Cytokinins in Corynebacterium fascians Cultures: Isolation and Identification of 6-(4-Hydroxy-3-methyl-cis-2-butenylamino)-2-methylthiopurine

In addition to the four cytokinins, 6-(3-methyl-2-butenylamino)purine, 6-methylaminopurine and the cis and trans isomers of 6-(4-hydroxy-3-methyl-2-butenylamino)purine, reported earlier from our laboratories, three cytokinin-active fractions have been obtained from the aqueous medium of 6-day-old Corynebacterium fascians cultures. One of these has been identified as 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-2-methylthiopurine (2-methylthio-cis-zeatin, c-ms²io⁶ Ade).     

Thermodynamics for base binding to four atropisomers cobalt(II) picket fence porphyrin. Intramolecular ligand–ligand interactions

Thermodynamic parameters for base binding to four atropisomers of meso-tetrakis(o-pivalamidophenyl)porphyrinatocobalt(II) were determined by spectrophotometry in toluene. The order of the affinities of the four isomers with 1-methylimidazole and pyridine is α⁴<α³<cis-α²<trans-α². The higher base affinities of the trans-α² complex compared with the α⁴ complex are due to an increase in the binding energies of the bases, although a substantial decrease of entropy changes also occurs; the differences of thermodynamic values on both complexes are −ΔΔG = 1.49 and 1.36 kcal/mol, −ΔΔH = 3.4 and 3.1 kcal/mol and −ΔΔS = 6.4 and 6.1 eu, with 1-methylimidazole and pyridine, respectively. With saturated bases pyrrolidine and piperidine, the affinities of the trans-α² complex are comparable to those of the α⁴ complex, and those of the cis-α² complex are the lowest. The increased steric repulsion between the pickets and ligated pyrrolidine or piperidine may cancel out the stabilizing effect on the base binding to the α² complexes. Proton NMR study suggests the preferential solvation of the four-coordinate species of the trans-α² complex to that of the α⁴ complex. It could be concluded that the stabilization of the base binding by the pickets is attributed to an intramolecular ligand–ligand interaction between the ligated base and the pickets rather than to the inhibition of the undesirable solvation on the active sites.     

Comparative metabolism of the cis and trans isomers of N-nitroso-2-6-dimethylmorpholine in rats,hamsters and guinea pigs

The in vivo metabolism of the cis and trans isomers of N-[3,5-³H] nitroso-2,6-dimethylmorpholine (NDMM) was studied in female Fischer rats, Syrian golden hamsters and guinea pigs by analysis of urinary metabolites using high pressure liquid chromatography (HPLC). Animals were treated by gavage with 12 mg/kg body wt. of NDMM, composed of both isomers and 12 μCi/kg body wt. of either of the separated radioactive isomers (cis or trans). Control animals received 12 mg, 12 μCi/kg body wt. NDMM with both isomers labeled in their natural proportion.There was a substantial increase in the excretion of a particular metabolite, 2-(2-hydroxyl-methyl)ethoxy propanoic acid, in the urine of rats, hamsters and guinea pigs 24 h after received the trans isomer (24, 22 and 13% of the total dose excreted, respectively). A minor metabolite was determined to be 2,6-dimethylmorpholine-3-one, another product of α-oxidation. The metabolite 1-amino-2-hydroxypropanol was identified, indicating that NDMM was metabolized by both α-and β-oxidation.In all three species, animals administered the cis isomer excreted larger amounts of N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) and N-nitroso-bis(2-hydroxypropyl)amine (BHP) products of beta oxidation, than those treated with the trans isomer. Hamsters and guinea pigs treated with the more carcinogenic cis isomer in these species, also excreted twice as much of two other metabolites than was found in the urine of animals given the trans isomer.The trans isomer of NDMM appeared to be preferentially metabolized by α-oxidation and from earlier studies this metabolic pathway seemed to be important in carcinogenesis by NDMM in the rat. The cis isomer might be in a conformation more favorable for β-oxidation and this pathway may be of primary importance in carcinogenesis by NDMM in hamsters and guinea pigs.     

Cytokinins in tRNA Obtained from Spinacia oleracea L. Leaves and Isolated Chloroplasts

Cytokinin-active ribonucleosides have been isolated from tRNA of whole spinach (Spinacia oleracea L.) leaves and isolated spinach chloroplasts. The tRNA from spinach leaf blades contained: 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine (cis and trans isomers), 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine, and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-d -ribofuranosylpurine (cis and trans isomers). A method for isolation of large amounts of intact chloroplasts was developed and subsequently used for the isolation of chloroplast tRNA. The chloroplast tRNA contained 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-d -ribofuranosylpurine (the cis isomer only). The structures of these compounds were assigned on the basis of their chromatographic properties and mass spectra of trimethylsilyl derivatives which were identical with those of the corresponding synthetic compounds. The results of this study indicate that ribosylzeatin was present in spinach leaf tRNA, but absent from the purified chloroplast tRNA preparation.     

Synthesis and NMR characterization of the novel mixed-ligand Pt(II) complexes cis- and trans-Pt(Ypy)(pyrimidine)Cl2 and trans,trans-Cl2(Ypy)Pt(μ-pyrimidine)Pt(Ypy)Cl2 (Ypy = pyridine derivative)

Mixed-ligand complexes of the type cis- and trans-Pt(Ypy)(pm)Cl₂ where Ypy = pyridine derivative and pm = pyrimidine were synthesized and characterized by IR spectroscopy and by multinuclear (¹⁹⁵Pt, ¹H and ¹³C) magnetic resonance spectroscopy. The cis compounds were prepared from the reaction of K[Pt(Ypy)Cl₃] with pyrimidine (1:1 proportion) in water, while most of the trans isomers were synthesized from the isomerization of the cis compounds. The cis isomers could not be isolated with the Ypy ligands containing two -CH₃ groups in ortho positions. When the aqueous reaction of K[Pt(Ypy)Cl₃] with pyrimidine was performed in a Pt:pm ratio = 2:1, the pyrimidine-bridged dinuclear species were formed. Only the most stable trans-trans isomers could be isolated pure. In IR spectroscopy, the cis monomers showed two ν(Pt-Cl) bands, while the trans monomers and dimers showed only one ν(Pt-Cl) band. The ¹⁹⁵Pt NMR signals of the cis monomers were found at slightly higher fields than those of the corresponding trans isomers. The δ(¹⁹⁵Pt) of the dimers were found close to those of the trans monomers. The NMR results were interpreted in relation to the solvent effect, which seems important in these complexes. The coupling constants J(¹⁹⁵Pt-¹H) and J(¹⁹⁵Pt-¹³C) are larger in the cis geometry. The crystal structures of the compounds cis-Pt(2,4-lut)(pm)Cl₂, trans-Pt(2,6-lut)(pm)Cl₂ and trans,trans-Cl₂(2,6-lut)Pt(μ-pm)Pt(Ypy)Cl₂ were studied by X-ray diffraction methods and the results have confirmed the configurations suggested by IR and NMR spectroscopies.     

Identification of cis/trans isomers of bis(acetylacetonato)nickel(II) complexes in solution based on electronic spectra

Electronic spectra of Ni(acac)₂ were studied in acetone, DMF, and some other solvents for the purpose of identifying the cis/trans isomers from the spectra (acac⁻ = acetylacetonate anion). The spectral components were investigated in the spin-allowed transition bands, and a relationship was found between the spectral pattern and the cis/trans isomers. According to this relationship, it was concluded that the cis isomer was formed in DMF and in N-methylformadide, whereas the trans isomer was formed in acetone and in pyridine. Based on the DFT computation, the cis-[Ni(acac)₂(DMF)₂] was found to be stabilized by intramolecular hydrogen bonds between acetylacetonate and DMF.     

Synthesis and NMR characterization of the novel mixed-ligands Pt(II) complexes Pt(amine)(pyrimidine)X2 and trans,trans-X2(amine)Pt(μ-pyrimidine)Pt(amine)X2 (X = I and Cl)

Mixed-ligand complexes of the type Pt(amine)(pm)I₂, (pm = pyrimidine) were synthesized and characterized by IR spectroscopy and by multinuclear (¹⁹⁵Pt, ¹H and ¹³C) magnetic resonance spectroscopy. The cis compounds were prepared from the reaction of I(amine)Pt(μ-I)₂Pt(amine)I with pyrimidine (1:2 proportion) in water, while the trans isomers were synthesized from the isomerization of the cis complexes in acetone. The cis isomers could not be isolated with several amines, especially the more bulky ones. In ¹H NMR, the pyrimidine protons of the cis compounds were found at lower fields than those of the trans analogs and the J(¹⁹⁵Pt-¹H) coupling constants are slightly larger in the cis geometry. For n-butylamine, the reaction produced also I₂(n-butylamine)Pt(μ-pm)Pt(n-butylamine)I₂. No such dimer could be isolated with the other amines. The compounds Pt(amine)(pm)Cl₂ were also prepared (amine = methylamine and t-butylamine) from the ionic complex K[Pt(amine)Cl₃] using an excess of pyrimidine. The IR and NMR characterization showed that the methylamine compound was a cis-trans mixture, while only the trans isomer was isolated with t-butylamine. When the same reaction was performed using a Pt:pm ratio of 2:1, Cl₂(amine)Pt(μ-pm)Pt(amine)Cl₂ was isolated. The pyrimidine-bridged dimers were identified by IR and multinuclear magnetic resonance spectroscopies as the trans-trans isomers. The trans monomers and dimers showed only one ν(Pt-Cl) band. The ¹⁹⁵Pt NMR signals of the dimers were found close to those of the monomer trans-Pt(amine)(pm)Cl₂.     

Synthesis of 2-methylthio-cis- and trans-ribosylzeatin and their isolation from Pisum tRNA

The cis isomer of 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthiopurine and its 9-β- and 9-α-d-ribofuranosyl derivatives have been synthesized and their physical and spectroscopic properties are described. The biological activities of these compounds have been determined in the tobacco bioassay and are compared with those of 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-2-methylthiopurine and its β-ribofuranoside. The 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-d-ribofuranosylpurine (ms-ribosylzeatin) isolated from a Pisum tRNA preparation was shown to consist of both isomers, which were separated by TLC and identified by comparisons of UV and MS with those of the synthetic compounds.     

Electron spin resonance and electron nuclear double resonance studies of cation radicals derived from tocopherol model compounds

Electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) measurements were performed for the cation radicals obtained from the model compounds of α-, β-, γ- and δ-tocopherol (vitamin E) by oxidizing the tocopherol precursors in an AlCl₃-CH₂Cl₂ solution. The proton hyperfine coupling constants g-values were precisely determined. The ENDOR spectra of the cation radicals of α-, β-, γ- and δ-tocopherol models in CH₂Cl₂ at ?100°C clearly show 10, 6, 6 and 12 different proton hyperfine couplings, respectively. By varying the temperature, the ESR spectra of the α- and δ-tocopherol model cations exhibit line-width alternation phenomena characteristic of the hindered rotation of the OH group. However, neither the β- nor the γ-tocopherol model cation radical ESR spectra show any sign of an alternative line-width effect. These results are interpreted by assuming that the β- and γ-tocopherol model cations are stabilized in the trans and cis conformations, respectively. On tocopherol model cations are stabilized in the trans and cis conformations, respectively. On the other hand, both the α- and δ-tocopherol model cations exist as cis and trans isomers.     

Redox potentials of the oriented film of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins

The redox potentials of the oriented films of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins (bR), prepared by adsorbing purple membrane (PM) sheets or its mutant on a Pt electrode, have been examined. The redox potentials (V) of the wild-type bR were −470 mV for the 13-cis configuration of the retinal Shiff base in bR and −757 mV for the all-trans configuration in H₂O, and −433 mV for the 13-cis configuration and −742 mV for the all-trans configuration in D₂O. The solvent isotope effect (ΔV=V(D₂O)−V(H₂O)), which shifts the redox potential to a higher value, originates from the cooperative rearrangements of the extensively hydrogen-bonded water molecules around the protonated CN part in the retinal Schiff base. The redox potential of bR was much higher for the 13-cis configuration than that for the all-trans configuration. The redox potentials for the E194Q mutant in the extracellular region were −507 mV for the 13-cis configuration and −788 mV for the all-trans configuration; and for the E204Q mutant they were −491 mV for the 13-cis configuration and −769 mV for the all-trans configuration. Replacement of the Glu¹⁹⁴ or Glu²⁰⁴ residues by Gln weakened the electron withdrawing interaction to the protonated CN bond in the retinal Schiff base. The E204 residue is less linked with the hydrogen-bonded network of the proton release pathway compared with E194. The redox potentials of the D96N mutant in the cytoplasmic region were −471 mV for the 13-cis configuration and −760 mV for the all-trans configuration which were virtually the same as those of the wild-type bR, indicating that the D to N point mutation of the 96 residue had no influence on the interaction between the D96 residue and the CN part in the Schiff base under the light-adapted condition. The results suggest that the redox potential of bR is closely correlated to the hydrogen-bonded network spanning from the retinal Schiff base to the extracellular surface of bR in the proton transfer pathway.     

Alloprenols: novel alpha-trans-polyprenols of Allophylus caudatus

A novel type of polyprenols, alloprenols, with an α-trans-isoprenoid unit was found in the leaves of Allophylus caudatus (Sapindaceae) besides typical α-cis-polyprenols. The polyprenol family (Prenol-11-13, Prenol-12 dominating) was accompanied by traces of dolichols of the same chain-length. Prenol α-cis- and α-trans-isomers were chromatographically separated and their structure was analyzed by HPLC/ESI-MS, HR-ESI-MS and ¹H and ¹³C NMR spectroscopy. Model compounds, semi-synthetic α-isomers of all-trans-Pren-9 and mainly-cis-Pren-11, were obtained using an oxidation-reduction procedure. Comparison of their NMR spectra confirmed the structure of the newly identified polyprenols. The observed pattern of NMR signal shifts may be applied for elucidation of isoprenoid structure.     

Synthesis and properties of methyl 9,12,15-octadecatrienoate geometric isomers

The eight geometrically isomeric methyl 9,12,15-octadecatrienoates were prepared by using the Wittig reaction to couple cis- or trans-3-hexyenyltriphenylphosphonium bromide and methyl 12-oxo-cis- or trans-9-dodecenoate. Pairs of geometric triene isomers formed were separated by partial silver resin chromatography. Physical constants including melting points, percent trans by infrared, equivalent chain lengths (ECL), and ¹³C nuclear magnetic resonance (NMR) chemcial shifts are tabulated for the individual isomers.     

Cytokinins in immature seeds of Dolichos lablab

Five cytokinins, trans-zeatin, 9-β-d-ribofuranosyl-trans-zeatin, 9-β-d-ribofuranosyl-cis-zeatin, 6-(trans-4-O-β-d-glucopyranosyl-3-methyl-2-butenylamino)purine and 6-(trans-4-O-β-d-glucopyranosyl-3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine were identified from immature seeds of Dolichos lablab.     

Identification of enriched conjugated linoleic acid isomers in cultures of ruminal microorganisms after dosing with 1-<Superscript>13</Superscript>C-linoleic acid

Most studies of linoleic acid biohydrogenation propose that it converts to stearic acid through the production of cis-9 trans-11 CLA and trans-11 C18:1. However, several other CLA have been identified in ruminai contents, suggesting additional pathways may exist. To explore this possibility, this research investigated the linoleic acid biohydrogenation pathway to identify CLA isomers in cultures of ruminai microorganisms after dosing with a ¹³C stable isotope. The ¹³C enrichment was calculated as [(M+1/M)×100] in labeled minus unlabeled cultures. After 48 h incubation, significant ¹³C enrichment was observed in seven CLA isomers, indicating their formation from linoleic acid. All enriched CLA isomers had double bonds in either the 9,11 or 10,12 position except for trans-9 cis-11 CLA. The cis-9 trans-11 CLA exhibited the highest enrichment (30.65%), followed by enrichments from 21.06 to 23.08% for trans-10 cis-12, cis-10 trans-12, trans-9 trans-11, and trans-10 trans-12 CLA. The remaining two CLA (cis-9 cis-11 and cis-10 cis-12 CLA) exhibited enrichments of 18.38 and 19.29%, respectively. The results of this study verified the formation of cis-9 trans-11 and trans-10 cis-12 CLA isomers from linoleic acid biohydrogenation. An additional five CLA isomers also contained carbons originating from linoleic acid, indicating that pathways of linoleic acid biohydrogenation are more complex than previously described.     

Synthesis and study of Pt(II)-aromatic amines complexes of the types cis- and trans-Pt(amine)2(NO3)2 and cis-Pt(amine)2(R(COO)2)

Complexes of the types cis- and trans-Pt(amine)₂(NO₃)₂ with amines containing a phenyl group were synthesized and studied mainly by IR and multinuclear magnetic resonance spectroscopies. The cis complexes could be synthesized pure only with the amines of the type Ph-R-NH₂ (R = alkyl), while pure trans compounds were synthesized with all the studied amines. In ¹⁹⁵Pt NMR spectroscopy, the dinitrato complexes of the amines Ph-R-NH₂ were observed around −1700 ppm for the cis isomers and at about −1580 for the trans complexes. For the other amines, where a phenyl ring is directly attached to the amino group, the signals were observed at lower fields, −1528 ppm for cis-Pt(PhNH₂)(NO₃)₂ and around −1450 ppm for all the trans isomers. There is a linear relationship between the δ(Pt) of the Pt(amine)₂(NO₃)₂ complexes and the pK_a of the protonated amines. The coupling constants ²J(¹⁹⁵Pt-¹HN) are larger in the cis compounds (ave. 76 Hz) than in the trans isomers (ave. 63 Hz). The complexes cis-Pt(amine)₂(R(COO)₂) with bidentate dicarboxylato ligands were also synthesized and characterized mainly by IR spectroscopy. The compounds apparently decompose in DMF and are too insoluble in other solvents for solution studies.     

Cytokinins: synthesis and biological activity of geometric and position isomers of zeatin

Geometric and position isomers of zeatin and of ribosylzeatin and other compounds closely related to zeatin have been tested in the tobacco (Nicotiana tabacum var. Wisconsin No. 38) bioassay. None was more active than zeatin itself. There was a much greater difference in activity (> 50-fold) between trans- and cis-zeatin than between trans-isozeatin [6-(4-hydroxy-2-methyl-trans-2-butenylamino) purine] and cis-isozeatin [6-(4-hydroxy-2-methyl-cis-2-butenylamino) purine], the latter being less active than cis-zeatin and trans-isozeatin. Higher concentrations were required for equivalent callus growth stimulated by the 9-ribosyl derivatives, which followed an order of decreasing activity: ribosyl-trans-zeatin > ribosyl-cis-zeatin > ribosyl-trans-isozeatin > ribosyl-cis-isozeatin, corresponding roughly to that of the bases. The effect of side chain, double bond saturation was to diminish the activity, and in the dihydro series the shift of the methyl group from the 3- to the 2-position in going from dihydrozeatin to dihydroisozeatin [6-(4-hydroxy-2-methylbutylamino) purine] resulted in a 70-fold decrease in activity. cis-Norzeatin [6-(4-hydroxy-cis-2-butenylamino) purine], which was less than one-fifth as active as cis-zeatin, showed the effect of complete removal of the side chain methyl group, and cyclic-norzeatin [6-(3,6-dihydro-1,2-oxazin-2-yl) purine] was about 1/100 as active as cis-norzeatin. These findings delineate completely the effect on the cytokinin activity of zeatin of variation in side chain geometry, presence and position of the methyl substituent, presence and geometry of hydroxyl substitution, presence of the double bond, and of side chain cyclization.     

Enzymatic separation of cis and trans isomers of alicyclic alcohols

It has been discovered that phosphatases [alkaline phosphatase, orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1, and acid phosphatase, orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] display a remarkable geometric specificity in the hydrolysis of cis and trans isomers of monoorthophosphate esters of substituted alicy clicalcohols. While steric hindrances prevent potato acid phosphatase from hydrolysing cis-2-methylcyclohexyl and cis-2-methylcyclopentyl phosphates, the corresponding trans isomers are readily hydrolysed by the enzyme (non-enzymatic, acid-catalysed or base-catalysed hydrolyses of the cis and trans isomers occur at similar rates). Cis isomers of methylcyclohexyl phosphates, in which the methyl group is remote from the hydrolysed ester bond, 3- or 4-, have nearly the same reactivities to phosphatases as their trans counterparts. However, if the methyl group in position 4 is replaced by a bulky substituent, e.g. tert-butyl, phosphatases again hydrolyse only the trans and not the cis isomer. These phenomena afford a simple method for preparative separation of cis and trans isomers of alicyclic alcohols: a mixture of the isomers is first phosphorylated with POCl₃ and then hydrolysed by phosphatase. The trans alcohol formed is extracted with CCl₄, followed by alkaline hydrolysis of the remaining cis-tester and subsequent extraction of the cis alcohol produced.     

Geometric specificity of porcine liver carboxylesterase and its application for the production of cis-arylacrylic esters

Porcine liver carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) hydrolyses trans isomers of three different methyl 3-arylacrylates approximately one order of magnitude faster than the corresponding cis isomers. This phenomenon can be used for preparative production of cis esters from their trans counterparts as exemplified by methyl cinnamate. A solution of commercial, predominantly trans methyl cinnamate was irradiated by ultraviolet light and the resultant mixture of trans and cis esters was passed through a column packed with immobilized esterase. The effluent contained mainly trans cinnamic acid and cis methyl cinnamate. The latter was then extracted with methylene chloride, and the cis ester was isolated by evaporating the solvent. By esterifying the co-produced trans acid, the process can be made continuous.     

A rapid method for the preparation of (125I)alpha-bungarotoxin

Electrofocusing followed by chromatography on Sephadex G-50 proved to be a fast method for the purification of the neurotoxin, α-bungarotoxin (α-BGT) from the venom of the krait (Bungarus multicinctus). The purity of the isolated α-BGT was checked by electrofocusing and ion-exchange chromatography. Blockade of the binding of [³H]ACh to the acetylcholine receptor in a membrane preparation of Torpedo electroplax was a convenient biochemical assay to identify the α-neurotoxin polypeptide. Iodination, accomplished by lactoperoxidase attached to Sepharose 4B, was simple and rapid, with a 60% recovery of [¹²⁵I]α-BGT. Biological activity of the α-BGT was determined by its toxicity to mice as well as by autoradiography of [¹²⁵I]α-BGT which proved its localization in the endplates of the mouse diaphragms.     

Agents related to a potent activator of the acetylcholine receptor of Electrophorus electricus

The synthesis of a number of compounds related to trans-3,3′-bis[α-(trimethylammonium)methyl]azobenzene dibromide (trans-3,3′-BisQ) (1)1 is described. Among the compounds are: [¹⁴C]-trans-3,3′-BisQ (1)1 diiodide, cis-3,3′-BisQ (2)1 dibromide, the trans-2,2′ (7)1 and 4,4′ (11)1 isomers of BisQ, 2,2′, (12)1, 3,3′ (13)1 and 4,4′ (14)1 isomers of bis-benzyldimethylammonium analogues, and related compounds in which the azo bridge between the two aromatic rings is replaced by diketo and amide bridges. Of them all, trans-3,3′-BisQ (1)1 was the most active cholinergic compound in the electroplax system of Electrophorus electricus; the pure cis isomer (2)1 was without activity. Intermediate activities were found for some of the other compounds and others were inhibitors. The relationship of the structure of these agents to a proposed conformation and topography of the binding site of the acetylcholine receptor (AChR) is discussed.