RU2624439C1 - 5-oxo-pro-arg-pro labeled by tritium on all amino acid residues - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: 5-oxo-Pro-Arg-Pro labeled by tritium on all amino acid residues, which can be used in analytical chemistry and biological studies, is proposed.

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EFFECT: compound has beneficial biological activity.

1 tbl, 1 ex

Description

The invention relates to the field of organic chemistry and may find application in analytical chemistry and biological research.

When studying physiologically active compounds, their labeled analogues are necessary.

It is known that the replacement of atoms of compounds with their labeled analogues does not lead to a change in any properties of the starting compound (Evans EA - Tritium and its compounds London Butterworths, 1974, p. 48) [1].

In addition, it was shown that the reactions of deuterium and tritium with substances deposited on the catalyst surface occur with similar rates and the same selectivity, and the ratio of the rates of these reactions approaches unity (Zolotarev Yu.A., Dadayan A.K., Kost N. V., Voevodina M.E., Sokolov O.Yu., Kozik BC, Scar S.I., Azev V.N., Bocharov E.V., Bogachuk A.P., Lipkin V.M., Myasoedov N .F. Quantitative analysis of the peptide HLDF-6-amide and its metabolites in the tissues of laboratory animals using their labeled with tritium and deuterium derivatives // Bioorganic chemistry I, 2015, Volume 41, No. 6, pp. 644-656 and Zolotarev Yu. A., Dadayan A.K., Borisov Yu. A., Kozik VS, Nazimov IV, Ziganshin RH, Bocharov EV, Chizhov AO, Myasoedov NF New Development in the Solid-State Isotope Exchange with Spillover Hydrogen in Organic Compounds // J. Phys. Chem. C. 2013. V. 117. P. 16878-16884) [2, 3].

It was also shown in [1] that in biological studies, the distribution of tritium in the molecules of labeled preparations plays a crucial role. The work emphasizes that in the study of drug metabolism, the stability of the position of tritium is one of the most important conditions of a biological experiment. For each specific study, a certain distribution of tritium in the molecules of the labeled preparations is required. A chemically stable bound tritium atom can become mobile upon the interaction of a tritium-containing compound with an enzyme. As a result, the tritium atom can be lost, despite the complete safety of the carbon skeleton of the molecule. If tritium is in another fragment of the molecule of the substance or there are several of them in the molecules of the substance, then the probability of obtaining more reliable data is significantly increased. It is important not only the distribution of tritium, but even its stereochemical conformation. For example, it is shown that the conversion of cholesterol-4- ^{14 C-3} H 7a- cholic acid in the body in vivo rat leads to a loss of at least 93% of the tritium position 7. However, the use of ^{cholesterol-14} C 4- 7β- ³ N all tritium remained in cholic acid.

Known 5-oxo-Pro-Arg-Pro formulas:

5-oxo-Pro-Arg-Pro is a universal anxiolytic, exhibits a hypoglycemic effect in animals, even against the background of a pathology such as metabolic syndrome, reduces platelet aggregation, restores normal values of total cholesterol, lipid profile even while eating food leading to increased blood cholesterol. The anticoagulant-fibrinolytic and antiplatelet effects of this peptide were established, as well as that 5-oxo-Pro-Arg-Pro is active against glutamatergic neurons and will cause biological effects associated with a change in the functional activity of the glutamatergic system of the brain (Glazova N.Yu., Levitskaya N.G., Andreeva L.A., Kamensky A.A., Myasoedov N.F. // Report of the Academy of Sciences of Physiology. 1999. T 367. No. 1, P. 137-140 [4]).

However, its analogue, labeled with tritium for all amino acid residues, is not described.

The technical result achieved by the present invention is to expand the range of labeled analogues of physiologically active compounds.

This technical result is achieved by obtaining tritium-labeled 5-oxo-Pro-Arg-Pro formula for all amino acid residues

The following is an example implementation of the invention.

Example I.

A solution of 5 mg of 5-oxo-Pro-Arg-Pro in 30 μl of water was impregnated with 50 mg of 5% Pd / BaSO ₄ and lyophilized.

40 mg of the mixture was placed in an ampoule, evacuated, and filled with gaseous tritium to a pressure of 400 hPa. The reaction was carried out at 190 ° C for 10 min. Then the ampoule was evacuated, the catalyst was removed on the filter, the substance was extracted with methanol (5 × 2 ml). The extracts were evaporated, the residue was dissolved in methanol (3 × 1 ml) and again evaporated to remove labile tritium. The final methanol solution contained 450 mCi of peptide.

Tritium-labeled 5-oxo-Pro-Arg-Pro was purified by preparative high performance liquid chromatography. After purification, the radiochemical purity of the labeled 5-oxo-Pro-Arg-Pro is not less than 97-98%, the yield is 75%, the molar radioactivity is 60 Ci / mmol (table).

Analysis of the reaction mixtures was performed on a Milichrom-A02 chromatograph, wavelength (nm): 200, 210, 220, 230, 240, using a ProntoSIL-120-5-C ₁₈ AQ DB-2003 column (2 × 75 mm, particle size 5 μm), in a gradient methanol-buffer (0.2 M LiClO ₄ +0.005 M HClO ₄ , pH 2.24) for 12 min at a temperature of 35 ° C. Analysis of 5-oxo-Pro-Arg-Pro was carried out by increasing the concentration of methanol from 1 to 30%. The flow rate of the eluent is 0.2 ml / min, the retention time of 5-oxo-Pro-Arg-Pro is 8.11 minutes.

Preparative isolation of the labeled peptide was carried out on a Kromasil 100 C18 column (8 × 150 mm, 7 μm), in a gradient of methanol and 0.1% aqueous trifluoroacetic acid for 30 min, increasing the methanol concentration from 0 to 50%. The flow rate of the eluent is 2 ml / min, the retention time of 5-oxo-Pro-Arg-Pro is 20.9 minutes.

Thus, tritium-labeled 5-oxo-Pro-Arg-Pro was obtained for all amino acid residues.

Claims (2)

Tritium-labeled for all amino acid residues of 5-oxo-Pro-Arg-Pro of the formula: