Kurt H. Scheller

Macrochelate formation in monomeric metal ion complexes of nucleoside 5'-triphosphates and the promotion of stacking by metal ions. Comparison of the self-association of purine and pyrimidine 5'-triphosphates using proton nuclear magnetic resonance

The concentration dependence of the chemical shifts of the protons H-2, H-8, and H-I’ or H-5, H-6, and H-1’ of A T P , I T P , and G T P or C T P and U T F ( = N T P ) , respectively, and of the corresponding nucleosides has been measured. The results are consistent with the isodesmic model of indefinite noncooperative stacking; the association constants for NTP4are between 1.3 (ATP4-) and about 0.4 M-’ (UTP4-) and for the nucleosides between 15 (adenosine) and 1.2 M-’ (uridine). The self-stacking tendency decreases within the series adenosine > guanosine > inosine > cytidine uridine. Due to the repulsion of the negatively charged phosphate moieties, this trend is much less pronounced for the corresponding N T F series. The charge effect also governs the series adenosine >> AMP2> ADP3ATP4-. Likewise the self-association tendency of ATP4-, ITP, and GTP‘is promoted by a factor of about 3-5 by the coordination of Mg2+ to the phosphate moiety, which neutralizes part of the negative charge a t this residue. However, the self-association tendency of Zn(ATP)2and Cd(ATP)2is much larger than of Mg(ATP)’-; this is explained by an increased tendency to form an intermolecular metal ion bridge in the dimeric stacks in which Zn2+ or Cd2+ is coordinated to the phosphate moiety of one ATP4and to N-7 of the adenine residue of the other A T P . The shifts of H-8 for complete stacking (6,) agree with this interpretation. There is no significant increase in stability in Zn(ITP)2and Zn(GTP)2-: Le., the stability of these stacks is governed only by the charge neutralization-the effect of Zn2+ is the same as that of Mg2+. Comparison of the shifts of H-8 at infinite dilution (6,) for several systems reveals that an M2+/N-7 interaction exists in the monomeric Zn2+ and CdZt complexes of the purine 5’-triphosphates; Le., a macrochelate is formed through an inrramolecular coordination of the metal ion to the phosphate moiety and to N-7. The position of this concentration-independent equilibrium between the open isomer (with phosphate coordination only) and the macrochelated isomer is estimated by comparing 6o of M(NTP)2with the shifts of H-8 for complete complex formation of the corresponding metal ion-nucleoside complexes, which were also determined. The N M R study gives no hint for such an N-7 interaction either for the corresponding Mg(NTP)2complexes or for a base interaction in any of the pyrimidine 5’-triphosphate complexes. These N M R results prompted an evaluation of stability data (obtained earlier under conditions where no self-association occurs), which give further evidence that macrochelate formation also occurs in the M(NTP)’complexes of purine nucleotides with Mn2+ Co2+, Ni2+, Cu2+, and Zn2+; no evidence for such an interaction is observed in the pyrimidine nucleotide complexes. Ionizkon of the base moiety in I T P , G T F , UTF, and TTP favors, however, the base-metal ion interaction and therefore also the formation of macrochelates in the M(NTP-H)3complexes. The percentage of the macrochelated isomer is estimated for all these systems: the whole range from nearly 100% ring back-bonding to only insignificant traces is observed. The ambivalent coordinating properties of nucleotides and their structural versatility are discussed. I t is now well-known tha t metal ions a r e essential in a large variety of biological processes, including those with nucleic acids and their der ivat ives4 For example, D N A polymerase contains tightly bound Zn2+, and there is evidence that this metal ion binds the enzyme to DNA.5s6 To fulfill its function the enzyme must also be activated by a divalent cation such as Mg2+ or Mn2+, and these metal ions bind the nucleoside triphosphate substrates to the enzyme.’,* Thus the interplay between metal ions and nucleotides, or their derivatives, is receiving much attention at

Metal ion/buffer interactions. Stability of alkali and alkaline earth ion complexes with triethanolamine (tea), 2-amino-2(hydroxymethyl)-1,3-propanediol (tris)and 2-[bis(2-hydroxyethyl)-amino] 2(hydroxymethyl)-1,3-propanediol (Bistris) in aqueous and mixed solvents☆

Abstract The acidity constants of the protonated buffers given in the title, i.e. of H(Tea) + , H(tris) + and H(Bistris) + , have been measured at 25 °C in water, 50% aqueous dioxane or methanol, and in 75 or 90% dimethylsulfoxide (Dmso) with tetramethylammonium nitrate as background electrolyte. The interaction of Tea, Tris, or Bistris (L) with the alkali or alkaline earth ions (M n+ was studied by potentiometric pH titrations in the same solvents and the stability constants of the ML n+ complexes were determined. The stability constants, log K M ML , of the Na + complexes with the several buffer-ligands in the given solvents vary from −1.05 [Na(Tea) + in H 2 O; I = 1.0] to 0.54 log units [Na(Bistris) + in 90% Dmso; I = 0.25]; the corresponding values for the Mg 2+ complexes range from 0.24 [Mg(Tea) 2+ in H 2 O; I = 1.0] to 0.91 log units [Mg(Bistris) 2+ in 90% Dmso; I = 0.25]. Unexpectedly, Ca(Bistris) 2+ is the most stable among the alkaline earth ion complexes in aqueous solution (log K Ca Ca(Bistris) = 2.25; the corresponding values for the Mg 2+ , Sr 2+ and Ba 2+ complexes are 0.34, 1.44 and 0.85, respectively; I = 1.0), while in 90% Dmso Sr(Bistris) 2+ is most stable (log K Sr Sr(Bistris) = 1.87; the corresponding values for the Mg 2+ , Ca 2+ and Ba 2+ complexes are 0.91, 1.64 and 1.14, respectively; I = 0.25). A similar, but less pronounced pattern is observed for the M(Tea) n+ complexes. obviously, the stabilities of the alkaline earth ion complexes with Bistris and Tea follow neither the order of the ionic radii nor that of the hydrated radii of the cations. In contrast, in all solvents the stability of the alkali ion complexes increases with decreasing ionic radii; this being also true for the alkaline earth iron complexes of Tris in aqueous solution. The possible reasons for these observations, the structures of the complexes in solution, and some biological implications are discussed. Calculations of the extent of complex formation show that in the physiological pH range the concentration of certain complexes may be quite pronounced; hence reservations should be exercised in employing these buffers in systems which also contain metal ions.

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.

Metal ion complexes of d-biotin in solution. Stability of the stereoselective thioether coordination

Abstract By spectrophotometry and 1 H nmr, several of the stability constants of the thioether complexes between Mg 2+ , Ca 2+ , Mn 2+ , Cu 2+ , Zn 2+ , Cd 2+ , or Ag + and d -biotin (Bio), tetrahydrothiophene (Tht), and dimethyl sulfide (Dms) have been measured in 50% aqueous ethanol, 96% N,N -dimethylformamide (DMF), 98% d 6 -dimethyl sulfoxide, or in D 2 O. With decreasing concentration of water, the thioether interaction increases with the biologically important metal ions, whereas, e.g., Ag + behaves in the opposite way. The stability of these complexes is, in general, quite small: for example, with d -biotin in 96% DMF ( I = 1.0; 25°C) log K M(Bio) M = 0.03 and 1.64 for Cu 2+ and Ag + , respectively; in D 2 O ( I = 0.5 for Ag + , all others 2–5; 27°C) log K M(Bio) M ≅ −1.0, −1.4, −1.2, −0.9, or 4.20 for Mg 2+ , Ca 2+ , Zn 2+ , Cd 2+ , or Ag + . In those cases where the difference log K M(Tht) M − log K M(Bio) M can be calculated, it is in the order of 0.3 log units; this observation, as well as the chemical shifts measured, confirm the earlier suggestion that the interaction at the sulfur of biotin is stereoselective: the metal ion coordinates from “below” the tetrahydrothiophene ring of biotin to the sulfur atom, i.e., trans to the urea ring. It is emphasized that despite the low stability of these complexes with the biologically meaningful metal ions, the extent of the interaction is enough to create specific structures.

1H-NMR study on self-association and macrochelate formation in metal ion systems of nucleoside 5′-diphosphates

Abstract Nucleotide and their complexes are substrates for many enzymic reactions [1]. The self-association via aromatic ring stacking of nucleoside 5′-mono- [2] and 5′-triphosphates [3] is now well established and the structures for the complexes of these nucleotide (=N) in solution are relatively well characterized [3, 4]. Much less is known about nucleoside 5′-diphosphates (NDP3−). Therefore, the concentration dependance of the chemical shifts for the protons H-2, H-8, and H-1′ of ADP3− and IDP3−, H-8 and H-i′ of GDP3−, and H-5, H-6, and H-1′ of CDP3− and UDP3− has been measured in D2O at 27°C. The results are consistent with the isodesmic model [3] of indefinite non-cooperative stacking (eqn. 1); the association constants for NDP3− are between 1.8 (ADP3−) and about 0.6M−1 (UDP3−. In agreement with earlier results [3] obtained under the same conditions for nucleosides and nucleoside 5′-triphosphates, the self-stacking tendency of the base moieties of the nucleic acids decreases in the series adenine > guanine ≳ hypoxanthine > cytosine ∼ uracil. Due to the repulsion of the negatively charged phosphate moieties, the self-association is always less pronounced for NDP3− compared with the corresponding nucleoside. Accordingly, the addition of Mg2+ to ADP3− favors self-stacking, as is obvious from Fig. 1: the curvature of the lines is more pronounced for the Mg(ADP)− system (K = 6.4 M−1) than for the ADP3− system. Generally, the self-stacking tendency of the NDP3− systems is promoted by a factor of about 2–3 by the coordination of Mg2+ to the phosphate moiety. However, the self-association tendency of Zn(ADP)− and Cd(ADP)− or Zn(IDP)− and Cd(IDP)− is much larger than of Mg(ADP)−; this is explained [5] by an increased tendency to form an intermolecular metal ion bridge in dimeric stacks, which may then further associate. In these dimeric stacks Zn2+ or Cd2+ is coordinated to the phosphate moiety of one NDP3− and to N-7 of the purine residue of the other NDP3−. The shifts of H-8 (and H-2) for complete stacking (δ∞) agree with this interpretation. Comparison of the shifts of H-8 at infinite dilution (δo) reveals that an M2+/N-7 interaction exists in the monomeric Zn2+ and Cd2+ complexes of purine NDP3−; i.e. a macrochelate is at least partially formed by an intramolecular coordination of the metal ion to the phosphate moiety and to N-7. The NMR study gives no hint for such an interaction in the corresponding Mg(NDP)− complexes or in any of the pyrimidine-NDP3− complexes [5]. An evaluation of the stability data available [6] gives further evidence for the existence of the concentration-independent equilibrium 2 between an open and a macrochelated isomer in purine-NDP3− complexes. For the M(ADP)− complexes of Mn2+, Co2+, Ni2+, Cu2+, and Zn2+, about 60, 70, 80, 95, and 75 percent respectively exist in the macrochelated form [5]. No evidence for such an isomer is found for Mg(ADP)− and Ni(CDP)− The ambivalent coordinating properties of nucleotide and their structural versatility is evident from these results. It should be emphasized that in studies aiming to evaluate the properties of monomeric nucleotide and their complexes, low concentrations must be employed (often

Influence of solvent and ligand-structure on the extent of intramolecular stacking interactions in mixed ligand complexes

Abstract The importance of aromatic-ring stacking for the creation of certain structural arrangements in large bio-molecules has often been emphasized ( e.g. [1]). However, the fact that at low concentrations stacking interactions between smaller molecules like amino acids and nucleotides can be promoted by the formation of a metal ion-bridge has only recently been recognized [2]. For example, the stacking between the indole moiety of tryptophanate (Trp − and the purine system of adenosine 5′-triphosphate (ATP 4− ) is facilitated in ternary M(ATP)(Trp) 3− complexes [3, 4]. Similarly, the hydrophobic interaction between the isopropyl moiety of leucinate (Leu − ) and the purine residue of ATP 4− is also promoted in M(ATP)(Leu) 3− complexes [4]. Based on the stability constants of the complexes and 1 H-NMR shift experiments the percentage of M(A)(B) 3− cl was estimated for both types of ternary complexes: The formation of the species M(ATP)(Trp) 3− cl was first shown [5] in 1974; its occurrence was subsequently confirmed by studies in several laboratories using different methods [6]. To learn more about the factors which govern the position of the intramolecular equilibrium (1), we have now used the following simple systems, since with them structural alterations are easily achieved: In 50% aqueous dioxane (I = 0.1, 25 °C) the stacking interaction is most pronounced for n = 1; i.e. for the ternary complex Cu(Phen)(C 6 H 5 CH 2 COO) + about 60 percent exists in the closed form. If phenylacetate is replaced by 2-(β-naphthyl)acetate the concentration of the closed isomer increases to about 80 percent. Ligands R(CH 2 ) n COO − with n = 0 or n > 1 form ternary complexes with a less pronounced intramolecular stacking interaction. Variation of the solvent composition also leads to a change in the percentage of [Cu(Phen)(C 6 H 5 CH 2 COO + ] cl : solvent H 2 O 30% diox. 60% diox. 90% diox. % closed isomer: 48 56 64 48 The observation of a maximal degree of formation for the closed isomer in about 60% aqueous dioxane is very surprising, because the stability of binary adducts like (Phen)(RCOO − ) decreases with increasing dioxane concentration. Hence, two opposite effects must be operating in the presence of metal ions. This puzzling result is of interest for biological systems, because in these the water activity may also be altered, e.g. at the surface or in grooves of proteins. Hydrophobic interactions are important, e.g. , in adduct formation between carboxypeptidase A and the inhibitor β-phenylpropionate [7]. Presently we are repeating the experiments in several ethanol/water mixtures to provide a broader generalization.

Comparison of the ligating properties of disulphides and thioethers: dimethyl disulphide, dimethyl sulphide, and related ligands

The stability constants of 1 : 1 complexes between dimethyl disulphide (dmds) or dimethyl thioether (dms) and Ca2+, Zn2+, Cd2+, Pb2+, or Ag+(KML for Mn++ L ⇌[ML]n+) have been determined in aqueous solution by 1H n.m.r. shift measurements. The results [e.g. log KCa(dmds)ca.–1.4, log KCd(dmds)ca.–1.4, log KAg(dmds)= 2.01 ± 0.09; log KCa(dms)ca.–1.6, log KCd(dms)=–0.3 ± 0.2, and log KAg(dms)= 3.7 ± 0.3] show that the complexes with soft metal ions are stronger than those with borderline or hard metal ions. It is also evident that the ligating properties of the thioether moiety are somewhat more pronounced towards borderline and soft metal ions than those of the disulphide group. Spectrophotometrically determined stability constants (in 50% aqueous ethanol) for the complexes between Mn2+, Cu2+, or one of the above metal ions and dmds, diethyl sulphide, or tetrahydrothiophen accord with this. In addition, for several complexes of tetrahydrothiophen-2-carboxylate (thtc–) and 1,2-dithiolan-3-carboxylate [= tetranorlipoate (tnl–)], the dimensionless constants for the intramolecular equilibrium between the chelated isomer (which is bonded to the metal by the sulphur atom and the carboxylate group) and the simple carboxylate-co-ordinated isomer have been calculated, together with the percentages of the chelated isomer {e.g. 93 ± 1 for [Cu(thtc)]+ and 41 ± 7 for [Cu(tnl)]+; 55 ± 5 for [Cd(thtc)]+ and ⩽20% for [Cd(tnl)]+; ⩽20% for both [Mn(thtc)]+ and [Mn(tnl)]+}. Possible biological implications of such weak interactions and the resulting intramolecular equilibria are briefly discussed.

Promotion of the hydrolysis of purine-nucleoside 5′-triphosphates by metal ions

Abstract Enzyme-catalyzed transfer of nucleotidyl and phosphoryl groups depends on the presence of metal ions. In consequence, model systems involving metal ion-promoted dephosphorylation of nucleoside 5′-triphosphates (NTP) (with transfer of a phosphoryl group to water) continue to be the focus of considerable attention. While early studies provided useful comparisons on the influence of various metal ions [1], additional investigations have uncovered more subtle but essential features, including (a) the formation and participation of complexes containing more than one metal ion per NTP [2–4], (b) the importance of metal ion interactions with purine bases [2, 5], and (c), the involvement of dimeric purine-nucleotide complexes which arise through base-stacking [2, 5]. For pyrimidine-NTP systems containing Ni 2+ , Cu 2+ or Zn 2+ the reactive species is a complex of type M 2 (NTP)(OH) − , where the nucleic base moiety of the pyrimidine-nucleotide is not involved [4]; this conclusion is confirmed by observations with methyl-triphosphate. In the case of purine-NTP systems the general situation is more complicated: for example, for ATP with Ni 2+ , Cu 2+ or Zn 2+ the reaction proceeds by way of dimeric complexes such as [M 2 (ATP)] 2 [2], which is possibly better formulated as [M 2 (ATP)] 2 -(OH) −1/−2 1 or 2 . [5]. The participation of dimers is in accord with the well-known self-association by base-stacking of purine-nucleotides [6]. The M 2+ /N-7 interaction [6] is also crucial for rapid dephosphorylation [2]. Thus, if the Cu 2+ /N-7 interaction in the Cu 2+ /ATP system is prevented by addition of one equivalent of 2,2′-bipyridyl (Bpy), with formation of a ternary Cu(Bpy)(ATP) 2− complex, the reactivity is reduced markedly and to the level shown by corresponding pyrimidine-NTP systems. The properties of binary Cu 2+ /purine-NTP systems also vary, depending on the stacking properties of the base residues and the extent of the Cu 2+ /N-7 interaction. Further, from 1 H-NMR shift experiments on the Cd 2+ /ATP system, it is evident that the release of N-7 from the coordination sphere of the metal ion by formation of hydroxo-complexes in the upper pH range parallels the decreasing dephosphorylation reactivity of the same system. It appears that one of the nucleotides in the dimer is needed to facilitate transfer of the other into the reactive state. Accordingly, the reactivity of the Cu 2+ /ATP 1:1 system is inhibited by adenosine and phosphate, while addition of AMP 2− further promotes the reactivity. We suggest that AMP 2− is able to take over the role of the ‘structuring’ ATP 4− in the reactive dimer, thus bringing more ATP into the reactive form. We postulate further that in the most reactive species one metal ion is coordinated to the α and β positions of the triphosphate moiety and a second metal ion is coordinated to the γ phosphate group, providing in this way a structure in which the terminal γ group is activated toward dephosphorylation. The metal ion/N-7 interaction seems to facilitate the formation of this reactive intermediate, a conclusion in accord with the lower reactivity of the pyrimidine-NTP and methyltriphosphate systems.