RU2073665C1 - 15-hydroxy-5z,8z,11z,13e-eicozatetraenic acid multiple labeled with tritium at double bonds and method of its synthesis - Google Patents

Abstract

FIELD: biochemistry. SUBSTANCE: product: 15-hydroxy-5Z,8Z,11Z,13E-[³H]-eicozatetraenic acid multiple labeled with tritium. [³H]-Arachidonic acid (substrate) multiple labeled with tritium at double bonds is incubated with enzyme 15-lipoxygenase (specific activity is 10.9 mcmole/min/mg) isolated from soybean. After 10-15 min incubation intermediate substance is reduced by addition of two-fold molar excess of NaBH₄, reaction mixture is acidified to pH = 3.5 and the end product is isolated. Substrate taken at amount 12-16 mcg is dissolved in 0.7-1.0 ml 0.1 M sodium-borate buffer, pH = 9.0, and enzyme is added at amount providing the ratio substrate : enzyme = 1: (15-20) (mcg/mcg). Incubation is carried out at 20-22 C. Synthesized product is used in medicine and veterinary science. EFFECT: improved method of synthesis. 2 cl, 4 tbl

Description

 The invention relates to the chemistry of natural and physiologically active compounds and may find application in medicine and veterinary medicine.

 Acyclic metabolites of polyunsaturated acids - hydroxyeycosozetetraenoic and hydroperoxyeykozatetraenoic acids, leukotrienes, lipoxins, etc. formed in the body during the enzymatic transformation of polyenoic acids, which are part of the lipids of cell membranes, play a key role in the development of inflammatory and allergic conditions.

 Being precursors of leukotrienes and lipoxins, hydroxyeycosatetraenoic and hydroperoxyeycosatetraenoic acids can be used to diagnose pre-inflammatory and pre-allergic pathologies. Moreover, it is necessary to develop methods for the determination of these eicosanoids in biological objects. Among these methods, radioimmunoassay and enzyme immunoassay methods are widely used. To implement these analytical approaches, it is necessary to develop preparative methods for the synthesis of tritium-labeled hydroxyeicosatetraenoic acids.

 Enzymatic synthesis is most acceptable, in which two main components are needed: a substrate and an enzyme.

 A known method of enzymatic synthesis of 15-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid using as an substrate the ammonium salt of arachidonic acid, and as the enzyme 15-lipoxygenase isolated from soybeans (drug company Fluka Switzerland), and the process is carried out under conditions of saturation of the reaction medium with oxygen (Hamberg M. Samuelsson BJ Biol. Chem. 1967, v. 242, p. 5329) (1).

 However, 15-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid short-labeled with tritium in double bonds was not obtained by this known method. Moreover, the known method is described uninformatively, which does not allow to calculate the yield of the target product. In addition, the process of obtaining it by this known method is long: the incubation of the enzyme with the substrate is carried out for 30 minutes.

 A known method of enzymatic synthesis of 15-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid. The method used a substrate in the form of arachidonic acid, and as an enzyme, 15-lipoxygenase isolated from soybeans (preparation of the company Serva, Germany).

 Incubation of the enzyme with the substrate according to this known method lasts 10 minutes The yield of the target product is 40% (Belosludtsev Yu.Yu. et al. Bioorganic chemistry, 1989, v. 15, No. 9, p. 1224 1231) (2).

 However, the yield of the target product when implementing this known method is low (40%). However, tritium-labeled 15-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid was not obtained by this known method.

 Known to be double-labeled with tritium in double bonds is 5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid (arachidonic acid) (Myagkova G.I. et al. Bioorganic chemistry, 1982, v. 8, N 2, p. 255 260) (3).

 However, the enzymatic synthesis of short-labeled tritium in double bonds of 15-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid, based on this known substrate, is not described.

The technical result achieved by the present invention is to obtain a new double-labeled tritium double bond of an acyclic metabolite of polyunsaturated acids - 15-hydroxy-5Z, 8Z, 11Z, 13E- [ ³ H] -ecosatetraenoic acid, increasing the yield of this target product in comparison with in a known manner (2) the enzymatic synthesis of 15-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid, starting from arachidonic acid, simplifying the method by carrying out the synthesis under conditions that do not require oxygen saturation of the reaction medium.

 Example 1

 Getting the enzyme.

100 g of soy flour are extracted with hexane, the precipitate is filtered, extracted with 50 mM sodium phosphate buffer (pH 7.0) at + 4 ^° C. The suspension is filtered, centrifuged for 10 min at 13000 g. The supernatant was adjusted to 30% saturation of (NH ₄ ) ₂ SO ₄ , centrifuged for 10 min at 13000 g. The supernatant was adjusted to 60% saturation of (NH ₄ ) ₂ SO ₄ , centrifuged, the precipitate was dissolved in 10 ml of 50 mM sodium phosphate buffer (pH 7.0) and dialyzed against the buffer. Protein purification is carried out by ion-exchange chromatography on DEAE-Sephadex. The protein solution after purification is subjected to freeze drying.

 Example 2

 Preparative enzymatic synthesis of uniformly labeled with tritium at double bonds of 15-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid.

50 ml of 0.1 M sodium borate buffer pH 9.0 was saturated with oxygen, 50 μl of a 0.5 M solution of [ ³ H] -arachidonic acid in hexane (up to a concentration of 500 μM), 1 ml of a solution of 15-lipoxygenase with a concentration of 12 were added, 5 mg / ml. Incubated for 10 min at room temperature (20-22 ^° C) in a stream of oxygen, then the resulting intermediate 15-hydroperoxy- [ ³ H] -ecosatetraenoic acid was reduced by adding a two-fold molar excess of NaBH ₄ . The reaction was stopped by adding 1.5 ml of a 1 M citric acid solution to pH 3.5, extracted with ethyl acetate twice in 100 ml, the extract was washed with distilled water, dried with anhydrous sodium sulfate, evaporated, and dissolved in 1 ml of isopropyl alcohol-hexane mixture (2:50 ); the target product was isolated by HPLC on silica gel in isopropyl alcohol-hexane eluent (2:50). Retention time 15 min. 5.6 mg of 15-hydroxy-5Z, 8Z, 11Z, 13E- ³ H-eicosatetraenoic acid (75%) with a purity of 99% (HPLC) were obtained from 7.5 mg of [ ³ H] -arachidonic acid.

UV spectrum: conjugated diene chromophore with λ _maX 234.5 nm
PMR spectrum δ, ppm (spectrum of unlabeled compound obtained under similar conditions: 0.88 (t, J 7 Hz, 3H, C-20), 1.00 2.08 (m, 10H, C-3 + C-16 + C-17 + C-18 + C-19), 2.13 (dt, J 7 Hz and 7 Hz, 2H C-4), 2.35 (t, J 7 Hz, 2H, C-2), 2.82 (dd , J 6 Hz, 2H, C-7), 2.98 (m, 2H, C-10), 4.27 (dt, J 6 Hz and 6 Hz, 1H, C-15), 5.39 (m 5H, C-5 + C-6 + C-8 + C-9 + C-11), 5.75 (dd, J 15.0 Hz and 6 Hz, 1H, C-14), 5.98 ( dd, J 11 Hz, 11 Hz, 1H, C-12), 6.61 (ddt, J 16.0; 11; 1.5 Hz, 1H, C-13).

Example 3
According to the method of example 2, the ratio of the substrate-enzyme was determined. The results are shown in table 1.

 Example 4

Studying the influence of the conditions of the enzymatic reaction on the yield of 15-hydroxy- [ ³ H] -eicosatetraenoic acid.

When working with milligram quantities of [ ³ H] -arachidonic acid, its molar radioactivity cannot be maximum. So, with a molar radioactivity of 6.9 Pbq / mol, one would have to operate with a radioactivity of 150,200 TBq (4.0 5.4 K), which is not caused by commercial necessity, requires large quantities of expensive ³ H-arachidonic acid and causes the risk of radioactive contamination. Therefore, it is advisable to work with [ ³ H] -arachidonic acid with a radioactivity of not higher than 70 TBq / mol (0.07 PBq / mol).

When using highly labeled [ ³ H] -arachidonic acid, it is necessary to work with microgram amounts of it. In this regard, the conditions for the analysis and purification of reaction mixtures were specially selected.

So, HPLC was performed on a Milichrom chromatograph (Russia) on a 2 × 60 column, Silabsorb 5C ₁₈ in a stepwise gradient of solvent mixtures of methanol, water and acetic acid in the ratios:
a) 85: 15: 1 (0.8 ml);
b) 75: 25: 1 (0.4 ml);
c) 70: 30: 1 (0.8 ml),
a speed of 100 μl / μl with multi-wave detection; on a Gilson chromatograph (France) in a solvent mixture: methanol-water-acetic acid (70: 30: 0.1) on a 3.3x150 mm column, Separon SGX 5C ₁₈ , flow rate 0.5 ml / min, retention time for 15-hydroxyeycosatetraenoic acid 33.74 min.

Tables 2, 3, 4 show the dependence of the yield of 15-hydroxy- [ ³ H] -eicosatetraenoic acid on the conditions of the enzymatic reaction.

 The dependences presented in tables 2, 3, 4 allowed us to conclude that when using microgram amounts of labeled arachidonic acid, the following conditions are optimal: the use of 12 16 μg of labeled acid (substrate) in 0.7 1.0 ml of buffer at a ratio substrate enzyme 1: 2 (μg / μg). It turned out that when using microgram amounts of labeled arachidonic acid, it was not necessary to saturate the medium with oxygen and conduct the reaction in an oxygen stream, the oxygen content in the buffer in excess is enough for biosynthesis. In addition, the reaction in highly diluted solutions significantly reduces the radiolysis of labeled eicosanoids, which ensures an increase in the yield of the target product.

 Example 5

Enzymatic synthesis of short-labeled 15-hydroxy- [ ³ H] -eycososetetraenoic acid.

12 16 μg of [ ³ H] arachidonic acid in 3 ml of methanol was added to a 5 ml reaction flask, evaporated and suspended on a shaker in 1.0 ml of borate buffer. Then add 30 to 40 μl of a solution of 1,5-lipoxygenase (0.8 mg / ml) in the same buffer. The reaction and purification are carried out as described in example 2 without the use of oxygen, but with vigorous stirring. The yield of labeled preparation is 60 65% with a molar radioactivity of 6.0 6.2 PBq / mol.

 Thus, when using a soybean enzyme with a specific activity of 10.9 μmol / min • mg and a substrate with a molar radioactivity of 6.9 Pbq / mol and a radioactivity of no higher than 0.07 Pbq / mol, the new method allows to obtain the target product with 60 65% yield and for a short incubation time with the possibility of using microgram amounts of substrate.

Claims (2)

 1. Multiple-labeled tritium in double bonds of 15-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid. 2. A method of producing 15-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid double-labeled with tritium in double bonds, comprising incubating a substrate with 15-lipoxygenase from soybeans for 10 15 minutes, recovering the intermediate product, acidifying the medium to pH 3, 5, isolation and purification of the target product, characterized in that the enzyme is used with a specific activity of 10.9 μmol / (min • mg), and [ ³ H] -arachidonic acid is used as a substrate, and the substrate is dissolved in 0 before incubation, 7 1.0 ml 0.1 M sodium borate buffer, pH 9.0 and incubation for odyat at 20 22 ^o C at an enzyme substrate ratio of 1: 1.5 2.0 (mg / g).

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