PHOTOSENSITIZATION BY ANTICANCER AGENTS—10. ortho-SEMIQUINONE AND SUPEROXIDE RADICALS PRODUCED DURING ANTHRAPYRAZOLE-SENSITIZED OXIDATION OF CATECHOLS

Photosensitized oxidation of catechol, 3,4-dihydroxybenzoic acid (DHBA), 3,4-dihydroxy-dihydrocinnamic acid (DHCA), and 3,4-dihydroxy-phenylalanine (DOPA) by novel anticancer agents, anthrapyrazoles (AP), has been studied employing EPR and the spin trapping technique. The formation of o-semiquinone radicals, the one-electron oxidation products of the catechols, stabilized in the form of zinc ion complexes, has been demonstrated. Rate constants for the disproportionation of the semiquinone radical/Zn2+ complexes in (DMSO)/acetate buffer (pH 4.5, 1:1 vol/vol; 100 mM Zn2+) mixture have been determined to be 0.35 x 10(4), 14 x 10(4), 8.8 x 10(4) and 3 x 10(4) M-1 s-1 for catechol, DHBA, DHCA and DOPA respectively. The presence of oxygen enhanced rather than inhibited the photogeneration of the o-semiquinone radicals and facilitated their EPR detection. The EPR spectrum of the superoxide radical adduct with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide was observed for the first time during photosensitized oxidation of the catechols in acidic aqueous solutions and in DMSO/acetate buffer mixture.     

Determination of dopa, dopamine, dopac, epinephrine, norepinephrine, alpha-monofluoromethyldopa and alpha-difluoromethyldopa in various tissues of mice and rats using reversed-phase ion-pair liquid chromatography with electrochemical detection

A method for the determination of catecholic amino acids and amines by reversed-phase ion-pair high-performance liquid chromatography with electrochemical detection has been developed. B using octanesulfonic acid for ion pairing and by optimising ionic strength, pH and methanol concentration of the mobile phase, separation was achieved of 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), norepinephrine (NE), epinephrine (EPI), and dopamine (DA). Alpha-Difluoromethyldopa (DFMD) and alpha-monofluoromethyldopa (MFMD), two potent enzyme-activated irreversible inhibitors of aromatic amino acid decarboxylase were also separated from the natural catechols. Concentrations of catechols and inhibitors were measured in brains, hearts and kidneys of mice treated with small repeated doses of MFMD. The method has also been applied to the determination of catechols in other organs such as prostates and seminal vesicles of rats and in smaller tissues like mesenteric arteries. A semi-automated procedure making use of an automatic sample processor and a digital integrator permitted the analysis of as many as sixty samples per day.     

Vibrational spectra and quantum chemical calculations of 3,4-diaminobenzoic acid.

The Fourier Transform Raman and Fourier Transform infrared spectra of 3,4-diaminobenzoic acid (3,4-DABA) were recorded in the solid phase. Geometry optimizations were done without any constraint and harmonic-vibrational wave numbers and several thermodynamic parameters were calculated for the minimum energy conformer at ab initio and DFT levels invoking 6-311++G(d,p) basis set. The results were compared with the experimental values with the help of specific scaling procedures, the observed vibrational wavenumbers in FT-IR and FT-Raman spectra were analyzed and assigned to different normal modes of the molecule. Most of the modes have wavenumbers in the expected range, the error obtained was in general very low. The appropriate theoretical spectrograms for the IR and Raman spectra of the title molecule were also constructed.     

Studies of the dissociation and the protonation of TB-chlorosulphophenol

The dissociation constant of each step for TB-chlorosulphophenol has been determined by potentiometric method, and the thermodynamic constants, △G°, △H° and △S°, of the dissociation process have been calculated. The protonation constants were measured by the spectrophotometric method. The pH values of various forms of anions of the chromogenic reagent at their concentrations were also calculated.     

Binding characteristics of dopa (3,4-dihydroxyphenylalanine) and its metabolites to bovine serum albumin as measured by ultrafiltration technique.

The interaction between 3,4-dihydroxyphenylalanine (DOPA), dopamine, 3-methoxytyramine and homovanillic acid and bovine serum albumin (BSA) was investigated by the ultrafiltration technique. The apparent binding constants were determined assuming the equivalence and independence of the binding sites on the BSA molecule. The binding constants were in the range of log K = 2.85 to 3.77 with 1 to 2 binding sites. The affinity of ligands to BSA strengthened with progression of the metabolism in the order of DOPA less than dopamine less than 3-methoxytyramine less than homovanillic acid.     

混合溶剂中标准pH参考值的研究——KHPh(0.05 mol/kg)+KCl+异丙醇+水体系

应用电动势法在10%、14.93%和20%异丙醇+水混合溶剂中测定了0.05mol/kg邻苯二甲酸氢钾(KHPh)缓冲溶液在278.15～318.15K范围内的标准pH参考值,根据Pitzer理论,提出了确定各离子活度系数的新方法,计算了邻苯二甲酸的第二级热力学解离常数和相应的热力学量。     

Determination of limiting ionic mobilities and dissociation constants of some local anaesthetics.

The limiting ionic mobilities and thermodynamic acid dissociation constants were calculated from isotachophoretic experiments for the local anaesthetics procaine, tetracaine, lidocaine, trimecaine, bupivacaine, cinchocaine, diperodone, diocaine, cocaine, psicaine-neu, tropacocaine, amylocaine, beta-eucaine and leucinocaine. The pH values at which the local anaesthetics with very similar limiting ionic mobilities can be isotachophoretically separated were determined from simulated mobility curves. The measuring apparatus employed a high-frequency contactless conductivity detector.     

Thermodynamics of mixture of boric acid with lithium borate and chloride

The first thermodynamic dissociation constants of boric acid in aqueous solution of lithium chloride with borate at five different temperatures from 278.15 to 318.15 K were evaluated from emf values of the cell without liquid junction potential with the improved extrapolation and polynomial approximation on the basis of Pitzer's theory. Values obtained from both methods are in good agreement with each other. Pitzer's parameters β(0) and β(1) of LiB(OH)4 and other thermodynamic quantities of dissociation process of boric acid are obtained. Results have been discussed.     

The dissociation of ethylenedithiodiacetic acid in aqueous solution

The dissociation of ethylenedithiodiacetic acid (H₂Edtda) was studied by potentiometric titration at 298.15 K and ionic strength values of 0.5, 1.0, and 1.5 against the background of sodium and lithium nitrates. The concentration and thermodynamic dissociation constants were determined.     

The heat effects of dissociation of N-(carboxymethyl)aspartic acid

The heat effects of dissociation of N-(carboxymethyl)aspartic acid were determined calorimetrically at 298.15 K and various ionic strength values. The standard thermodynamic characteristics of dissociation of the complexone at fixed and zero ionic strengths were calculated.     

Mechanisms of acid decomposition of dithiocarbamates. 3. Aryldithiocarbamates and the torsional effect

The acid decomposition of some p-substituted aryldithiocarbamates (arylDTCs) was observed in 20% aqueous ethanol at 25 degrees C, mu = 1.0 (KCl, for pH > 0). The pH-rate profiles showed a dumbell shape with a plateau where the observed first-order rate constant k(obs) was equal to k(o), the rate constant of the decomposition of the dithiocarbamic acid species. The acid dissociation constants of the dithiocarbamic acids (pK(a)) and their conjugate acids (pK(+)) were calculated from the pH-rate profiles. Comparatively, k(o) was more than 10(4)-fold faster than alkyldithiocarbamates (alkDTCs) with similar pK(N) (the acid dissociation constant of the parent amine). It was observed that the values of pK(a) and pK(+)were 5 and 8 units of pK, respectively, higher than the expected values from the pK(N) of alkylDTCs. The higher values were attributed to the inhibition of the delocalization of the nitrogen electron pair into the benzene ring because of the strong electron withdrawal effect of the thiocarbonyl group. Comparison of the activation parameters showed that the rate acceleration was due to a decrease in the enthalpy of activation. Proton inventory indicated the existence of a multiproton transition state, and it was consistent with an S to N proton transfer through a water molecule. There are two hydrogens contributing to a secondary SIE, and there are also two protons that are being transferred at the transition state to form a zwitterion followed by fast C-N bond cleavage. The mechanism could also be a concerted asynchronic process where the N-protonation is more advanced than the C-N bond breakdown. The kinetic barrier is similar to the torsional barrier of thioamides, suggesting that the driving force to reach the transition state is the needed torsion of the C-N bond that inhibits the resonance with the thiocarbonyl group and the aromatic moiety, increasing the basicity of the nitrogen and making the proton transfer thermodynamically favorable.     

Spectroscopic determination of acid dissociation constants of N-substituted-6-acylbenzothiazolone derivatives

The acid dissociation constants of twelve novel drug precursor N-substituted-6-acylbenzothiazolone derivatives were determined by using the UV-vis spectroscopic technique. The protonation and deprotonation behaviors of the investigated molecules were researched from the super basic to super acid regions (i.e., 8 mol·L(-1) KOH to 98% H(2)SO(4)) including the pH region. It is observed that all of the molecules are protonated in the super acidic region. The calculated relative stability values of possible tautomer structures indicate that the keto form of investigated molecules is favored over the enol form. It was predicted that protonation occurs at the amide (oxo) group found in the keto form.     

Dissociation constants of some derivatives of 5-amino isophthalic acid in mixed solvents

The dissociation constants of some derivatives of 5-amino isophthalic acid have been measured by Calvin Bjerrum pH titration method in DMF-water and DMSO-water (60: 40 v/v) systems at 308.15 K. It is observed that dissociation constant depends on the solvent and substituents group present in the compound.     

Kinetics and mechanism of the oxidation of a heterocyclic thioamide by silver(II)-cyclam

The kinetics of the oxidation of 4,6-dimethyl-2-mercaptopyrimidine (DMP) by Ag(cyclam)²⁺ were studied in buffer solutions from pH 5.8 to 7.2 at constant ionic strength of 0.10?M?(NaClO₄). The reaction is observed to be first-order with respect to [Ag(cyclam)²⁺] and to [DMP]. However, the reaction rate is affected by the pH of the solution owing to the acid–base equilibrium of the thiol. The mechanism postulated to account for the kinetics includes an acid–base equilibrium and oxidation of thiol (RSH) and thiolate ion (RS^?) by Ag(cyclam)²⁺ to RS^· radicals which undergo rapid dimerization to form disulfide (RSSR). From the postulated mechanism and the observed kinetics a rate expression was derived, and second-order rate constants and activation parameters were calculated. The pK _a values of the acid dissociation reaction of DMP were also determined at four temperatures using spectrophotometric methods, and thermodynamic parameters calculated from the K _a values.     

Study of catecholamines in patient urine samples by capillary electrophoresis.

Capillary zone electrophoresis with photodiode array detection at 220 nm was used for analysis of catechol compounds in human urine. The method was optimized with reference compounds 3,4-dihydroxybenzylamine, adrenaline, noradrenaline, normetanephrine, dopamine, dopac (homogensitic acid), methanephrine, vanillyl-mandelic acid, 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid and 3-methoxytyramic acid at pH 4.0 and 8.0 for their electrophoretic separation. The UV spectra of the catechols were detected at a concentration of 20 microM. Repeatability of the method calculated using the absolute migration times of the catechols was below 1.5% and using the peak areas below 5%. The patient samples were hydrolyzed by 0.5 M acid or base solutions. In the studies, a few patient samples were analyzed using 3,4-dihydroxybenzylamine as an internal standard. In the hydrolysis steps needed for their detection in urine, all the other catecholamines, except 5-HIAA, did not decompose to detectable species at 220 or 254 nm. The concentrations of the catecholamines observed in real samples were at nM levels.     

The heat effects of dissociation of N,N-bis-(carboxymethyl)aspartic acid

The heat effects of dissociation of N,N-bis-(carboxymethyl)aspartic acid were measured calorimetrically at 298.15 K and various solution ionic strengths. The standard thermodynamic characteristics of dissociation of the complexone studied were calculated at fixed and zero ionic strength values.     

Dissociation kinetics of macrocyclic trivalent lanthanide complexes of 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A)

The [H(+)]-catalyzed dissociation rate constants of several trivalent lanthanide (Ln) complexes of 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (LnDO2A(+), Ln = La, Pr, Eu, Er and Lu) have been determined in two pH ranges: 3.73-5.11 and 1.75-2.65 at four different temperatures (19-41.0 °C) in aqueous media at a constant ionic strength of 0.1 mol dm(-3) (LiClO(4)). For the study in the higher pH range, i.e. pH 3.73-5.11, copper(II) ion was used as the scavenger for the free ligand DO2A in acetate/acetic acid buffer medium. The rates of Ln(III) complex dissociation have been found to be independent of [Cu(2+)] and all the Ln(III) complexes studied show [H(+)]-dependence at low acid concentrations but become [H(+)]-independent at high acid concentrations. Influence of the acetate ion content in the buffer on the dissociation rate has also been investigated and all the complexes exhibit a first-order dependence on [Acetate]. The dissociation reactions follow the rate law: k(obs) = k(Ac)[Acetate] + K'k(lim)[H(+)]/(1 + K'[H(+)]) where k(AC) is the dissociation rate constant for the [Acetate]-dependent pathway, k(lim) is the limiting rate constant, and K' is the equilibrium constant for the reaction LnDO2A(+) + H(+) ? LnDO2AH(2+). In the lower pH range, i.e. pH 1.75-2.65, the dye indicator, cresol red, was used to monitor the dissociation rate, and all the Ln(III) complexes also show [H(+)]-dependence dissociation pathways but without the rate saturation observed at higher pH range. The dissociation reactions follow the simple rate law: k(obs) = k(H)[H(+)], where k(H) is the dissociation rate constant for the pathway involving monoprotonated species. The absence of an [H(+)]-independent pathway in both pH ranges indicates that LnDO2A(+) complexes are kinetically rather inert. The obtained k(AC) values follow the order: LaDO2A(+) > PrDO2A(+) > EuDO2A(+) > ErDO2A(+) > LuDO2A(+), whereas the k(lim) and k(H) values follow the order: LaDO2A(+) > PrDO2A(+) > ErDO2A(+) > EuDO2A(+) > LuDO2A(+), mostly consistent with their thermodynamic stability order, i.e. the more thermodynamically stable the more kinetically inert. In both pH ranges, activation parameters, ΔH*, ΔS* and ΔG*, for both acetate-dependent and proton-catalyzed dissociation pathways have been obtained for most of the La(III), Pr(III), Eu(III), Er(III) and Lu(III) complexes, from the temperature dependence measurements of the rate constants in the 19-41 °C range. An isokinetic (linear) relationship is found between ΔH* and ΔS* values, which supports a common reaction mechanism.     

Rapid procedure for chromatographic isolation of DOPA, DOPAC, epinephrine, norepinephrine and dopamine from a single urinary sample at endogenous levels

A three-step procedure has been investigated to extract 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), epinephrine (E), norepinephrine (NE) and dopamine (DA) from a single urinary sample with the object of obtaining extracts pure enough for specific fluorimetric assay. The procedure described in this paper results from the combination of urine purification on an aluminum oxide column, separation by ion-exchange chromatography of the DOPA-DOPAC fraction from catecholamines, and ether isolation of DOPAC from DOPA. The whole procedure is rapid and easily performed in one work day. Extraction recoveries were 72.4 +- 3.5%, 76 +- 2%, 85.7 +- 3.3%, 85.6 +- 1.4% and 92.4 +- 5.5% for DOPA, DOPAC, E, NE and DA respectively (n=6). The lowest amounts of the five catechols that could be detected in urinary samples by a combination of this extraction procedure and the methods of assay used in our laboratory were 15 ng for DOPA, 40 ng for NE, 20 ng for E, 152 ng for DA and 2.95 micrograms for DOPAC. Urinary volumes convenient for accurate estimation of each compound were 20 ml for healthy human subjects. For pathological or pharmacological purposes, 5 ml of human urine were sufficient. The daily excretion of DOPA, DOPAC, E, NE and DA found by this procedure agrees with data obtained by other authors in healthy subjects. In pathological samples, our three-step procedure led to lower amounts than methods using alumina purification only. The discrepancies between the two methods are discussed in terms of development of internal standards, relative specificity of fluorimetric assays, values of blank eluates, and the possibility of interference from unknown abnormal body metabolites or pharmacological drugs not eliminated by a single-step alumina purification.     

Development of novel antioxidants: design,synthesis, and reactivity

We are attempting to develop novel synthetic antioxidants aimed at retarding the effects of free-radical induced cell damage. In this paper we discuss the design strategy and report the synthesis of seven novel antioxidants, including six catechols and a benzylic phenol. The bond dissociation enthalpy (BDE) for the most active (weakest) OH bond in each molecule was calculated by theoretical methods, as well as the BDE for the semiquinone radical. Reaction rates with the nitrogen-centered free radical DPPH(*) were measured in ethyl acetate. The log of k(DPPH) for bimolecular reaction correlated well with the primary BDE. The correlation between rate constants and calculated BDEs shows that the BDE is a good predictor of antioxidant activity with DPPH(*), suggesting that our design criteria are useful and that these compounds should undergo further testing in cell cultures and in animal models.     

Convenient synthesis of acetonide-protected 3,4-dihydroxyphenylalanine (DOPA) for Fmoc solid-phase peptide synthesis

We report a facile approach to the synthesis of acetonide and Fmoc-protected 3,4-dihydroxyphenylalanine (DOPA), Fmoc-DOPA(acetonide)-OH. By protecting the amino group of DOPA with a phthaloyl group and the carboxyl group as a methyl ester, acetonide protection of the catechol of DOPA derivative was realized in the presence of p-toluenesulfonic acid. Following removal of protecting groups, the intermediate was converted to Fmoc-DOPA(acetonide)-OH, which was successfully incorporated into a short DOPA-containing peptide, derived from marine tubeworm cement proteins Pc1 and Pc2.