Theoretical calculation of the NMR spin-spin coupling constants and the NMR shifts allow distinguishability between the specific direct and the water-mediated binding of a divalent metal cation to guanine

The calculated intermolecular and intramolecular indirect NMR spin-spin coupling constants and NMR shifts were used for the discrimination between the inner-shell and the outer-shell binding motif of hydrated divalent cations Mg(2+) or Zn(2+) with a guanine base. The intermolecular coupling constants (1)J(X,O6) and (1)J(X,N7) (X = Mg(2+), Zn(2+)) can be unambiguously assigned to the specific inner-shell binding motif of the hydrated cation either with oxygen O6 or with nitrogen N7 of guanine. The calculated coupling constants (1)J(Mg,O6) and (1)J(Zn,O6) were 6.2 and -17.5 Hz, respectively, for the inner-shell complex of cation directly interacting with oxygen O6 of guanine. For the inner-shell coordination of the cation at nitrogen N7, the calculated coupling constants (1)J(Mg,N7) and (1)J(Zn,N7) were 5.6 and -36.5 Hz, respectively. When the binding of the cation is water-mediated, the coupling constant is zero. To obtain reliable shifts in NMR parameters, hydrated guanine was utilized as the reference state. The calculated change of NMR spin-spin coupling constants due to the hydration and coordination of the cation with guanine is caused mainly by the variation of Fermi-contact coupling contribution while the variation of diamagnetic spin-orbit, paramagnetic spin-orbit, and spin-dipolar coupling contributions is small. The change of s-character of guanine sigma bonding, sigma antibonding, and lone pair orbitals upon the hydration and cation coordination (calculated using the Natural Bond Orbital analysis) correlates with the variation of the Fermi-contact term. The calculated NMR shifts delta(N7) of -15.3 and -12.2 ppm upon the coordination of Mg(2+) and Zn(2+) ion are similar to the NMR shift of 19.6 ppm toward the high field measured by Tanaka for N7 of guanine upon the coordination of the Cd(2+) cation (Tanaka, Y.; Kojima, C.; Morita, E. H.; Kasai. Y.; Yamasaki, K.; Ono, A.; Kainosho, M.; Taira, K. J. Am. Chem. Soc. 2002, 124, 4595-4601). The present data indicate that measurements of NMR intermolecular coupling constants may be used to discriminate between the specific inner- and outer-shell binding of divalent cations to nucleobases in DNA and RNA.     

Evidence for a thermodynamically distinct Mg2+ ion associated with formation of an RNA tertiary structure

A folding strategy adopted by some RNAs is to chelate cations in pockets or cavities, where the ions neutralize charge from solvent-inaccessible phosphate. Although such buried Mg(2+)-RNA chelates could be responsible for a significant fraction of the Mg(2+)-dependent stabilization free energy of some RNA tertiary structures, direct measurements have not been feasible because of the difficulty of finding conditions under which the free energy of Mg(2+) chelation is uncoupled from RNA folding and from unfavorable interactions with Mg(2+) ions in other environments. In a 58mer rRNA fragment, we have used a high-affinity thermophilic ribosomal protein to trap the RNA in a structure nearly identical to native; Mg(2+)- and protein-stabilized structures differ in the solvent exposure of a single nucleotide located at the chelation site. Under these conditions, titration of a high affinity chelation site takes place in a micromolar range of Mg(2+) concentration, and is partially resolved from the accumulation of Mg(2+) in the ion atmosphere. From these experiments, we estimate the total and site-specific Mg(2+)-RNA interaction free energies over the range of accessed Mg(2+) concentrations. At 0.1 mM Mg(2+) and 60 mM K(+), specific site binding contributes ～-3 kcal/mol of the total Mg(2+) interaction free energy of ～-13 kcal/mol from all sources; at higher Mg(2+) concentrations the site-binding contribution becomes a smaller proportion of the total (-4.5 vs -33 kcal/mol). Under approximately physiological ionic conditions, the specific binding site will be saturated but will provide only a fraction of the total free energy of Mg(2+)-RNA interactions.     

Predicting the coordination geometry for Mg2+ in the p53 DNA-binding domain: insights from computational studies

Zn(2+) in the tumor-suppressor protein p53 DNA-binding domain (DBD) is essential for its structural stability and DNA-binding specificity. Mg(2+) has also been recently reported to bind to the p53DBD and influence its DNA-binding activity. In this contribution, the binding geometry of Mg(2+) in the p53DBD and the mechanism of how Mg(2+) affects its DNA-binding activity were investigated using density functional theory (DFT) calculations and molecular dynamics (MD) simulations. Various possible coordination geometries of Mg(2+) binding to histidines (His), cysteines (Cys), and water molecules were studied at the B3LYP/6-311+g** level of theory. The protonation state of Cys and the environment were taken into account to explore the factors governing the coordination geometry. The free energy of the reaction to form the Mg(2+) complexes was estimated, suggesting that the favorable binding mode changes from a four- to six-coordinated geometry as the number of the protonated Cys increases. Furthermore, MD simulations were employed to explore the binding modes of Mg(2+) in the active site of the p53DBD. The simulation results of the Mg(2+) system and the native Zn(2+) system show that the binding affinity of Mg(2+)to the p53DBD is weaker than that of Zn(2+), in agreement with the DFT calculation results and experiments. In addition, the two metal ions are found to make a significant contribution to maintain a favorable orientation for Arg248 to interact with putative DNA, which is critically important to the sequence-specific DNA-binding activity of the p53DBD. However, the effect of Mg(2+) is less marked. Additionally, analysis of the natural bond orbital (NBO) charge transfer reveals that Mg(2+) has a higher net positive charge than Zn(2+), leading to a stronger electrostatic attractive interaction between Mg(2+) and putative DNA. This may partly explain the higher sequence-independent DNA-binding affinity of p53DBD-Mg(2+) compared to p53DBD-Zn(2+) observed in experiment.     

Competition between Li+ and Mg2+ in metalloproteins. Implications for lithium therapy

Lithium is used (in the form of soluble salts) to treat bipolar disorder and has been considered as a possible drug in treating chronic neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases. One of the proposed mechanisms of Li(+) action involves a competition between the alien Li(+) and native Mg(2+) for metal-binding sites and subsequent inhibition of key enzymes involved in specific neurotransmission pathways, but not vital Mg(2+) proteins in the cell. This raises the following intriguing questions: Why does Li(+) replace Mg(2+) only in enzymes involved in bipolar disorder, but not in Mg(2+) proteins essential to cells? In general, what factors allow monovalent Li(+) to displace divalent Mg(2+) in proteins? Specifically, how do the composition, overall charge, and solvent exposure of the metal-binding site as well as a metal-bound phosphate affect the selectivity of Li(+) over Mg(2+)? Among the many possible factors, we show that the competition between Mg(2+) and Li(+) depends on the net charge of the metal complex, which is determined by the numbers of metal cations and negatively charged ligands, as well as the relative solvent exposure of the metal cavity. The protein itself is found to select Mg(2+) over the monovalent Li(+) by providing a solvent-inaccessible Mg(2+)-binding site lined by negatively charged Asp/Glu, whereas the cell machinery was found to select Mg(2+) among other competing divalent cations in the cellular fluids such as Ca(2+) and Zn(2+) by maintaining a high concentration ratio of Mg(2+) to its biogenic competitor in various biological compartments. The calculations reveal why Li(+) replaces Mg(2+) only in enzymes that are known targets of Li(+) therapy, but not in Mg(2+) enzymes essential to cells, and also reveal features common to the former that differ from those in the latter proteins.     

Unraveling the aflatoxin-FAPY conundrum: structural basis for differential replicative processing of isomeric forms of the formamidopyrimidine-type DNA adduct of aflatoxin B1

Aflatoxin B1 (AFB) epoxide forms an unstable N7 guanine adduct in DNA. The adduct undergoes base-catalyzed ring opening to give a highly persistent formamidopyrimidine (FAPY) adduct which exists as a mixture of forms. Acid hydrolysis of the FAPY adduct gives the FAPY base which exists in two separable but interconvertible forms that have been assigned by various workers as functional, positional, or conformational isomers. Recently, this structural question became important when one of the two major FAPY species in DNA was found to be potently mutagenic and the other a block to replication [Smela, M. E.; Hamm, M. L.; Henderson, P. T.; Harris, C. M.; Harris, T. M.; Essigmann, J. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6655-6660]. NMR studies carried out on the AFB-FAPY bases and deoxynucleoside 3',5'-dibutyrates now establish that the separable FAPY bases and nucleosides are diastereomeric N5 formyl derivatives involving axial asymmetry around the congested pyrimidine C5-N5 bond. Anomerization of the protected beta-deoxyriboside was not observed, but in the absence of acyl protection, both anomerization and furanosyl --> pyranosyl ring expansion occurred. In oligodeoxynucleotides, two equilibrating FAPY species, separable by HPLC, are assigned as anomers. The form normally present in duplex DNA is the mutagenic species. It has previously been assigned as the beta anomer by NMR (Mao, H.; Deng, Z. W.; Wang, F.; Harris, T. M.; Stone, M. P. Biochemistry 1998, 37, 4374-4387). In single-stranded environments the dominant species is the beta anomer; it is a block to replication.     

1H and (113)Cd NMR Investigations of Cd(2+) and Zn(2+) Binding Sites on Serum Albumin: Competition with Ca(2+), Ni(2+), Cu(2+), and Zn(2+)

1H and (113)Cd NMR studies are used to investigate the Cd(2+) binding sites on serum albumin (67 kDa) in competition with other metal ions. A wide range of mammalian serum albumins possess two similar strong Cd(2+) binding sites (site A 113-124 ppm; site B 24-28 ppm). The two strong sites are shown not to involve the free thiol at Cys34. Ca(2+) influences the binding of Cd(2+) to isolated human albumin, and similar effects due to endogenous Ca(2+) are observed for intact human blood serum. (1)H NMR studies show that the same two His residues of human serum albumin are perturbed by Zn(2+) and Cd(2+) binding alike. Zn(2+) displaces Cd(2+) from site A which leads to Cd(2+) occupation of a third site (C, 45 ppm). The N-terminus of HSA is not the locus of the two strong Cd(2+) binding sites, in contrast to Cu(2+) and Ni(2+). After saturation of the N-terminal binding site, Cu(2+) or Ni(2+) also displaces Cd(2+) from site A to site C. The effect of pH on Cd(2+) binding is described. A common Cd(2+)/Zn(2+) binding site (site A) involving interdomain His residues is discussed.     

Influence of Mg2+ and Cd2+ on the interaction between sparfloxacin and calf thymus DNA

Mg(2+) and Cd(2+) have different binding capacity to sparfloxacin, and have different combination modes with calf thymus DNA. Selecting these two different metal ions, the influence of them on the binding constants between SPFX and calf thymus DNA, as well as the related mechanism have been studied by using absorption and fluorescence spectroscopy. The result shows that Cd(2+) has weak binding capacity to SPFX in the SPFX-Cd(2+) binary system, but can decrease the binding between SPFX and DNA obviously in SPFX-DNA-Cd(2+) ternary system. Mg(2+) has strong binding capacity to SPFX. It can increase the binding between SPFX and DNA at concentrations <0.01 mM, and decrease the binding between them at concentrations >0.01 mM. Referring to the different modes of Mg(2+) and Cd(2+) binding to DNA, the mechanism of the influence of metal ions on the binding between SPFX and DNA has been proposed. SPFX can directly bind to DNA by chelating DNA base sites. If a metal ion at certain concentration mainly binds to DNA bases, it can decrease the binding constants between SPFX and DNA through competing with SPFX. While if a metal ion at certain concentration mainly binds to phosphate groups of DNA, it can increase the binding constants by building a bridge between SPFX and DNA. The influence direction of metal ions on the binding between quinolone and DNA relays on their binding ratio of affinity for bases to phosphate groups on DNA. Our result supports Palumbo's conclusion that the binding between SPFX and the phosphate groups is the precondition for the combination between SPFX and DNA, which is stabilized through stacking interactions between the condensed rings of SPFX and DNA bases.     

Direct NMR detection of alkali metal ions bound to G-quadruplex DNA

We describe a general multinuclear (1H, 23Na, 87Rb) NMR approach for direct detection of alkali metal ions bound to G-quadruplex DNA. This study is motivated by our recent discovery that alkali metal ions (Na+, K+, Rb+) tightly bound to G-quadruplex DNA are actually "NMR visible" in solution (Wong, A.; Ida, R.; Wu, G. Biochem. Biophys. Res. Commun. 2005, 337, 363). Here solution and solid-state NMR methods are developed for studying ion binding to the classic G-quadruplex structures formed by three DNA oligomers: d(TG4T), d(G4T3G4), and d(G4T4G4). The present study yields the following major findings. (1) Alkali metal ions tightly bound to G-quadruplex DNA can be directly observed by NMR in solution. (2) Competitive ion binding to the G-quadruplex channel site can be directly monitored by simultaneous NMR detection of the two competing ions. (3) Na+ ions are found to locate in the diagonal T4 loop region of the G-quadruplex formed by two strands of d(G4T4G4). This is the first time that direct NMR evidence has been found for alkali metal ion binding to the diagonal T4 loop in solution. We propose that the loop Na+ ion is located above the terminal G-quartet, coordinating to four guanine O6 atoms from the terminal G-quartet and one O2 atom from a loop thymine base and one water molecule. This Na+ ion coordination is supported by quantum chemical calculations on 23Na chemical shifts. Variable-temperature 23Na NMR results have revealed that the channel and loop Na+ ions in d(G4T4G4) exhibit very different ion mobilities. The loop Na+ ions have a residence lifetime of 220 micros at 15 degrees C, whereas the residence lifetime of Na+ ions residing inside the G-quadruplex channel is 2 orders of magnitude longer. (4) We have found direct 23Na NMR evidence that mixed K+ and Na+ ions occupy the d(G4T4G4) G-quadruplex channel when both Na+ and K+ ions are present in solution. (5) The high spectral resolution observed in this study is unprecedented in solution 23Na NMR studies of biological macromolecules. Our results strongly suggest that multinuclear NMR is a viable technique for studying ion binding to G-quadruplex DNA.     

Differential effects of Mg(ii) and N(alpha)-4-tosyl-l-arginine methyl ester hydrochloride on the recognition and catalysis in ATP hydrolysis

The supramolecular interactions of Mg(ii) and N(alpha)-4-tosyl-l-arginine methyl ester hydrochloride (TAME) with ATP have been investigated using (1)H and (31)P NMR spectra. Furthermore, the hydrolysis of ATP catalyzed by Mg(ii) and TAME has been studied at 60 degrees C and pH 7 using (31)P NMR spectra. In the Mg(ii)-ATP-TAME ternary system, the binding interaction of Mg(2+) with ATP involves not only N1 and N7 in the adenine ring but also beta- and gamma-phosphate of ATP. The binding forces are mainly electrostatic interaction and cation (Mg(2+))-pi interaction. The guanidinium group and the aromatic ring of TAME interacts with ATP by beta and gamma phosphate and the adenine ring of ATP. The binding forces are mainly electrostatic interactions and pi-pi stacking. A significant difference between the binary and the ternary system indicates that TAME is essential to the stablization of the intermediate. Kinetic studies show that the hydrolysis rate constant of ATP is 2.16 x 10(-2) h(-1) at pH 7 in the Mg(ii)-TAME-ATP ternary system. The Mg(ii) ion and TAME can accelerate the ATP hydrolysis process. A possible mechanism has been proposed that the hydrolysis occurs through an addition-elimination, in which the phosphoramidate intermediate was observed at 3.21 ppm in the (31)P NMR of the ternary system. These results provide further information concerning the effect of the key amino acid residue and metal ions as cofactors of ATPase on ATP synthesis/hydrolysis at the molecular level.     

Relationship of solution and protein-bound structures of DNA duplexes with the major intrastrand cross-link lesions formed on cisplatin binding to DNA.

DNA bases in the three-base-pair (3bp) region of duplexes with the two major lesions of cisplatin (cis-PtCl(2)(NH(3))(2)) with DNA, namely d(XGG) and d(XAG) ( = N7-platinated base), differ in their relative positions by as much as approximately 3.5 A in structures in the literature. Such large differences impede drug design and assessments of the effects of protein binding on DNA structure. One recent and several past structures based on NMR-restrained molecular dynamics (RMD) differ significantly from the reported X-ray structure of an HMG-bound XGG 16-mer DNA duplex (Ohndorf, U.-M.; Rould, M. A.; He, Q.; Pabo, C. O.; Lippard, S. J. Nature 1999, 399, 708). This 16-mer structure has several significant novel and unique features (e.g., a bp step with large positive shift and slide). Hypothesizing that novel structural features in the XGG or XAG region of duplexes elude discovery by NMR methods (especially because of the flexible nature of the 3bp region), we studied an oligomer with only G.C bp's in the XGGY site by NMR methods for the first time. This 9-mer gave a 5'-G N1H signal with a normal shift and intensity and showed clear NOE cross-peaks to C NHb and NHe. We assigned for the first time (13)C NMR signals of a duplex with a GG lesion. These data, by adding NMR-based criteria to those inherent in NOESY and COSY data, have more specifically defined the structural features that should be present in an acceptable model. In particular, our data indicated that the sugar of the X residue has an N pucker and that the GG cross-link should have a structure similar to the original X-ray structure of cis-Pt(NH(3))(2)(d(pGpG)) (Sherman S. E.; Gibson, D.; Wang, A. H.-J.; Lippard, S. J. J. Am. Chem. Soc. 1988, 110, 7368). With these restrictions added to NOE restraints, an acceptable model was obtained only when we started our modeling with the 16-mer structural features. The new X-ray/NMR-based model accounted for the NOESY data better than NOE-based models, was very similar in structure to the 16-mer, and differed from solely NOE-based models. We conclude that all XGG and XAG (X = C or T) duplexes undoubtedly have structures similar to those of the 16-mer and our model. Thus, protein binding does not change greatly the structure of the 3bp region. The structure of this region can now be used in understanding structure-activity relationships needed in the design of new carrier ligands for improving Pt anticancer drug activity.     

Metal ion catalysis in the beta-elimination reactions of N-[2-(4-pyridyl)ethyl]quinuclidinium and N-[2-(2-pyridyl)ethyl]quinuclidinium in aqueous solution

Catalysis of the beta-elimination reaction of N-[2-(4-pyridyl)ethyl]quinuclidinium (1) and N-[2-(2-pyridyl)ethyl]quinuclidinium (2) by Zn(2+) and Cd(2+) in OH(-)/H(2)O (pH = 5.20-6.35, 50 degrees C, and mu = 1 M KCl) has been studied. In the presence of Zn(2+), the elimination reactions of both isomers occur from the Zn(2+)-complexed substrates (C). The equilibrium constants for the dissociation of the Zn(2+)-complexes are as follows: K(d) = 0.012 +/- 0.003 M (isomer 1) and K(d) = 0.065 +/- 0.020 M (isomer 2). The value of k(C)(H2O) for isomer 1 is 4.81 x 10(-6) s(-1). For isomer 2 both the rate constants for the "water" and OH(-)-induced reaction of the Zn(2+)-complexed substrate could be measured, despite the low concentration of OH(-) in the investigated reaction mixture [k(C)H2O)= 1.97 x 10(-6) s(-1) and k(C)(OH-)= 21.9 M(-1) s(-1), respectively]. The measured metal activating factor (MetAF), i.e., the reactivity ratio between the complexed and the uncomplexed substrate, is 8.1 x 10(4) for the OH(-)-induced elimination of 2. This high MetAF can be compared with the corresponding proton activating factor (Alunni, S.; Conti, A.; Palmizio Errico, R. J. Chem. Soc., Perkin Trans. 2 2000, 453), PAF = 1.5 x 10(6) and is in agreement with an E1cb irreversible mechanism (A(xh)D(E)* + D(N)) (Guthrie, R. D.; Jencks, W. P. Acc. Chem. Res. 1989, 22, 343). A value of k(C)(H2O)>or= 23 x 10(-7) s(-1) is estimated for the Cd(2+)-complexed isomer 2, while catalysis by Cd(2+) has not been observed for isomer 1.     

Common semiopen conformations of Mg2+-free Ras, Rho, Rab, Arf, and Ran proteins combined with GDP and their similarity with GEF-bound forms

A computational study was performed on the Mg(2+)-free conformations of the small guanine nucleotide-binding proteins (GNBPs): Ras, Rho, Rab, Arf, and Ran, which were complexed with GDP. Molecular dynamics (MD) simulation was executed for each complex for the duration of 3.0 ns to investigate the effects of Mg(2+) ions on the GNBPs' structure. The results indicated that all Mg(2+)-free GNBPs formed a groove between the switch region and the nucleotide-binding site. In some GNBP families, the release of Mg(2+) was reported to play an important role in binding the guanine nucleotide-exchanging factor (GEF) promoting the GDP/GTP exchange reaction. Interestingly, the grooves, which appeared in the MD simulations, were similar to the grooves experimentally observed in the GNBP-GEF complex. We also calculated the Mg(2+)-bound GNBPs to compare with the Mg(2+)-free forms. No groove was observed in the Mg(2+)-bound GNBPs. These results demonstrated a regulatory role of Mg(2+) ion to prepare a template for the GEF binding. Moreover, the results suggested that the release of Mg(2+) ion lead to the GEF-GNBP binding.     

Novel tailormade Bi4MO4(PO4)2 structural type (M = Mg, Zn)

In the Bi(2)O(3)-MO-P(2)O(5) ternary system, the commonly observed sizable 1D ribbon-like units have been extended to their 2D infinite end member, leading to the novel tailormade Bi(4)MO(4)(PO(4))(2) compounds. It contains planar [Bi(2)O(2)](2+) derivatives, separated by two slabs of PO(4), which create channels hosting the M(2+) cations (M = Mg, Zn). For both compounds, supercell orderings occur comparatively to the predicted ideal crystal structure (V(Mg) = 2V(ideal) and V(Zn) = 8V(ideal)). In the Mg case a transition into the ideal lattice occurs above 450 °C. In spite of the conceptual assembly of 2D motifs, the final architecture is three-dimensional due to strong interbonds. Thus, our work gives new insights on the possibility for versatile organization of original secondary building units (SBUs) able to self-assemble into predicted structural edifices. Single-crystal and powder XRD versus temperature, high-temperature (31)P NMR, as well as transmission electron microscopy were used for structural characterization. Preliminary electric characterization is also reported.     

Acid-base and metal ion binding properties of guanylyl(3'-->5')guanosine (GpG-) and 2'-deoxyguanylyl(3'-->5')-2'-deoxyguanosine [d(GpG)-] in aqueous solution

The acidity constants of guanylyl(3'-->5')guanosine (GpG(-)) and 2'-deoxyguanylyl(3'-->5')-2'-deoxyguanosine [d(GpG)(-)] for the deprotonation of their (N1)H sites were measured by potentiometric pH titrations in aqueous solution (25 degrees C; I = 0.1 M, NaNO(3)). The same method was used for the determination of the stability constants of the 1:1 complexes formed between Mg(2+), Ni(2+), or Cd(2+) (= M(2+)) and (GG-H)(2-), and in the case of Mg(2+) also of (GG-2H)(3-), where GG(-) = GpG(-) or d(GpG)(-). The stability constants of the M(GG)(+) complexes were estimated. The acidity constants of the H(dGuo)(+) and dGuo species (dGuo = 2'-deoxyguanosine) and the stability constants of the corresponding M(dGuo)(2+) and M(dGuo-H)(+) complexes were also measured. Comparison of these and related data allows the conclusion that N7 of the 5'G unit in GG(-) is somewhat more basic than the one in the 3'G moiety; the same holds for the (N1)(-) sites. On the basis of comparisons with the stability constants measured for the dGuo complexes, it is concluded that M(2+) binding of the GG dinucleoside monophosphates occurs predominantly in a mono-site fashion, meaning that macrochelate formation is not very pronounced. Indeed, it was a surprise to find that the stabilities of the complexes of dGuo or (dGuo-H)(-) and the corresponding ones derived from GG(-) are so similar. Consequently, it is suggested that in the M(GG)(+) and M(GG-H) complexes the metal ion is mainly located at N7 of the 5'G unit since this is the more basic site allowing also an outer-sphere interaction with the C6 carbonyl oxygen and because this coordination mode is also favorable for an electrostatic interaction with the negatively charged phosphodiester bridge. It is further suggested that Mg(2+) binding (which is rather weak compared to that of Ni(2+) and Cd(2+)) occurs mainly in an outer-sphere mode, and on the basis of the so-called Stability Ruler it is concluded that the binding properties of Zn(2+) to the GG species are similar to those of Ni(2+) and Cd(2+).     

N-methylmonothiocarbamatopentamminecobalt(III): restricted C-N bond rotation and the acid-catalyzed O- to S-bonded rearrangement

High-resolution (1)H and (13)C NMR studies on the linkage isomers [(NH(3))(5)CoOC(S)NHCH(3)](2+) and [(NH(3))(5)CoSC(O)NHCH(3)](2+) reveal that the O-bonded form exists as a 5:1 mixture of Z and E isomers arising from restricted rotation about the C-N bond. Similarly, restricted rotation is observed (at 20 degrees C) for the S-bonded isomer (Z/E ca. 18:1), but not for the isoelectronic carbamate ion [(NH(3))(5)CoOC(O)NHCH(3)](2+), nor for the unsubstituted carbamato complex [(NH(3))(5)CoOC(O)NH(2)](2+). An analysis of the variable-temperature NMR data for the O-bonded carbamato and urea complexes has provided quantitative data on the rotational barriers, and these ions involve much faster C-N bond rotations than the thiocarbamato complexes. The acid-catalyzed reaction of [(NH(3))(5)CoOC(S)NHCH(3)](2+) is confirmed, but there is much less parallel hydrolysis (ca. 2%) than previously reported (40 +/- 10%) for 0.1 M HClO(4). In 1 M HClO(4), [(NH(3))(5)CoSC(O)NHCH(3)](2+) and [(NH(3))(5)CoOH(2)](3+) are formed in parallel as an 83:17 mixture. The kinetic data suggest that the protonated form is at least 20-fold more reactive than the free ion and that the linkage isomerization and hydrolysis pathways are both acid-catalyzed, the latter clearly more so than the rearrangement.     

Influence of the environment on the specificity of the Mg(II) binding to uracil

Interactions of uracil with the Mg(2+) ion were studied theoretically in the gas phase and in solution. The bare Mg(2+) prefers bidentate N-C═O binding sites stabilizing rare keto-enol forms of the base. Hydration and/or phosphate binding of the Mg(2+) ion shield its positive charge, which leads to preference of monodentate binding to the oxygen keto atoms, shifting fully the equilibrium between the tautomers back toward the canonical diketo tautomer. In solution, a direct inner-sphere metal binding to uracil is not clearly advantageous compared to the outer-sphere metal binding. Similar trends were also obtained for the Ca(2+) ion. Results are supported by the natural bond orbital (NBO) and atoms in molecule (AIM) analyses and the combined extended transition-state energy decomposition analysis and natural orbitals for chemical valence (ETS-NOCV).     

A new multifunctional ferrocenyl-substituted ferrocenophane derivative: optical and electronic properties and selective recognition of Mg2+ ions

The synthesis, characterisation, and X-ray structure of a new strained asymmetric diferrocene derivative (2) is reported. Compound 2 acts as a highly specific electrochemical and optical Mg(2+) ion sensor, as revealed by spectroscopic and electrochemical techniques. Thus, in the presence of Mg(2+), a new redox peak appears in the cyclic voltammogram (CV) that is anodically shifted compared to the E(1/2) of the free receptor (DeltaE(1/2)=340 mV). Diferrocene derivative 2 also gives a highly visual response upon addition of Mg(2+), namely a change of colour from orange to deep purple. In addition, compound 2 does not show any significant sensing activity in the presence of Ca(2+) or alkaline ions. On protonation, it is converted into the stable diferrocenylcarbenium salt 4, in which two different modes of stabilisation of the alpha-carbocationic centre are clearly demonstrated by a combination of (1)H NMR and (57)Fe M?ssbauer spectroscopic measurements. Finally, by a partial (chemical or electrochemical) oxidation, compound 2 forms the asymmetric mixed-valence species 2(+), which can be isolated as the solid salt 6 by using CF(3)SO(3) (-) as a counterion. This mixed-valence species shows a fast intramolecular electron-transfer process, as ascertained by several spectroscopic techniques.     

Influence of DNA structure on adjacent site cooperative binding

Previous NMR studies of Hoechst 33258 with the d(CTTTTGCAAAAG)2 sequence have shown very strong (K2 > K1) cooperativity between two adjacent binding sites (Searle, M. S.; Embrey, K. J. Nucleic Acids Res. 1990, 18 (13), 3753- 3762). In contrast, surface plasmon resonance (SPR) results with the hairpin analog of the same sequence show significantly reduced cooperativity. In an effort to explain the difference, two-dimensional (2-D) NMR experiments were done on both duplex and hairpin. Hoechst 33258 and an amidine analog, DB183, show very strong cooperativity with the duplex DNA but much weaker cooperativity with the hairpin. The significantly lower thermal melting temperature (Tm) of the duplex (34.8 degrees C) in comparison to its hairpin analog (62.3 degrees C) supports the idea of a dynamic difference between the two DNA structures that can influence cooperativity in binding. These results confirm the role of conformational entropy in positive cooperativity in some DNA interactions.     

Displacement of Mn2+ from RNA by K+, Mg2+, neomycin B,and an arginine-rich peptide: indirect detection of nucleic acid/ligand interactions using phosphorus relaxation enhancement

We have developed a novel method to study the interactions of nucleic acids with cationic species. The method, called phosphorus relaxation enhancement (PhoRE), uses (1)H-detected (31)P NMR of exogenous probe ions to monitor changes in the equilibrium between free Mn(2+) and Mn(2+) bound to the RNA. To demonstrate the technique, we describe the interactions of four RNA molecules with metal ions (K(+) and Mg(2+)), a small molecule drug (neomycin b), and a cationic peptide (RSG1.2). In each case, cationic ligand binding caused Mn(2+) to be displaced from the RNA. Free Mn(2+) was determined from its effect on the T(2) NMR relaxation rate of either phosphite (HPO(3)(2-)) or methyl phosphite (MeOPH, CH(3)OP(H)O(2-)). Using this method, the effects of [RNA] as low as 1 microM could be measured in 20 min of accumulation using a low field (200 MHz) instrument without pulsed field gradients. Cation association behavior was sequence and [RNA] dependent. At low [K(+)], Mn(2+) association with each of the RNAs decreased with increasing [K(+)] until approximately 40 mM, where saturation was reached. While saturating K(+) displaced all the bound Mn(2+) from a 31-nucleotide poly-uridine (U(31)), Mn(2+) remained bound to each of three hairpin-forming sequences (A-site, RRE1, and RRE2), even at 150 mM K(+). Bound Mn(2+) was displaced from each of the hairpins by Mg(2+), allowing determination of Mg(2+) dissociation constants (K(d,Mg)) ranging from 50 to 500 microM, depending on the RNA sequence and [K(+)]. Both neomycin b and RSG1.2 displaced Mn(2+) upon binding the hairpins. At [RNA] approximately 3 microM, RRE1 bound a single equivalent of RSG1.2, whereas neither RRE2 nor A-site bound the peptide. These behaviors were confirmed by fluorescence polarization using TAMRA-labeled peptide. At 2.7 microM RNA, the A-site hairpin bound a single neomycin b molecule. The selectivity of RSG1.2 binding was greatly diminished at higher [RNA]. Similarly, each hairpin bound multiple equivalents of neomycin at the higher [RNA]. These results demonstrate the utility of the PhoRE method for characterizing metal binding behaviors of nucleic acids and for studying RNA/ligand interactions.