Metal Ion-Coordinating Properties in Aqueous Solution of the Antivirally Active Nucleotide Analogue (S)-9-[3-Hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA). Quantification of Complex Isomeric Equilibria

Acyclic nucleoside phosphonates are of medical relevance and deserve detailed chemical characterization. We focus here on ( S )‐9‐[3‐hydroxy‐2‐(phosphonomethoxy)propyl]adenine (HPMPA) and include for comparison 9‐[2‐(phosphonomethoxy)ethyl]adenine (PMEA), as well as the nucleobase‐free (phosphonomethoxy)ethane (PME) and ( R )‐hydroxy‐2‐(phosphonomethoxy)propane (HPMP). The acidity constants of H 3 (HPMPA) + were determined and compared with those of the related phosph(on)ate derivatives; they are also needed to understand the properties of the metal ion complexes. Given that in vivo nucleotides and their analogues participate in reactions typically as divalent metal ion (M 2+ ) complexes, the stability constants of the M(H;HPMPA) + and M(HPMPA) species with M 2+ = Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , and Cd 2+ were measured. Comparisons between the results for HPMPA 2- and the previous data for PMEA 2- , HPMP 2- and PME 2- revealed that for most M(HPMPA) complexes the enhanced stability (the enhancement relative to the stability of a simple phosphonate‐M 2+ coordination), can solely be explained by the formation of 5‐membered chelates involving the ether oxygen. These chelates occur in equilibrium with simple ′open′ phosphonate‐M 2+ species, the phosphonate group being the primary binding site. The only exceptions are the M(HPMPA) complexes of Ni 2+ , Cu 2+ , and Zn 2+ , which show an additional stability enhancement; in these instances not only the indicated 5‐membered chelates are formed, but M 2+ coordinates in addition to N3 of the adenine residue forming a 7‐membered chelate ring. This observation regarding N3 is important because it emphasizes the metal ion affinity of this site (which is often ignored). Note that in the DNA double helix N3 is exposed to the solvent in the minor groove. The stability data for the monoprotonated M(H;HPMPA) + complexes suggest that these carry H + at the phosphonate group whereas M 2+ is partly at the nucleobase and partly also at the phosphonate group. The ratios of these isomers depend on the metal ion involved, e.g., for Cu(H;HPMPA) the ratio of the isomers is about 1:1

Metal Ion Complexes of Nuceloside Phosphorothioates Reflecting the Ambivalent Properties of Lead (II)

This Perspective outlines the coordinating properties of lead( II ), to some extent in comparison with related metal ions like Ca 2+ , Zn 2+ or Cd 2+ . It is worth noting that the affinity of Pb 2+ towards phosphate residues corresponds to that of Cu 2+ . Furthermore, the binding tendency of Pb 2+ towards thiophosphate groups as present in methyl thiophosphate (MeOPS 2− ) or uridine 5′- O -thiomonophosphate (UMPS 2− ) is compared with that of the parent ligands, that is, methyl phosphate (CH 3 OPO 3 2− ) and uridine 5′-monophosphate (UMP 2− ). The replacement of an O by a S atom makes the monoprotonated thiophosphate group considerably more acidic [compared to ROP(O) 2 − (OH)], but at the same time its affinity for Pb 2+ increases tremendously: more than 99% of Pb 2+ is S-bound. This is very different if the coordinating properties of uridylyl-(5′→3′)-[5′]-uridylate (pUpU 3− ) and P -thiouridylyl-(5′→3′)-[5′]-uridylate (pUp (S) U 3− ) are compared. The phosphate-coordinated Pb 2+ forms a 10-membered chelate with one of the two terminal O atoms of the phosphodiester linkage, which reaches a formation degree of about 90% in Pb(pUpU) − . However, in Pb(pUp (S) U) − the formation degree of the chelate is reduced to about half in accordance with the fact that now only one terminal O atom is available in the thiophosphate diester bridge, that is, Pb 2+ coordinates to this O showing no affinity for S in ROP(O)(S) − OR′. These observations are ascribed to the properties of the Pb 2+ lone pair, which shapes the Pb 2+ coordination sphere; its role is discussed further in this Perspective and a caveat is made regarding Pb 2+ binding to a thiophosphate diester linkage

Extent of intramolecular π stacks in aqueous solution in mixed-ligand copper(II) complexes formed by heteroaromatic amines and the anticancer and antivirally active 9-[2-phosphonomethoxy)ethyl]guanine (PMEG).✩ a comparison with related acyclic nucleotide analogues

The acyclic nucleoside phosphonate (ANP2– ) 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) is anticancer and antivirally active. The acidity constants of the threefold protonated H3(PMEG)+ were determined by potentiometric pH titrations (aq. sol.; 25°C; I = 0.1 M, NaNO3). Under the same conditions and by the same method, the stability constants of the binary Cu(H;PMEG)+ and Cu(PMEG) complexes as well as those of the ternary ones containing a heteroaromatic N ligand (Arm), that is, of Cu(Arm)(H;PMEG)+ and Cu(Arm)(PMEG), where Arm = 2,2'-bipyridine (Bpy) or 1,10-phenanthroline (Phen), were measured. The corresponding equilibrium constants, taken from our earlier work for the systems with 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) and 9-[2-(phosphonomethoxy)ethyl]-2,6-diamino-purine (PMEDAP) as well as those for Cu(PME) and Cu(Arm)(PME), where PME2– = (phosphonomethoxy)ethane = (ethoxymethyl)phosphonate, were used for comparisons. These reveal that in the monoprotonated ternary Cu(Arm)(H;PE)+ complexes, the proton and Cu(Arm)2+ are at the phosphonate group; the ether oxygen of the -CH2-O-CH2-P(O) 2 ! (OH) residue also participates to some extent in Cu(Arm)2+ coordination. Furthermore, the coordinated Cu(Arm)2+ forms a bridge with the purine moiety undergoing π-π stacking which is more pronounced with H·PMEDAP– than with H·PMEA– . Most intense is π stack formation (st) with the guanine residue of H·PMEG– ; here the bridged form Cu(Arm)(H·PMEG) st + occurs next to an open (op), unbridged (binary) stack, formulated as Cu(Arm)2+/(H·PMEG) op ! . – The unprotonated and neutral ternary Cu(Arm)(PE) complexes are considerably more stable than the corresponding Cu(Arm)(R-PO3) species, where R-PO3 2! represents a phosph(on)ate ligand with a group R that is unable to participate in any intramolecular interaction. The observed stability enhancements are mainly due to intramolecular stack formation (st) between the aromatic rings of Arm and the purine residue in the Cu(Arm)(PE) complexes and also, to a smaller extent, to the formation of fivemembered chelates involving the ether oxygen of the -CH2-O-CH2-PO 3 2! residue (cl/O) of the PE2– species. The quantitative analysis of the intramolecular equilibria reveals three structurally different Cu(Arm)(PE) isomers; e.g., of Cu(Phen)(PMEG) ca. 1.1% exist as Cu(Phen)(PMEG)op, 3.5% as Cu(Phen)(PMEG)cl/O, and 95% as Cu(Phen)(PMEG)st. Comparison of the various 3 formation degrees reveals that within a given Cu(Arm)(PE) series the stacking tendency decreases in the order PMEG2– ≥ PMEDAP2– > PMEA2– . Furthermore, stacking is more pronounced in the acyclic Cu(Arm)(PE) complexes compared with that in the Cu(Arm)(NMP) species, where NMP2– = corresponding parent (2'-deoxy)nucleoside 5'-monophosphate. Here is possibly one of the reasons for the biological activity of the ANPs. One is tempted to speculate that the pronounced stacking tendency of PMEG2– , together with a different H-bonding pattern, leads to enhanced binding in the active site of nucleic acid polymerases, thus being responsible for the pronounced anticancer and antiviral activity of PMEG

Complex Formation of Lead(II) with Nucleotides and Their Constituents

Lead is widely distributed in the environment; it is known to mankind for thousands of years and its toxicity is nowadays (again) well recognized, though on the molecular level only partly understood. One of the reasons for this shortcoming is that the coordination chemistry of the biologically important lead(II) is complicated due to the various coordination numbers it can adopt (CN = 4 to 10) as well as by the 6s² electron lone pair which, with CN = 4, can shield one side of the Pb²⁺ coordination sphere. The chapter focuses on the properties of Pb²⁺ complexes formed with nucleotides and their constituents and derivatives. Covered are (among others) the complexes formed with hydroxy groups and sugar residues, the interactions with the various nucleobases occurring in nucleic acids, as well as complexes of phosphates. It is expeced that such interactions, next to those like with lipids and proteins, are responsible for the toxic properties of lead. To emphasize the special properties of Pb²⁺ complexes, these are compared as far as possible with the corresponding properties of the Ca²⁺, Fe²⁺, Cu²⁺, Zn²⁺, and Cd²⁺ species. It needs to be mentioned that the hard-soft rule fails with Pb²⁺. This metal ion forms complexes with ligands offering O donors of a stability comparable to that of Cu²⁺. In contrast, with aromatic N ligands, like imidazole or N7 sites of purines, complex stability is comparable to that of the corresponding Fe²⁺ complexes. The properties of Pb²⁺ towards S donor sites are difficult to generalize: On the one hand Pb²⁺ forms very stable complexes with nucleoside 5′-O-thiomonophosphates by coordinating to nearly 100% at S in the thiophosphate group; however, on the other hand, once a sulfur atom replaces one of the terminal oxygen atoms in the phosphodiester linkage, macrochelate formation of the phosphate-bound Pb²⁺ occurs with the O and not the S site. Quite generally, the phosphodiester linkage is a relatively weak binding site, but the affinity increases further to the mono-and then to the di-and triphosphate. The same holds for the corresponding nucleotides, though the Pb²⁺ affinity had to be estimated via that of the Cu²⁺ complexes for some of these ligands. Complex stability of the pyrimidine-nucleotides (due to their anti conformation) is solely determined by the coordinating tendency of the phosphate group(s); this also holds for the Pb²⁺ complex of adenosine 5′-monophosphate. For the other purinenucleotides macrochelate formation takes place by the interaction of the phosphate-coordinated Pb²⁺ with the N7/(C6)O site of, e.g., the guanine residue. The extents of the formation degrees of these chelates are summarized. Unfortunately, information about mixed ligand (ternary) or other higher order comlexes is missing, but still it is hoped that the present overview will help to understand the interaction of Pb²⁺ with nucleotides and nucleic acids, and especially that it will facilitate further research in this fascinating area

Probing the Stacking Properties of Nucleosides and Nucleotides with Heteroaromatic Amines and the Effect of Metal Ion-Bridging on the Stability of the Stacks. Implications for biological systems.

Noncovalent interactions play important roles in modern chem. research encompassing also bio- systems. Among these interactions arom.-ring stacking is esp. pronounced as, next to hydrogen bonding, hydrophobic and ionic interactions, it is crucial for the three-dimensional structure of nucleic acids (RNA, DNA) and proteins (enzymes). The authors have now measured by 1H-NMR shift expts. the stability of binary stacks formed between purine nucleosides or nucleotides (N) and the "indicator" ligand 1,10-phenanthroline (Phen), and the authors also reviewed the related literature. Surprisingly, the stability of the (Phen)(N) adducts is largely independent of the type of purine residue involved, including deprotonation, e.g., at (N1)H of a guanine moiety, and also of the location of the phosphate group at the ribosyl ring. This contrasts with the self-stacking tendency which decreases within the series adenosine < guanosine < inosine < cytidine approx. uridine. The formation of an ionic (+/-) or metal ion (M2+) bridge stabilizes the formation of stacks as obsd., e.g., in mixed ligand Cu2+ complexes formed between Phen and AMP (5'-AMP2-); yet, in these instances the position of the phosphate group at the ribosyl ring affects the stability of the stacks: It decreases in the order 2'-AMP2- < 5'-AMP2- < 3'-AMP2- in the Cu(Phen)(AMP) complexes demonstrating a significant steric discrimination. The stability of stacks also depends on the size of the arom.-ring systems involved; as one would expect, purines stack better than pyrimidines and Phen generally better than 2,2'-bipyridine. Results obtained with various mixed ligand metal ion complexes contg. ATP (ATP4-) and an amino acid anion (Aa-) lead to the conclusion: The recognition of the adenine residue by the amino acid side chain in M(ATP)(Aa)3- complexes decreases in the series tryptophan (indole residue) < histidine (imidazole residue) < leucine (iso-Pr residue) < alanine (Me group). This type selectivity is certainly of relevance for amino acid/protein interactions with nucleotides/nucleic acids. The addn. of an org. solvent like 1,4-dioxane reduces the solvent polarity and decreases the stability of binary stacks like (Phen)(ATP)4- dramatically; in contrast, intramol. stacks, as present in Cu(Phen)(ATP)2-, are much less affected. Because the "effective" dielec. const. or permittivity in the active site cavity of an enzyme or a ribozyme is lower than in bulk water, Nature has here a further tool to achieve selectivity. Here it needs to be noted that the involved changes in free energy (ΔG0) are small; e.g., a formation degree of 20% of an intramol. stack in a mixed ligand complex corresponds only to -0.6 kJ mol-1 allowing Nature to shift such equil. easily

Intramolecular pi-pi stacking interactions in aqueous solution in mixed-ligand copper(II) complexes formed by heteroaromatic amines and the nucleotide analogue 9-[2-(phosphonomethoxy)ethyl]-2-aminopurine (PME2AP), an isomer of the antivirally active 9-[2-( phosphonomethoxy)ethyl]adenine (PMEA)

The stability constants of the mixed-ligand complexes formed between Cu(Arm)(2+), where Arm = 2,2`-bipyridine (Bpy) or 1,10- phenanthroline (Phen), and the monoanion or the dianion of 9-[2-(phosphonomethoxy)ethyl]-2-aminopurine (PME2AP), a structural isomer of the antivirally active 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), were determined by potentiometric pH titrations in aqueous solution at 25 degrees C and I = 0.1M (NaNO3). Detailed stability constant comparisons reveal that in the monoprotonated ternary Cu(Arm)(H;PME2AP)(+) complexes the proton is at the phosphonate group and that stacking between Cu(Arm)(2+) and H(PME2AP)(-) plays a significant role. The ternary Cu(Arm)(PME2AP) complexes are considerably more stable than the corresponding Cu(Arm)(R-PO3) species, where R-PO32- represents a phosph(on)ate ligand with a group R that is unable to participate in any kind of interaction within the complexes. The increased stability is attributed to intramolecular stack formation in the Cu(Arm)(PME2AP) complexes and also, to a smaller extent, to the formation of 5-membered chelates involving the ether-oxygen present in the -CH2-O-CH2-PO32- residue of PME2AP(2-). This latter interaction was previously quantified by studying ternary Cu(Arm)(PME) complexes (PME2- = dianion of (phosphonomethoxy)ethane), which can form the 5-membered chelates but where no intramolecular ligand-ligand stacking is possible. Application of these results allows a quantitative analysis of the intramolecular equilibria involving three structurally different Cu(Arm)(PME2AP) species; e. g., about 5% of the Cu(Bpy)(PME2AP) system exist with the metal ion solely coordinated to the phosphonate group, 15% as a 5-membered chelate involving the ether-oxygen atom of the -CH2-O-CH2-PO32- residue, and 80% with an intramolecular pi-pi stack between the purine moiety of PME2AP(2-) and the aromatic rings of Bpy. Finally, comparison of the stacking properties of PME2AP(2-) and PMEA(2-) in their ternary complexes reveals that stacking is somewhat more pronounced in the Cu(Arm)(PMEA) than in the Cu(Arm)(PME2AP) species. Speculatively, this reduced stacking intensity, together with a different hydrogen-bonding pattern, could well lead to a different positioning of the 2- aminopurine moiety (compared to the adenine residue) in the active site cavity of nucleic acid polymerases and thus be responsible for the reduced antiviral activity of PME2AP compared with that of PMEA. (C) 2008 Elsevier B. V. All rights reserved

Acid–base and metal ion-binding properties of thiopyrimidine derivatives

The thionucleoside 2-thiocytidine (C2S) as well as the thiouridines (US) occur in Nature, especially in transfer RNAs, and they also receive attention in diverse fields like nanotechnology and drug research. If (C2)O in cytidine (Cyd) is replaced by (C2)S to give the thio analogue C2S, the release of H + from (N3)H in H(C2S) + (p K a = 3.44) is facilitated somewhat [H(Cyd) + ; p K a = 4.24], yet, the deprotonation of the (C4)NH 2 group is much more affected: the p K a decreases from ca. 16.7 in Cyd to 12.65 in C2S. This is because the amino-thione tautomer dominating in the neutral C2S, transfers into the imino-thioate form, which has the charge largely localized on (C2)S − . As a consequence, the M(C2S) 2+ species (M 2+ = Zn 2+ or Cd 2+ ) transfer very easily into their deprotonated M(C2S − H) + forms. This reaction is extremely facilitated by M 2+ coordination at (C2)S − and occurs already at a pH slightly above 3. It is shown that the (C2)S M 2+ coordination dominates to more than 99% in both the M(C2S) 2+ and the M(C2S − H) + complexes; their structures, including chelate formation with the participation of N3, are evaluated. In 2-thiouridine (U2S), 4-thiouridine (U4S), and 2,4-dithiouridine (U2S4S), the release of H + from (N3)H, compared to Urd (p K a = 9.18), is facilitated by ca. 1 to 2 p K units, the charge being largely localized on the (C)S sites; this leads with (U4S − H) − and (U2S4S − H) − to the reduction of Cu(II) to Cu(I), transforming the thiolate into a disulfide. In Cu(U2S − H) + Cu(II) is stable, most likely due to steric constraints inhibiting disulfide formation. The stability of the M(US − H) + complexes with Ni 2+ , Cu 2+ or Cd 2+ is enhanced by about 1.3 to 2 log units compared to the corresponding uridinate complexes. The properties of the biologically relevant Zn(US − H) + are expected to be between those with Ni 2+ and Cd 2+ . The relatively high affinity of the (C)S sites for these M 2+ is reflected in the 2-thiouridine 5′-monophosphate (U2SMP 2− ) and 4-thiouridine 5′-monophosphate (U4SMP 2− ) complexes, M 2+ being located to more than 99% at the thiouracil residue and only traces are coordinated at the phosphate group. In the N3-deprotonated Cu[(U2S − H)MP] − species the anti conformer is partly turned into the syn one allowing thus a formation degree of about 60% of the macrochelate formed by (C2)S − and the phosphate group. The corresponding coordination pattern also seems to hold for Cd[(U2S − H)MP] − , though the formation degree of the macrochelate is lower. No macrochelate formation is detected for Ni[(U2S − H)MP] − , as well as for Ni[(U4S − H)MP] − and Cd[(U4S − H)MP] − . The reasons for the indicated coordination patterns are discussed, as well as the biological implications of the summarized results, especially with regard to tRNAs

Metal Ion-Binding Properties of (S)-1-[3-Hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC, Cidofovir). A Nucleotide Analogue with Activity Against DNA Viruses

The acyclic nucleoside phosphonate ( S )-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC; Cidofovir) has been approved for clinical use in antiviral therapy. We determined the acidity constants of H 2 (HPMPC) ± , as well as that of the nucleobase-free (hydroxy-2-(phosphonomethoxy)propane (H(HPMP) − ) ( I = 0.1 M, NaNO 3 ; 25 °C). Given that in vivo nucleotides and their analogues participate in reactions typically as metal ion (M 2+ ) complexes, the stability constants of the M(H;HPMPC) + , M(HPMPC), and M(HPMP) complexes with M 2+ = Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , and Cd 2+ were measured. Comparisons between results for HPMP 2− and previous data for PME 2− ( CH3CH2OCH2PO32-"&gt; ; phosphonomethoxyethane) revealed the hydroxyl-group effect. The hydroxyl group stabilizes only complexes with the heavier alkaline earth metal ions (Ca 2+ , Sr 2+ , Ba 2+ ). For all other complexes, the enhanced stability can solely be explained by the formation of 5-membered chelates involving the ether oxygen; these occur in equilibrium with simple 'open' phosphonate-M 2+ species. The stability of the M(HPMPC) complexes is also higher than expected for a phosphonate-only coordination, indicating that chelates are formed, but comparison with the HPMP 2− data shows that the cytosine base does not affect complex stability. Similar observations were made previously with related cytosine derivatives. The stability data for the monoprotonated M(H;HPMPC) + complexes suggest that these carry H + predominantly on the phosphonate group, and M 2+ on the nucleobase

Extent of metal ion-sulfur binding in complexes of thiouracil nucleosides and nucleotides in aqueous solution

Previously published stability constants of several metal ion (M2+) complexes formed with thiouridines and their 5`-monophosphates, together with recently obtained log K-M(U)(M) versus pK(U)(H) plots for M2+ complexes of uridinate derivatives (U-) allowed now a quantitative evaluation of the effect that the exchange of a (C)O by a (C)S group has on the stability of the corresponding complexes. For example, the stability of the Ni2+, Cu2+ and Cd2+ complexes of 2-thiouridinate is increased by about 1.6, 2.3, and 1.3 log units, respectively, by the indicated exchange of groups. Similar results were obtained for other thiouridinates, including 4-thiouridinate. The structure of these complexes and the types of chelates formed (involving (N3)(-) and (C)S) are discussed. A recently advanced method for the quantification of the chelate effect allows now also an evaluation of several complexes of thiouridinate 5`-monophosphates. In most instances the thiouracilate coordination dominates the systems, allowing only the formation of small amounts of phosphate-bound isomers. Among the complexes studied only the one formed by Cu2+ with 2-thiouridinate 5`-monophosphate leads to significant amounts of the macrochelated isomer, which means that in this case Cu2+ is able to force the nucleotide from the anti to the syn conformation, allowing thus metal ion binding to both potential sites and this results in the formation of about 58 isomer. The remaining 42 coordinated to the thiouracilate residue; Cu2+ binding to the phosphate group occurs in this case only in trace amounts. (c) 2007 Elsevier Inc. All rights reserved