EA015276B1 - (22R,23R)-2α,3α,22,23-TETRAHYDROXY-26-(HEMISUCCINATE)-B-HOMO-7-OXA-5α-HOLESTAN-6-ONE AS HAPTEN IN SYNTHESIS OF CONJUGATE 28-NORBRASSINOLID WITH BOVINE SERUM ALBUMIN - Google Patents

Abstract

The invention relates to bioorganic chemistry, namely to a novel chemical compound (22R,23R)-2α,3α,22,23-tetrahydroxy-26-(hemisuccinate)-B-homo-7-oxa-5α-holestan-6-one which can be used as hapten in the synthesis of immunogen –conjugate of 28-norbrassinolid with bovine serum albumin for preparing antibodies, specific to 7-oxa-6-oxobrassinosteroids. The task of present invention is (22R,23R)-2α,3α,22,23-tetrahydroxy-26-(hemisuccinate)-B-homo-7-oxa-5α-holestan-6-one as hapten in the synthesis of immunogen (conjugate of 28-norbrassinolid with bovine serum albumin). The proposed invention is solved by a claimed compound as hapten which is produced by a scheme comprising interaction pentaacetate is (22R,23R)-2α,3α,22,23,26-pentahydroxy-5α-holestan-6-one with triflouroperacedic acid with subsequent saponification of protective classifications of prepared lactone by potassium hydroxide and relactonization under hydrochloric acid forming pentahydroxylactone, protection of vicinal secondary hydroxyl group as acetonides, conjunction of succinic acid remainder by primary hydroxyl group of formed diacetanide and hydrolysis of acetonide grouppings.

Description

The invention relates to the field of bioorganic chemistry, namely, a new chemical compound - (22K, 23K) -2a, 3a, 22,23-tetrahydroxy-26- (hemisuccinate) -B-homo-7-oxa-5a-cholestan-6-one with structural formula 1

The claimed compound can be used as a hapten in the synthesis of an immunogen - a conjugate of 28-norbrassinolide with bovine serum albumin to obtain antibodies specific for 7-oxa-6-oxobrassinosteroids.

Brassinosteroids (BS) are natural low molecular weight bioregulators that are plant hormones [1, 2]. They are present in all plant objects and have growth modulating and adaptogenic effects. The content of brassinosteroid 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 in doses of 5-20 mg per hectare of crops, a noticeable growth-stimulating 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 [38]. 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 most active are the brassinosteroids, which have 7-oxa-6-oxo grouping in their structure (the so-called brassinosteroid lactones) [1]. The low BS content in natural objects and the vanishingly low doses that cause their effect make it necessary to develop methods for the analysis of brassinosteroids, in particular brassinosteroid lactones. As an available method, an enzyme-linked immunosorbent assay for the determination of 7-oxa-6-oxobrassinosteroids is proposed, which, first of all, requires the presence of an appropriate immunogen. Brassinosteroids themselves do not have immunogenicity, therefore, to obtain an immune response in animals, they should be used for immunization in the form of conjugates with proteins, which, in turn, can be obtained through the corresponding haptens.

At present, haptens based on brassinosteroids containing a complete set of unsubstituted characteristic functions in the cyclic part and in the side chain are unknown.

Closest to the claimed hapten based on the brassinolide formula

which is an ester of succinic acid at the C3-hydroxyl group of brassinolide [9], which is one of the key structural elements necessary for the manifestation of biological activity. The conjugate synthesized based on it did not allow to obtain antibodies specific even to the initial 248-methylbrassinosteroids.

Also known are haptens of castasterone of formula 3 and 24-epicastasterone of formula 4

which were proposed by the authors to obtain conjugates with a protein for the production of antibodies used in the radioimmunoassay of brassinosteroids [10] and the immunochemical analysis of 24K brassinosteroids [11, 12], respectively. However, conjugate 3 synthesized in this way did not allow the production of antibodies specific even to the 248-methyl-brassinosteroid group, for analysis of which

- 1 015276 ryh it was intended. Using conjugate 4, it was possible to obtain antibodies specific only to the group of 24K-methylbrassinosteroids and unable to distinguish structural features of cycle B of the molecule.

Haptens and, therefore, immunogens based on 28-norbrassinosteroids are unknown.

The objective of the present invention is (22K, 23K) -2a, 3a, 22,23-tetrahydroxy-26- (hemisuccinate) B-homo-7-oxa-5a-cholestan-6-one (1) as a hapten in the synthesis of conjugate 28 norbrassinolide with bovine serum albumin.

The problem is solved by the claimed compound as a hapten, which is obtained according to scheme 1 by the interaction of acetate (22K, 23K) -2a, 3a, 22,23,26-pentahydroxy-5a-cholestan-6-one 5 [13] with trifluoroacetic acid followed by saponification of the protective groups of the obtained lactone 6 with potassium hydroxide and relaxation under the action of hydrochloric acid with the formation of pentahydroxylactone 7, protection of the vicinal secondary hydroxyl groups in the form of acetonides, addition of the succinic acid residue on the primary hydroxyl group formed diatsetanida 8 and hydrolysis of the acetonide groups.

Example 1. Mixture (22K, 23K) -2a, 3a, 22,23,26-pentahydroxy-5a-cholestan-6-one 5 (405 mg, 0.868 mmol), Ac2O (6 ml, 63.6 mmol), ΏΜΆΡ (39 mg, 0.319 mmol) and pyridine (10 ml) were kept at 65 ° C for 24 hours. The reaction mixture was diluted with water and extracted with EyAc (3x40 ml). The combined organic extracts were dried over Mg _: 8 () |, the solvent was distilled off in vacuo, and the residue was chromatographed on a silica gel column. The obtained pentaacetate was dissolved in C1 GHA (20 ml) and added at -15 ° C to a solution of trifluoroacetic acid prepared from trifluoroacetic anhydride (3.0 ml) and 30% hydrogen peroxide (0.87 ml) in 0 ° (14 ml). The reaction mixture was stirred at -15 ° C for 5 hours, then diluted with saturated NaHCO3 solution and extracted with 1 GHA (3x40 ml). The combined organic extracts were dried over Mg _: 8 (D, the solvent was distilled off in vacuo, the residue was applied to a silica gel column and eluted with a mixture of petroleum ether and ethyl acetate (20: 1 5: 1). 400 mg (66%) were obtained (22K, 23K). -2a, 3a, 22,23,26-pentaacetoxy-B-homo-7-oxa-5a-cholestan-6-one 6.

^! H NMR (500 MHz) δ: 0.71 (3H, s, 18-CH ₃ ), 0.96 (3H, s, 19-CH ₃ ), 1.98 (3H, s, acetate), 2.00 (3H, s, acetate), 2.02 (3H, s, acetate), 2.04 (3H, s, acetate), 2.09 (3H, s, acetate), 2.97 (1H, dd, 1 = 4.6, 12.3 Hz, C5-H), 3.824.12 (4H , m, С26-Н and С7-Н), 4.85 (m, 1Н, С2-Н), 4.97 (1Н, m, С22-Н or С23-Н), 5.21 (1Н, m, С22-Н or С23- H), 5.34 (1H, m, C3-H).

¹³ C NMR (125 MHz) δ: 11.56, 12.73, 12.86, 15.40, 15.95, 18.15, 20.76, 20.81, 20.89, 20.99, 21.07, 22.21, 24.65, 28.01, 28.90, 28.97, 29.26, 34.86, 37.41, 38.35, 38.84 , 39.12, 39.41, 41.97, 42.46, 51.28, 52.31, 52.41, 58.30, 67.88, 67.99, 68.88, 69.18, 70.33, 70.41, 71.20, 71.81, 76.31, 76.53, 169.88, 170.17, 170.31, 170.39, 170.45, 170.95, 174.95 .

To a solution of lactone 6 (95 mg) in methanol (4 ml) was added KOH (200 mg) and boiled for 1 h, then acetic acid (0.5 ml) was added and the mixture was kept for 1.5 h. Silica gel was added to the reaction mixture ( 3 ml) and evaporated. The residue was applied to a silica gel column and eluted with a mixture of chloroform and methanol (15: 1 6: 1). Get 62 mg (93%) (22K, 23K) -2a, 3a, 22,23,26-pentahydroxy-B-homo-7oxa-5a-cholestan-6-one 7. So pl. 261-263 ° С (СНС13-МеОН).

^! H NMR (SPS13 + SP3OC 500 MHz) δ: 0.71 (3H, s, 18-CH ₃ ), 0.88 (3H, s, 19-CH ₃ ), 2.97 (1H, dd, 1 = 3.9, 12.3 Hz, C5- H), 3.28-3.61 (5Н, m, С2-Н, С22-Н, С23-Н, С26-Н), 3.92 (1Н, m, С3-Н), 4.12 (2Н, m, С7-Н).

¹³ C NMR (SPS 13 + CB3OO, 125 MHz) δ: 10.73, 11.00, 14.45, 15.28, 17.14, 21.54, 23.96, 26.81, 30.71, 31.28, 31.76, 36.67, 36.75, 36.79, 37.48, 38.54, 39.07, 40.37, 40.43 , 41.75, 50.61, 51.61, 51.66, 57.48, 65.99, 67.11, 67.21, 67.49, 70.06, 70.16, 75.71, 76.16, 177.32.

A mixture of pentaol 7 (106 mg, 0.214 mmol), TUNN ^ O (1 mg, 5.2 μmol), 2,2-dimethoxypropane (0.3

- 20155276 ml, 2.4 mmol), CH ₂ Cl ₂ (4 ml) and MeOH (0.6 ml) were stirred for 3 hours, then triethylamine (0.1 ml) was added to the reaction mixture and evaporated. The residue was applied to a silica gel column and eluted with a mixture of cyclohexane and ethyl acetate (10: 1 2: 1). 79 mg (60%) (22Κ, 23Κ.) - 2α, 3α, 22.23bis (isopropylidenedioxy) -26-hydroxy-B-homo-7-oxa-5a-cholestan-6-one 8 are obtained.

'H NMR (500 MHz) δ: 0.70 (3H, m, 18-CH ₃ ), 0.86 (3H, s, 19-CH ₃ ), 1.29 (3H, s, acetonide), 1.346 (3H, s, acetonide) , 1.351 (3H, s, acetonide), 1.49 (3H, s, acetonide), 3.27 (1H, dd, 1 = 3.4, 10.7 Hz, C ₅ -H), 3.38-3.86 (4H, m), 4.07 (2H , m) 4.36 (2H, m).

¹³ C NMR (125 MHz) δ: 11.92, 12.64, 16.77, 18.11, 19.62, 22.91, 23.54, 24.36, 26.39, 27.02, 27.28, 27.48, 27.83, 32.94, 33.35, 34.95, 35.41, 35.46, 35.82, 36.32, 38.39 , 39.29, 39.51, 40.13, 43.04, 51.72, 53.04, 54.61, 67.29, 68.37, 72.40, 73.01, 74.43, 76.52, 82.71, 82.83, 107.52, 107.90, 108.09, 176.64.

A mixture of alcohol 8 (115 mg, 0.199 mmol), succinic anhydride (110 mg, 1.1 mmol), ΌΜΆΡ (30 mg, 0.25 mmol) and pyridine (4 ml) was kept at a temperature of 90 ° C for a day. After this, the reaction mixture was evaporated, the residue was applied to a column of silica gel and eluted with a mixture of cyclohexane and ethyl acetate (10: 1 1: 2). Receive 120 mg (90%) (22K, 23K.) - 2a, 3a, 22,23-bis (isopropylidenedioxy) -26 (hemisuccinate) -B-homo-7-oxa-5a-cholestan-6-one 9.

'H NMR (500 MHz) δ: 0.71 (3H, m, 18-CH ₃ ), 0.86 (3H, s, 19-CH ₃ ), 1.30 (3H, s, acetonide) 1.34 (6H, br.s, acetonide ), 1.49 (3Н, s, acetonide), 2.64 (4Н, m, СО ₂ СН ₂ СН2СО ₂ Н), 3.27 (1Н, dd, 1 = 3.8, 10.7 Hz, С ₅ -Н), 3.61 (1Н, t , 1 = 9 Hz, С _23- Н), 3.79 (1Н, m, С ₂₂ -Н), 3.91-4.11 (4Н, m), 4.37 (2Н, m).

¹³ C NMR (125 MHz) δ: 11.90, 11.93, 12.69, 12.76, 16.61, 17.84, 19.62, 22.93, 23.55, 24.38, 26.39, 27.01, 27.33, 27.48, 27.86, 28.79, 28.84, 28.90, 30.26, 30.32, 33.37 , 35.59, 35.69, 35.85, 36.77, 36.84, 39.30, 39.52,

40.16, 43.07, 51.72, 53.05, 54.64, 68.97, 69.78, 71.19, 72.42, 73.02, 75.00, 75.22, 82.82, 83.01, 107.58, 107.93, 107.99, 172.12, 172.15, 176.75, 176.97.

A mixture of diacetonide 9 (23 mg, 41.4 μmol), acetic acid (2.5 ml) and water (0.5 ml) was kept at 110 ° C for 15 minutes, then evaporated, the residue was applied to a silica gel column and elute with a mixture of chloroform and methanol (15: 1 8: 1). Obtain 14 mg (80%) (22Κ., 23Κ.) - 2α, 3α, 22,23 tetrahydroxy-26- (hemisuccinate) -B-homo-7-oxa-5a-cholestan-6-one (1). T. pl. 225-227 ° C (BULE-Meon).

'|| NMR (СОС1 ₃ + СП ₃ OD, 500 MHz) δ: 0.71 m (3Н, 18-СН ₃ ), 0.87 s (3Н, 19-СН ₃ ), 2.58 (4Н, m, СО ₂ СН ₂ СН2СО ₂ Н) 3.13 (1H, dd, 1 = 4.4, 12.2 Hz, C ₅ -H), 3.27 (1H, m), 3.57 (1H, m), 3.90-4.13 (5H, m).

¹³ C NMR (СОС1 ₃ + СП ₃ OD, 125 MHz) δ: 10.99, 11.01, 11.12, 11.19, 14.68, 15.05, 17.68, 21.66, 24.10, 26.93, 26.96, 28.32, 28.50, 28.64, 30.75, 35.78, 36.10, 36.83, 36.92, 37.63, 38.61, 39.13, 39.15, 40.47, 40.52, 41.87, 50.71, 51.66, 51.68, 57.53, 67.26, 67.33, 68.00, 69.69, 67.73, 70.04, 70.19, 76.17, 76.22, 172.60, 172.62,

174.16, 174.24, 177.39.

The synthesized hapten 1 is used to obtain a conjugate of 28-norbrassinolide with bovine serum albumin (BSA), which is confirmed by an example of a specific implementation.

Example 2. Dissolve 30 mg (55.7 mmol) (22K, 23K.) - 2a, 3a, 22,23-tetrahydroxy-26- (hemisuccinate) B-homo-7-oxa-5a-cholestan-6-one ( 1) in 6 ml abs. dioxane, add 8 mg of dry Ν-oxisuccinimide (69.6 mmol) and drop it at 1 = 8-10 ° С 13 mg (63.1 mmol) of dicyclohexyl carbodiimide in 4 ml of abs. dioxane. Stirred for 0.5 hours at 1 = 10 ° C and 6 hours at room temperature. The reaction progress is monitored by TLC in an ethyl acetate system: methanol = 9: 1 (P, - (1) = 0.05; K. _£ - (conjugate) = 0.67). The precipitated dicyclohexylurea is filtered off and the dioxane solution is added to a solution of BSA in 0.1 M NaHCO ₃ , pH 8.35, containing 60 mg of protein in 10 ml of buffer. They stand it for 20 hours at room temperature and the excess of activated ether and dioxane are removed by dialysis against a 0.005M solution No. C1. The resulting conjugate (1-BSA) was lyophilized and stored frozen at -18 ° C.

The resulting conjugate is used to obtain antibodies specific for 7-oxa-6-oxobrassinosteroids, which is confirmed by an example of a specific implementation.

Example 3. A group of six rabbits is immunized with a conjugate of 28-norbrassinolide-BSA with a density of 25 molecules of the steroid per 1 molecule of protein. Each rabbit is injected subcutaneously at 10-15 points of the back with 1 mg of conjugate, previously dissolved in 0.5 ml of phosphate-buffered saline (pH 7.4) and emulsified in an equal volume of Freund's complete adjuvant. The intervals between injections were 4 weeks. Immunization is continued for 6 months, performing periodic sampling of blood from the ear vein of animals. The obtained serum samples are tested for binding ability with respect to 24-norbrassinolide and their titers and values of association constants (K _a ) are determined. The titer is defined as the working dilution of the antiserum, providing maximum sensitivity of the test system with such conjugate binding, which allows you to build a calibration graph in the range of 2.5-0.2 OE.

- 3 015276

Characteristics of antisera obtained by immunization of animals with a conjugate of 24-norbrassinolide with BSA

Antiserum K _and x 10 'M' ¹ Titer A1 1,5 1: 1,000,000 A2 1.9 1: 1 200 000

Thus, the determination of the optimal titer of antiserum for use in ELISA showed that its dilution of 1,000,000 (A1) or 1,200,000 (A2) times provides good conditions for the determination of 28-norbrassinolide.

Literature

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Claims (1)

Formula of the Invention 28-Norbrassinolide with bovine serum albumin.