RU2175317C1 - Poly-(para-dihydroxy-para-phenylene)-thiosulfoacid sodium salt eliciting superoxidase activity and method of its synthesis - Google Patents

Abstract

FIELD: organic chemistry, biochemistry. SUBSTANCE: invention relates to poly-(para-dihydroxy-para-phenylene)-thiosulfoacid sodium salt of the general formula (I)

eliciting superoxidase activity. Indicated compound provides to broaden the spectrum of substances which are able to carry out the reaction of dismutation of O₂ and they can be used as antioxidants in biology, microbiology and medicine. Invention describes also method of synthesis of said compound that involves the interaction of para-benzoquinone with sodium thiosulfate in an aqueous medium in the mole ratio of components from 3:1 to 10:1. EFFECT: improved method of synthesis, broadened spectrum of antioxidants. 4 cl, 5 dwg, 3 ex

Description

 The invention relates to the chemistry of polymers, and the proposed product can find application as an antioxidant in biology, microbiology and medicine.

BACKGROUND OF THE INVENTION
In biology, cases of the damaging effect of oxygen on biological tissues are widely known, in particular, with an increase in its partial pressure. More thorough studies have shown that the toxic effects on biological objects are not provided by oxygen itself, but by intermediate products of its metabolism (Medvedev Yu.V., Tolstoy A.D. Hypoxia and free radicals in the development of pathological conditions of the body. - M .: 000 " Terra-Calender and Promotion ", 2000. - 232 p., [1]). In the case of a complete reduction of oxygen, water is formed, while its partial recovery results in the appearance of a number of reactive oxygen species (ROS) in the tissues that can have a damaging effect on various vital cell elements, including the genetic apparatus, enzymes, membrane and other structures. Biological life forms could survive in such conditions by developing an effective system of protection against ROS - a system of various antioxidants, the main tasks of which are to minimize the processes of nonphysiological production of ROS and remove them from the biological medium in case of occurrence.

As a result of the addition of the first electron to the oxygen molecule, the superoxide radical anion O ₂ • ^- (CAP) is formed, the subsequent reduction of which is accompanied by the appearance of hydrogen peroxide and a very active hydroxyl radical. In the process of subsequent oxidative reactions in biological tissues, alkane hydroperoxides, alkyl, alkoxyl and peroxyl radicals appear. To reduce the concentration of O ₂ • ^- formed inside the cells and generating the whole gamut of other ROS, a special enzyme, superoxide dismutase (SOD), functions, which disproportionates CAP ions according to the following scheme:
O ₂ • ^- + O ₂ • ^- + 2H ⁺ _ → H ₂ O ₂ + O ₂ (I)
The exceptional role of SOD in protecting cells from the toxic effects of ROS is confirmed by the fact that the rate constant of the reaction of dismutation of the radical anions O ₂ • ^- on this enzyme exceeds at least 2 orders of magnitude the same constants for all other enzymes (Fridovich I. V: Free radicals in biology / Ed. W. Prior, T. 1.-M .: Mir, 1979, S. 272-314 [2]).

 Superoxide dismutases are divided into several types, depending on the type of biological objects and the location of the enzyme in the cell. Common to all types of SOD is a complex polypeptide chain, consisting of 300-400 amino acid residues, and the presence in the polypeptide chain of metal ions of variable valency, in particular ions of copper, zinc, manganese or iron [2].

In the process of SOD functioning, the role of metal ions of variable valency is reduced, firstly, to capture and hold O ₂ • ^- radical anpion radicals near the active centers and, secondly, to participate in electron donation and acceptance processes. The function of the polypeptide bond is reduced to the organization of the proper geometry of the active center of the enzyme and the modification of its activity due to the formation of the corresponding ligand environment of the central ion of the enzyme.

 Despite the record high activity of SOD, a significant decrease in the activity of SOD is observed in various tissues in various pathological conditions of the body, which inevitably leads to the activation of pathological processes of free radical oxidation in tissues and their damage (Kamyshentsev M.V., Volchek I.V., Platonov V G.G. et al. Dokl. AN, 1992, v. 326, No. 4, pp. 722-724 [3]). Therefore, at present, as compounds with superoxide dismutase activity, various compounds of synthetic origin are proposed, in particular metal complexes of variable valency, such as manganese (RF patent application N 98104277, [4]).

SUMMARY OF THE INVENTION
The objective of the present invention is to expand the range of products capable of, like SOD, to carry out the O ₂ • ^- dismutation reaction. Of particular interest is the possibility of implementing such a reaction on a non-protein product, since proteins are hydrolytically unstable, and their synthesis is time-consuming and has not yet been realized in the case of SOD. It is also highly desirable to exclude the presence of metal ions of variable valency in such a product, which often exhibit high prooxidant activity, being present in biological media even in low concentrations.

Based on the foregoing, the authors of the present invention suggested that a molecule of a different nature compared to SOD will be able to carry out the O ₂ • ^- dismutation reaction if it has the ability to accept radical anions and participate in reversible electron and proton transfer processes.

,
где n = 2-6.Therefore, to achieve this objective, the authors of the present invention synthesized the sodium salt of poly (para-dihydroxy-para-phenylene) thiosulfonic acid (hereinafter also referred to as para-NPF for brevity), having the general structural formula:

,
where n = 2-6.

The gross formula is C _6n H _{4n + 1} O _{2n + 3} S ₂ Na product.

The product was obtained by reacting para-benzoquinone with sodium thiosulfate in an aqueous medium by heating from 50 ^° C to 100 ^° C. The product formed during the reaction was separated from the solvent and purified from low molecular weight impurities by extraction with diethyl ether. The synthesis conditions and properties of the resulting product are described in more detail in the following examples.

The closest structural analogue of the proposed compound is the sodium salt of [poly- (2,5-dihydroxy-phenylene)] -4-thiosulfonic acid (meta NPF), obtained by the interaction of para-benzoquinone with sodium thiosulfate in an aqueous-alcoholic medium at a temperature above 65 ^o C (RF Patent N 2105000, IPC ⁶ C 07 C 381/02, C 08 G 61/10, A 61 K 31/05 [5]). In a known product, the polymer chain consists of a set of hydroquinoid units interconnected in a meta-position (meta-NPF).)
The fundamental difference between the compound proposed in accordance with the present invention from its analogue is that in the proposed compound, the hydroquinoid units form a polymer chain due to attachment in the para position, and not in the meta position, as in the analogue. As you know, a change in the chemical structure of a substance is inevitably associated with a change in its properties. It is known from the chemistry of aromatic compounds that substituents located in the meta-position of the aromatic nucleus have a weak effect on each other, while substituents located in the para-position strongly affect each other and the molecule as a whole. The most striking manifestation of the special properties of the product in accordance with the present invention compared to the analogue is the manifestation of a characteristic signal recorded using the method of electron paramagnetic resonance spectroscopy (EPR spectroscopy), indicating the presence of unpaired electrons in para-NPFs (Fig. 1), which were not fixed in analog samples. The spectrum does not change the intensity during the monthly storage of the sample in air. The presence of the EPR signal in para-NPF samples and its stability during the monthly storage process indicate a strong interaction between all phenyl cores of the polymer chain connected to each other in the para-position. As a result, a fairly extended conjugation system is formed, capable of effectively retaining an unpaired electron recorded by EPR spectroscopy. The EPR spectrum is observed both for a solid sample (curve 1, shown by a solid line in Fig. 1), and for an aqueous solution of a substance (curve 2, shown by a dashed line in Fig. 1).

Study of the superoxide dismutase activity of the sodium salt of poly (para-dihydroxy-para-phenylene) thiosulfonic acid (para-NPF)
To confirm the superoxide dismutase activity (SOD activity) of the proposed product, we used a method based on the competitive interaction of O ₂ • ^- with the known dye tetranitrotetrazolium blue (TNT) and the test substance (Beauchamp C., Fridovich 1., Anal.Biochem., 44, 276 (1971) [6]).

The experimental design and the results obtained are presented in FIG. 2, on which are conventionally marked:
ON - light on;
OFF - turn off the light;
TNT - the introduction of dye;
para-NPF - the introduction of the sodium salt of poly (para-dihydroxy-para-phenylene) thiosulfonic acid.

Образовавшиеся анион-радикалы самопроизвольно диссоциируют на кислород и перекись водорода, которая в присутствии каталазы расщепляется на кислород и воду:

Убыль кислорода в ячейке регистрировали с помощью кислородного датчика (участок А кривой). При выключении света генерация O₂•^- прекращается и кислород не расходуется (начало участка В кривой). При выключении света в ячейку вводили краситель ТНТ, который при повторном генерировании анион-радикалов вызывает их интенсивное окисление, превращаясь в формазан в соответствии со следующей реакцией:

При этом количество окисленных анион-радикалов строго соответствует количеству регенерированного кислорода и концентрация регенерированного кислорода в ячейке не меняется.The generation of O ₂ • ^- in the cell was carried out by the photochemical method in the presence of riboflavin and tetramethylenediamine:

The formed radical anions spontaneously dissociate into oxygen and hydrogen peroxide, which in the presence of catalase is split into oxygen and water:

A decrease in oxygen in the cell was recorded using an oxygen sensor (section A of the curve). When the light is turned off, the generation of O ₂ • ^- stops and oxygen is not consumed (the beginning of the section In the curve). When the light was turned off, a TNT dye was introduced into the cell, which upon the repeated generation of radical anions causes their intense oxidation, turning into formazan in accordance with the following reaction:

The amount of oxidized radical anions strictly corresponds to the amount of regenerated oxygen and the concentration of regenerated oxygen in the cell does not change.

Внесение пара-НПФ в среду, где генерируются анион-радикалы O₂•^-, ускоряет процесс их дисмутации, при этом эффективность данного процесса зависит от концентрации пара-НПФ. Результаты исследования СОД-активности предложенного соединения представлены в Таблице 1.The introduction of para-NPF into the cell and the inclusion of light is accompanied by repeated absorption of oxygen, but at a lower rate (section C of the curve), which indicates the occurrence of competitive interaction of O ₂ • ^- with the dye (without oxygen consumption by reaction (IV)) and with para- NPF by reaction (III) with the consumption of oxygen. The obtained results confirm that para-NPF exhibits superoxidase activity, realizing the process of dismutation by reaction

The introduction of para-NPFs into the environment where O ₂ • ^- radical anions are generated accelerates the process of their dismutation, while the effectiveness of this process depends on the concentration of para-NPFs. The results of the study of the SOD activity of the proposed compounds are presented in Table 1.

Substances capable of decreasing the concentration of O ₂ • ^- radical anions responsible for the appearance of the remaining gamma of ROS are of significant practical interest as antioxidants. The introduction of para-NPF in the composition of the glucose-mineral nutrient medium containing (g / l): glucose - 4, Na ₂ HPO ₄ - 7, KH ₂ PO ₄ - 3, NH ₄ Cl - 1, NaCl - 0.5, MgSO ₄ - 0.2, CaCl ₂ - 0.02, nicotinic acid - 0.005, para-NPF - 0.03, allowed the cultivation of Escherichia coli strain M-17 to increase the number of viable cells after completion of the cultivation process to (88.2 ± 4.3)% instead of (75.8 ± 3.3)% in the control, where no para-NPF was administered. The antioxidant properties of the substance proposed in accordance with the present invention are manifested not only in aqueous media, but also in dry products (see Table 2).

 If the content of viable cells after cultivation of microorganisms is taken as 100%, then in the process of freeze drying, about 15% of the cells lose their viability. The grinding process is accompanied by significant inactivation of microorganisms and the number of viable cells is about half. In the case of using para-NPF, after grinding, about 75% of the cells from the initial culture remain viable.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS
The invention is illustrated in graphic materials on which:
FIG. 1 is an EPR spectrum of a powder of para-NPF (curve 1) and a 5% solution of para-NPF in water (curve 2);
FIG. 2 is a diagram of a process for determining the SOD activity of para-NPFs;
FIG. 3 is an IR spectrum of para-NPF in a KBr tablet;
FIG. 4 is a UV absorption spectrum of a solution of para-NPF in water;
FIG. 5 is a ¹³ C-NMR spectrum of a para-NPF in a solid phase.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION
For a better understanding of the invention, the following examples are given.

 Example 1

76.6 g (0.31 M) of sodium thiosulfate are placed in a 2 liter apparatus equipped with a stirrer and a jacket, and 1 liter of distilled water is poured with stirring. The solution is heated to a temperature of 60 ^° C. and 100 g (0.93 M) of para-benzoquinone are added with an intensively working stirrer. After 1.5 hours, the process is stopped and the resulting product is dried on a spray dryer. The dry powdery product is placed in a Soxhlett apparatus and the impurities are removed using diethyl ether as an extractant. The extraction time is 24 hours. The product is dried under vacuum at a temperature of 50 ^o C. The yield of the target product is 109.2 g. The molecular weight determined by the cryoscopic method is 400.

The IR spectrum of the sample (Fig. 3) has a wide band of stretching vibrations of hydroxyl groups in the region of 3600 - 2000 cm ^-1 , bands at 1200 and 1040 cm ^-1 confirm the presence of sulfo groups, the band in the region of 1600-1550 cm ^-1 refers to the so-called "breathing" vibrations of C = C bonds of the benzene ring. Another characteristic vibrational band of the C = C ring is observed at 1500 cm ^-1 . The absorption bands of 1370, 1448, and 1520 cm ⁻¹ relate to vibrations of the CC bonds of the ring mixed with deformation of the angles of СОН and CCH. The band in the region of 830 cm ^-1 refers to non-planar deformation vibrations of the CH bond in the tetra-substituted benzene ring.

In the electronic absorption spectrum of para-NPF (Fig. 4), there is continuous absorption from the ultraviolet to the visible region with two weakly pronounced maxima of y of 40,000 and 33,000 cm ^-1 . These bands relate to π-π ^* transitions in aromatic nuclei with two chromophore groups located in a para position relative to each other.

 Para-NPF introduced into the bacterial suspension of Escherichia coli strain M-17 increases the viability of microbial cells dried by contact drying during storage at room temperature. The results are presented in Table 3.

From table 3 it is seen that when storing a dry bacterial culture for a month at room temperature, there is a loss of viability of 80% of the original cells, which is associated with slowly occurring oxidation reactions. The introduction of para-NPF into the cell suspension before the drying stage reduces the losses during the monthly storage by 5–20%, which may be related to the termination of the free radical oxidation chain process at the first stage — the stage of formation of O ₂ • ^- radical anions.

 Example 2

38.3 g (0.15 M) of sodium thiosulfate and 1 L of distilled water are placed in the apparatus described in Example 1, the solution is heated to 70 ^° C. and 100 g (0.93 M) of para-benzoquinone are added with effective stirring. After 2 hours, the reaction was stopped and the contents were dried on a spray dryer. After extracting impurities with diethyl ether for a day, the product is dried under vacuum at 50 ^o C. The yield of the target product is 98.7 g. According to elemental analysis, the sulfur content is 11.7%, and the molecular weight is 650.

подтверждена на модели активированных полиморфно ядерных лейкоцитов (нейтрофилов). Известно, что нейтрофилы являются одним из звеньев клеточного иммунитета, обеспечивающим защиту организма от чужеродных объектов. Одним из эффективных механизмов бактерицидного воздействия на чужеродные клетки является способность нейтрофилов быстро перестроить свой метаболизм на массовую продукцию анион-радикалов O₂•^- оказывающих токсический эффект на патогены (Кузнецов В. Ф., Черешков В. А. Патофизиологическая дисфункция нейтрофилов. - Киров, 1998, - 119 с. [7]). Результаты сравнительных испытаний СОД-активности синтезированного образца и СОД представлены в Таблице 4.The ability of the obtained sample to contribute to disproportionation

confirmed on the model of activated polymorphic nuclear leukocytes (neutrophils). It is known that neutrophils are one of the links of cellular immunity, providing protection of the body from foreign objects. One of the effective mechanisms of bactericidal effect on foreign cells is the ability of neutrophils to quickly rebuild your metabolism for mass production of radical anions O ₂ • ^- has a toxic effect on pathogens (Kuznetsov, VF, petioles VA pathophysiological dysfunction of neutrophils -. Kirov, 1998, 119 p. [7]). The results of comparative tests of the SOD activity of the synthesized sample and SOD are presented in Table 4.

Excessive production of O ₂ • ^- neutrophils in a number of pathologies is responsible for damage to the vascular bed. The introduction of drugs that provide the ability to effectively disproportionate O ₂ • ^- will reduce the pathological effect of activated neutrophils on blood vessels.

 Example 3

Into the apparatus described in Example 1, 23 g (0.093 M) of sodium thiosulfate are placed in 1 L of distilled water, the solution is heated to 90 ^° C. and 100 g (0.93 M) of para-benzoquinone are introduced with vigorous stirring. After 1.5 hours, the reaction was stopped and the contents were dried on a spray dryer. The isolated dry product is extracted with diethyl ether for 24 hours, then dried under vacuum at a temperature of 50 ^o C. The yield of the target product is 80.2 g. According to elemental analysis, the sulfur content of 10.8%, molecular weight 750.

The addition of hydroquinoid units in the synthesized sample in the para position relative to each other is confirmed by ¹³ C-NMR data on the presence of an absorption band at 147.87 ppm, characteristic of the C _arom- C _arom bond between benzene rings (Fig. 5).

Claims (4)

1. The sodium salt of poly (para-dihydroxy-para-phenylene) thiosulfonic acid of the General formula

where n = 2 - 6,
possessing superoxidase activity.  2. A method for producing the sodium salt of poly (para-dihydroxy-para-phenylene) thiosulfonic acid as defined in claim 1, comprising the reaction of parabenzoquinone with sodium thiosulfate in an aqueous medium at a molar ratio between reactants of 3: 1 to 10: 1.  3. The method according to claim 2, characterized in that it further includes cleaning the reaction product from low molecular weight impurities by extracting the specified product with diethyl ether.  4. The method according to p. 3, characterized in that it further includes drying the specified product from the extractant.

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