Roger Tribolet

The Imidazole Group and Its Stacking Properties in Mixed Ligand Metal Ion Complexes

Abstract The imidazole group is an important metal ion binding site in biosystems. However, the fact that this group is also able to undergo stacking and hydrophobic interactions with other aromatic or aliphatic residues is less well known. In this Comment several examples are summarized for the solid state, as well as for solutions of low-molecular-weight mixed ligand complexes with an intramolecular ligand-ligand interaction involving the imidazole ring. Such an interaction occurs, e.g., in Cu(histidinate)(AA) complexes, where AA - = tryptophanate, phenylalaninate or valinate, and the formation degree of the intramolecular adduct decreases in this order. In addition, evidence is presented for a purine-imidazole stack in M(adenosine 5′-triphosphate)(imidazole)2- complexes. It is thus becoming obvious that the imidazole group does not only bind metal ions but also has additional and rather significant structuring properties via the possibility to undergo stacking and to form hydrophobic adducts with other...

Influence of decreasing solvent polarity (dioxane–water mixtures) on the stability and structure of binary and ternary complexes of adenosine 5′-triphosphate and uridine 5′-triphosphate

The influence of dioxane on the complex equilibria involving the reactants adenosine 5′-triphosphate (ATP4–), uridine 5′-triphosphate (UTP4–), Cu2+, 2,2′-bipyridyl (bipy), and 1,10-phenanthroline (phen) has been determined. The concentration dependence of the chemical shifts of the protons of bipy and phen has been measured and the self-association property of these aromatic ligands (arm) was quantified with the isodesmic model of indefinite non-co-operative stacking. The stacking tendency is considerably diminished by dioxane: e.g., in D2O Kphenself= 31.1 ± 3.4 l mol–1 and in 50%(v/v)[2H8]dioxane–D2O kphenself= 0.63 ± 0.13 l mol–1. Similarly, the formation of the binary stacks (arm)(ATP)4– is also inhibited by dioxane: the stability of these adducts decreases by factors of Ca. 1/20 (or more) by changing the solvent from water to 50%(v/v) dioxane–water. The acidity constants of the mentioned (two-fold protonated) nucleoside 5′-triphosphates (NTP) and the stability constants of their binary and ternary complexes have been determined by potentiometric pH titrations in water, 30 and 50%(v/v) dioxane–water. By using the results obtained for the UTP systems mainly for comparisons, the following three intramolecular equilibria have been evaluated. (i) The proton in Cu(H-ATP)–may be located at N-1 or at the terminal γ-phosphate group: in water the isomer with the proton at N-1 occurs in significant amounts (∼50%), while in the dioxane–water mixtures the phosphate-protonated isomer strongly dominates. (ii) Cu(ATP)2– exists in two forms: one isomer has a phosphate co-ordination only, while the other is a macrochelate involving in addition N-7; the macrochelated isomer decreases from 68% in water to about 24% in 50% dioxane–water. (iii) While there is evidence that intramolecular stacks may also be formed in Cu(arm)(H-NTP)– and Cu(arm)(UTP)2–(and as far as possible their formation was quantified), the extent of stacking in the Cu(arm)(ATP)2– systems could be well characterized: e.g., with Cu(phen)(ATP)2– in water, ca. 92% exists in the stacked form and in 50%(v/v) dioxane–water ca. 49% of the ternary complex still remains stacked. This means, by going from water to 50% dioxane–water the stability of the metal-bridged Cu(arm)(NTP)2– stacks decreases only by a factor of Ca. 1/2, while the stability of unbridged binary (arm)(NTP)4– stacks decreases by Ca. 1/20 (or more). Similar trends are expected for the corresponding equilibria with other metal ions; the related search for selectivity regarding biological systems is discussed.

Solvent effects on intramolecular hydrophobic ligandligand interactions in binary and ternary complexes

Abstract The stability constants of mixed ligand complexes of the type M(Phen)(ACA) + , where M = Cu 2+ or Zn 2+ , Phen = 1,10-phenanthroline and ACA − = propionate, valerate and 2-cyclohexylacetate, were determined by potentiometric pH titration in 50% (v/v) dioxane water and were compared with the stabilities of the corresponding ternary complexes formed with formate and acetate. The ternary complexes containing the alkanecar☐ylates (ACA − ) are significantly more stable, due to intramolecular hydrophobic interactions between the alkyl residue of the ACAt¯ligands and the 1,10-phenanthroline molecule. For Zn(Phen)(valerate) + this intramolecular ligand-ligand interaction was confirmed by 1 H NMR shift measurements. The formation degree of the intramolecular adducts in the ternary Cu 2+ and Zn 2+ complexes was calculated and the position of the intramolecular equilibrium between the opened and closed isomer was determined: the closed isomer occurs between about 10 to 35 percent. Comparisons with related data show that the extent of this interaction is about the same in water and in 50% aqueous dioxane; this contrasts with the experience made with simple unbridged adducts, which are destabilized by the addition of dioxane (or other organic solvents). Furthermore, evaluation of the available stability data for the Cu 2+ /leucinate (Leu − ) system shows that addition of some dioxane to an aqueous solution (in which of the closed isomer exists to about 20%) favors the intramolecular interaction between the two isopropyl residues in Cu(Leu) 2 considerably: in 40 to 50% aqueous dioxane the formation degree of the closed isomer reaches about 80%. Higher concentrations of the organic solvent destabilize the hydrophobic interaction. The overall stability of Cu(Leu) + and Cu(Leu) 2 , as well of Cu(alaninate) + and Cu(alaninate) 2 , is governed by the polarity of the solvent while the extent of the intramolecular ligand-ligand interaction is influenced by the hydrophobic properties of the solvent molecules. Based on the stability data it is shown that intramolecular ligand ligand interactions are quite a common feature for many binary and ternary amino acid complexes: e.g. , M(norvalinate) 2 , M(phenyl-alaninate) 2 , M(tyrosinate) 2 [M = Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ ] or Cu(tyrptophanate) 2 and M(phenylalaninate)(norvalinate) or M(phenylalaninate)(tyrosinate) [M = Co 2+ , Ni 2+ , Cu 2+ ]. In addition, evidence is presented that direct M 2+ -aromatic interactions are of no significance in these amino acid complexes in solution. The relevance of the present results with regard to biological systems is indicated.

Synergism between different metal ions in the dephosphorylation of adenosine 5'-triphosphate (ATP) in mixed metal ion/ATP systems, and influence of a decreasing solvent polarity (dioxane-water mixtures) on the dephosphorylation rate. Effects of Mg2+, Na+, and NH4+ ions

The initial rates of the dephosphorylation (i.e., v0 = d[PO4]/dt) of adenosine 5′-triphosphate (ATP) (=10−3 M) in the mixed metal ion systems Cu2+/ATP/Mg2+, Ni2+, Zn2+, or Cd2+ with the ratios 1:1:1 and 1:1:5 have been measured and compared at pH 5.5 and 50°C (I = 0.1, NaClO4) with the corresponding binary ATP/M2+ 1:2 and 1:6 systems. The remarkable result is that addition of Mg2+ to a Cu2+/ATP 1:1 system accelerates the dephosphorylation rate significantly more than the same amounts of Mg2+ accelerate the reaction in a Mg2+/ATP 1:1 system. The same synergism, based also on the Cu2+/ATP 1:1 system, is observed with Ni2+, but not with Zn2+ and Cd2+. This observation is attributed to the formation of a prereactive state in the Cu2+/ATP 1:1 system, i.e., of a [Cu(ATP)]24− dimer which involves purine-stacking and a Cu2+/N-7 interaction; the inherent reactivity in this dimer may be triggered by the addition of Mg2+ or Ni2+. In Zn2+ or Cd2+/ATP 1:1 systems also a prereactive state is formed and therefore no synergism is observed in a comparison with the corresponding mixed Cu2+ systems. In agreement herewith, there is a rather pronounced synergism in the Zn2+/ATP Mg2+ system at pH 7.5, and a somewhat smaller one under the same conditions in the Zn2+/ATP/Na+ system. In the latter system the synergism may be considerably favored by reducing the solvent polarity, i.e., by changing the solvent from water to 50% (v/v) dioxane-water; similar effects, though less pronounced, are observed with Zn2+/UTP. In connection with the solvent effects it is recalled that the polarity in the active-site cavities of enzymes is also lower than in the bulk water. By experiments with ATP and Mg2+/ATP systems it is shown that Na+ and NH4+ have corresponding effects; this observation is important regarding cationic side chains of amino acid residues in proteins. Some further implications of the present results for biosystems are also indicated. The phosphate groups in TNP are labeled as α, β, and γ, where the latter refers to the terminal phosphate group (Fig. 1). If nothing else is specified, the formula PO4 reperesents all related species which may be present in solution, i.e., H3PO4, H2PO4−, HPO42−, and PO43−. The term “dephosphorylation” is used for the transfer of a phosphoryl group to a water molecule; the term “hydrolysis” can also refer to this, but for the most part we use it in connection with the formation of hydroxo complexes of metal ions. The terms monomeric or dimeric complexes mean that one or two NTP4− together with at least the corresponding equivalents of M2+ are within the considered complex; hence, e.g., M2(NTP) is a monomeric (but dinuclear) nucleotide complex whereas [M(NTP)]24− is a dimeric one.

Influence of dioxane on the extent of intramolecular hydrophobic ligand-ligand interactions in the binary Cu2+ 1:2 complexes of L-leucinate, L-valinate and L-norvalinate

Abstract The stability constants of the binary Cu(AA)+ and Cu(AA)2 complexes, where AA− = L-alaninate (Ala−), L-leucinate (Leu−), L-valinate (Val−) or L-norvalinate, have been determined by potentiometric pH titrations in water, and in 30, 50, 70 and 80% (v/v) dioxane-water mixtures (I = 0.1 M, NaNO3; 25 °C). The overall stability of Cu(AA)+ and Cu(AA)2 is governed for all four amino acetates (AA−) by the polarity of the solvent, while the extent of the intramolecular hydrophobic ligand-ligand interaction between the aliphatic side-chains in Cu(Val)2, Cu(Leu)2 and Cu(Nva)2 is obviously influenced by the hydrophobic solvation properties of the organic solvent molecules. Based on the stability difference Δ log KAA* = log KCu(AA)2Cu(AA) - log KCu(AA)Cu it is shown that Cu(Val)2, Cu(Leu)2 and Cu(Nva)2 are more stable than Cu(Ala)2, and this increased stability is taken as evidence for hydrophobic side- chain interactions in Cu(Val)2, Cu(Leu)2 and Cu(Nva)2; such interactions are not possible in Cu(Ala)2 due to the small size of the methyl side-chain. By using the stability data of the Cu2+/Ala− system as a basis for the evaluation, the extent of the hydrophobic ligand-ligand interaction (= closed form) in the other three Cu(AA)2 complexes is calculated: the percentages of the closed forms vary between about 10 and 30% (based on Cu(AA)2/tot). The formation degree of the closed species is influenced by the solvent: addition of some dioxane to an aqueous solution favors their formation, contrary to the experience with simple unbridged hydrophobic adducts which are destabilized. Such a destabilization of the closed Cu(AA)2 species occurs only at high concentrations of the organic solvent (usually more than 70%). The general relevance of the present results, especially with regard to biological systems, is indicated.

An estimation of the equivalent solution dielectric constant in the active‐site cavity of metalloenzymes

The stability constants of the 1:1 complexes between Cu2+ and Zn2+ with formate, acetate and several phenylalkanecarboxylates, i.e. C6H5-(CH2)n-COO- with n = 0 to 5, are summarized for water, 50% aqueous ethanol and 50% aqueous dioxane (I = 0.1 M; 25 degrees C): Complex stability depends upon carboxylate group basicity. The influence of varying amounts of ethanol or dioxane (up to 90%) on the stability of the Cu2+ and Zn2+ (M2+) complexes with formate and acetate (CA) was measured by potentiometric pH titrations. The values for pKHH(CA) and log KMM(CA) increase, as expected, with increasing amounts of the organic solvents, i.e. with decreasing solvent polarity. The changes in the equilibrium constants are also evaluated with regard to the mole fractions of the organic solvents and the corresponding dielectric constants. These results may be used to estimate for low dielectric cavities in proteins the equivalent solution dielectric constant on the basis of enhanced carboxylate basicity or metal ion binding capability (method 1). Furthermore, the measured stability constants are used for comparisons of the coordination tendency of carboxylate ligands towards zinc(II)-metalloenzymes (method 2); in this way the equivalent solution dielectric constants in the active-site cavities of bovine carbonic anhydrase and carboxypeptidase A are estimated: the values are of the order of 35 and 70, respectively. This method seems to be generally applicable to metalloproteins.

Intramolecular ionic interactions in ternary amino acid complexes [1]

Abstract The so-called ‘noncovalent’ interactions [4] between biomolecules are not only crucial for the structural organization of high molecular weight biological systems, but also determine to a large extent the structure of amino acid and nucleotide containing low molecular weight metal ion complexes in solution. For example, the intramolecular equilibria between different isomers of mixed ligand complexes are well established for aromatic-ring stacking and hydrophobic interactions [3, 5, 6]. Ionic interactions between oppositely charged side-chains of two amino acids coordinated to the same metal ion are also known [7], but the extent of this interaction has so far hardly been characterized [6]. Therefore, the ionized forms of the following two amino acids were selected for such a study: In the (CH3)3 N + -residue the positive charge is somewhat shielded, but the advantage of this residue is that no hydrogen bonds can be formed with −O3S−, as would have been the case with the more common H3 N + - group. In addition, the latter group and the use of a carboxylate group (instead of the sulfonate residue in HC) would have led to additional proton equilibria, thus complicating the systems significantly. For comparison DL-alanine (Ala) with its non-reactive side chain was also employed. As a representative metal ion Cu2+ was used, because it forms rather stable complexes with the glycinate-like structural unit. That ionic interactions between the (CH3)3 N + - and −O3S-residues are possible was proven by 1H-NMR shift experiments with benzenesulfonate and the tetramethylammonium ion; the binary adduct has a stability constant of 0.7 M−1 in aqueous solution at 34 °C (I = 0.1, NaNO3). The results for the binary amino acid parent systems are given in Table I [8]. There are indications that the sulfonate group of HC interacts with an apical position of the Cu2+ coordination sphere and that decreasing ionic strength and the addition of dioxane favor this interaction. Such apical interactions are also known for related ligands [10]. The percentage of the ‘closed’ isomer of the ternary Cu(TMO)(HC) complex, i.e. of the isomer with an intramolecular ionic ligand-ligand interaction, was calculated [5, 6, 11] using the results obtained for the Ala−/Cu2+/HC2− system as a basis. From Table II it is evident that the ‘intensity’ of the ionic interaction increases with decreasing ionic strength and also with the decreasing polarity of the solvent; in other words, with the decreasing water activity. This result is exactly the behavior expected for ionic interactions, and it also holds if the optically pure ligand systems are used. The described ionic interactions are relatively weak (ΔG° ⋍ −3 kJ/mol), but they are strong enough to allow ‘flexible structural organizations’ in biological systems. t001 . Negative Logarithms of the Acidity Constants for the Protonated Ligands and Logarithms of the Stability Constants for the Binary Amino Acid Cu2+ Complexes (25 °C). Solvent Ligand (A) pKHH2A pKHHA log KCuCuA log KCuACuA2 H2O/I = 0.1 Ala− 2.39 9.81 8.23 6.82 TMO 1.94 8.81 7.41 6.29 HC2− 2.22 9.04 8.03 6.47 H2O/I = 0.01 Ala2− 2.36 9.86 8.42 6.95 TMO 1.88 a 8.71 7.25 6.16 HC2− 2.33 9.19 8.36 6.47 60% dioxane (40% H2O; v/v) I = 0.01 Ala2− 3.49 10.31 10.84 8.81 TMO 2.58 8.84 9.20 7.69 HC2− 3.65 10.06 11.30 8.18 a This value was measured for experimental reasons at I = 0.02, NaCl4. t002 . Logarithms of the Stability Constants for the Ternary Amino Acid Cu2+ Complexes and Percentage of the Isomer with an Intramolecular Ionic Ligand-Ligand Interaction (25 °C). Solvent Ligands A/B log βCuCuAB Δlog KCu a2 %[CuAB]cl H2O/I = 0.1 HC2−/Ala2− 15.22 ± 0.01 −1.04 HC2−/TMO 14.57 ± 0.01 −0.87 32 H2O/I = 0.01 HC2−/Ala− 15.57 ± 0.01 −1.21 HC2−/TMO 14.77 ± 0.01 −0.84 57 60% dioxane (40% H2O; v/v) I = 0.01 HC2−/Ala− 20.03 ± 0.02 −2.11 HC2−/TMO 18.99 ± 0.02 −1.51 75 a2 Δ KCu = log βCuCuAB − (log KCuCuA + log KCuCuB) = log KCuACuAB − log KCuCuB (see [2, 3, 5, 6, 11]).