Mechanism of the pyrophosphate migration in the enzymatic cyclization of geranyl and linalyl pyrophosphates to (+)- and (-)-bornyl pyrophosphates

Soluble enzymes from sage (Salvia officinalis) and tansy (Tanacetum vulgare), which catalyze the cyclization of geranyl pyrophosphate and the presumptive intermediate linalyl pyrophosphate to the (+) and (-) enantiomers, respectively, of 2-bornyl pyrophosphate, were employed to evaluate mechanistic alternatives for the pyrophosphate migration in monoterpene cyclization reactions. Separate incubation of [1-3H2,alpha-32P]- and [1-3H2,beta- 32P]geranyl and (+/-)-linalyl pyrophosphates with partially purified preparations of each enantiomer-generating cyclase gave [3H, 32P]bornyl pyrophosphates, which were selectively hydrolyzed to the corresponding bornyl phosphates. Measurement of 3H:32P ratios of these monophosphate esters established that two ends of the pyrophosphate moiety retained their identifies in the cyclization of both precursors to both products and also indicated that there was no appreciable exchange with exogenous inorganic pyrophosphate in the reaction. Subsequent incubations of each cyclase with [8,9-14C,1-18O]geranyl pyrophosphate and with (1E)-(+/-)-[1-3H,3-18O]linalyl pyrophosphate gave the appropriate (+)- or (-)-bornyl pyrophosphates, which were hydrolyzed in situ to the corresponding borneols. Analysis of the derived benzoates by mass spectrometry demonstrated each of the product borneols to possess an 18O enrichment essentially identical with that of the respective acyclic precursor. The absence of P alpha-P beta interchange and the complete lack of positional 18O isotope exchange of the pyrophosphate moiety are compatible with tight ion pairing of intermediates in the coupled isomerization-cyclization of geranyl pyrophosphate and establish a remarkably tight restriction on the motion of the transiently generated pyrophosphate anion with respect to its cationic terpenyl reaction partner.     

Syntheses and spectroscopic characterization of some phosphoramidates as reversible inhibitors of human acetylcholinesterase and determination of their potency

The ability of phosphoramidates Me2NP(O)(Cl)(p-NHC6H4NO2) 1, Me2NP(O)(p-NHC6H4NO2)2 2, (CH3C6H4O-p)P(O)(p-NHC6H4NO2)2 3 and (CH3C6H40-p)2P(O)(p-NHC6H4NO2) 4 to inhibit human acetylcholinesterase (hAChE) has been evaluated by a modified Ellman's method and spectrophotometric measurements. Results showed that compounds 1 and 2 do not have any inhibitory potency, whereas compounds 3 and 4 were reversible mixed inhibitors. The IC50 values for inhibitors 3 and 4 were 0.143 and 0.581 mM, respectively. The previously unknown compounds 3 and 4 were synthesized and characterized by 1H, 13C, 31P NMR and IR spectroscopy and elemental analysis.     

Single diastereomers of unsymmetrical tris-spirocyclic cyclotriphosphazenes based on 1,1'-bi-2-naphthol--synthesis and structures

Diastereoselective synthesis and characterization of chiral unsymmetrical tris-spirocyclic cyclotriphosphazenes based on chiral 1,1'-bi-2-naphthol (BINOL) are reported. Specifically, the chiral compounds (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)Cl(2) [(-)-4] and (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](OCH(2)CH(2)NMe)(2) [(-)-5] are prepared by starting with the chiral mono-spiro compound (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)]Cl(4) [(-)-3]. Synthesis of four other chiral spirocyclics, N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](OCH(2)CH(2) NMe)(O-2,2'C(6)H(4)-C(6)H(4)O)[(-)-6 and (+)-6], N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](NMe(2))(4) [(-)-7], N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)(NMeCH(2)CH(2)OH)(2) [(-)-8 and (+)-8], and N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)[NHCH(2)CH(2)CH(2)Si(OEt)(3)](2) (9) is also reported herein. Compounds 4-6 are obtained in the solid state diastereoselectively and their X-ray structures have been determined and discussed. The diastereoselectivity is also shown by structural characterization of two distinct isomers in the case of 6 [(-)-6 and (+)-6, respectively] by starting with precursor of 3 having (R) or (S)-BINOL residue. The (1)H NMR spectra of 7 and 8 exhibit doublets with virtual coupling for the methyl protons, consistent with the chiral nature of the binaphthoxy residue. The potential of 9, which hydrolyzes readily in CDCl(3) solution, as a useful precursor for chiral polymer applications is highlighted.     

Structure-activity study of phosphoramido acid esters as acetylcholinesterase inhibitors

Phosphoramido acid esters (CH(3))(2)NP(O)X(p-OC(6)H(4)-CH(3)) (containing P-Cl (1), P-O (2), P-F (3), P-CN (5), and P-N (4,6) bonds, X for 2, 4 and 6 is OCH(3), (C(2)H(5))(2)N and morpholin) have been synthesized to investigate the structure-activity study of AChE enzyme inhibition, through the parameters logP, delta(31)P and IC(50). After their characterization by (31)P, (31)P{(1)H}, (13)C, (1)H NMR, IR and mass spectroscopy, the parameters logP and delta(31)P ((31)P chemical shift in NMR) were used to evaluated the lipophilicity and electronical properties. The ability of compounds to inhibit human AChE was predicted by PASS software (version 1.193), and experimentally evaluated by a modified Ellman's assay.     

Anticholinesterase activity of some major intermediates in carbacylamidophosphate synthesis: preparation, spectral characterization and inhibitory potency determination

Carbacylamidophosphates with the general formula RC(O)NHP(O)R1R2 constitute organophosphorus compounds that are used as insecticides, pesticides and ureas inhibitors. In this work, we studied the inhibition potency of CCl3-C(O)NHP(O)Cl21, CHCl2C(O)NHP(O)Cl(2)2, CH2ClC(O)NHP(O)Cl23 and CF3C(O)NHP(O)Cl(2)4, which are the major intermediates for carbacylamidophosphates synthesis towards human erythrocyte acetylcholinesterase (hAChe) activity using Ellman's modified kinetic method. Unexpectedly, it was observed that they were not only hydrolytically unstable but also inhibited hAChE in a similar manner to that produced by organophosphorus insecticides. Enzymatic data, bimolecular inhibition rate constants (ki) and IC50 values for inhibition of hAChE demonstrated that they are irreversible inhibitors and the inhibition potency of compound 2 (IC50 = 88 microM) was the greatest in comparison with compounds 1, 3 and 4. Also the electropositivity of the phosphorus atom and the hydrophobicity of the compounds demonstrated that these two factors play an additional effect and different role in the inhibitory activity of these compounds. Hydrolytic stability of the compounds was determined by 31P NMR monitoring of the loss of the parent molecules with D2O as a function of time. This study considers antiacetylcholinesterase activity according to the structural and the electronic aspects of compounds 1-4, according to IR, 1H, 13C and 31P NMR spectral data.     

X-ray studies on crystalline complexes involving amino acids and peptides. Part XX. Crystal structures of DL-arginine acetate monohydrate and DL-lysine acetate and a comparison with the corresponding L-amino acid complexes

Crystals of DL-arginine acetate monohydrate, C6H15N4O2+C2H3O2-.H2O, are monoclinic, P2(1)/c, with a = 13.552(2), b = 5.048(2), c = 18.837(3) A, beta = 101.34(2) degrees and Z = 4, and those of DL-lysine acetate, C6H15N2O2+.C2H3O2- are triclinic, P1, with a = 5.471(2), b = 7.656(2), c = 12.841(2) A, alpha = 94.48(1), beta = 94.59(2), gamma = 98.83(2) degrees and Z = 2. The structures have been solved by direct methods and refined to R = 0.058 and 0.077 for 1522 and 1259 observed reflections respectively. The difference in the number and the nature of proton donors leads to a difference in hydrogen bond density in the two structures. The basic elements of aggregation in both the structures are pairs of amino acid molecules, each pair stabilized by two centrosymmetrically related hydrogen bonds involving alpha-amino and alpha-carboxylate groups, stacked along the shortest dimension to form columns. The pairs are held together in each column by head-to-tail sequences. The columns stack along a crystallographic axis to form layers. Adjacent layers are bridged by acetate ions. The amino acid-acetate interactions are primarily through side chains and involve specific interactions and characteristic interaction patterns. The gross features of molecular aggregation are nearly the same in DL-arginine acetate monohydrate and L-arginine acetate whereas they are substantially different in the lysine complexes. In both cases, one of the two head-to-tail sequences in the L complex is replaced by a hydrogen bonded loop involving alpha-amino and alpha-carboxylate groups, in the DL complex. This may have implications for prebiotic condensation during chemical evolution.     

Thiourea derivatives and their nickel(II) and platinum(II) complexes: antifungal activity

We have synthesized two thiourea derivatives of methyl anthranilate (1, 2) and their complexes with nickel (3) and platinum(II) (4). We have also prepared the complexes of nickel(II) with two benzoylthiourea derivatives (5, 6). The obtained compounds were characterized by elemental analysis, spectroscopic methods (FT-IR, UV-vis, NMR), mass spectrometry and thermal analysis. Compound 1, C(20)H(23)N(3)O(2)S, crystallizes in monoclinic space group P21/n, with Z=4, and unit cell parameters, a=8.8042(4) A, b=7.6608(3) A, c=28.834(2) A, alpha=gamma=90 degrees, beta=90.94(1) degrees. Compound 2, C(20)H(21)N(3)O(3)S, crystallizes in monoclinic space group P21/c, with Z=4, and unit cell parameters, a=7.7345(4) A, b=8.6715(4) A, c=29.113(2) A, alpha=gamma=90 degrees, beta=90.67(1) degrees. Compound 5, C(24)H(30)N(4)NiO(2)S(2), crystallizes in monoclinic space group P21/n, with Z=4, and unit cell parameters, a=10.4317(8) A, b=18.517(2) A, c=13.299(1) A, alpha=gamma=90 degrees, beta=104.53(1) degrees. Compound 6, C(25)H(28)Cl(2)N(4)NiO(4)S(2), crystallizes with a molecule of CH(2)Cl(2) in triclinic space group P-1, with Z=2, and unit cell parameters, a=10.362(1) A, b=11.849(2) A, c=12.536(2) A, alpha=90.04(2) degrees, beta=84.73(1) degrees, gamma=113.43(2) degrees. Compounds 1 and 2 show antifungal activity against the major pathogens responsible for important plant diseases (Botrytis cinerea, Colletotrichum fragariae, Fusarium oxysporum and Phoma betae). The antifungal activity is practically the same for morpholine and ethyl derivatives.     

Structural and spectroscopic characterization of aqua-diargininate-copper(II)-carbonate monohydrate

The molecular structure of the title complex, [Cu(C6H14N4O2)2(H2O)]CO3.H(2)O, was determined by single crystal X-ray diffractometry. It crystallizes in the monoclinic space group P2(1), with Z = 2. The Cu(II) ion is in a square pyramidal environment, trans coordinated at the basis by two argininate groups acting as bidentate ligands through their amino nitrogen and carboxylate oxygen atoms. The coordination around copper is completed by a water molecule at the pyramid apex. The infrared, Raman and electronic spectra are briefly discussed on the basis of the structural peculiarities of the complex.     

Mixed ligand complexes of platinum(II) and palladium(II) with cytokinin-derived compounds Bohemine and Olomoucine: X-ray structure of [Pt(BohH+-N7)Cl(3)].9/5H2O [Boh=6-(benzylamino)-2-[(3-(hydroxypropyl)-amino]-9-isopropylpurine,Bohemine

The new square-planar Pt(II) and Pd(II) complexes with cytokinin-derived compounds Bohemine and Olomoucine, having the formulae [Pt(BohH(+))Cl(3)].H(2)O (1), [Pt(Boh)(2)Cl(2)].3H(2)O (2), [Pt(Boh-H)Cl(H(2)O)(2)].H(2)O (3), [Pt(OloH(+))Cl(3)].H(2)O (4), [Pd(BohH(+))Cl(3)].H(2)O (5), [Pd(Boh)Cl(2)(H(2)O)] (6), [Pd(Boh-H)Cl(H(2)O)].EtOH (7) and [Pd(OloH(+))Cl(3)].H(2)O (8), where Boh=6-(benzylamino)-2-[(3-(hydroxypropyl)amino]-9-isopropylpurine and Olo=6-(benzylamino)-2-[(2-(hydroxyethyl)amino]-9-methylpurine, have been synthesized. The complexes have been characterized by elemental analyses, IR, FAB+ mass, 1H, 13C and 195Pt NMR spectra, and conductivity data. The molecular structure of the complex [Pt(BohH(+)-N7)Cl(3)].9/5H(2)O has been determined by an X-ray diffraction study. Results from physical studies show that both Bohemine and Olomoucine are coordinated to transition metals through the N(7) atom of purine ring in all the complexes. The prepared compounds have been tested in vitro for their possible cytotoxic activity against G-361 (human malignant melanoma), HOS (human osteogenic sarcoma), K-562 (human chronic myelogenous leukemia) and MCF-7 (human breast adenocarcinoma) cell lines and IC(50) values have been also determined for all the complexes. IC(50) values estimated for the Pt(II)-Bohemine complexes (2.1-16 microM) allow us to conclude that they could find utilization in antineoplastic therapy. Thus, from a pharmacological point of view, Pt(II) complexes of Bohemine may represent compounds for a new class of antitumor drugs.     

N9-Substituted N⁶-[(3-methylbut-2-en-1-yl)amino]purine derivatives and their biological activity in selected cytokinin bioassays

Rational design is one of the latest ways how to evaluate particular activity of signal molecules, for example cytokinin derivatives. A series of N(6)-[(3-methylbut-2-en-1-yl)amino]purine (iP) derivatives specifically substituted at the N9 atom of purine moiety by tetrahydropyran-2-yl, ethoxyethyl, and C2-C4 alkyl chains terminated by various functional groups were prepared. The reason for this rational design was to reveal the relationship between specific substitution at the N9 atom of purine moiety of iP and cytokinin activity of the prepared compounds. The synthesis was carried out either via 6-chloro-9-substituted intermediates prepared originally from 6-chloropurine, or by a direct alkylation of N9 atom of N(6)-[(3-methylbut-2-en-1-yl)amino]purine. Selective reduction was implemented in the preparation of compound N(6)-[(3-methylbut-2-en-1-yl)amino]-9-(2-aminoethyl-amino)purine (12) when 6-[(3-methylbut-2-en-1-yl)amino]-9-(2-azidoethyl)purine (7) was reduced by zinc powder in mild conditions. The prepared derivatives were characterized by C, H, N elemental analyses, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), melting point determinations (mp), CI+ mass spectral measurement (CI+ MS), and by (1)H NMR spectroscopy. Biological activity of prepared compounds was assessed in three in vitro cytokinin bioassays (tobacco callus, wheat leaf senescence, and Amaranthus bioassay). Moreover, the perception of prepared derivatives by cytokinin-sensitive receptor CRE1/AHK4 from Arabidopsis thaliana, as well as by the receptors ZmHK1 and ZmHK3a from Zea mays, was studied in a bacterial assay where the response to the cytokinin treatment could be specifically quantified with the aim to reveal the way of the perception of the above mentioned derivatives in two different plant species, that is, Arabidopsis, a model dicot, and maize, a model monocot. The majority of cytokinin derivatives were significantly active in both Amaranthus as well as in tobacco callus bioassay and almost inactive in detached wheat leaf senescence assay. N9-Substituted iP derivatives remained active in both in vitro bioassays in a broad range of concentrations despite the fact that most of the derivatives were unable to trigger the cytokinin response in CRE1/AHK4 and ZmHK1 receptors. However, several derivatives induced low but detectable cytokinin-like activation in maize ZmHK3a receptor. Compound 6-[(3-methylbut-2-en-1-yl)amino]-9-(tetrahydropyran-2-yl)purine (1) was also recognized by CRE1/AHK4 at high concentration ≥ 50 μM.     

Synthesis and structural characterization of rhenium(I) tricarbonyl complexes with the bidentate ligands o-(diphenylphosphino)benzaldehyde (P∩O) and o-[(diphenylphosphino)benzylidene]analine (P∩N)

A series of rhenium(I) tricarbonyl complexes with bidentate P,O donor ligand o-(diphenylphosphino)benzaldehyde (P∩O) and its Schiff base P,N donor ligand o-[diphenylphosphino)benzylidene]analine (P∩N) have been synthesized and structurally characterized. All the complexes of the type [ReX(CO)(3)(LL)] (where LL = P∩O, P∩N) reveal a distorted octahedral structure with the three carbonyl ligands arranged in the facial fashion. Crystal data for 1, C(22)H(15)ClO(4)PRe·1/2C(6)H(14): triclinic, P1, a=8.7430(4), b=9.5767(4), c=13.9449(6) ?, α=93.651(1), β=101.265(1), γ=93.048(1); V=1140.20(9) ?(3), Z=2.2, C(22)H(15)BrO(4)Pre: monoclinic, C2/c, a=31.6800(16), b=8.8880(4), c=18.4517(9) ?, β=124.990(1); V=4256.4(4) ?(3), Z=8. 3, C(28)H(20)ClNO(3)Pre: monoclinic, C2/c, a=22.675(4), b=8.803(2), c=28.218(5) ?, β=100.192(3); V=5543.5(16) ?(3), Z=8.4, C(28)H(20)BrNO(3)Pre: monoclinic, C2/c, a=23.035(1), b=8.7561(4), c=28.269(1) ?, β=100.811(1); V=5600.4(5) ?(3), Z=8.     

Protonated nucleobase ligands: synthesis, structure and characterization of 9-methyladeninium hexachloroplatinate and pentachloro(9-methyladeninium)platinum(IV)

Na(2)[PtCl(6)] was found to react with (9-MeAH)Cl(.)H(2)O (2) (9-MeA=9-methyladenine) in aqueous solution yielding (9-MeAH)(2)[PtCl(6)](.)2H(2)O (3). The same compound was obtained from hexachloroplatinic acid and 9-methyladenine. Performing this reaction at 60 degrees C, complex formation took place yielding the 9-methyladeninium complex [PtCl(5)(9-MeAH)](.)2H(2)O (4a). An analogous complex, [PtCl(5)(9-MeAH)](.)1/2(18C6)(.)H(2)O (4b, 18C6=crown ether 18-crown-6), was formed in the reaction of aquapentachloroplatinic acid (H(3)O)[PtCl(5)(H(2)O)](.)2(18C6)(.)6H(2)O (1) with 9-methyladenine in 1:1 ratio. All complexes were isolated in moderate to good yields as yellow powder (4b) and crystals (3, 4a), respectively. They were fully characterized by microanalysis, IR and NMR ((1)H, (13)C, (195)Pt) spectroscopies, and in part (2, 3, 4a) also by single-crystal X-ray diffraction analysis. Molecular structure of complex 4a exhibited that the 9-methyladeninium ligand is N1 protonated and coordinated through N7 to platinum(IV).     

Cloning and characterization of a chromosome-encoded catechol 2,3-dioxygenase gene from Pseudomonas aeruginosa ZD 4-3

Catechol 2,3-dioxygenase (C23O), one of extradiol-type dioxygenases cleaving the aromatic C-C bond at the meta-position of dihydroxylated aromatic substrates, catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde. Based on curing experiment, PCR identification, and Southern hybridization, the gene responsible for C23O was localized on a 3.5-kb EcoRI/BamHI fragment and cloned from P. aeruginosa ZD 4-3 able to degrade both single and bicyclic compounds via the meta-cleavage pathway. A complete nucleotide sequence analysis of the C23O revealed that it had one ORF, which showed a strong amino acid sequence similarity to the known C23Os of mesophilic gram-negative bacteria. The alignment analysis indicated that distinct difference existed between the C23O in this study and the 2,3-dihydroxybiphenyl dioxygenases cleaving bicyclic aromatic compounds. The heterogenous expression of the pheB gene in Escherichia coli BL21(DE3) demonstrated that this C23O possessed a meta-cleavage activity.     

Pulse sequences for detection of NH2···N hydrogen bonds in sheared G⋅A mismatches via remote, non-exchangeable protons

The non-detectability of NH...N hydrogen bonds in nucleic acids due to exchange broadened imino/amino protons has recently been addressed via the use of non-exchangeable protons for detecting internucleotide 2hJ(NN) couplings. In these applications, the appropriate non-exchangeable proton is separated by two bonds from the NH...N bond. In this paper, we extend the scope of this approach to protons which are separated by four bonds from the NH...N moiety. Specifically, we consider the case of the commonly occurring sheared G x A mismatch alignment, in which we use the adenine H2 proton to report on the (A)N6H6(1.2)...N3(G) hydrogen bond, in the presence of undetectable, exchange broadened N6H6(1.2) protons. Two sequences, the 'straight-through' (H6)N6N3H2 and 'out-and-back' H2N6N3 experiments, are presented for observing these correlations in H2O and D2O solution, respectively. The sequences are demonstrated on two uniformly 15N,13C labelled DNA samples: d(G1G2G3T4T5C6A7G8G9)2, containing a G3 x (C6-A7) triad involving a sheared G3 x A7 mismatch, and d(G1G2G3C4A5G6G7T8)4, containing an A5 x (G3 x G6 x G3 x G6) x A5 hexad involving a sheared G3 x A5 mismatch.     

Coordination compounds derived from the interaction of streptomycin and cobalt, nickel, copper, and calcium salts characterized by 13C NMR and spectroscopic studies. Structure and bonding properties of the streptidine fraction

The coordination compounds of streptomycin (St), Co2(St)Cl4.13H2O (2), Co2(St)(NO3)4.7H2O (3), Ni2(St)Cl4.14H2O (4), Ni2(St)(NO3)4.14H2O (5), Cu2(St)Cl4.6H2O (6), and Ca(St)Cl2.8H2O (7) have been synthesized by the reaction of streptomycin sulfate (1) with three equivalents of the corresponding inorganic salt. The compounds (2)-(7) were characterized by electronic spectroscopy (in the solid state and in solution) by conductivity measurements and by 13C NMR in solution. The reaction of streptomycin with CuCl2 in water hydrolyzed the molecule giving the copper complex of the streptidine fraction (Std), Cu(Std)Cl.H2O (8). This compound was characterized by the same techniques. Detailed x-ray diffraction and 13C NMR studies of streptidine sulfate (9) were carried out.     

Influence of electron transport proteins on the reactions catalyzed by Fusarium fujikuroi gibberellin monooxygenases

The multifunctional cytochrome P450 monooxygenases P450-1 and P450-2 from Fusarium fujikuroi catalyze the formation of GA14 and GA4, respectively, in the gibberellin (GA)-biosynthetic pathway. However, the activity of these enzymes is qualitatively and quantitatively different in mutants lacking the NADPH:cytochrome P450 oxidoreductase (CPR) compared to CPR-containing strains. 3beta-Hydroxylation, a major P450-1 activity in wild-type strains, was strongly decreased in the mutants relative to oxidation at C-6 and C-7, while synthesis of C19-GAs as a result of oxidative cleavage of C-20 by P450-2 was almost absent whereas the C-20 alcohol, aldehyde and carboxylic acid derivatives accumulated. Interaction of the monooxygenases with alternative electron transport proteins could account for these different product distributions. In the absence of CPR, P450-1 activities were NADH-dependent, and stimulated by cytochrome b5 or by added FAD. These properties as well as the decreased efficiency of P450-1 and P450-2 in the mutants are consistent with the participation of cytochrome b5:NADH cytochrome b5 reductase as redox partner of the gibberellin monooxygenases in the absence of CPR. We provide evidence, from either incubations of GA12 (C-20 methyl) with cultures of the mutant suspended in [18O]H2O or maintained under an atmosphere of [18O]O2:N2 (20:80), that GA15 (C-20 alcohol) and GA24 (C-20 aldehyde) are formed directly from dioxygen and not from hydrolysis of covalently enzyme-bound intermediates. Thus these partially oxidized GAs correspond to intermediates of the sequential oxidation of C-20 catalyzed by P450-2.     

Estimation of the irreversible inhibition reaction rate constants and of the concentration of cholinesterase active sites under commensurable concentrations of enzyme and inhibitors]

Kinetic analysis of the interaction of butyrylcholinesterase and the phosphoorganic inhibitor GT-161 [(C2H2O)2P(O)SC2H4+N(CH3)2C6H5-1-] is carried out. Short time incubations of an enzyme and an inhibitor (1-3 sec), even under commensurable concentrations, were shown to enable the rate constants of irreversible enzyme inhibition to be calcualted by the formula for pseudomonomoleuclar reactions. A simple analytical method for the estimation of the enzyme active sites concentrations is proposed.     

Heterometallic manganese/zinc-phytate complex as a model compound for metal storage in wheat grains

Myo-inositol-1,2,3,4,5,6-hexakisphosphate, also known as phytate, is a natural metal chelate present in cereals, an important feedstock worldwide. This article reports the characterization of three metal storage model complexes: the homometallic Mn(II) myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6), the heterometallic Zn(II), Mn(II) analogue Na4MnZn4(C6H6O24P6) x (NO3)2 x 8H2O (MnZn4IP6) and the homometallic Zn(II) metal complex Na3Zn5(C6H6O24P6)OH x 9H2O (Zn5IP6). The techniques of high-resolution 23Na, 13C and 31P NMR, electron paramagnetic resonance (EPR) and X-ray photoelectron spectroscopy (XPS) were applied in this study. The complexation of Zn(II) and Mn(II) by phosphate groups of IP6 is demonstrated by NMR and XPS results. 13C NMR results show a conformation for IP6 consisting of five equatorial phosphate groups to one axial group showing only one chemical environment for Zn and two for Mn, when characterized by XPS and EPR, in both Mn complexes. These results support, for the first time, a probable supramacromolecular structure for phytate complexes of transition metals. Based on the similarity between the EPR spectra of wheat seeds and that of the MnZn4IP6 compound, the manganese storage centers in wheat grains can be assigned to similar heterometallic phytate complexes.     

Prebiotic synthesis in atmospheres containing CH4, CO,and CO2

The prebiotic synthesis of organic compounds using a spark discharge on various simulated primitive earth atmospheres at 25 degrees C has been studied. Methane mixtures contained H2 + CH4 + H2O + N2 + NH3 with H2/CH4 molar ratios from 0 to 4 and pNH3 = 0.1 torr. A similar set of experiments without added NH3 was performed. The yields of amino acids (1.2 to 4.7% based on the carbon) are approximately independent of the H2/CH4 ratio and whether NH3 was present, and a wide variety of amino acids are obtained. Mixtures of H2 + CO + H2O + N2 and H2 + CO2 + H2O + N2, with and without added NH3, all gave about 2% yields of amino acids at H2/CO and H2/CO2 ratios of 2 to 4. For a H2/CO2 ratio of 0, the yield of amino acids is extremely low (10(-3)%). Glycine is almost the only amino acid produced from CO and CO2 model atmospheres. These results show that the maximum yield is about the same for the three carbon sources at high H2/carbon ratios, but that CH4 is superior at low H2/carbon ratios. In addition, CH4 gives a much greater variety of amino acids than either CO or CO2. If it is assumed that an abundance of amino acids more complex than glycine was required for the origin of life, then these results indicate the requirement for CH4 in the primitive atmosphere.     

Structural studies of O6-methyldeoxyguanosine and related compounds: a promutagenic DNA lesion by methylating carcinogens.

O6-Methylation of guanine residues in DNA can induce mutations by formation of base mispairing due to the deprotonation of N(1). The electronic, geometric and conformational properties of three N(9)-Substituted O6-methylguanine derivatives, O6-methyldeoxyguanosine (O6mdGuo), O6-methylguanosine (O6mGuo) and O6, 9-dimethylguanine (O6mdGua), were investigated by X-ray and/or NMR studies. O6mdGuo crystallizes in the monoclinic space group P2(1) with cell parameters a = 5.267(1), b = 19.109(2), c = 12.330(2) A, beta = 92.45(1) degrees, V = 1239.8(3) A3, z = 4 (two nucleosides per asymmetric unit), and O6mGua in the monoclinic space group P2(1)/n with cell parameters a = 10.729(2), b = 7.640(1) c = 10.216(1) A, beta = 92.17(2) degrees, V = 836.7(2) A3, z = 4. The geometry and conformation of O6-methylguanine moieties observed in both crystals and are very similar. Furthermore, the molecular dimensions of the O6methylguanine residue resemble more closely those of adenine than those of guanine. The methoxy group is coplanar with the purine ring, the methyl group being cis to N(1). The conformation of O6-methylguanine nucleosides is variable. The glycosidic conformation of O6mdGuo is anti for molecule (a) and high-anti for molecule (b) in the crystal, while that of O6mGuo is syn [Parthasarathy, R & Fridey, S. M. (1986) Carcinogenesis 7, 221-227]. The sugar ring pucker of O6mdGuo is C(2')-endo for molecule (a) and C(1')-exo for molecule (b). The C(4')-C(5') exocyclic bond conformation in O6mdGuo is gauche- for molecule (a) but trans for molecule (b), in contrast with gauche+ for O6mGuo. The hydrogen bonds exhibited by O6-methylguanine derivatives differ from those in guanine derivatives; the amino N(2) and ring N(3) and N(7) atoms of O6-methylguanine residues are involved in hydrogen bonding. 1H-NMR data for O6mdGuo and O6mdGuo reveal the predominance of a C(2')-endo type sugar puckering. In O6mdGuo, however, a contribution of a C(1')-exo sugar puckering is significant. The NOE data also indicate that O6mdGuo molecules exist with nearly equal population for anti (including high anti) and syn glycosidic conformations. These observations and their biological implications are discussed.