Observations on the use of guaiacol and 2,2′-azino-di(3-ethylbenzthiazoline-6-sulfonic acid) as peroxidase substrates

Aging of aqueous guaiacol (o-methoxyphenol) solutions over a period of several months led to the spontaneous formation of peroxidatic compound(s) and other unidentified oxidation products of guaiacol. This accelerated the oxidation of guaiacol catalyzed by lactoperoxidase (LPO) severalfold depending on the pH of the reaction mixture. The peroxide(s) acted like H₂O₂ while the aromatic oxidation products may be more reactive than guaiacol. Five- to 12-month-old 20 mm stock solutions contained even 0.05-0.3% of H₂O₂ equivalents. The formation of the peroxidatic compound(s) was found to be a photochemical process which progressed in a few hours at 254 nm and slowly (detectable in 2-week-old solutions) in regular glass bottles kept under normal laboratory illumination. The kinetics and pH dependence of the oxidation of aged guaiacol solutions by LPO were distinctly different from those found with fresh substrate. The spontaneously formed peroxidatic compound is possibly a better oxygen donor in LPO assays than H₂O₂. The spontaneously formed aromatic oxidation products of guaiacol may include compounds that contain diphenoquinone groups. The complexity of the oxidation of guaiacol and the multitude of reaction products formed require special consideration in kinetic studies of LPO. The use of 2,2′-azino-di(3-ethylbenzthiazoline-6-sulfonic acid) as a LPO substrate was studied. The published method utilizing this substrate was modified into a more sensitive procedure by readjusting some of the reaction conditions.     

Molecular orbital studies on nucleoside antibiotics,VII. Conformation of 2′-amino- 2′-deoxyguanosine

Conformational properties of the nucleoside antibiotic 2-amino-2-deoxyguanosine have been investigated by the PCILO method along with those of its parent nucleoside, guanosine. This antibiotic, formed by replacement of the 2-hydroxyl group by an amino group in guanosine, shows anti-tumor activity and also inhibits RNA and protein syntheses. Both C(2)-endo and C(3)-endo sugar conformations have been considered in the computations. The results indicate striking similarity between the conformations of the antibiotic and the parent nucleoside, particularly in simulated aqueous environment. The biological implication of this result in terms of the antibiotic activity is discussed.     

New Enzymatic Colorimetric Reactions of Benzoic Acid Derivatives with ABTS [2,2′-Azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt] in the Presence of Laccase

New enzymatic colorimetric reactions of benzoic acid derivatives with 2,2′-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) in the presence of laccase are described. These laccase-catalyzed colorimetric reactions exhibited the characteristic colors arising from a strong absorption maximum in the visible region. Using these colorimetric reactions, benzoic acids e.g., 4-hydroxy-, 3-hydroxy-, and 3,5-diamino-benzoic acids, could be determined in micromolar amounts. These methods will offer broad applicability to those enzymatic assays which are designed to release benzoic acids.     

Terminal-sequence studies of high-molecular-weight ribonucleic acid. The reaction of periodate-oxidized ribonucleosides, 5′-ribonucleotides and ribonucleic acid with isoniazid

1. The reaction products of isoniazid with periodate-oxidized ribonucleosides and 5′-ribonucleotides have been characterized as the monohydrazones. 2. The stability, chromatographic and electrophoretic properties of the hydrazones are described. 3. ³H-labelled isoniazid was shown to react with the 5′-linked terminal adenosine and cytidine groups of periodate-oxidized Escherichia coli transfer RNA. One mole of isoniazid reacts with 27×10³g. of the transfer RNA. 4. One mole of ³H-labelled isoniazid reacts with approx. 10⁶g. of rabbit-reticulocyte ribosomal RNA. After fractionation of the RNA into its two components and treating the fractionated material with pancreatic ribonuclease and ribonuclease T₁ evidence is presented for the existence of two 5′-linked terminal sequences in the 30s fraction and only one sequence in the 17s fraction. 5. The application of this method to determining terminal sequences of high-molecular-weight RNA is discussed.     

Biosynthetic origin of the saw-toothed profile in δC and δΗ of n-alkanes and systematic isotopic differences between n-, iso- and anteiso-alkanes in leaf waxes of land plants

The n-fatty acids containing an even number of carbons (ECN-n-FAs) in higher plants are biosynthesised by repetitive addition of a two carbon unit from malonyl-ACP. The n-alkanes containing an odd number of carbon atoms (OCN-n-alkanes) are generally formed by the decarboxylation of ECN-n-FAs, but it is unknown how the less abundant even-carbon-numbered alkanes (ECN-n-alkanes) are biosynthesised in higher plants.There is a distinctive compositional pattern of incorporation of stable carbon (¹³C) and hydrogen (²H) isotopes in co-existing ECN- and OCN-n-alkanes in leaves of higher plants, such that the OCN n-alkanes are relatively enriched in ¹³C but relatively depleted in ²H against the ECN-n-alkanes. This is consistent with the OCN-n-fatty acids having a propionate precursor which is derived from reduction of pyruvate. A tentative pathway is presented with propionate produced by enzymatic reduction of pyruvate which is then thio-esterified with CoSH (coenzyme A thiol) in the chloroplast to form the terminal precursor molecule propionyl-CoA. This is then repetitively extended/elongated with the 2-carbon unit from malonyl-ACP to form the long chain OCN-n-fatty acids.The anteiso- and iso-alkanes in Nicotiana tabacum leaf waxes have previously been found to be systematically enriched in ¹³C compared with the n-alkanes by Grice et al. (2008). This is consistent with the isotopic composition of their putative respective precursors (pyruvate as precursor for n-alkanes, valine for iso-alkanes and isoleucine for anteiso-alkanes). The current study complements that of Grice et al. (2008) and looks at the distribution of hydrogen isotopes. The n-alkanes were found to be more enriched in deuterium (²H) than the iso-alkanes which in turn were more enriched than the anteiso-alkanes. We propose therefore that the depletion of ²H in the iso-alkanes, relative to the n-alkanes is the consequence of accepting highly ²H-depleted hydrogen atoms from NADPH during their biosynthesis. The anteiso-alkanes are further depleted again because there are three NADPH-derived hydrogen atoms in their precursor isoleucine, as compared with only one NADPH-derived hydrogen in valine, the precursor of the iso-alkanes.     

Inhibition and Substrate Specificity of Adenosine Deaminase. Interaction with 2′-, 3′- and/or 5′-Substituted Adenine Nucleoside Derivatives

Abstract

A series of adenine nucleoside derivatives, most of them prepared for the first time, have been evaluated as substrates or inhibitors of adenosine deaminase. The best inhibitory results were obtained with the 3′, 5′-di-O-benzoyl esters of 9-β-D-pentofuranosyladenines.     

Reactions of l-ergothioneine and some other aminothiones with 2,2′- and 4,4′-dipyridyl disulphides and of l-ergothioneine with iodoacetamide. 2-Mercaptoimidazoles, 2- and 4-thiopyridones, thiourea and thioacetamide as highly reactive neutral sulphur nucleophiles

1. The reactions of 2,2'- and 4,4'-dipyridyl disulphide (2-Py-S-S-2-Py and 4-Py-S-S-4-Py) with l-ergothioneine (2-mercapto-l-histidine betaine), 2-mercaptoimidazole, 1-methyl-2-mercaptoimidazole, thiourea, thioacetamide, 2-thiopyridone (Py-2-SH) and 4-thiopyridone (Py-4-SH) were investigated spectrophotometrically in the pH range approx. 1-9. 2. These reactions involve two sequential reversible thiol-disulphide interchanges. 3. The reaction of l-ergothioneine with 2-Py-S-S-2-Py and/or with the l-ergothioneine-Py-2-SH mixed disulphide, both of which provide Py-2-SH, is characterized by at least three reactive protonic states. This provides definitive evidence that neutral l-ergothioneine is a reactive nucleophile, particularly towards the highly electrophilic protonated disulphides. 4. A similar situation appears to obtain in the reactions of l-ergothioneine and Py-2-SH with 4-Py-S-S-4-Py and in the reactions of the other 2-mercaptoimidazoles, thiourea and Py-4-SH with 2-Py-S-S-2-Py. The nucleophilic reactivity of Py-4-SH suggests that general base catalysis provided by the disulphide in a cyclic or quasi-cyclic transition state is not necessary to generate nucleophilic reactivity in the other amino-thiones whose geometry could permit such catalysis. 5. The existence of a positive deuterium isotope effect in the l-ergothioneine-2-Py-S-S-2-Py system at pH6-7 provides no evidence for general base catalysis but is in accord with a mechanism involving specific acid catalysis and post-transition-state proton transfer. 6. The pH-dependences of the overall equilibrium positions of the various thiol-disulphide interchanges are described. 7. Reaction of thioacetamide with a stoicheiometric quantity of 2-Py-S-S-2-Py at pH1 provides 2 molecules of Py-2-SH per molecule of thioacetamide and elemental sulphur; these findings can be accounted for by thiol-disulphide interchange to provide a thioacetamide-Py-2-SH mixed disulphide followed by fragmentation to provide CH(3)CN, S and Py-2-SH. 8. Provision of high reactivity in the neutral forms of the members of this series of sulphur nucleophiles by electron donation by the amino group is compared with the well known alpha effect that provides enhanced nucleophilicity in compounds containing an electronegative atom adjacent to the nucleophilic atom. 9. The decrease in the u.v. absorption of l-ergothioneine at 257nm consequent on transformation of its aminothione moiety into an S-alkyl-2-mercaptoimidazole moiety provides a convenient method of following the alkylation of l-ergothioneine by iodoacetamide. 10. The pH dependence of the extinction coefficient of l-ergothioneine at 257nm is described by epsilon(257)={8x10(3)/(1+K(a)/[H(+)]} +6x10(3)m(-1).cm(-1) in which pK(a)=10.8. 11. In the pH range 3-11 the reaction is characterized by two reactive protonic states (X and XH). 12. The X state, reaction of the ionized 2-mercaptoimidazole moiety of the l-ergothioneine dianion with neutral iodoacetamide, is characterized by the second-order rate constant 4.0m(-1).s(-1) (25.0 degrees C, I=0.05). The XH state, characterized by the second-order rate constant 0.03m(-1).s(-1), is interpreted as reaction of the thione form of the neutral 2-mercaptoimidazole moiety of the l-ergothioneine monoanion with neutral iodoacetamide. 13. The XH state of the alkylation reaction does not exhibit a deuterium isotope effect.     

TheS,X-acetals in nucleoside chemistry. I. The synthesis of 2′- and 5′-O-methylthiomethylribonucleosides

Ribonucleoside 2′- and 5′-O-methylthiomethyl derivatives were synthesized from selectively protected nucleosides by the action of a dimethyl sulfoxide-acetic anhydride-acetic acid mixture.     

Complexes of magnesium(II) and other divalent metal ions with adenosine 5′-triphosphate and 2,2′-dipyridylamine in aqueous solution

Binary and ternary systems involving adenosine 5′-triphosphate (ATP), 2,2′-dipyridylamine (DPA) and magnesium, calcium, strontium, manganese, cobalt, copper, and zinc(II) metal ions have been investigated in aqueous media by potentiometric titrations. The analysis of the titration curves shows the existence of M(ATP)²⁻, M(ATP)(H)⁻, and M(ATP)₂(H)₂⁴⁻ species for alkaline-earth metal ions, while no ternary complex can be detected. For transition metal ions both binary and ternary species are found. Binary M(ATP)₂(H)₂⁴⁻ complexes are present in solutions containing manganese and cobalt(II) metal ions but these species cannot be revealed in the case of copper and zinc(II). Ternary complexes as M(ATP)(DPA)²⁻ and M(ATP)(DPA)(H)⁻ are common to all transition metals. Binuclear and hydroxo complexes as M₂(ATP)(OH)⁻ and M(ATP)(OH)³⁻ are found only for copper and zinc(II). A hypothesis on the possible role of the species M-ATP in 1:2 ratio in the dephosphorylation mechanism is advanced on the basis of a comparison between the equilibrium data in the solution phase and the solid state structures of the magnesium, calcium, and manganese(II)- ATP-DPA systems.     

Synthesis,cytotoxicity and DNA-binding properties of Pd(II), Cu(II) and Zn(II) complexes with 4′-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-2,2′:6′,2″-terpyridine

Pd(II), Cu(II) and Zn(II) complexes (1–3) based on 4′-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-2,2′:6′,2″-terpyridine were synthesized and characterized by UV, IR, NMR, EPR, HRMS, elemental analyses, and molar conductivity measurements. The cytotoxicity of these complexes against HL-60, BGC-823, KB, Bel-7402, A549, Hela, K562 and MCF-7 cell lines in vitro was measured by MTT method. The DNA binding property of the complexes was evaluated by UV, fluorescence, CD spectroscopies and thermal denaturation. The cytotoxicity of complexes 1 and 3 against all the tested cell lines is better than that of cisplatin. Complexes 1 and 2 exhibit 7- and 4-folds higher cytotoxicity than cisplatin against Bel-7402 cell line. Complex 3 displays the highest cytotoxicity against all the cell lines tested, and shows 7-, 14-, 8-, 11- and 8-folds higher cytotoxicity than cisplatin against Bel-7402, A549, Hela, K562 and MCF-7 cell lines. The complexes bind to DNA via intercalation mode and complex 3 stabilizes the G-quadruplex. The results reveal that all the complexes display high cytotoxicity against all the tested cancer cell lines, and complex 3 is selective for G-quadruplex over duplex DNA.     

Nucleosides. IL. Synthesis and Properties of 2,4-Quinazolinedione N-1-2′ Deoxy-, 3′-Deoxy- and 2′,3′-Dideoxynucleosides

Abstract

A series of 6- and/or 7-substituted 2,4-quinazoline-dione N-1-deoxyribofuranosides have been synthesized and characterized. The 2′-deoxy-β-D-ribofuranosides 23–28 have been prepared by transformation of the corresponding ribofuranosides by chemical deoxygenation. Direct glycosidation to the β-anomers with a 2′-deoxyribofuranosyl donor to pure anomers failed due to missing diastereoselectivity and difficult separation of the reaction products. The synthesis of the 3′-deoxy-β-D-ribofuranosides 54–58, however, was achieved by glycosidation of the trimethylsilylated 2,4-quinazolinediones 43–47 with an appropriate 3′-deoxyribofuranosyl donor (48). The 2′,3′-dideoxy-β-D-ribofuranosyl derivatives 63–66 were again obtained by chemical deoxygenation of the corresponding 2′-deoxy-β-D-nucleosides, since all experiments of direct glycosidation with a 2′,3′-dideoxyribofuranosyl donor as well as the chemical conversion of the corresponding ribonucleosides into the 2′,3′-dideoxynucleosides failed due to side reactions. The newly synthesized compounds have been identified by UV and ¹H-NMR spectra as well as elemental analyses.     

The amino acid conjugate formed by the interaction of the anion transport inhibitor 4,4′-diisothiocy ano-2,2′- stilbenedisulfonic acid (DIDS) with band 3 protein from human red blood cell membranes

The specific anion transport inhibitor 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and its reduced analog (H₂DIDS), when irreversibly bound to band 3 protein of the red blood cell membrane, form amino acid conjugates through interaction with the ?-amino group of a particular lysine residue. The specific residue is located in a transmembrane segment of band 3 protein and appears to be a close neighbor of the transport site.     

Metabolic activation of a mutagen, 2-amino-6-methyldipyrido-[1,2-a:3′,2′-d]imidazole. Identification of 2-hydroxyamino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole and its reaction with DNA

A potent mutagen, 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole(Glu-P-1), isolated from pyrolysates of L-glutamic acid and casein, was metabolically activated and bound to DNA. An activated form was identified as 2-hydroxyamino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole(N-OH-Glu-P-1). Synthetic N-OH-Glu-P-1 reacted with DNA only after O-acetylation to give a modified DNA, which on hydrolysis gave 2-(C⁸-guanyl)amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole(gua-Glu-P-1). The same adduct was isolated from DNA modified with Glu-P-1 by microsomes in vitro, as reported earlier.     

Computer-augmented modeling studies of Pb(II) and Cd(II) complexes with maleic acid in ethylene glycol–water mixture

Chemical speciation of binary complexes of Pb(II) and Cd(II) ions with maleic acid have been studied pH metrically in the concentration range of 0–50% v/v ethylene glycol (EG)–water mixtures maintaining an ionic strength of 0.16 molL^?1 at 303 K. Alkalimetric titrations were carried out in different relative concentrations of metal and maleic acid. Stability constants of various models of binary complexes were refined with MINIQUAD75. The best-fit chemical models were selected based on statistical parameters and residual analysis. The species detected are ML₂, ML₃, and ML₂H for Pb(II) and Cd(II). The chemical speciation, metal bioavailability, and transportation are explained based on the distribution diagrams.     

3′-Azido-3′-deoxy-5′-O-isonicotinoylthymidine: A Novel Antiretroviral Analog of Zidovudine. II. Stability in Aqueous Media and Experimental and Theoretical Ionization Constants

Abstract

Degradation of 3′-azido-3′-deoxy-5′-O-isonicotinoylthymidine (AZT-Iso), an antiretroviral derivative of zidovudine, was investigated in buffer pH 7.4, µ = 300 mOsm at 37, 50 and 60°C, and in water (pH 6.6, 37°C), giving zidovudine (AZT) and isonicotinic acid (INA) as products. The rate constants were determined by reversed-phase HPLC showing pseudo-first-order kinetics related to the residual amount of AZT-Iso. In this way, the studied compound was demonstrated to be 153 times more stable in water than in buffer solution at 37°C. The analytical method was conveniently validated demonstrating to be a rapid and accurate stability-indicating technique. In addition, experimental and theoretical values of pKa were determined.     

Assimilation of exogenous 2′-14C-indole-3-acetic acid and 3′-14C-tryptophan exposed to the roots of three wheat varieties

This study was conducted to determine if plants can assimilate indole-3-acetic acid (IAA) from rooting media and if exogenous L-tryptophan (L-TRP) can be assimilated and converted by plants into auxins. The addition of 2-¹⁴C-IAA (3.7 kBq plant^-1) to wheat (Triticum aestivum L.) seedlings of three varieties grown in nutrient solution resulted in the uptake (avg.=7.6%) of labelled IAA. Most of the label IAA was recovered in the shoot (avg.=7.2%) with little accumulation in the root (avg.=0.43%). A portion of the assimilated IAA-label in the plant was identified by co-chromatography and UV spectral confirmation as IAA-glycine and IAA-aspartic acid conjugates. Little of the assimilated IAA label was found as free IAA in the wheat plants. These same assimilation patterns were observed when 2-¹⁴C-IAA was added to wheat plants grown in sterile and nonsterile soil. In contrast, the wheat varieties assimilated considerably less (avg.=1.3%) of the added microbial IAA precursor, 3-¹⁴C-L-TRP (3.7 kBq plant^-1) and thus much lower amounts of IAA conjugates were detected. Glasshouse soil experiments revealed that 2 out of 3 wheat varieties had increased growth rates and increased yields when L-TRP (10^-5 and 10^-7 M) was added to the root zone. It is surmised that this positive response is a result of microbial auxin production within the rhizosphere upon the addition of the precursor, L-TRP. The amino acid composition of the root exudates plays a critical role in microbial production of auxins in the rhizosphere. This study showed that wheat roots can assimilate IAA from their rooting media, which will supplement the endogenous IAA levels in the shoot tissue and may positively influence plant growth and subsequent yield.