EA015684B1 - 24-epicastasterone dansylhydrazide as labeled antigen for immunochemical analysis of (24r)-methylbrassinosteroids - Google Patents

Abstract

The invention relates to immunochemical analysis of brassinosteroidal phytohormones, namely to novel chemical composition – 24-epicastasterone dansylhydrazide. The aim of the invention is 24-epicastasterone conjugate with dansylhydrazide which can be used as labeled antigen in immunofluorescence determination of (24R) methylbrassinosteroids. The assigned task is achieved by claimed 24-epicastasterone conjugate with dansylhydrazine obtained by interacting 24-epicastasterone in mixture of dimethyl formamide and acetic acid and subsequent isolation of targeted conjugate by a method of column chromatography.

Description

The invention relates to the field of immunochemical analysis of phytohormones of brassinosteroids, and in particular to a new chemical compound - dansylhydrazide 24-epicastasterone of the formula I he

which can be used as a labeled antigen in an analytical test system for immunofluorescence determination of (24K) methyl brassinosteroids.

Brassinosteroids are natural low molecular weight bioregulators that are plant hormones [1]. They are present in all plant objects and have growth modulating and adaptogenic effects. The content of brassinosteroids in plants is very low - 10 ⁵ -10 ^-9 % or less. In very small concentrations, they also exhibit their biological effect. It has been established, for example, that when various crops are treated with brassinosteroids in doses of 5-20 mg per hectare of crops, a noticeable growth-promoting and adaptogenic effect is observed, leading to an increase in yield, an increase in the quality of products and plant resistance to adverse conditions and diseases [2-7]. These properties of brassinosteroids make them attractive as a basis for agricultural products. One of the most famous is the epin drug, the active ingredient of which is the phytohormone 24-epibrassinolide. The availability of the latter (as well as other brassinosteroids) can provide only chemical synthesis.

All of the above puts on the agenda the development of methods for the analysis of brassinosteroids, which should have high sensitivity, selectivity, ease of execution and be accessible. The recent development of immunofluorescence assays requires the presence of both an immunogen and an immunofluorescence label, which may be a substrate labeled with dansyl hydrazine.

The conjugates of brassinosteroids with dansilhydrazine (or another fluorescent label) that were obtained and could be used in immunoassay systems to determine 24-epicastasterone and epibrassinolide are currently unknown.

The aim of the invention is a previously unknown conjugate of 24-epicastasterone and dansilhydrazine dansilhydrazide 24-epicastasterone of formula I, which can be used as a labeled antigen in immunofluorescence analysis of (24B) -methyl brassinosteroids.

This goal is achieved by the claimed conjugate I, which is obtained by the interaction of 24epicastasterone with dansylhydrazine in a mixture of dimethylformamide and acetic acid.

The invention is confirmed by examples of specific performance.

Example 1. Epicastasterone (0.051 g, 0.11 mol) and dansilhydrazine (0.0318 g, 0.12 mol) are dissolved in a mixture of dimethylformamide and acetic acid (1 ml, 1/1), then the reaction mixture is stirred at room temperature to disappearance of the starting compound (TLC control). The solvent was removed in vacuo, the residue was chromatographed on a silica gel column. The yield of conjugate I is 77%. T. pl. 160-163 ° C (EJAc-hexane). Spectrum '11 ΝΜΒ: Ή-NMR (δ, ppm): 0.50 (8, 1Н, Н18), 0.84-0.97 (t, 15Н, Н19, Н21, Н-26, Н27, Н-28), 2.41 (άά, 1H, 1 = 3.6, 13.7 Hz, H-5), 2.88 (8, 6H, -ΝΜ ^), 3.41 (t, 1H), 3.54 (t, 1H), 3.69 (t, 1H), 3.90 (t, 1H), 7.15 (ά, 1H, 1 = 7.5 Hz), 7.55 (t, 3H), 8.30-8.36 (t, 2H), 8.54 (ά, 1H, 1 = 8.4Hz). ¹³ C-NMR (δ, ppm): 11.3, 12.7, 12.8, 13.2, 17.6, 22.2, 22.7, 23.7, 25.0, 28.1, 28.9, 29.6, 33.1, 33.2, 37.5, 40.8, 40.9, 41.7, 42.8 , 44.0, 45.9, 46.1, 54.2, 55.4, 57.7, 69.3, 69.8, 73.5, 77.4, 116.5, 120.7, 124.4, 129.4, 131.1, 131.2, 131.5, 132.0, 135.7, 153.0, 162.8.

The synthesized dansyl hydrazide of 24-epicastasterone can be used as a labeled antigen for the quantitative determination of (24B) methyl brassinosteroids (24-epibrassinolide and 24 picicastasterone) by immunofluorescence analysis, which is confirmed by an example of a specific implementation.

Example 2. As components of the analytical system use: antiserum to 24epicastasterone obtained according to the method described previously [8]; claimed dansylhydrazide 24epicastasterone; calibration samples - buffer solutions containing 24-epibrassinolide 0 μg / ml; 1 μt / ml, 10 μg / ml and 100 μg / ml; buffer solution for the dilution of dansyl hydrazide 24epicastasterone; wash buffer. Antibodies to 24-epibrassinolide are immobilized on the inner surface of 96-well microplates, commonly used in enzyme-linked immunosorbent assay. For analysis, 0.05 ml of calibration samples are added to the wells of the plate. Then, 0.1 ml of dansyl hydrazide 24-epicastasterone solution is added to all wells. Incubate the mixture for 2 hours at 37 ° C. Then, the contents of the wells are transferred to opaque plates (Otshet) to measure fluorescence. Fluorescence measurements are carried out on an instrument! PPpCs Μ200 (Tesap, Austria) at 20 ° C.

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Luminescence excitation is carried out at a wavelength of 340 nm. Fluorescence spectra are recorded in the range of 370-620 nm.

The fluorescence intensity in the calibration samples is for 10 μg / ml - 10 relative units, 1 μg / ml - 50 relative units, 10 μg / ml - 400 relative units, 100 μg / ml - 1200 relative units. for an analytical sample of 20 μg / ml - 570 rel. Thus, based on a comparison of the fluorescence intensity of the analyzed sample and calibration samples, it is possible to establish the concentration of brassinosteroid in the analyzed sample.

The above results confirm the possibility of using the inventive dansylhydrazide 24-epicastasterone for the quantitative determination of (24K) -methylbrassinosteroids with a sensitivity of 0.5 μg / ml.

The inventive dansylhydrazide 24-epicastasterone is obtained in high yield, provides the necessary immunogenicity tags and allows the quantification of (24K) methylbrassinosteroids by immunofluorescence analysis. It is a stable compound and does not show signs of decomposition for at least several months at room temperature. For comparison, the conjugate of 24-epibrassinolide with horseradish peroxidase, used for similar purposes in an enzyme-linked immunosorbent assay, is characterized by insufficient stability (storage at -18 ° C) and multi-stage synthesis [9].

Literature

1. Khripach V.A., Lakhvich F.A., Zhabinsky V.N. Brassinosteroids. Minsk: Navuka Tekhshka, 1993.288 s.

2. Patent of the Republic of Belarus No. 2806 of 11.23.1995. Means for reducing the accumulation of radionuclides by plants and the method of its use. / Khripach V.A., Zhabinsky V.N., Litvinovskaya R.P., Zavadskaya M.I., Deeva V.P., Vedeneev A.P. // Of. Bull. Belgos-pat., 2000, N2 (26), p. 159.

3. Patent of the Republic of Belarus No. 3488 dated July 4, 1996. A way to increase the nutritional value of potatoes. / Khripach V.A., Zhabinsky V.N., Litvinovskaya R.P., Zavadskaya M.I., Savelyeva E.A., Karas I.I. (Belarus), Vakulenko V.V. (Russia). // Of. Bull. Belgos-pat., 2000, N3 (27), p. 74.

4. Patent of the Republic of Belarus No. 3400 dated July 4, 1996. A method of protecting potatoes from late blight. / Khripach V.A., Zhabinsky V.N., Litvinovskaya R.P., Zavadskaya M.I., Savelyeva E.A., Karas I.I., Kilchevsky A.V., Titova S.N. // Of. Bull. Bel-gos-pat., 2000, N2 (25), p.75.

5. Patent RB 5168. / Khripach V.A., Litvinovskaya R.P., Zhabinsky V.N., Zavadskaya M.I., Volynets A.P., Prokhorchik R.A., Pshenichnaya L.A., Manzhelesova N.E., Morozik G.V. // Of. Bull. NCIS RB, 2003 2 (37), p. 95.

6. Patent RB 5212. A way to increase the yield of flax and improve the quality of flax fiber. / Voskresenskaya L.G., Khripach V.A., Zhabinsky V.N., Zavadskaya M.I., Litvinovskaya R.P. // Of. Bull. NCIS RB, 2003 2 (37), p. 93.

7. Patent RB N5698. The method of propagation of healthy seed material of potatoes. / Bobrik A.O., Khripach V.A., Zhabinsky V.N., Zavadskaya M.I., Litvinovskaya R.P. // Of.Byul.NTsIS RB, 2003 4 (39).

8. Khripach V.A., Sviridov O.V., Pryadko A.G., Litvinovskaya R.P., Drach S.V., Matveentsev V.D., Novik T.V., Mikhaylopulo K.I., Zhabinsky V.N., Zavadskaya M.I., Averkova M.A., Drachenova O.A., Chashchina N.M. Bioorgan. Chemistry, 33 (2007) 371-378.

Claims (1)

CLAIM 24-epicastasterone dansyl hydrazide of formula I as a labeled antigen for immunofluorescence determination of (24H) methyl brassinosteroids. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2