as the latter mentioned situation is concerned, PMEApp 4is favored over (d)ATP 4in accord with studies showing that PMEApp 4is initially a better substrate for polymerases. [ 3] The explanation [ 21] for this observation is that the participation of the ether oxygen in metal ion binding, [ 4,32] giving rise to 5-membered chelates, and the enhanced basicity of the phosphonate group (as shown now) favor M(o)-M(,) coordination [ 21]  Of course, once the PMEA moiety is incorporated in the growing nucleic acid chain, this is terminated due to the lack of a 3'-hydroxy group, thus leading to the antiviral action of PMEA.