Synthesis of tritium-labeled phytohormones

A method has been developed for obtaining tritium-labeled analogues of -indolylacetic acid and of 6-benzylaminopurine by the method of isotopic catalytic exchange. The localization of cytokinin- and auxin-binding proteins in cotton seedlings has been investigated.A. S. Sadykov Institute of Bioorganic Chemistry, Uzbekestan Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 699–702, November–December, 1992.     

Synthesis of tritium-labeled PAM-43

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Synthesis of tritium-labeled compounds using a fibroid carbon carrier

The results of tritium labeling of organic compounds by solid phase catalysis, using fibrous carbon, were compared with those, obtained by using activated carbon purchased from Norit. It was found that utilization of multi-wall carbon nanotubes lead to a considerable growth of the yield (10–20 times) and molar radioactivity (1.5–2 times) of the labeled compounds. Using such a carrier it became possible to obtain a tritium-labeled biologically active compound like diazepam, Etomidate, and phenylalanine with molar reactivity 10–25 Ci/mmol.     

Synthesis of tritium-labeled components of nucleic acids of the hypoxanthine series

The synthesis of [8-³H] and [2,8-³H]hypoxanthine, inosine, inosine 5-mono-, -di-and -triphosphates, and also of 2-deoxy[2,2-8-³H]inosine from the corresponding labeled compounds of the adenine series with the aid of the de-amination reaction is described. De-amination was carried out with sodium nitrite in the presence of acetic acid. In the case of bases and nucleosides, the separation of the reaction mixtures with simultaneous desalting of the final products was achieved by column chromatography on Sephadex G-10 or SE C-25 with elution by water. For nucleotides, the isolation process included chromatography on DEAE-Sephadex A-25 (HCOO^– or Cl^–) and Dowex 1×8 (Cl^–) followed by desalting with the aid of reprecipitation or adsorption on Carboraffin activated carbon. The molar radioactivities of the compounds synthesized amounted to 370–2220 TBq/mole (10–60 kCi/mole) and corresponded to the molar radioactivities of the initial compounds of the adenine series.V. G. Khlopin Radium Institute, Leningrad. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 128–131, January–February, 1987.     

Synthesis of tritium-labeled nucleoside 5′-triphosphates and nucleoside 5′-diphosphates

Tritium-labeled nucleoside 5′-triphosphates (NTPs) nucleoside 5′-diphosphates (NDPs) containing the tritium label in positions 8 (in the purine nucleus) and 5 (in the pyrimidine nucleus) have been obtained by the dehalogenation of the corresponding bromine derivatives with gaseous tritium. The dehalogenation of the Br-NTPs and Br-NDPs was carried out at atmospheric pressure in an aqueous alkaline medium using palladium catalysts (5% Pd/BaSO₄ or α-Pd). The possibility of introducing a tritium label into nucleotides of the adenine series by the heterogenecus isotope exchange reaction with gaseous tritium in the presence of 5% Pd/BaSO₄ has been investigated. For the compounds synthesized, the compositions of the eluents used for the chromatographic isolation of the desired products are given. The molar activities of the compounds synthesized were between 370 and 740 TB_q/mole (10–20 kCi/mole).     

Preparation of tritium-labeled prostaglandin E3

Conditions for the biosynthesis of PGE₃ from eicosa-5,8,11,14,17-pentaenoic acid using an enzyme system isolated from ram seminal vesicles have been worked out. Tritium-labeled PGE₃ has been obtained in good yield with a molar radioactivity sufficient for performing many biological investigations.Institute of Molecular Genetics, Academy of Sciences of the USSR, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 319–322, May–June, 1985.     

Investigations into synthetic phytohormones

Conclusions Trialkylsilyl esters of ring-substituted aryloxyacetic acids have been prepared for the first time, and their PMR and IR spectra studied.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 5, pp. 1132–1134, May, 1977.     

Synthesis of a tritium-labeled, fipronil-based, highly potent, photoaffinity probe for the GABA receptor

3-[4-[1-(2,6-dichloro-4-trifluoromethylphenyl)pyrazolo]]-3-(trifluoromethyl)diazirine is a fipronil-based (i.e. fiprole), high-affinity probe for the GABA receptor. For synthesis of the tritium-labeled version of this trifluoromethyldiazirinylfiprole ([(3)H]TDF) the required intermediate, 3-[4-[1-(2,6-dichloro-3-iodo-4-trifluoromethylphenyl)-5-iodopyrazolo]]-3-(trifluoromethyl)diazirine, was prepared in 10 steps from pyrazole and 3,5-dichloro-4-fluorobenzotrifluoride. One of the key transformations was lithiation and subsequent iodination of the 4-(2,2,2-trifluoro-1-hydroxyethyl)pyrazole intermediate. The last step involved reduction of the diiodofiprole with tritium, Pd/C, and triethylamine in ethyl acetate and afforded [(3)H]TDF with a specific activity of 15 Ci/mmol and 99% radiopurity.     

Preparation of tritium-labeled biogenic amines and their analogs

The possibility has been shown of introducing a tritium label into biogenic amines and their analogs in nonaqueous systems. Labeled compounds possessing a specific activity sufficient for performing many biological investigations have been obtained in good yields. Institute of Molecular Genetics, Academy of Sciences of the USSR, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 657–663, September–October, 1980.     

植物样品中内源性植物激素时空分布的研究进展

内源性植物激素是在植物体中合成，并在整个植物生命周期内调控其生长发育、应对外界刺激等过程中发挥重要作用的一类微量或痕量有机小分子化合物。随着植物激素分析方法的发展，分析样品用量逐渐减少，不同植物组织（或器官）中植物激素的种类和含量差异不断呈现，极大促进了植物激素生理作用的研究。近年来，植物样品中内源性植物激素的时空分布研究已成为植物激素分析的一大热点。该文总结了近五年来，内源性植物激素时空分布的研究进展，主要从分析难点、分析方法、主要植物激素在植物体内的时空分布情况等方面进行讨论和总结，相关内容围绕本研究组在植物激素检测方面的工作展开。最后，展望了植物激素时空分析这一研究领域的未来发展方向。     

毛细管液相色谱法分离植物内源激素

建立了毛细管液相色谱法(CLC)同时分离测定4种植物内源激素的方法.采用硅胶基质ODS整体柱(27 cm×100 μm i.d.)作为分离柱,以带有光程为3 mm的光纤检测池的紫外检测器作为检测手段,在室温下以含10 mmol/L乙酸(pH 3.0)的V(甲醇):V(水)＝35:65为流动相,以0.6 μL/min的流速进行等度洗脱.采用溶剂梯度效应和扩展光程的检测池来提高检测灵敏度,该方法测定4种植物内源激素的检出限(S/N=3)在27.7～196.1 ng/mL之间,峰面积和保留时间的相对标准偏差(RSD)小于2.8%.方法已用于不同种类玉米样品的测定.     

Biological radiation effects of carbon-14- and tritium-labeled 5-fluorouracil

This paper describes the biological radiation effects of carbon-14 and tritium labeled 5-fluorouracils (5-FU) which were given intraperitoneally at reasonable dose (14C: 1.85 MBq/kg (0.05 microCi/kg), 3H: 3.7 MBq/kg (0.1 microCi/kg)) and its forty times dose once a day for 3 days in mice. The results indicated that the change of body weight, urine and feces volume and its radioactivity, and organ weight and its residual radioactivity except pancreas were not observed any significant difference between three groups of both 14C and 3H, but white blood cell number markedly decreased proportionally with radioisotope dose of both 14C and 3H and also red blood cell number decreased by [6-3H]-5-FU 148 MBq/kg i.p. The results suggest that high level radioisotope dose of both 14C and 3H 5-FUs have no biological radiation effects on the studies of drug metabolism in vivo, however that in the case of hematogenic organ, the biological radiation effect is not disregarded.     

On-line concentration and pressurized capillary electrochromatographic analysis of phytohormones in corn

A pressurized CEC (pCEC) method was developed for the separation of phytohormones, in which UV absorbance was used as the detector and a monolithic silica-ODS column as the separation column. The parameters (including the concentration of organic solvent in the mobile phase, pH of the electrolyte buffer, applied voltage) affecting the separation resolution were evaluated. Two on-line concentration techniques, namely, solvent gradient zone sharpening effect and field-enhanced sample stacking, were utilized to improve detection sensitivity. The combination of the two techniques proved to be beneficial to enhance the detection sensitivity by enabling the injection of large volumes of samples. Compared to the conventional injection mode, the enhancement in the detection sensitivities of phytohormones using the on-line concentration technique is in the range from 9- to 23-fold. The developed pCEC method was applied to evaluate phytohormones in corns.     

Interaction of abscisic-acid-binding cotton (Gossypium hirsutum) protein and phytohormones

The interaction of representatives of three classes of phytohormones, abscisic acid (ABA), the synthetic auxin 1-NAA, and the synthetic cytokinin 6-BAP, with ABA-binding cotton protein was studied. Binding of ABA with its protein was shown to be specific and receptor-like. Competitive protein binding showed that 1-NAA (15±3%), 6-BAP (82±3%), and ABA (95±3%) replace³H-ABA from the protein complex. The possibility of reacting ABA-binding protein with various classes of intracellular phytohormones is discussed.Institute of Genetics and Experimental Biology of Plants, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (99871) 64 22 30. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 252–253, May–June, 2000.     

Enzymatic synthesis of multiple tritium-labeled nucleosides and nucleotides from nitrogen bases and ribose

A wide range of highly active nucleotides containing tritium labels in the heterocycle nucleus and ribose residue has been synthesized with the aid of an enzyme preparation fromE. coli, starting from tritium labeled nitrogen bases and ribose. In individual cases, the complex enzyme preparation was modified by the addition of nucleotide kinases or phosphatases.V. G. Khlopin Radium Institute and Biological Scientific Research Institute, A. A. Zhdanov Leningrad State University. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 258–264, March–April, 1988.     

Comprehensive chemical derivatization for gas chromatography-mass spectrometry-based multi-targeted profiling of the major phytohormones

In the present investigation we report selection of the N-methyl-N-(tert.-butydimethylsilyl)trifluoroacetamide (MTBSTFA) reagent as the most comprehensive derivatization protocol among 17 tested reactions covering trifluoroacetylation, pentafluorobenzylation, methylations, and trimethylsilylations. MTBSTFA allowed easy and robust tert.-butyldimethylsilyl derivatization of 1-aminocyclopropane-1-carboxylic acid, indole-3-acetic acid, (+/-)-jasmonic acid, salicylic acid, (+/-)-abscisic acid, meta-topolin, and trans-zeatin. Detection limits as analysed by selected ion monitoring quadrupole GC-MS were 0.2, 0.01, 1.0, 0.02, 0.3, 0.3, and 0.9 pmol of injected substance, respectively. Analysis of gibberellic acid A3, trans-zeatin riboside and (+/-)-abscisic acid-beta-D-glucopyranosyl ester was best when coupled by splitting extracts and trimethysilylation. The MTBSTFA derivatization protocol was optimised, and validated. The preparation was insensitive to 2% residual water and to < or = 1 day storage at room temperature. The final scheme was highly reproducible and successfully applied to extracts from approximately 300 mg (fresh mass) of tobacco (Nicotiana tabacum) root and Arabidopsis thaliana seedling.     

Molecular mechanisms of the regulatory action of phytohormones and defoliants of the cytokinin type

A comparative study has been made of the interaction of a number of phytohormones and defoliants with a cytokinin-binding protein from cotton shoots by the method of a competitive receptor test. Methodological approaches to the molecular modeling of processes involved in the interaction of a fragment of the structure of a cytokinin-binding protein with the compounds under investigation have been developed.A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (371) 162 70 71. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 507–509, July–August, 1999.     

QuEChERS-based extraction procedure for multifamily analysis of phytohormones in vegetables by UHPLC-MS/MS

A new method has been developed and validated for the simultaneous analysis of different phytohormones (auxins, cytokinins and gibberellins) in vegetables. The compounds were extracted using a QuEChERS-based method (acronym of quick, easy, cheap, effective, rugged and safe). The separation and determination of the selected phytohormones were carried out by ultra high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), using electrospray ionization source (ESI) in positive and negative ion modes. The method was validated and mean recoveries were evaluated at three concentration levels (50, 100 and 250 μg/kg), ranging from 75 to 110% at the three levels assayed. Intra- and interday precisions, expressed as relative standard deviations (RSDs), were lower than 20 and 25%, respectively. Limits of quantification (LOQs) were equal or lower than 10 μg/kg. The developed procedure was applied to seven courgette samples, and naphthylacetic acid, naphthylacetamide and benzyladenine were found in some of the analysed samples.