Binding constant measurement by hyper-Rayleigh scattering: bilirubin-human serum albumin binding as a case study

In this paper, a new application of the hyper-Rayleigh scattering technique in determining multiple binding constants of a small molecule like bilirubin to a macromolecule like the protein human serum albumin has been demonstrated. Human serum albumin has two binding sites for bilirubin, and the binding constants have been measured by carrying out a second harmonic titration of the protein against bilirubin and vice versa. The measured binding constants K(1) = 1.5 +/- 0.43 x 10(7) M(-1) and K(2) = 1.01 +/- 0.16 x 10(6) M(-1) agree well with the reported values obtained by other methods.     

Large-scale millisecond intersubunit dynamics in the B subunit homopentamer of the toxin derived from Escherichia coli O157

We report here solution NMR relaxation measurements that show millisecond time-scale intersubunit dynamics in the homopentameric B subunit (VTB) of the toxin derived from Escherichia coli O157. These data are consistent with interconversion between an axially symmetric form and a low-abundance ( approximately 10%, 45 degrees C) higher energy form. The higher energy state is depopulated on binding of a novel bivalent analogue (P(k) dimer) of the natural carbohydrate acceptor globotriaosylceramide. The isothermal titration calorimetry isotherm for the binding of P(k) dimer to VTB is consistent with a five-site sequential binding model which assumes that cooperative effects arise through communication only between neighboring binding sites. The resulting thermodynamic parameters (K(a1) = 114 +/- 2.2 M(-1), K(a2) = 283 +/- 4.5 M(-1), DeltaH(1) degrees = -116.3 +/- 0.55 kJ/mol, and DeltaH(2) degrees = -50.3 +/- 0.11 kJ/mol) indicate favorable entropic cooperativity that has not previously been observed in multivalent systems.     

A comparison between electrokinetic capillary chromatography and absorption spectroscopy for the analysis of peptide-micelle association by weak hydrophobic interactions

Micellar electrokinetic capillary chromatography (MEKC) was compared to absorption spectroscopy to estimate equilibrium association constans (K(as)) for peptide-micelle systems involving three peptides (leucine-enkephalin, methionine-enkephalin and leucine-phenylalanine (LF)) and two surfactant micelles (sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB)). Buffer pH was chosen to minimize purely electrostatic interactions between peptides and micelles that could not be interrogated by absorption spectroscopy. Viscosity-corrected MEKC mobilities gave reasonably similar estimates of K(as) between the two methods for all three peptide-SDS micelle systems, with K(as) values ranging from 13.7 +/- 0.3 to 49 +/- 1 M(-1). For CTAB, estimates of K(as) for LF-CTAB micelle association were of the same order of magnitude as the SDS micelle by the two methods of estimation. On the other hand, enkephalin-CTAB micelle binding was about 10 times stronger (K(as) = 122 +/- 3 M(-1) to 311 +/- 9 M(-1)) than the enkephalin-SDS micelle binding. In addition, MEKC underestimated the K(as) values relative to spectroscopy by a factor of 2-3 for the enkephalin-CTAB system.     

Kinetics and mechanism of methane, methanol, and dimethyl ether C-H activation with electrophilic platinum complexes

The relative rates of C-H activation of methane, methanol, and dimethyl ether by [(N-N)PtMe(TFE-d(3))](+) ((N-N) = ArN=C(Me)-C(Me)=NAr; Ar = 3,5-di-tert-butylphenyl, TFE-d(3) = CF(3)CD(2)OD) (2(TFE)) were determined. Methane activation kinetics were conducted by reacting 2(TFE)-(13)C with 300-1000 psi of methane in single-crystal sapphire NMR tubes; clean second-order behavior was obtained (k = 1.6 +/- 0.4 x 10(-3) M(-1) s(-1) at 330 K; k = 2.7 +/- 0.2 x 10(-4) M(-1) s(-1) at 313 K). Addition of methanol to solutions of 2(TFE) rapidly establishes equilibrium between methanol (2(MeOD)) and trifluoroethanol (2(TFE)) adducts, with methanol binding preferentially (K(eq) = 0.0042 +/- 0.0006). C-H activation gives [(N-N)Pt(CH(2)OD)(MeOD)](+) (4), which is unstable and reacts with [(RO)B(C(6)F(5))(3)](-) to generate a pentafluorophenyl platinum complex. Analysis of kinetics data for reaction of 2 with methanol yields k = 2.0 +/- 0.2 x 10(-3) M(-1) s(-1) at 330 K, with a small kinetic isotope effect (k(H)/k(D) = 1.4 +/- 0.1). Reaction of dimethyl ether with 2(TFE) proceeds similarly (K(eq) = 0.023 +/- 0.002, 313 K; k = 5.5 +/- 0.5 x 10(-4) M(-1) s(-1), k(H)/k(D) = 1.5 +/- 0.1); the product obtained is a novel bis(alkylidene)-bridged platinum dimer, [(diimine)Pt(mu-CH(2))(mu-(CH(OCH(3)))Pt(diimine)](2+) (5). Displacement of TFE by a C-H bond appears to be the rate-determining step for all three substrates; comparison of the second-order rate constants (k((methane))/k((methanol)) = 1/1.3, 330 K; k((methane))/k((dimethy)(l e)(ther)) = 1/2.0, 313 K) shows that this step is relatively unselective for the C-H bonds of methane, methanol, or dimethyl ether. This low selectivity agrees with previous estimates for oxidations with aqueous tetrachloroplatinate(II)/hexachloroplatinate(IV), suggesting a similar rate-determining step for those reactions.     

Reactive intermediates relevant to the carbonylation of CH(3)Mn(CO)(5). Activation parameters for key dynamic processes

Reported is a time-resolved infrared and optical kinetics investigation of the transient species CH(3)C(O)Mn(CO)(4) (I(Mn)) generated by flash photolysis of the acetyl manganese pentacarbonyl complex CH(3)C(O)Mn(CO)(5) (A(Mn)) in cyclohexane and in tetrahydrofuran. Activation parameters were determined for CO trapping of I(Mn) to regenerate A(Mn) (rate = k(CO) [CO][I(Mn)]) as well as the methyl migration pathway to form methylmanganese pentacarbonyl CH(3)Mn(CO)(5) (M(Mn)) (rate = k(M)[I(Mn)]). These values were Delta H(++)(CO) = 31 +/- 1 kJ mol(-1), Delta S(++)(CO) = -64 +/- 3 J mol(-1) K(-1), Delta H(++)(M) = 35 +/- 1 kJ mol(-1), and Delta S(++)(M) = -111 +/- 3 J mol(-1) K(-1). Substantially different activation parameters were found for the methyl migration kinetics of I(Mn) in THF solutions where Delta H(++)(M) = 68 +/- 4 kJ mol(-1) and Delta S(++)(M) = 10 +/- 10 J mol(-1) K(-1), consistent with the earlier conclusion (Boese, W. T.; Ford, P. C. J. Am. Chem. Soc. 1995, 117, 8381-8391) that the composition of I(Mn) is different in these two media. The possible isotope effect on k(M) was also evaluated by studying the intermediates generated from flash photolysis of CD(3)C(O)Mn(CO)(5) in cyclohexane, but this was found to be nearly negligible (k(M)(h)/k(M)(d) (298 K) = 0.97 +/- 0.05, Delta H(++)(M)(d) = 37 +/- 4 kJ mol(-1), and Delta S(++)(M)(d) = -104 +/- 12 J mol(-1) K(-1)). The relevance to the migratory insertion mechanism of CH(3)Mn(CO)(5), a model for catalytic carbonylations, is discussed.     

Providing a chemical basis toward understanding the histidine base-on motif of methylcobalamin-dependent methionine synthase: an improved purification of methylcobinamide,plus thermodynamic studies of methylcobinamide binding exogenous imidazole and pyridine bases

Reported herein are the synthesis and improved purification of MeCbi(+).BF(4)(-) leading to 95% pure product. The availability of this higher purity MeCbi(+).BF(4)(-) has, in turn, allowed a study of the K(assoc), DeltaH, and DeltaS for exogenous imidazole and pyridine bases binding to MeCbi(+) in ethylene glycol and buffered aqueous solution. The results show that (1) the bases studied have larger K(assoc) values (where measurable) when binding to MeCbi(+) than when binding to AdoCbi(+) under analogous conditions; (2) comparison of the thermodynamic binding parameters for py and N-MeIm show that these bases bind similarly, within experimental error to MeCbi(+), contrary to what was seen earlier with AdoCbi(+); (3) the bases follow the expected trend, with the base with the highest pK(a) of those studied, 4-Me(2)Npy, exhibiting the highest K(assoc) value (K(assoc)(25 degrees C) = 18.0 +/- 0.3 M(-1)) and the base of lowest pK(a), py, exhibiting the lowest detectable K(assoc) value (K(assoc) (25 degrees C) = 6.2 +/- 0.4 M(-1)); (4) there is no detectable binding (K(assoc) = 0.07 M(-1)) for 2-Mepy or 2,6-Me(2)py with MeCbi(+); and (5) the base that is closest to the biologically relevant axial His759 residue in methionine synthase, N-MeIm, exhibits an unusual DeltaH value for the formation of MeCbi(+).N-MeIm, results interpreted as offering further support for the presence of sigma plus pi effects when imidazole bases bind to alkylcobinamides. The results of these studies allow the percentage of base-on methylcobinamide, MeCbi(+).base, to be calculated as a function of temperature and added base. As such, they provide necessary background information for RS(-) + MeCbi(+).base and other methionine synthase chemical precedent studies.     

Mechanism of CO exchange on cis-[M(CO)2X2]- complexes (M=Rh,Ir;X=Cl, Br, or I)

The CO exchange on cis-[M(CO)2X2]- with M = Ir (X = Cl, la; X = Br, 1b; X = I, 1c) and M = Rh (X = Cl, 2a; X = Br, 2b; X = I, 2c) was studied in dichloromethane. The exchange reaction [cis-[M(CO)2X2]- + 2*CO is in equilibrium cis-[M(*CO)2X2]- + 2CO (exchange rate constant: kobs)] was followed as a function of temperature and carbon monoxide concentration (up to 6 MPa) using homemade high gas pressure NMR sapphire tubes. The reaction is first order for both CO and cis-[M(CO)2X2]- concentrations. The second-order rate constant, k2(298) (=kobs)[CO]), the enthalpy, deltaH*, and the entropy of activation, deltaS*, obtained for the six complexes are respectively as follows: la, (1.08 +/- 0.01) x 10(3) L mol(-1) s(-1), 15.37 +/- 0.3 kJ mol(-1), -135.3 +/- 1 J mol(-1) K(-1); 1b, (12.7 +/- 0.2) x 10(3) L mol(-1) s(-1), 13.26 +/- 0.5 kJ mol(-1), -121.9 +/- 2 J mol(-1) K(-1); 1c, (98.9 +/- 1.4) x 10(3) L mol(-1) s(-1), 12.50 +/- 0.6 kJ mol(-1), -107.4 +/- 2 J mol(-1) K(-1); 2a, (1.62 +/- 0.02) x 10(3) L mol(-1) s(-1), 17.47 +/- 0.4 kJ mol(-1), -124.9 +/- 1 J mol(-1) K(-1); 2b, (24.8 +/- 0.2) x 10(3) L mol(-1) s(-1), 11.35 +/- 0.4 kJ mol(-1), -122.7 +/- 1 J mol(-1) K(-1); 2c, (850 +/- 120) x 10(3) L mol(-1), s(-1), 9.87 +/- 0.8 kJ mol(-1), -98.3 +/- 4 J mol(-1) K(-1). For complexes la and 2a, the volumes of activation were measured and are -20.9 +/- 1.2 cm3 mol(-1) (332.0 K) and -17.2 +/- 1.0 cm3 mol(-1) (330.8 K), respectively. The second-order kinetics and the large negative values of the entropies and volumes of activation point to a limiting associative, A, exchange mechanism. The reactivity of CO exchange follows the increasing trans effect of the halogens (Cl < Br < I), and this is observed on both metal centers. For the same halogen, the rhodium complex is more reactive than the iridium complex. This reactivity difference between rhodium and iridium is less marked for chloride (1.5: 1) than for iodide (8.6:1) at 298 K.     

Equilibrium and Kinetics of Bromine Hydrolysis

Equilibrium constants for bromine hydrolysis, K(1) = [HOBr][H(+)][Br(-)]/[Br(2)(aq)], are determined as a function of ionic strength (&mgr;) at 25.0 degrees C and as a function of temperature at &mgr; approximately 0 M. At &mgr; approximately 0 M and 25.0 degrees C, K(1) = (3.5 +/- 0.1) x 10(-)(9) M(2) and DeltaH degrees = 62 +/- 1 kJ mol(-)(1). At &mgr; = 0.50 M and 25.0 degrees C, K(1) = (6.1 +/- 0.1) x 10(-)(9) M(2) and the rate constant (k(-)(1)) for the reverse reaction of HOBr + H(+) + Br(-) equals (1.6 +/- 0.2) x 10(10) M(-)(2) s(-)(1). This reaction is general-acid-assisted with a Br?nsted alpha value of 0.2. The corresponding Br(2)(aq) hydrolysis rate constant, k(1), equals 97 s(-)(1), and the reaction is general-base-assisted (beta = 0.8).     

Hydroxylation of phenolic compounds by a peroxodicopper(II) complex: further insight into the mechanism of tyrosinase

The dicopper(I) complex [Cu2(MeL66)]2+ (where MeL66 is the hexadentate ligand 3,5-bis-{bis-[2-(1-methyl-1H-benzimidazol-2-yl)-ethyl]-amino}-meth ylbenzene) reacts reversibly with dioxygen at low temperature to form a mu-peroxo adduct. Kinetic studies of O2 binding carried out in acetone in the temperature range from -80 to -55 degrees C yielded the activation parameters DeltaH1(not equal) = 40.4 +/- 2.2 kJ mol(-1), DeltaS1)(not equal) = -41.4 +/- 10.8 J K(-1) mol(-1) and DeltaH(-1)(not equal) = 72.5 +/- 2.4 kJ mol(-1), DeltaS(-1)(not equal) = 46.7 +/- 11.1 J K(-1) mol(-1) for the forward and reverse reaction, respectively, and the binding parameters of O2 DeltaH degrees = -32.2 +/- 2.2 kJ mol(-1) and DeltaS degrees = -88.1 +/- 10.7 J K(-1) mol(-1). The hydroxylation of a series of p-substituted phenolate salts by [Cu2(MeL66)O2]2+ studied in acetone at -55 degrees C indicates that the reaction occurs with an electrophilic aromatic substitution mechanism, with a Hammett constant rho = -1.84. The temperature dependence of the phenol hydroxylation was studied between -84 and -70 degrees C for a range of sodium p-cyanophenolate concentrations. The rate plots were hyperbolic and enabled to derive the activation parameters for the monophenolase reaction DeltaH(not equal)ox = 29.1 +/- 3.0 kJ mol(-1), DeltaS(not equal)ox = -115 +/- 15 J K(-1) mol(-1), and the binding parameters of the phenolate to the mu-peroxo species DeltaH degrees(b) = -8.1 +/- 1.2 kJ mol(-1) and DeltaS degrees(b) = -8.9 +/- 6.2 J K(-1) mol(-1). Thus, the complete set of kinetic and thermodynamic parameters for the two separate steps of O2 binding and phenol hydroxylation have been obtained for [Cu2(MeL66)]2+.     

Complexation of Nickel(II) with Guanosine 5'-Monophosphate and Inosine 5'-Monophosphate: A Potentiometric and Calorimetric Study

The constants (K(s)) and enthalpies (DeltaH(s)) for stacking interactions between purine nucleoside monophosphates were determined by calorimetry; the values thus obtained were guanosine as follows: K(s) = 2.1 +/- 0.3 M(-)(1) and DeltaH(s) = -41.8 +/- 0.8 kJ/mol for adenosine 5'-monophosphate (5'AMP); K(s) = 1.5 +/- 0.3 M(-1) and DeltaH(s) = -42.0 +/- 1.5 kJ/mol for guanosine 5'-monophosphate (5'GMP); and K(s) = 1.0 +/- 0.2 M(-1) and DeltaH(s) = -42.3 +/- 1.1 kJ/mol for inosine 5'-monophosphate (5'IMP). The interaction of nickel(II) with purine nucleoside monophosphates was studied using potentiometric and calorimetric methods, with 0.1 M tetramethylammonium bromide as the background electrolyte, at 25 degrees C. The presence in solution of the complexes [Ni(5'GMP)(2)](2)(-) and [Ni(5'IMP)(2)](2)(-) was observed. The thermodynamic parameters obtained were log K(ML) = 3.04 +/- 0.02, log K(ML2) = 2.33 +/- 0.02, DeltaH(ML) = -18.4 +/- 0.9 kJ/mol and DeltaH(ML2) = -9.0 +/- 1.9 kJ/mol for 5'GMP; and log K(ML) = 2.91 +/- 0.01, log K(ML2) = 1.92 +/- 0.01, DeltaH(ML) = -16.2 +/- 0.9 kJ/mol and DeltaH(ML2) = -0.1 +/- 2.3 kJ/mol for 5'IMP. The relationships between complex enthalpies and the degree of macrochelation, as well as the stacking interaction between purine bases in the complexes are discussed in relation to previously reported calorimetric data.     

Seven-coordinate iron complex as a ditopic receptor for lithium salts: study of host-guest interactions and substitution behavior

Interactions between the seven-coordinate tweezerlike [Fe(dapsox)(H2O)2]ClO4 complex (H2dapsox = 2,6-diacetylpyridine-bis(semioxamazide)) with different lithium salts (LiOTf, LiClO4, LiBF4, and LiPF6) in CH3CN have been investigated by electrochemical, spectrophotometric, 7Li and 19F NMR, kinetic, and DFT methods. It has been demonstrated that this complex acts as ditopic receptor, showing spectral and electrochemical ion-pair-sensing capability for different lithium salts. In general, the apparent binding constants for lithium salts increase in the order LiOTf < LiClO4 < LiBF4. From the electrochemical measurements, the apparent lithium salt binding constants for the Fe(III) and Fe(II) forms of the complex have been obtained, suggesting a stronger host-guest interaction with the reduced form of the complex. In the presence of LiPF6, the solution chemistry is more complex because of the hydrolysis of PF6-. The kinetics of the complexation of [Fe(dapsox)(CH3CN)2]+ by thiocyanate at -15 degrees C in acetonitrile in the presence of 0.2 M NBu4OTf shows two steps with the following rate constants and activation parameters: k(1) = 411 +/- 14 M(-1) s(-1); DeltaH(1) not equal = 9 +/- 2 kJ mol(-1); DeltaS1 not equal = -159 +/- 6 J K(-1) mol(-1); k(2) = 52 +/- 1 M(-1) s(-1); DeltaH(2) not equal = 4 +/- 1 kJ mol(-1); DeltaS(2) not equal = -195 +/- 3 J K(-1) mol(-1). The very negative DeltaS not equal values are consistent with an associative (A) mechanism. Under the same conditions but with 0.2 M LiOTf, k1Li and k2Li are 1605 +/- 51 and 106 +/- 2 M(-1) s(-1), respectively. The increased rate constants for the {[Fe(dapsox)(CH3CN)2] x LiOTf}+ adduct are in agreement with an associative mechanism. Kinetic and spectrophotometric titration measurements show stronger interaction between the lithium salt and the anion-substituted forms, [Fe(dapsox)(CH3CN)(NCS)] and [Fe(dapsox)(NCS)2]-, of the complex. These experiments demonstrate that in nonaqueous media lithium salts cannot be simply used as supporting electrolytes, since they can affect the kinetic behavior of the studied complex. DFT calculations revealed that the negatively charged alpha-oxyazine oxygen atoms are responsible for cation binding (electrostatic interactions), whereas the two terminal amide groups bind the anion via hydrogen bonding.     

A cucurbit[6]uril analogue: host properties monitored by fluorescence spectroscopy

This paper describes the host properties of a new cucurbit[6]uril analogue, studied by fluorescence and 1H NMR spectroscopy. This host has an elongated cavity with oval-shaped portals. It is intrinsically fluorescent, and more importantly, this fluorescence is sensitive to guest encapsulation, allowing for the study of the inclusion of nonfluorescent guests by fluorescence spectroscopy. In the case of benzene as guest, significant enhancement of the cucurbit[6]uril analogue host fluorescence was observed upon addition of benzene; this allowed for the determination of the binding constant for 1:1 host-guest complexation, yielding a value of K = 6900 +/- 1100 M(-1). This complexation was also studied by 1H NMR, yielding a similar value of K = 8980 +/- 500 M(-1). The binding of a much larger guest, the dye Nile Red, was also studied, but in this case using guest fluorescence. Significant suppression of the Nile Red fluorescence was observed upon 1:1 complexation with the cucurbit[6]uril analogue, with an extremely large binding constant of 8.2 +/- 0.5 x 10(6) M(-1), indicating a very strong host-guest interaction and an excellent size and shape match. In both cases, binding was much stronger than in the case of the same guests with cucurbit[6]uril itself, and in the case of Nile Red, binding was also much stronger than with modified beta- or gamma-cyclodextrins. This is partly a result of the partial aromatic nature of the host walls, which allow for pi-pi interactions not possible in cucurbiturils or cyclodextrins. The ability to study its inclusion complexes using either host or guest fluorescence, and the very high binding constants observed, illustrates the versatility and potential usefulness of this new host compound.     

Evaluation of the parameters of 1:1 charge transfer complexes from spectrophotometric data by non-linear numerical method

The non-linear numerical method for evaluation of equilibrium constants and molar extinction coefficients of molecular complexes from a spectrophotometric experiment is described, which in contrast to linear models has no limitations with respect to concentrations of the components. The proposed procedure is applied to donor-acceptor interaction in solution between N-ethyl carbazole (EtCz) and 7,7,8,8-tetracyanoquinodimethane (TCNQ) or n-hexyl 2,5,7-trinitro-9-dicyanomethylenefluorene-4-carboxylate (HexDTFC) to evaluate the method and to obtain the parameters of charge transfer complexes (CTCs) formation. Association constants (K) and molar extinction coefficients (epsilon) of CTCs derived from non-linear approach (EtCz-TCNQ: K = 2.49+0.19 M(-1); epsilon = 2950 +/- 160 M(-1) cm(-1). EtCz-HexDTFC: K = 12.1 +/- 0.3 M(-1); epsilon = 1335 +/- 24 M(-1) cm(-1)) are close to that from linear models but show lower standard errors in parameter estimations.     

Formation of Metallo-6(A)-((2-(bis(2-aminoethyl)amino)ethyl)amino)-6(A)-deoxy-beta-cyclodextrins and Their Complexation of Tryptophan in Aqueous Solution

A pH titration study shows that 6(A)-((2-(bis(2-aminoethyl)amino)ethyl)amino)-6(A)-deoxy-beta-cyclodextrin (betaCDtren) forms binary metallocyclodextrins, [M(betaCDtren)](2+), for which log(K/dm(3) mol(-)(1)) = 11.65 +/- 0.06, 17.29 +/- 0.05, and 12.25 +/- 0.03, respectively, when M(2+) = Ni(2+), Cu(2+), and Zn(2+), where K is the stability constant in aqueous solution at 298.2 K and I = 0.10 mol dm(-)(3) (NaClO(4)). The ternary metallocyclodextrins [M(betaCDtren)Trp](+), where Trp(-) is the tryptophan anion, are characterized by log(K/dm(3) mol(-)(1)) = 8.2 +/- 0.2 and 8.1 +/- 0.2, 9.5 +/- 0.3 and 9.4 +/- 0.2, and 8.1 +/- 0.1 and 8.3 +/- 0.1, respectively, where the first and second values represent the stepwise stability constants for the complexation of (R)- and (S)-Trp(-), respectively, when M(2+) = Ni(2+), Cu(2+), and Zn(2+). From comparisons of stabilities and UV-visible spectra, the binary and ternary metallocyclodextrins appear to be six-coordinate when M(2+) = Ni(2+) and Zn(2+) and five-coordinate when M(2+) = Cu(2+). The factors affecting the stoichiometries and stabilities of the metallocyclodextrins, are discussed and comparisons are made with related systems.     

Iron(III) complex of a crown ether-porphyrin conjugate and reversible binding of superoxide to its Iron(II) form

The synthesis and characterization of the Fe(III) complex of a novel crown ether-porphyrin conjugate, 52-N-(4-aza-18-crown-6)methyl-54,104,154,204-tetra-tert-butyl-56-methyl-5,10,15,20-tetraphenylporphyrin (H2Porph), as well as the corresponding hydroxo, dimeric, Fe(II), and peroxo species are reported. The crystal structure of [FeIII(Porph)Cl].H3O+.FeCl4-.C6H6.EtOH is also reported. [FeIII(Porph)(DMSO)2]+ and K[FeIII(Porph)(O22-)] are high-spin species (M?ssbauer data: delta = 0.38 mm s(-1), DeltaEq = 0.83 mm s(-1) and delta = 0.41 mm s(-1), DeltaEq = 0.51 mm s(-1), respectively), whereas in a solution of reduced [FeIII(Porph)(DMSO)2]+ complex the low-spin [FeII(Porph)(DMSO)2] (delta = 0.44 mm s(-1), DeltaEq = 1.32 mm s(-1)) and high-spin [FeII(Porph)(DMSO)] (delta = 1.27 mm s(-1), DeltaEq = 3.13 mm s(-1)) iron(II) species are observed. The reaction of [FeIII(Porph)(DMSO)2]+ with KO2 in DMSO has been investigated. The first reaction step, involving reduction to [FeII(Porph)(DMSO)2], was not investigated in detail because of parallel formation of an Fe(III)-hydroxo species. The kinetics and thermodynamics of the second reaction step, reversible binding of superoxide to the Fe(II) complex and formation of an Fe(III)-peroxo species, were studied in detail (by stopped-flow time-resolved UV/vis measurements in DMSO at 25 degrees C), resulting in kon = 36 500 +/- 500 M(-1) s(-1), koff = 0.21 +/- 0.01 s(-1) (direct measurements using an acid as a superoxide scavenger), and KO2- = (1.7 +/- 0.2) x 10(5) (superoxide binding constant kinetically obtained as kon/koff), (1.4 +/- 0.1) x 10(5), and (9.0 +/- 0.1) x 10(4) M(-1) (thermodynamically obtained in the absence and in the presence of 0.1 M NBu4PF6, respectively). Temperature-dependent kinetic measurements for kon (-40 to 25 degrees C in 3:7 DMSO/CH3CN mixture) yielded the activation parameters DeltaH = 61.2 +/- 0.9 kJ mol(-1) and DeltaS = +48 +/- 3 J K(-1) mol(-1). The observed reversible binding of superoxide to the metal center and the obtained kinetic and thermodynamic parameters are unique. The finding that fine-tuning of the proton concentration can cause the Fe(III)-peroxo species to release O2- and form an Fe(II) species is of biological interest, since this process might occur under very specific physiological conditions.     

Thermodynamic and kinetic studies on the reaction between the vitamin B12 derivative beta-(N-methylimidazolyl)cobalamin and N-methylimidazole: ligand displacement at the alpha axial site of cobalamins

The equilibria and kinetics of substitution of the 5,6-dimethylbenzimidazole at the alpha site of beta-(N-methylimidazolyl)cobalamin by N-methylimidazole have been investigated, and the product, bis(N-methylimidazolyl)cobalamin, has been characterized by visible and 1H NMR spectroscopies. The equilibrium constant for (N-MeIm)Cbl+ + N-MeIm right harpoon over left harpoon (N-MeIm)2Cbl+ was determined by 1H NMR spectroscopy (9.6 +/- 0.1 M(-1), 25.0 degrees C, I = 1.5 M (NaClO4)). The observed rate constant for this reaction exhibits an unusual inverse dependence on N-methylimidazole concentration, and it is proposed that substitution occurs via a base-off solvent-bound intermediate. Activation parameters typical for a dissociative ligand substitution mechanism are reported at two different N-MeImT concentrations, 5.00 x 10(-3) M (DeltaH++ = 99 +/- 2 kJ x mol(-1), DeltaS++ = 39 +/- 5 J x mol(-1) x K(-1), DeltaV++ = 15.0 +/- 0.7 cm3 x mol(-1), and 1.00 M (DeltaH++ = 109.4 +/- 0.8 kJ x mol(-1), DeltaS++ = 70 +/- 3 J x mol(-1) x K(-1), DeltaV++ = 16.8 +/- 1.1 cm3 x mol(-1)). According to the proposed mechanism, these parameters correspond to the equation of (N-MeIm)2Cbl+ and the ring-opening reaction of the alpha-DMBI of (N-MeIm)Cbl+ to give the solvent-bound intermediate in both cases, respectively.     

Influence of terpyridine as pi-acceptor ligand on the kinetics and mechanism of the reaction of NO with ruthenium(III) complexes

The kinetics of the unusually fast reaction of cis- and trans-[Ru(terpy)(NH3)2Cl]2+ (with respect to NH3; terpy=2,2':6',2"-terpyridine) with NO was studied in acidic aqueous solution. The multistep reaction pathway observed for both isomers includes a rapid and reversible formation of an intermediate Ru(III)-NO complex in the first reaction step, for which the rate and activation parameters are in good agreement with an associative substitution behavior of the Ru(III) center (cis isomer, k1=618 +/- 2 M(-1) s(-1), DeltaH(++) = 38 +/- 3 kJ mol(-1), DeltaS(++) = -63 +/- 8 J K(-1) mol(-1), DeltaV(++) = -17.5 +/- 0.8 cm3 mol(-1); k -1 = 0.097 +/- 0.001 s(-1), DeltaH(++) = 27 +/- 8 kJ mol(-1), DeltaS(++) = -173 +/- 28 J K(-1) mol(-1), DeltaV(++) = -17.6 +/- 0.5 cm3 mol(-1); trans isomer, k1 = 1637 +/- 11 M(-1) s(-1), DeltaH(++) = 34 +/- 3 kJ mol(-1), DeltaS(++) = -69 +/-11 J K(-1) mol(-1), DeltaV(++) = -20 +/- 2 cm3 mol(-1); k(-1)=0.47 +/- 0.08 s(-1), DeltaH(++)=39 +/- 5 kJ mol(-1), DeltaS(++) = -121 +/-18 J K(-1) mol(-1), DeltaV(++) = -18.5 +/- 0.4 cm3 mol(-1) at 25 degrees C). The subsequent electron transfer step to form Ru(II)-NO+ occurs spontaneously for the trans isomer, followed by a slow nitrosyl to nitrite conversion, whereas for the cis isomer the reduction of the Ru(III) center is induced by the coordination of an additional NO molecule (cis isomer, k2=51.3 +/- 0.3 M(-1) s(-1), DeltaH(++) = 46 +/- 2 kJ mol(-1), DeltaS(++) = -69 +/- 5 J K(-1) mol(-1), DeltaV(++) = -22.6 +/- 0.2 cm3 mol(-1) at 45 degrees C). The final reaction step involves a slow aquation process for both isomers, which is interpreted in terms of a dissociative substitution mechanism (cis isomer, DeltaV(++) = +23.5 +/- 1.2 cm3 mol(-1); trans isomer, DeltaV(++) = +20.9 +/- 0.4 cm3 mol(-1) at 55 degrees C) that produces two different reaction products, viz. [Ru(terpy)(NH3)(H2O)NO]3+ (product of the cis isomer) and trans-[Ru(terpy)(NH3)2(H2O)]2+. The pi-acceptor properties of the tridentate N-donor chelate (terpy) predominantly control the overall reaction pattern.     

Cooperative ligation,back-bonding,and possible pyridine-pyridine interactions in tetrapyridine-vanadium(II): a visible and X-ray spectroscopic study

The binding of pyridine by V(II) in aqueous solution shows evidence for the late onset of cooperativity. The K(1) governing formation of [V(py)](2+) (lambda(max) = 404 nm, epsilon(max) = 1.43 +/- 0.3 M(-1) cm(-1)) was determined spectrophotometrically to be 11.0 +/- 0.3 M(-)(1), while K(1) for isonicotinamide was found to be 5.0 +/- 0.1 M(-1). These values are in the low range for 3d M(2+) ions and indicate that V(II).py back-bonding is not significant in the formation of the 1:1 complex. Titration of 10.5 mM V(II) with pyridine in aqueous solution showed an absorption plateau at about 1 M added pyridine, indicating a reaction terminus. Vanadium K-edge EXAFS analysis of 63 mM V(II) in 2 M pyridine solution revealed six first-shell N/O ligands at 2.14 A and 4 +/- 1 pyridine ligands per V(II). UV/vis absorption spectroscopy indicated that the same terminal V(II) species was present in both experiments. Model calculations showed that in the absence of back-bonding only 2.0 +/- 0.2 and 2.4 +/- 0.2 pyridine ligands would be present, respectively. Cooperativity in multistage binding of pyridine by [V(aq)](2+) is thus indicated. XAS K-edge spectroscopy of crystalline [V(O(3)SCF(3))(2)(py)(4)] and of V(II) in 2 M pyridine solution each exhibited the analogous 1s --> (5)E(g) and 1s --> (5)T(2g) transitions, at 5465.5 and 5467.5 eV, and 5465.2 and 5467.4 eV, respectively, consistent with the EXAFS analysis. In contrast, [V(py)(6)](PF(6))(2) and [V(H(2)O)(6)]SO(4) show four 1s --> 3d XAS transitions suggestive of a Jahn-Teller distorted excited state. Comparison of the M(II)[bond]N(py) bond lengths in V(II) and Fe(II) tetrapyridines shows that the V(II)[bond]N(py) distances are about 0.06 A shorter than predicted from ionic radii. For [VX(2)(R-py)(4)] (X = Cl(-), CF(3)SO(3)(-); R = 4-Et, H, 3-EtOOC), the E(1/2) values of the V(II)/V(III) couples correlate linearly with the Hammett sigma values of the R group. These findings indicate that pi back-bonding is important in [V(py)(4)](2+) even though absent in [V(py)](2+). The paramagnetism of [V(O(3)SCF(3))(2)(py)(4)] in CHCl(3), 3.8 +/- 0.2 mu(B), revealed that the onset of back-bonding is not accompanied by a spin change. Analysis of the geometries of V(II) and Fe(II) tetrapyridines indicates that the ubiquitous propeller motif accompanying tetrapyridine ligation may be due to eight dipole interactions arising from the juxtaposed C-H edges and pi clouds of adjoining ligands, worth about -6 kJ each. However, this is not the source of the cooperativity in the binding of multiple pyridines by V(II) because the same interactions are present in the Fe(II)-tetrapyridines, which do not show cooperative ligand binding. Cooperativity in the binding of pyridine by V(II) is then assigned by default to V(II)-pyridine back-bonding, which emerges only after the first pyridine is bound.     

Higher affinity quadruply hydrogen-bonded complexation with 7-deazaguanine urea

[structure: see text] UG forms a highly stable quadruply hydrogen-bonded heterocomplex with DAN, but the fidelity of the complex is lowered somewhat by the Hoogsteen-side oligomerization of UG (K(assoc) approximately 230 M(-)(1), CDCl(3)). DeUG was prepared as a more robust analogue of UG lacking the Hoogsteen nitrogen atom. Remarkably, the deaza analogue, DeUG, forms a much more stable complex with DAN (>10-fold higher K(assoc) for DeUG.DAN vs UG.DAN) but also dimerizes more strongly (K(dim) = 880 +/- 40 M(-)(1), CDCl(3)) by adopting a conformation preorganized for both binding and dimerization.     

A deconvolution method for the separation of specific versus nonspecific interactions in noncovalent protein-ligand complexes analyzed by ESI-FT-ICR mass spectrometry

A method to separate specific and nonspecific noncovalent interactions observed in ESI mass spectra between a protein and its ligands is presented. Assuming noncooperative binding, the specific ligand binding is modeled as a statistical distribution on identical binding sites. For the nonspecific fraction we assume a statistical distribution on a large number of "nonspecific" interacting sites. The model was successfully applied to the noncovalent interaction between the protein creatine kinase (CK) and its ligands adenosine diphosphate (ADP) and adenosine triphosphate (ATP) that both exhibit nonspecific binding in the mass spectrum. The two sequential dissociation constants obtained by applying our method are K(1,diss) = 11.8 +/- 1.5 microM and K(2,diss) = 48 +/- 6 microM for ADP. For ATP, the constants are K(1,diss) = 27 +/- 7 microM and K(2,diss) = 114 +/- 27 microM. All constants are in good correlation with reported literature values. The model should be valuable for systems with a large dissociation constant that require high ligand concentrations and thus have increased potential of forming nonspecific adducts.