Reactions of plant copper/topaquinone amine oxidases with N6-aminoalkyl derivatives of adenine

Plant copper/topaquinone-containing amine oxidases (CAOs, EC 1.4.3.6) are enzymes oxidising various amines. Here we report a study on the reactions of CAOs from grass pea (Lathyrus sativus), lentil (Lens esculenta) and Euphorbia characias, a Mediterranean shrub, with N6-aminoalkyl adenines representing combined analogues of cytokinins and polyamines. The following compounds were synthesised: N6-(3-aminopropyl)adenine, N6-(4-aminobutyl)adenine, N6-(4-amino-trans-but-2-enyl) adenine, N6-(4-amino-cis-but-2-enyl) adenine and N6-(4-aminobut-2-ynyl) adenine. From these, N6-(4-aminobutyl) adenine and N6-(4-amino-trans-but-2-enyl)adenine were found to be substrates for all three enzymes (Km approximately 10(-4)M). Absorption spectroscopy demonstrated such an interaction with the cofactor topaquinone, which is typical for common diamine substrates. However, only the former compound provided a regular reaction stoichiometry. Anaerobic absorption spectra of N6-(3-aminopropyl)adenine, N6-(4-amino-cis-but-2-enyl)adenine and N6-(4-aminobut-2-ynyl)adenine reactions revealed a similar kind of initial interaction, although the compounds finally inhibited the enzymes. Kinetic measurements allowed the determination of both inhibition type and strength; N6-(3-aminopropyl)adenine and N6-(4-amino-cis-but-2-enyl)adenine produced reversible inhibition (Ki approximately 10(-5) - 10(-4) M) whereas, N6-(4-aminobut-2-ynyl)adenine could be considered a powerful inactivator.     

Mass spectrometric analysis of cytokinins in plant tissue VII. Quantification of cytokinin bases by negative ion mass spectrometry

A study of derivatives of N⁶-(isopent-2-enyl)adenine formed by substitution at N-9 indicated that sensitivity of detection by chemical ionization mass spectrometry was maximized by a pentafluorobenzyl substituent and negative ion monitoring. O-t-Butyldimethylsilyl-9-pentafluorobenzyl derivatives of zeatin (Z),cis-zeatin (cis-Z), and dihydrozeatin (DZ) were characterized by mass spectrometry. A procedure was based on these stable derivatives and negative ion chemical ionization mass spectrometry for quantification of zeatin and dihydrozeatin in plant tissue.For part VI, see Letham and Singh (1989).     

Amines in unicellular green algae: 2. Amines in Scenedesmus acutus

Scenedesmus acutus contains about 10 major amines and at least 20 other amines which are present in very small quantities. The following amines were identified by mass spectrometry after separation of the trifluoroacetyl derivatives by gas-liquid chromatography and of the dansyl ² derivatives by thin-layer chromatography: methylamine, dimethylamine, ethylamine, ethanolamine, putrescine, cadaverine, spermidine, N-(3-aminopropyl)-1,3-diaminopropane, N-(4-aminobutyl)-1,4-diaminobutane, 2-phenylethylamine, tyramine, piperazine, adenine, and γ-butyrolactam. The methods applied for the analyses of these amines are described and discussed.     

Mass spectrometric analysis of cytokinins in plant tissue VII. Quantification of cytokinin bases by negative ion mass spectrometry

A study of derivatives of N⁶-(isopent-2-enyl)adenine formed by substitution at N-9 indicated that sensitivity of detection by chemical ionization mass spectrometry was maximized by a pentafluorobenzyl substituent and negative ion monitoring. O-t-Butyldimethylsilyl-9-pentafluorobenzyl derivatives of zeatin (Z),cis-zeatin (cis-Z), and dihydrozeatin (DZ) were characterized by mass spectrometry. A procedure was based on these stable derivatives and negative ion chemical ionization mass spectrometry for quantification of zeatin and dihydrozeatin in plant tissue.     

Cytokinins in shoots of the chestnut tree

Five cytokinins, trans-zeatin, 9-β-d-ribosyl-trans-zeatin, O-β-d-glucosyl-trans-zeatin, N⁶-(3-methyl-but-2-enyl)adenine and N⁶-(3-methyl-but-2-enyl)adenosine were identified in shoots of the chestnut tree.     

Isolation and Estimation of Cytokinins and Cytokinin-Containing Transfer RNAs from Cucumis sativus L. var. Guntur Seedlings

N⁶-(Δ²-Isopentenyl) adenosine antibodies were used for the isolation of free cytokinins and cytokinin-containing tRNAs from parts of Cucumis sativus L. var. Guntur seedlings and for the estimation of cytokinins in them. Immobilized N⁶-(Δ²-isopentenyl) adenosine antibodies retained tRNAs containing N⁶-(Δ²-isopentenyl) adenosine and N⁶-(4-hydroxy-3-methylbut-2-enyl) adenosine with equal efficiencies. There were at least five cytokinins in the free form in cucumber seedlings. N⁶-(4-Hydroxy-3-methylbut-2-enyl) adenosine, N⁶-(Δ²-isopentenyl) adenosine, and N⁶-(Δ²-isopentenyl) adenine were present at least to the extent of 80, 23, and 9 nanograms, respectively, in the cotyledons and 40, 6, and 3 nanograms, respectively, in the decotyledonated seedlings per gram of tissue. Only two cytokinins were found in the tRNAs of cucumber cotyledons, namely N⁶-(Δ²-isopentenyl) adenosine and N⁶-(4-hydroxy-3-methylbut-2-enyl) adenosine in amounts of 12 and 318 nanograms, respectively, per gram of tissue. Immunoaffinity chromatographic analysis of radiolabeled aminoacyl tRNAs from cucumber cotyledons showed that tRNA^Phe and tRNA^Tyr contained cytokinins whereas tRNA^Ala did not.     

Synthesis of monosaccharides by oxidation of furfural derivatives

dl-Ribose, dl-arabinose, and dl-xylose have been synthesized from 2-furaldehyde. Condensation of 2-furaldehyde with pinacol followed by oxidation of the product with bromine water or lead tetra-acetate and reduction with sodium borohydride gave dl-2-(1,4-dihydroxy-cis-but-2-enyl)-4,4,5,5-tetramethyl-1,3-dioxolane (4a). Hydroxylation of the dibenzoate of 4a followed by debenzoylation and hydrolysis gave dl-ribose and dl-arabinose. Epoxidation of 4a with monoperoxysuccinic acid followed by hydrolysis gave dl-xylose.     

A Facile Synthesis of cis-9-[4-(1,2-Dihydroxyethyl)-cyclopent-2-enyl]guanine and Its Derivative

Abstract

The synthesis of carbocyclic nucleosides, cis-9-[4-(1,2-dihydroxyethyl)-cyclopent-2-enyl]guanine (3) and cis-2-amino-6-cyclopropylamino-9-[4-(1,2-dihydroxyethyl)-cyclopent-2-enyl]guanine (4), was achieved from cyclopentadiene (5) in five and six steps, respectively. This route involves a hetero Diels-Alder reaction and a Pd(0)-catalyzed coupling reaction.     

Cis-avicennol,a new pyranocoumarin from the root bark of Zanthoxylum elephantiasis

A new pyranocoumarin has been isolated from the root bark of Zanthoxylum elephantiasis and identified as cis-avicennol (6-(3-hydroxy-3-methyl-cis-but-1-enyl)-5-methoxy-2,2-dimethyl- 2H-benzo [1,2b:3,4b′] dipyran-8-one) on the basis of comparison of spectral data with that of trans-avicennol and conversion to tetrahydroavicennol. The usefulness of the lanthanide shift reagent Eu(fod)₃ in the assignment of cis configuration to the butenyl sidechain is briefly discussed.     

Protein Folding: Grand Challenge of Nature

Abstract

Conformational preferences of the hypermodified nucleic acid bases N6-(Δ² -cis-hydroxyisopentenyl)adenine, cis-io⁶Ade also known as cis-zeatin, and N⁶-(Δ² -trans-hydroxyisopentenyl)adenine, trans-io⁶ade or trans-zeatin, and 2-methylthio derivatives of these cis-ms²io⁶Ade or cis-ms²zeatin, and trans-ms²io⁶Ade or trans-ms²zeatin have been investigated theoretically by the quantum chemical Perturbative Configuration Interaction with Localized Orbitals (PCILO) method. Automated geometry optimization using quantum chemical MNDO, AMI and PM3 methods has also been made to compare the salient features. The predicted most stable conformation of cis-io⁶Ade, trans-io⁶Ade, cis-ms²io⁶Ade and trans-ms²io⁶Ade are such that in each of these molecules the isopentenyl substituent spreads away (has “dista” conformation) from the five membered ring imidazole moiety of the adenine. The atoms N(6), C(10) and C(11) remain coplanar with the adenine ring in the predicted preferred conformation for each of these molecules. In cis-io⁶Ade as well as cis- ms²io⁶Ade the hydroxyl oxygen may participate in intramolecular hydrogen bonding with the H-C(10)-H group. In trans-io⁶Ade the hydroxyl group is oriented towards the H-C(2) instead. This orientation is retained in trans-ms²io⁶Ade, possible O-H…S hydrogen bonding may be a stabilizing factor. In all these four modified adenines C(11)-H is favourably placed to participate in intramolecular hydrogen bonding with N(1). In cis-ms²io⁶Ade as well as trans-ms²io⁶Ade the 2-methylthio group preferentially orients on the same side as C(2)-N(3) bond, due to this nonobstrusive placing, orientation of the hydroxyisopentenyl substituent remains unaffected by 2-methylthiolation. Thus the N(1) site remains shielded irrespective of the 2-methylthiolation status in these various cis-and trans-zeatin analogs alike. Firmly held orientation of hydroxyisopentenyl substituent in zeatin isomers and derivatives, in contrast to adaptable orientation of isopentenyl substituent in i⁶Ade and ms²i⁶Ade, may account for the increased efficiency of suppressor tRNA and reduced codon context sensitivity accompanied with the occurrence of ms²-zeatin (ms²io⁶Ade) modification.     

Incorporation of 3H-adenine into free cytokinins by cytokinin-autonomous tobacco callus tissue

Cytokinin-autonomous tobacco callus was incubated in defined mineral medium containing ³H-adenine for 60 minutes. Radioactivity was incorporated into the four predominant free cytokinins, ribosyl-$\text{trans}$-zeatin, $\text{trans}$-zeatin, N⁶-(Δ²-isopentenyl) adenosine and N⁶-(Δ²-isopentenyl) adenine. The bases were more abundant than their respective ribosides, N⁶-(Δ²-isopentenyl) adenine being the most abundant cytokinin. No discrete peaks of radioactivity could be detected on the HPLC column eluate corresponding to the elution volumes of $\text{cis}$-zeatin and ribosyl-$\text{cis}$-zeatin.     

Far-infrared spectra of poly(-α-amino acids) with basic alkyl group side chains

Far-infrared spectra in the region from 700 to 60 cm^?1 have been measured for the α-helix structures of poly(L -α-amino-n-butyric acid), poly-L -norvaline, poly-L -norleucine, and poly-L -leucine and for the β-form structures of poly(L -α-amino-n-butyric acid), poly-L -valine, poly(DL -amino-n-butyric acid), poly-DL -norvaline, and poly-DL -norleucine. The changes of the spectra on N-deuteration have been measured in the region between 700 and 400 cm^?1. It is concluded that, the α-helix has characteristic bauds near 690, 650, 610, 380, 150, and 100 cm^?1, and that the β-form has characteristic bands near 700, 240, and 120 cm^?1. The main-chain vibrations in the region from 600 to 200 cm^?1 are strongly coupled with the side-chain deformation vibrations.     

Acetyl-CoA Carboxylase Inhibitors from Avocado (Persea americana Mill) Fruits

A methanol extract of avocado fruits showed potent inhibitory activity against acetyl-CoA carboxylase, a key enzyme in fatty acid biosynthesis. The active principles were isolated and identified as (5E,12Z,15Z)-2-hydroxy-4-oxoheneicosa-5,12,15-trienyl (1), (2R,12Z,15Z)-2-hydroxy-4-oxoheneicosa-12,15-dienyl (2), (2R^*,4R^*)-2,4-dihydroxyheptadec-16-enyl (3) and (2R^*,4R^*)-2,4-dihydroxyheptadec-16-ynyl (4) acetates by instrumental analyses. The IC₅₀ of the compounds were 4.0×10^-6, 4.9×10^-6, 9.4×10^-6, and 5.1×10^-6M, respectively.     

Catabolism of polyamines in the rat: Polyamines and their non-α-amino acid metabolites

The metabolic fate of stable isotopically labeled polyamines was investigated after their first and second intraperitoneal injection in rats. Using gas chromatographic and mass fragmentographic analyses of acid-hydrolyzed 24-h urines, some aspects of the polyamine metabolism could be elucidated. After the injections with hexadeutero-1,3-diaminopropane, obly labeled 1,3-diaminopropane was recovered from the urine samples. The rat injected with tetradeuteroputrescine excreted labeled putrescine excreted labeled putrescine, γ-amino-n-butyric acid, 2-hydroxyputrescine and spermidine, while the urine samples of the rat after the injections with tetradeuterocadaverine contained labeled cadaverine and δ-aminovaleric acid. The injections of hexadeuterospermidine led to the appearance of labeled spermidine, isoputreanine, putreanine, N-(2-carboxyethyl)-4-amino-n-butyric acid, putrescine, γ-amino-n-butyric acid, 1,3-diaminopropane, β-alanine and spermine. After the injections with octadeuterospermine, labeled spermine, N-(3-aminopropyl)-N′-(2-carboxyethyl)-1,4-diaminobutane, N,N′-bis(2-carboxyethyl)-1,4-diaminobutane, spermidine, isoputreanine, putreanine, N-(2-carboxyethyl)-4-amino-n-butyric acid, putrescine, 1,3-diaminopropane, β-alanine, 2-hydroxyputrescine and possibly γ-amino-n-butyric acid were recovered. Clear differences between the metabolism after the first and second injection were noted for putrescine, spermidine and spermine, which is suggestive for enzyme induction and/or the existence of salvage pathways.     

N-(Delta-Isopentenyl)adenosine: Its Occurrence as a Free Nucleoside in an Autonomous Strain of Tobacco Tissue

Cytokinins from both the free nucleoside pool and the transfer RNA have been isolated and identified in a habituated strain of tobacco pith callus (Nicotiana tabacum [L] var. Wisconsin 38). The transfer RNA of this strain contains both N⁶-(Δ²-isopentenyl) adenosine and N⁶-(4-hydroxy-3-methylbut-2-cis-enyl) adenosine. The trans-hydroxylated derivative is absent from the transfer RNA of this dark-grown tissue. N⁶-(Δ²-Isopentenyl)-adenosine was identified as a component of the free nucleoside pool in concentrations of about 10 micrograms per kilogram of tissue.     

Conformational Preferences of Modified Nucleic Acid Bases N6-Methyl-N6-(N-Threonylcarbonyl) Adenine and 2-Methylthio-N6-(N-Threonylcarbonyl) Adenine by the Quantum Chemical PCILO Calculations

Abstract

Conformational preferences of the hypermodified nucleic acid bases N⁶-methyl-N⁶-(N- threonylcarbonyl) Adenine, m⁶ tc⁶ Ade, and 2-methylthio-N⁶-(N-threonylcarbonyl) Adenine, mS² tc⁶ Ade, have been studied theoretically using the quantum chemical PCILO (Perturbative Configuration Interaction using Localized Orbitals) method. The multidimensional conformational space has been searched using selected grid points formed by combining the various torsion angles which take the favoured values obtained from energy variation with respect to each torsion angle individually. In m⁶ tc⁶ Ade and mS² tc⁶ Ade alike the threonylcar- bonyl substituent preferably orients away (distal) from the imidazole moiety of the adenine ring. And as in the simpler N⁶-(N-threonylcarbonyl) Adenine, tc⁶ Ade, the atoms in the ureido group as well as the amino acid carbon atoms C(12) and C(13) remain coplanar with the purine base. As in tc⁶ Ade, this conformation is stabilized by the intramolecular hydrogen bond between N(11)H of the amino acid and N(l) of the adenine base.

The N⁶-methyl protons, in m⁶ tc⁶ Ade, take trans-staggered orientation with respect to the C(6)-N(6) bond. The preferred orientation of the 2-methylthio group is cis to the C(2)-N(3) bond in mS² tc⁶ Ade. This is in marked contrast to the modified nucleic acid base 2-methylthio-N⁶-(Δ²-isopentenyl) Adenine, mS² i⁶ Ade, where the 2-methylthio group orients trans to the C(2)- N(3) bond, causing a change in the preferred orientation of the isopentenyl component on methylthiolation. The present results thus indicate that unlike in the isopentenyl adenine the role of further chemical substitutions in threonylcarbonyl adenine may be indirect and less pronounced.     

Biochemical Characterization of Cytokinin Oxidases/Dehydrogenases from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Expressed in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.

Transgenic tobacco plants overexpressing single Arabidopsis thaliana cytokinin dehydrogenase (CKX, EC 1.5.99.12) genes AtCKX1, AtCKX2, AtCKX3, AtCKX4, AtCKX5, AtCKX6, and AtCKX7 under the control of a constitutive 35S promoter were tested for CKX-enzymatic activity with varying pH, electron acceptors, and substrates. This comparative analysis showed that out of these, only AtCKX2 and AtCKX4 were highly active enzymes in reaction with isoprenoid cytokinins (N ⁶ -(2-isopentenyl)adenine (iP), zeatin (Z)) and their ribosides using the artificial electron acceptors 2,6-dichlorophenol indophenol (DCPIP) or 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q₀). Turnover rates of these cytokinins by four other AtCKX isoforms (AtCKX1, AtCKX3, AtCKX5, and AtCKX7) were substantially lower, whereas activity of AtCKX6 was almost undetectable. The isoenzymes AtCKX1 and AtCKX7 showed significant preference for cytokinin glycosides, especially N ⁶ -(2-isopentenyl)adenine 9-glucoside, under weakly acidic conditions. All enzymes preferentially cleave isoprenoid cytokinins in the presence of an electron acceptor, but aromatic cytokinins are not resistant and are degraded with lower reaction rates as well. Cytokinin nucleotides, considered as resistant to CKX attack until now, were found to be potent substrates for some of the CKX isoforms. Substrate specificity of AtCKXs is discussed in this study with respect to the structure of the CKX active site. Further biochemical characterization of the AtCKX1, AtCKX2, AtCKX4 and AtCKX7 enzymes showed pH-dependent activity profiles.     

Four base recognition by triplex-forming oligonucleotides at physiological pH

We have achieved recognition of all 4 bp by triple helix formation at physiological pH, using triplex-forming oligonucleotides that contain four different synthetic nucleotides. BAU [2′-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine] recognizes AT base pairs with high affinity, ^MeP (3-methyl-2 aminopyridine) binds to GC at higher pHs than cytosine, while ^APP (6-(3-aminopropyl)-7-methyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one) and S [N-(4-(3-acetamidophenyl)thiazol-2-yl-acetamide)] bind to CG and TA base pairs, respectively. Fluorescence melting and DNase I footprinting demonstrate successful triplex formation at a 19mer oligopurine sequence that contains two CG and two TA interruptions. The complexes are pH dependent, but are still stable at pH 7.0. BAU, ^MeP and ^APP retain considerable selectivity, and single base pair changes opposite these residues cause a large reduction in affinity. In contrast, S is less selective and tolerates CG pairs as well as TA.     

Differential cytokinin structure-activity relationships in phaseolus

The activities of eight cytokinins in promoting callus growth were tested in two Phaseolus genotypes, P. vulgaris L. var. Great Northern, and P. lunatus L. var. Kingston. The structural feature which contributes to the major genotypic difference in cytokinin structure-activity relationships is the presence or absence of a double bond at the 2,3-position of the isoprenoid N⁶ side chain. In Kingston, trans-zeatin was 3-fold more active than dihydrozeatin and 30-fold more active than cis-zeatin. The activities of N⁶-(Δ²-isopentenyl)adenine and N⁶-isopentyladenine were nearly the same. In Great Northern, however, dihydrozeatin was at least 30-fold more active than both trans-zeatin and cis-zeatin, and N⁶-isopentyladenine was 100-fold more active than N⁶-(Δ²-isopentenyl)adenine. The results suggest the possibility of employing cytokinin structure-activity relationships in distinguishing genotypic differences in cytokinin function and metabolism.     

I. A unique deaminated metabolite of sulfamethazine [4-amino-N-(4, 6-dimethyl-2-pyrimidinyl) benezenesulfonamide] in swine

¹⁴C-Sulfamethazine [4-amino-$\text{N}$-(4, 6-dimethyl-2-pyrimidinyl)- ¹⁴C-benzenesulfonamide] was administered orally to young, 21-kg male swine. A unique metabolite, $\text{N}$-(4, 6-dimethyl-2-pyrimidinyl)- ¹⁴C-benzenesulfonamide (¹⁴C-desaminosulfamethazine), was identified in skeletal muscle collected 24 hr after dosing. The structure of the metabolite was confirmed by synthesis, comparative electron impact mass spectrometry, chemical ionization mass spectrometry, and infrared spectroscopy.