Active Species for Ce(IV)-Induced Hydrolysis of Phosphodiester Linkage in cAMP AND DNA

The hydrolysis of cyclic adenosine 3′,5′-monophosphate and 2′-deoxythymidylyl(3′-5′)2′-deoxythymidine by Ce(NH₄)₂(NO₃)₆ was kinetically studied. The rate of hydrolysis was fairly proportional to the concentration of [Ce ^IV ₂ (OH)₄]⁴⁺, showing that this is the catalytically active species. According to quantum-chemical calculation, the two Ce(IV) ions in this [Ce^IV ₂(OH)₄]⁴⁺ cluster are bridged by two OH residues. Upon the complex formation with H₂ PO₄ ^? (a model compound for the phosphodiesters), these two Ce(IV) ions bind the two oxygen atoms of the substrate and enhance the electrophilicity of the phosphorus atom. The catalytic mechanism of Ce(IV)-induced hydrolysis of phosphodiesters has been proposed on the basis these results.     

Hydrolysis of DNA by Ce(IV)/EDTA complexes--the mechanism of DNA cleavage and design of artificial restriction enzymes.

Homogeneous Ce(IV) complex of EDTA promptly hydrolyzes oligonucleotides under physiological conditions. Moreover, the activity of Ce(IV)/EDTA for DNA hydrolysis is promoted by the addition of amines. When [Ce(IV)/EDTA] = 5 mumol dm3 and [ethylenediamine] = 100 mmol dm3, the catalytic activity is about 50 times as large as that of Ce(IV)/EDTA. The combination of Ce(IV)/EDTA and amines is eminent tools for the future molecular biology and biotechnology.     

Synthesis and structure determination of some nonanomerically C-C-linked serine glycoconjugates structurally related to mannojirimycin

The Bucherer-Bergs reaction of methyl 2,3-O-isopropylidene-alpha-d-lyxo-hexofuranosid-5-ulose gave (4'S)-4'-carbamoyl-4'-[methyl (4R)-2,3-O-isopropylidene-beta-l-erythrofuranosid-4-C-yl]-oxazolidin-2'-one instead of expected hydantoins. A mixture of hydantoins--(5'R)-triphenylmethoxymethyl-5'-[methyl (4R)-2,3-O-isopropylidene-beta-l-erythrofuranosid-4-C-yl]-imidazolidin-2',4'-dione and (5'S)-triphenylmethoxymethyl-5'-[methyl (4R)-2,3-O-isopropylidene-beta-l-erythrofuranosid-4-C-yl]-imidazolidin-2',4'-dione was obtained from the 5-ulose having protected primary OH group at C-6. The 4'-S configuration of 2 as well as 5'-S configuration of (5'S)-hydroxymethyl-5'-[methyl (4R)-2,3-O-isopropylidene-beta-l-erythrofuranosid-4-C-yl]-imidazolidin-2',4'-dione (9) was confirmed by X-ray crystallography. Corresponding alpha-amino acid--methyl (5S)-5-amino-5-C-carboxy-5-deoxy-alpha-d-lyxo-hexofuranoside (alternative name: 2-[methyl (4R)-beta-l-erythrofuranosid-4-C-yl]-l-serine) (11) was obtained from the hydantoin 9 by acid hydrolysis of the isopropylidene and trityl groups followed by basic hydrolysis of the hydantoin ring. Analogous derivatives with 5-R configuration, formed in a minority, were also isolated and characterised.     

Reactions of gold(III) complexes with serum albumin.

The reactions of a few representative gold(III) complexes -[Au(ethylenediamine)2]Cl3, [Au(diethylentriamine)Cl]Cl2, [Au(1,4,8,11-tetraazacyclotetradecane)](ClO4)2Cl, [Au(2,2',2'-terpyridine)Cl]Cl2, [Au(2,2'-bipyridine)(OH)2][PF6] and the organometallic compound [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine-H)(OH)][PF6]- with BSA were investigated by the joint use of various spectroscopic methods and separation techniques. Weak metal-protein interactions were revealed for the [Au(ethylenediamine)2]3+ and [Au(1,4,8,11-tetraazacyclotetradecane)]3+ species, whereas progressive reduction of the gold(III) centre was observed in the cases of [Au(2,2'-bipyridine)(OH)2]+ and [Au(2,2',2'-terpyridine)Cl]2+. In contrast, tight metal-protein adducts are formed when BSA is reacted with either [Au(diethylentriamine)Cl]2+ and [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine-H)(OH)]+. Notably, binding of the latter complex to serum albumin results in the appearance of characteristic CD bands in the visible spectrum. It is suggested that adduct formation for both of these gold(III) complexes occurs through coordination at the level of surface histidines. Stability of these gold(III) complexes/serum albumin adducts was tested under physiologically relevant conditions and found to be appreciable. Metal binding to the protein is tight; complete detachment of the metal from the protein has been achieved only after the addition of excess potassium cyanide. The implications of the present results for the pharmacological activity of these novel cytotoxic agents are discussed.     

Bimetallic complexes of spiro-azacrown ligands as catalysts of phosphoester and phosphoric anhydride cleavage

The ability of bimetallic homo- and heteronuclear complexes of two spiro-linked ligands, viz. a biazacrown (i.e., 2,6,10,14,18,22-hexaazaspiro[11.11]tricosane (1)) and an azacrown-crown ether (i.e., 14,17,20,23,26-pentaoxa-2,6,10-triaza-spiro[11.15]heptacosane (2)), to promote the cleavage of the phosphoester linkage of dinucleoside 3',5'-phosphates and the phosphoric anhydride bridge of dinucleoside 5',5'-triphosphates was studied. In both reactions, the bimetallic homonuclear Cu2+ and Zn2+ complexes were better catalysts than their monometallic counterparts. The acceleration was two- to five-fold with the phosphoester cleavage and 3- to 20-fold with the phosphoric anhydride cleavage. Interestingly, the most-efficient catalyst of the phosphoester cleavage was the heterodinuclear Ni2+,Zn2+ complex of 1, the catalytic activity of which was up to 5- and 100-fold that of the homodinuclear Zn2+ and Ni2+ complexes, respectively. Moreover, this cooperative acceleration was observed to depend on the identity of the 5'-linked nucleoside: 3',5'-UpU and 3',5'-ApU were cleaved much faster than 3',5'-UpA, and no cooperative acceleration was observed with 3',5'-ApA. The reaction was second-order in hydroxide ion concentration, suggesting that a double deprotonation took place on going from the initial to the transition state. Evidently, in addition to deprotonation of the attacking 2'-OH group, N(3)H of the 5'-linked uridine was displaced by one of the metal ions of the cleaving agent. With the phosphoric anhydride cleavage, no similar cooperativity of two different metal ions was observed, but the greatest rate-acceleration was achieved with the homodinuclear Cu2+ complexes.     

Derivatives of methanopterin, a coenzyme involved in methanogenesis

Degradational studies of methanopterin, a coenzyme involved in methanogenesis, are reported. The results of these studies are in full accordance with the proposed structure of methanopterin as N-[1'-(2'-amino-4'-hydroxy-7' -methyl-6'-pteridinyl)ethyl]-4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl(5'-1' )O-alpha-ribofuranosyl-5'-phosphoric acid] aniline in which the phosphate group is esterified with alpha-hydroxyglutaric acid. Acid hydrolysis of methanopterin cleaved the 5'----1' glycosidic bond and yielded a 'hydrolytic product' which was identified as N-[1'-(2'-amino-4'-hydroxy-7' -methyl-6'-pteridinyl)ethyl]-4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl]aniline. Alkaline permanganate oxidation of methanopterin yielded 7-methylpterin-6-carboxylic acid. Catalytic (or enzymatic) hydrogenation of methanopterin gave a mixture of 6-ethyl-7-methyl-7,8-dihydropterin, 6-ethyl-7-methylpterin and a third compound, named methaniline which was identified as 4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl(5'----1')O-alpha -ribofuranosyl-5'-phosphoric acid]aniline, in which the phosphate group is esterified with alpha-hydroxyglutaric acid. Methanosarcina barkeri contains a closely related coenzyme called sarcinapterin, which was identified as a L-glutamyl derivative of methanopterin, where the glutamate moiety is attached to the alpha-carboxylic acid group of the alpha-hydroxyglutaric acid moiety of methanopterin via an amide linkage.     

Titanium(IV) targets phosphoesters on nucleotides: implications for the mechanism of action of the anticancer drug titanocene dichloride

Abstract Reactions between the anticancer drug titanocene dichloride (Cp2TiCl2) and various nucleotides and their constituents in aqueous solution or N,N-dimethylformamide (DMF) have been investigated by 1H and 31P NMR spectroscopy and in the solid state by IR spectroscopy. In aqueous solution over the pH* (pH meter reading in D2O) range 2.3-6.5, CMP forms one new species with Ti(IV) bound only to the phosphate group. In acidic media at pH*<4.6, three species containing titanocene bound to the phosphate group of dGMP, AMP, dTMP and UMP are formed rapidly. The bases also appear to influence titanocene binding. Only one of these Ti(IV)-bound species can be detected in the pH* range of 4.6-6.5 in each case. The order of reactivity towards Cp2TiCl2(aq) at pH* ca. 3 is GMP>TMP approximately AMP > CMP. At pH* > 7.0, hydrolysis of Cp2TiCl2 predominated and little reaction with the nucleotides was observed. Binding of deoxyribose 5'-phosphate and 4-nitrophenyl phosphate to Cp2TiCl2(aq) via their phosphate groups was detected by 31P NMR spectroscopy, but no reaction between Cp2TiCl2(aq) and deoxyguanosine, 9-ethylguanine or deoxy-D-ribose was observed in aqueous solution. The nucleoside phosphodiesters 3',5'-cyclic GMP and 2',3'-cyclic CMP did not react with Cp2TiCl2(aq) in aqueous solution; however, in the less polar solvent DMF, 3',5'-cyclic GMP coordination to [Cp2Ti]2+ via its phosphodiester group was readily observed. Binding of titanocene to the phosphodiester group of the dinucleotide GpC was also observed in DMF by 31P NMR. The nucleoside triphosphates ATP and GTP reacted more extensively with Cp2TiCl2(aq) than their monophosphates; complexes with bound phosphate groups were formed in acidic media and to a lesser extent at neutral pH. Cleavage of phosphate bonds in ATP (and GTP) by Cp2TiCl2(aq) to form inorganic phosphate, AMP (or GMP) and ADP (or GDP) was observed in aqueous solutions. In addition, titanocene binding to ATP was not inhibited by Mg(II), but the ternary complex titanocene-ATP-Mg appeared to form. These reactions contrast markedly with those of the drug cisplatin, which binds predominantly to the base nitrogen atoms of nucleotides and only weakly to the phosphate groups. The high affinity of Ti(IV) for phosphate groups may be important for its biological activity.     

Influence of dangling thymidine residues on the stability and structure of two DNA duplexes

We have employed temperature-dependent UV spectroscopy, circular dichroism (CD), 400-MHz proton nuclear magnetic resonance (NMR), and computer modeling to characterize both structurally and thermodynamically the influence of unpaired, dangling thymidine residues (T) on the thermal stability and melting behavior of two DNA core duplexes. The specific DNA double helices that we have investigated in this work are core duplexes [d(GC)3]2 (I) and [d(CG)3]2 (IV), 3' dangling T derivatives [d[(GC)3TT]]2 (II) and [d[(CG)3TT]]2 (V), and 5' dangling T derivatives [d[TT(GC)3]]2 (III) and [d[TT(CG)3]]2 (VI). Our experimental data allow us to reach the following conclusions: (1) For both core duplexes (I and IV), the addition of dangling T residues on either the 5' or 3' end causes an increase in the optical melting temperature tm. (2) For both core duplexes, 5' dangling T residues induce a greater increase in the optical tm's than 3' dangling T residues. (3) For both cores duplexes, the increase in tm induced by the addition of dangling T residues is enthalpic in origin, with 5' dangling T residues inducing a greater increase in the van't Hoff transition enthalpy than 3' dangling T's. (4) Dangling T residues cause downfield shifts in all of the nonexchangeable aromatic protons of the [d(GC)3]2 core duplex (I), with the 5' T residues inducing the largest shifts. For the most part, this trend does not hold with the [d(CG)3]2 core duplex (IV). (5) For both core duplexes, the addition of dangling T residues causes an increase in the NMR tm's of almost all the nonexchangeable aromatic protons of the core duplex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transition state complexes of the Klebsiella pneumoniae nitrogenase proteins. Spectroscopic properties of aluminium fluoride-stabilized and beryllium fluoride-stabilized MgADP complexes reveal conformational differences of the Fe protein.

Stable inactive 2 : 1 complexes of the Klebsiella pneumoniae nitrogenase components (Kp2/Kp1) were prepared with ADP or the fluorescent ADP analogue, 2'(3')-O-[N-methylanthraniloyl] ADP and AlF(4)(-) or BeF(3)(-) ions. By analogy with published crystallographic data [Schindelin et al. (1997) Nature 387, 370-376)], we suggest that the metal fluoride ions replaced phosphate at the two ATP-binding sites of the iron protein, Kp2. The beryllium (BeF(x)) and aluminium (AlF(4)(-)) containing complexes are proposed to correspond to the ATP-bound state and the hydrolytic transition states, respectively, by analogy with the equivalent complexes of myosin [Fisher et al. (1995) Biochemistry 34, 8960-8972]. (31)P NMR spectroscopy showed that during the initial stages of complex formation, MgADP bound to the complexed Kp2 in a manner similar to that reported for isolated Kp2. This process was followed by a second step that caused broadening of the (31)P NMR signals and, in the case of the AlF4- complex, slow hydrolysis of some of the excess ADP to AMP and inorganic phosphate. The purified BeFx complex contained 3.8 +/- 0.1 MgADP per mol Kp1. With the AlF(4)(-) complex, MgAMP and adenosine (from MgAMP hydrolysis) replaced part of the bound MgADP although four AlF(4)(-) ions were retained, demonstrating that full occupancy by MgADP is not required for the stability of the complex. The fluorescence emission maximum of 2'(3')-O-[N-methylanthraniloyl] ADP was blue-shifted by 6-8 nm in both metal fluoride complexes and polarization was 6-9 times that of the free analogue. The fluorescence yield of bound 2'(3')-O-[N-methylanthraniloyl] ADP was enhanced by 40% in the AlF(4)(-) complex relative to the solvent but no increase in fluorescence was observed in the BeFx complex. Resonance energy transfer from conserved tyrosine residues located in proximity to the Kp2 nucleotide-binding pocket was marked in the AlF(4)(-) complex but minimal in the BeFx fluoride complex, illustrating a clear conformational difference in the Fe protein of the two complexes. Our data indicate that complex formation during the nitrogenase catalytic cycle is a multistep process involving at least four conformational states of Kp2: similar to the free Fe protein; as initially complexed with detectable (31)P NMR; as detected in mature complexes with no detectable (31)P NMR; in the AlF(4)(-) complex in which an altered tyrosine interaction permits resonance energy transfer with 2'(3')-O-[N-methylanthraniloyl] ADP.     

Complexation of thiomalic acid with mercury (II) and lead (II) under physiological conditions

The complex formation of thiomalic acid (H3L) with Hg(II) and Pb(II) was investigated under physiological conditions of 37 degrees C and 0.15 mol dm-3 NaCl by potentiometric titrations using glass electrodes. From the analysis of the emf data in the two systems by use of computer program MIQUV it was concluded that the species formed in the two systems are [HgH4L2], [HgH3L]-, [HgH2L2]2-, [HgHL2]3-, [HgHL], [HgL]-, [HgL2]4-, [Hg(OH)L]2-, [Hg(OH)L2]5-, [PbH2L2]2-, [PbH2L]+, [PbHL2]3-, [PbHL], [PbL]-, [Pb(OH)L]2-, and [Pb(OH)2L]3-. The hydrolytic reactions of Hg(II), data on which were used in the analysis of the above system, were also studied by separate potentiometric titrations. Measurements of 13C NMR spectra of [HgL2]4- and [PbL]- and [PbHL2]3- in D2O solutions suggested that the ligand coordinates with both the metal ions through the sulfhydryl group and one of the two carboxylate groups in such a way that the five-membered chelate ring is formed within the complexes.     

Depolarizing stimuli and neurotransmitters utilize separate pathways to activate protein kinase C in sympathetic neurons.

Several types of extracellular signals affect the function of peripheral neurons. Depolarizing stimuli cause sudden increases in permeability to various ions leading to propagation of nerve impulses and release of transmitter substances. Neurons also receive external signals via neurotransmitter receptors located on the membrane. Different types of receptors present on sympathetic neurons are believed to modulate stimulation-evoked release of norepinephrine. We have investigated the effects of depolarizing stimuli and neurotransmitters on different signaling pathways in homogeneous cultures of chick sympathetic neurons. Depolarizing stimuli (35 mM KCl; electrical stimulation, 1 Hz for 5 min) and neurotransmitters (acetylcholine and 5-hydroxytrypatmine) enhanced membrane binding of protein kinase C by 2-5-fold. 35 mM KCl increased formation of 1,2-diacylglycerol and hydrolysis of [3H]phosphatidycholine without affecting [3H] phosphoinositide hydrolysis. Neurotransmitters increased [3H]inositol phosphates and 1,2-diacylglycerol without affecting the hydrolysis of [3H]phosphatidylcholine. 5-Hydroxytryptamine and acetylcholine (muscarinic component) did not increase Ca2+ concentration in the Indo-1-loaded neuronal cell body or the growth cone, but 35 mM KCl and electrical stimulation caused a marked increase in Ca2+ concentration in both regions of sympathetic neurons. We believe this to be the first demonstration of these two types of signalling mechanisms co-existing in sympathetic neurons; depolarization activate the phosphatidylcholine pathway and neurotransmitters activate the phosphatidylinositol pathway. The importance of two pathways in controlling neuronal Ca2+ concentration and the release of transmitter is discussed.     

Screening for tyrosinase inhibitors among extracts of seashore plants and identification of potent inhibitors from Garcinia subelliptica

The tyrosinase inhibitory activity of methanol extracts of the leaves of 39 plant species growing on the seashore of Iriomote island (Okinawa, Japan) was investigated. The extracts of Hibiscus tiliaceus, Carex pumila, and Garcinia subelliptica showed potent activity among them. The inhibitors in the extract of Garcinia subelliptica were purified by assay-guided fractionation to give two biflavonoids. These were known compounds (2R,3S-5,7,4',5',7',3',4'-heptahydroxy flavanone[3-8'] flavone and 5,7,4',5',7',3',4'-heptahydroxy[3-8'] biflavanone), although their strong inhibitory activity toward tyrosinase is revealed for the first time in this work. One of these biflavonoids (2R,3S-5,7,4',5',7',3',4'-heptahydroxy flavanone[3-8'] flavone) showed much stronger activity (IC50 2.5 microM) than that of kojic acid (IC50 9.1 microM) when L-tyrosine was used as the substrate.     

Investigations of the metal ion-binding sites of yeast inorganic pyrophosphatase

Yeast inorganic pyrophosphatase was found to bind two Mn2+ per subunit in the absence of phosphate and three Mn2+ per subunit in the presence of phosphate. Kinetic studies of the pyrophosphatase-catalyzed hydrolysis of Cr(NH3)4PP and Cr(H2O)4PP were carried out with Mn2+ and with Mg2+ as activators. The results from these studies suggest that three divalent cations per pyrophosphatase active site are required for catalysis. NMR and EPR studies were conducted to evaluate the relative location of the metal ion binding sites on the enzyme. The two Mn2+ ions bound to the free enzyme are in close enough proximity to magnetically interact. Analysis of the NMR and EPR data in terms of a dipolar relaxation mechanism between Mn2+ ions provides an estimate of the distance between them of 10-14 A. When the diamagnetic substrate analog [Co(NH3)4PNP]- or intermediate analog [Co(NH3)4 (P)2]- are bound to pyrophosphatase, two Mn2+ ions still bind to the enzyme and their magnetic interaction increases. In the presence of these Co3+ complexes, the Mn2+--Mn2+ separation decreases to 7-9 A. Several NMR and EPR experiments were conducted at low Mn2+ to pyrophosphatase ratios (approximately 0.3), where only one Mn2+ ion binds per subunit, in the presence of Cr3+ or Co3+ complexes of PNP or PP. Analysis of the Mn2+--Cr3+ dipolar relaxation evident in proton NMR and EPR data provided for the calculation of Mn2+--Cr3+ distances. When the substrate analog CrPNP was present, the Mn2+--Cr3+ distance was congruent to 7 A whereas, when Cr(P)2 was bound to pyrophosphatase, the Mn2+--Cr3+ distance was congruent to 5 A. These results strongly support a model for the catalytic site of pyrophosphatase that involves three metal ion cofactors.     

Site-specific OH attack to the sugar moiety of DNA: a comparison of experimental data and computational simulation.

Little computational or experimental information is available on site-specific hydroxyl attack probabilities to DNA. In this study, an atomistic stochastic model of OH radical reactions with DNA was developed to compute relative OH attack probabilities at individual deoxyribose hydrogen atoms. A model of the self-complementary decamer duplex d(CCAACGTTGG) was created including Na(+) counter ions and the water molecules of the first hydration layer. Additionally, a method for accounting for steric hindrance from nonreacting atoms was implemented. The model was then used to calculate OH attack probabilities at the various C-H sites of the sugar moiety. Results from this computational model show that OH radicals exhibit preferential attack at different deoxyribose hydrogens, as suggested by their corresponding percentage solvent-accessible surface areas. The percentage OH attack probabilities for the deoxyribose hydrogens [1H(5')+2H(5'), H(4'), H(3'), 1H(2')+2H(2'), H(1')] were calculated as approximately 54.6%, 20.6%, 15.0%, 8.5% and 1.3%, respectively, averaged across the sequence. These results are in good agreement with the latest experimental site-specific DNA strand break data of Balasubramanian et al. [Proc. Natl. Acad. Sci. USA 95, 9738-9742 (1998)]. The data from this stochastic model suggest that steric hindrance from nonreacting atoms significantly influences site-specific hydroxyl radical attack probabilities in DNA. A number of previous DNA damage models have been based on the assumption that C(4') is the preferred site, or perhaps the only site, for OH-mediated DNA damage. However, the results of the present study are in good agreement the experimental results of Balasubramanian et al. in which OH radicals exhibit preferential initial attack at sugar hydrogen atoms in the order 1H(5')+2H(5') > H(4') > H(3') > 1H(2')+2H(2') > H(1').     

Rates of platination of -AG- and -GA- containing double-stranded oligonucleotides: effect of chloride concentration

The kinetics of the reactions between 15N-labelled cisplatin and 14-base pair duplex oligonucleotides with either 5'-AG-3' or 5'-GA-3' groupings as the principal platination site are examined in the presence of 60-80 mM chloride by [1H,15N]HSQC 2D NMR spectroscopy. The presence of chloride at these concentrations results in a five-fold decrease in the rate of hydrolysis of cisplatin to cis-[PtCl(NH3)2(OH2)]+ and a two- to twenty-fold decrease in the rate of monofunctional adduct formation. The effects on the rate of closure from monofunctional to bifunctional adducts are less well established but some of these rates appear not to be significantly reduced by the presence of added chloride. The results provide a caution that the use of chloride to quench platination reactions may not be fully effective.     

Structural and functional characterization of seven spermicidal vanadium(IV) complexes: potentiation of activity by methyl substitution on the cyclopentadienyl rings.

In a systematic effort to identify and develop effective vanadocene(IV) complexes as a new class of contraceptive agents, the effect of methyl substitution in the cyclopentadienyl rings of Cpx2VCl2 on their spermicidal activity has been examined. The spermicidal activities of compounds Cpx2VCl2 [Cpx = Me5Cp (Cp*) (1), Cp (3), MeCp (Cp') (5)], as well as two of their corresponding vanadium(V) oxidation products Cp*V(O)Cl2 (2) and CpV(O)Cl2 (4), were examined by computer-assisted sperm analysis (CASA). These analyses have established that penta-substitution of the Cp ring by electron-donating methyl groups augments the spermicidal activity 10-fold. The corresponding V(V) oxo compounds, Cp*V(O)Cl2 (2) and CpV(O)Cl2 (4), tested under identical conditions did not show as effective spermicidal activity even though these complexes have a pseudo-tetrahedral geometry similar to the active vanadocene(IV) dichlorides. Two pseudo-octahedral V(IV) complexes with tris-pyrazolyl borate as ligand, (HBpz3)V(O)Cl.DMF (6) and (HB(3,5-Me2pz)3)V(O)Cl.DMF (7), were also found to exhibit potent spermicidal activity. Although some vanadium(IV) complexes may immobilize sperm due to the generation of .OH radicals, the lack of spermicidal activity of VOSO4 which generates .OH radicals, and the potent spermicidal activity of [Cp2V(acac)][O3SCF3] (8), and [Cp2V(DeDtc)][O3SCF3] (9) which do not generate .OH radicals, indicate that .OH radical mediated reactions may not be essential for the spermicidal activity of vanadium(IV) complexes.     

Synthesis of 1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol by calvarial cells. Characterization of the enzyme systems.

The synthesis of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3] from 25-hydroxycholecalciferol [25(OH)D3] has previously been shown to occur in cells isolated from bone. The main findings of the present study are that the enzyme systems which catalyse these syntheses are: (1) active at 'in vitro' substrate concentrations over the range of 2-50 nM; (2) regulatable in a complex way by 1,25(OH)2D3, 24,25(OH)2D3, 25,26-dihydroxycholecalciferol and 25(OH)D3, but not by cholecalciferol ('vitamin D3'); and (3) have relatively short half-lives (approx. 5 h).     

Preparation and structure determination of two sugar amino acids via corresponding hydantoin derivatives

(4R)-2,3-O-Isopropylidene-methylspiro[4,6-dideoxy-alpha-L-lyxo+ ++-hexopyranosid-4,5'-imidazolidin]-2',4'-dione and (4R)-2,3-O-isopropylidene-methylspiro[4,6-dideoxy-beta-D-ribo-h exopyranosid-4,5'-imidazolidin]-2',4'-dione were prepared under various reaction conditions starting from methyl 6-deoxy-2,3-O-isopropylidene-alpha-L-lyxo-hexopyranosid-4-++ +ulose. Corresponding alpha-amino acids methyl (4R)-4-amino-4-C-carboxy-4,6-dideoxy-alpha-L-lyxo-hexopyranosid e and methyl (4R)-4-amino-4-C-carboxy-4,6-dideoxy-beta-D-ribo-hexopyranoside were obtained from the above hydantoins by selective acid hydrolysis of the isopropylidene group, followed by basic hydrolysis of the hydantoin ring. The crystal structures of both hydantoin derivatives are also presented.     

Interaction of morphocycline with beryllium ions]

Behaviour of morphocycline (H5R) and its complex with beryllium ions in acqueous solutions was studied fluorimetrically. The ionization constants of H5R were estimated at pH 1.5-15 according to the data of fluorimetric determination with respect to OH-group: pK1 6.40, pK2 8.25, pK3 10.65, pK4 11.30. Two constants characterizing the deprotonization process with respect to the carbonylic group and nitrogen were also estimated: pK01--1.0 (greater than C = 0) and pK02 4.75 (--N=). Formation of an intensively fluorescing compound less than [Be3(OH)3(H2O2)5]2HR greater than 2+ was observed at pH 6.0-7.0. The cause of such intensive fluorescence was deformation of ion [Be3(OH)3(H2O)6]3+ because of its exclusion from the coordinating sphere of one molecule of water during the complex formation and decreasing of level H comes from II as compared to the morphocycline level II comes from n. A procedure for detecting morphocycline in the blood of humans and animals was developed.     

Generation of *OH initiated by interaction of Fe2+ and Cu+ with dioxygen; comparison with the Fenton chemistry

Iron and copper toxicity has been presumed to involve the formation of hydroxyl radical (*OH) from H2O2 in the Fenton reaction. The aim of this study was to verify that Fe2+-O2 and Cu+-O2 chemistry is capable of generating *OH in the quasi physiological environment of Krebs-Henseleit buffer (KH), and to compare the ability of the Fe2+-O2 system and of the Fenton system (Fe2+ + H2O2) to produce *OH. The addition of Fe2+ and Cu+ (0-20 microM) to KH resulted in a concentration-dependent increase in *OH formation, as measured by the salicylate method. While Fe3+ and Cu2+ (0-20 microM) did not result in *OH formation, these ions mediated significant *OH production in the presence of a number of reducing agents. The *OH yield from the reaction mediated by Fe2+ was increased by exogenous Fe3+ and Cu2+ and was prevented by the deoxygenation of the buffer and reduced by superoxide dismutase, catalase, and desferrioxamine. Addition of 1 microM, 5 microM or 10 microM Fe2+ to a range of H2O2 concentrations (the Fenton system) resulted in a H2O2-concentration-dependent rise in *OH formation. For each Fe2+ concentration tested, the *OH yield doubled when the ratio [H2O2]:[Fe2+] was raised from zero to one. In conclusion: (i) Fe2+-O2 and Cu+-O2 chemistry is capable of promoting *OH generation in the environment of oxygenated KH, in the absence of pre-existing superoxide and/or H2O2, and possibly through a mechanism initiated by the metal autoxidation; (ii) The process is enhanced by contaminating Fe3+ and Cu2+; (iii) In the presence of reducing agents also Fe3+ and Cu2+ promote the *OH formation; (iv) Depending on the actual [H2O2]:[Fe2+] ratio, the efficiency of the Fe2+-O2 chemistry to generate *OH is greater than or, at best, equal to that of the Fe2+-driven Fenton reaction.