CS271963B1 - Method of cu/ii catalytic activity affection - Google Patents

Description

The invention relates to a process for influencing the catalytic activity of Cu / II / ions for the dismutation of superoxide ion radicals Oj in in vitro systems by using as coordination compounds chelated by Schiff base anions of the composition. where O is R, H ⁺ , or Na ⁺ , derived from salicylaldehyde and L-glutamic acid , or D, L-glutamic.

It is known that some metalloenzymes, e.g. Cu / Zn - superoxide dismutase, EC 1.15.1.1. / CU / Zn-SOD / catalyze the dismutation of toxic anion radicals OJ, which are formed in organisms as one of the products of reduction of molecules 0 ₂ ·

According to current scientific knowledge, several pathological conditions and processes are associated with the deficiency of the enzyme Cu / Zn-SOD in organisms, or with disorders of its activity, which causes an increase in the level of superoxide ions OJ and in connection with other toxic anion radicals, 0H ^T ( B. Halliwell, Superoxide dismutase and the superoxide theory of oxygen toxicity, in: R. Lontie, Copper Proteins and Copper Enzymes, vol. II, CRC Press, Inc. Boca Raton Florida 1984, pp. 63-102).

Research into the metalloenzymes of this group, mainly Cu / Zn-SOD, as well as the elucidation of the significance and function of metal ions, in particular Cu / II / and their active center, has recently received increased attention. It has been found that the active center of Cu / Zn-SOD forms a pentacoordinated Cu / II / ion which binds four N-atoms from histidine residues as well as one O-atom from the HJO molecule. (M. Fielden, C. Rotilio, The structure and mechanism of Cu / Zn-superoxide dismutase, in: R. Lontie, Copper Proteins and Copper Enzymes, vol. II. CRC Press, Inc. Boca Raton Florida 1984, p.27 -61.) The research in this area as well as the use of its results in practice are also considerably expensive, given the high price relations of the isolated natural enzyme Cu / Zn-SOD on the international market. Therefore, great attention is paid to the research of coordination compounds of media with different ligands, as a simple model of the active center in Cu / Zn-SOD.

Coordinating compounds Cu (II) with various ligands of relatively low mol. weight of Mr, although they lack the protein component present in Cu / Zn-SOD, the composition and structure of the coordination polyhedron, resp. electron system on the central atom can to some extent actively mimic the center of the natural metalloenzyme Cu / Zn-SOD. The degree of dismutant activity against Oj in such coordination compounds depends on several factors, especially on the nature of bound ligands, the structure of the coordination polyhedron, including its distortions, the redox potential Cu / Cu ¹ in the complex, but also on the environment, especially the pH in which the complex acts on the substrate. However, experimentally verified findings have shown that the dismutant activity of Cu / II / in such (model) complexes represents only a fraction of its activity, characteristic of natural Cu / Zn-SOD.

In vitro, aquamadate cations themselves show the most significant dismutating activity, but only in the acidic pH range. However, in the neutral or weakly basic region, which characterizes the physiological conditions, the dismutation activity of aqueducted ions practically disappears. However, Cu (II) retains some of the catalytic activity of a given type when bound in complexes with lysine, histidine, indomethacin and the like. The dismutant activity of Cu (II) in its complex with ethylenediaminetetraacetate ligand is also more noticeable in the acidic pH range (W. F. Beyer, I. Fridovich, Anal. Biochem. 173, 160 (1988)).

Influencing the properties of Cu / II / ions by binding them to coordination compounds of suitable types maintaining at least a certain proportion of their dismutating activity even in conditions close to physiological (neutral or weakly basic pH range) is of particular importance for basic research (biochemistry, molecular biology, biocoordination chemistry) as well as in terms of

CS 271 963 B1 search for promising compounds for diagnostic purposes, possibly for use in pharmacotherapeutic practice (antiphlogistics, antirheumatics, radioprotectives, antiproliferatives, antineoplastics, etc.). From the point of view of the ease of use of the Cu (II) coordination compounds in research or when used as dismutant active substances, their sufficient solubility in water is also very important.

It is also known that the dianion of Schiff's base, derived from salicylaldehyde and L-glutamic acid of formula (sal-L-gluH), i.e. N-salicylidene-L-glutamate of formula ΖΪ, Η..CH = N-CH / COO ^- / 2 - 6

-CH ₂ -CH ₂ -COOH 7 forms a copper complex of formula Cu (sal-L-gluH) .3 H ₂ O, where sal-L-gluH is the same as above. (Y. Nakao, K. Sakurai and A. Nakahara, Bull. Chem. Soc. Japan 40, 1536 (1967)). In the square-pyramidal structure of this complex, in addition to the trifunctional Uganda of the formula (sal-L-gluH), two H ₂ O molecules are coordinated to Cu (II), so that it is diacva- (N-salicy-human-L-glutamate) copper monohydrate. complex (J. Soldánová, J. Kratsmár-šmogrovič, F. Pavelčík, V. Seressová and M. Žemlička, Proc. 11th Conf. Coord. Chem., Smolenice 1987, pp. 365 -370). The coordinated L-form of the Schiff base in this complex racemizes by the action of strong bases, which was used for the simple preparation of the complex of formula Cu (sal-D, L-gluH). .3H ₂ O (J. Kratsmár-šmogrovič, V. Seressová, M. Žemlička and L. Gážová, Czech AO No. PV-6023-88). By neutralizing the terminal, uncoordinated carboxyl group in the glutamate part of the Schiff base in the complex of formula (Cu) / sal-L-gluH // H ₂ O / ₂ /.H ₂ O, where sal-L-gluH is the same as above, good soluble complex salt: potassium- (N-salicylidene-L-glutamate) potassium mednate dihydrate (F. Jursík and B. Hájek, Collection Czechoslovak Chem. Commun. 37, 1652/1972), which can also be used to prepare its sodium salt , resp. D, L-forms of the complex: aqua-N-salicylidene-D, L-glutamate / potassium or sodium citrate.

To date, however, it has not been known that chelation by ligands of the above composition, formula 2- 2- 3 "3-.

(sal-L-gluH), (sal-D, L-gluH) and (sal-L-glu), or (sal-D, L-gluz) affects the superoxide dismutase activity of Cu (II) ions by maintaining it in usable degree also in neutral, resp. weakly basic environment, typical for physiological conditions.

The process according to the invention consists in using solutions of the L- or D, L-form of the N-salicylidene glutamate-copper complex or N-salicylidene glutamate-mandate of the above composition, in standard in vitro test systems with radical radium as substrate, under physiological-like conditions.

The solution results in the dismutation elimination of superoxide α7 radicals, or their capture by the action of Cu / II / coordination compounds of the above composition, which represents their new, hitherto unknown effect, which exceeds the similar effect of many other copper complexes.

The following examples illustrate but do not limit the use of the method of the invention.

Example 1

The dismutase activity of aqua- (N-salicylidene-L-glutamate) potassium dihydrate dihydrate and, for comparison, the metalloenzyme Cu / Zn-SOD (a commercial preparation from SIGMA isolated from bovine erythrocytes) was determined by an indirect method in an in vitro system of xanthine xanthine oxidase, based on spectrophotometric determination of the inhibition of cytochrome c reduction, at λ = 550 nm.

The reaction mixture (1 ml) each contained 0.05 mol / l phosphate buffer, pH = 7.8; Disodium -4--5--5-ethylenediaminetetraacetate (1.10 mol), xanthine (5.10 mol), cytochrome c (1.10 mol) and xatine oxidase (0.01 U).

The unit of dismutase activity (1U) in said system is defined as the 50% inhibition of cytochrome c reduction by the determined mass amounts of the enzyme Cu / Zn-SOD, resp. test compound. Under these conditions, dismutase activity was achieved by 1U effect

CS 271 963 Bl

3.66.10 ^-4 mg Cu / Zn-SOD, or 3.94.10 ^-1 mg of the tested K / Cu / sal-L-glu // H ₂ ° / J · .2H ₂ O complex (0.093%).

Example 2

By testing the complex / Cu / sal-L-gluH // H ₂ O / ₂ JH ₂ O under the same conditions as in Example 1, a comparable activity was found (0.091%) as in Example 1.

Example 3

By testing the K / 7Cu / sal-D, L-glu // H, O /] complex under conditions identical to Example ^z- 1, 1U dismutase activity was obtained by the action of 3.95.10 mg of this compound (0.093%).

Example 4.

Dismuthase activity of complex compounds, (Examples 1 and 3):

ΚΓ Cu / sal-L-glu // H ₂ O / J.2 H ₂ O and K £ 7Cu / sal-D, L-glu // H ₂ O / J were also determined by an indirect method in an in vitro xanthine- xanthine oxidase, based on spectrophotometric detection of inhibition of the reduction of 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyltetrazolium chloride (INT) to formazan, at λ = 485 nm. The reaction mixtures (1 ml) each contained 0.05 mol / l of phosphate buffer (pH = 7.8), (1.10 ^-4 mol), xanthine (5.10 ^-5 mol), INT (9.8.10 ^-2 mol) and xanthine oxidase (0.01 U). Unit dismutase activity (1 U), characterized by 50% inhibition of formazan formation in the system, was manifested by the effect of:

9.71.10 ^-5 mg Cu / Zn-SOD (100%)

5.95.10 mg K [Cu / sal-L-glu // H ₂ O / J.2 H ₂ O (0.163%), or

5.37.10 ^-2 mg KC Cu / sal-D, L-glu // H ₂ O / J (0.187%).

Example 5

In addition, the dismutase activity of the Cu ¹¹ chelate complexes of Examples 1 and 3 was determined using them as scavengers of OJ superoxide anions in determining their formation by human polymorphonuclear leukocytes (PMN Leu) in zymosan phagocytosis.

The reaction mixtures (1 mL) contained heparinized (5 U / mL) phosphate buffer with 0.1% glucose, pH 7.4; 3.10 ⁵ PMN Leu, 25 moles of cytochrome c, 0.05 mg (150 U) Cu / Zn-SOD, or 0.05 mg, of test compound and suspended zymosan (20 particles per PMN Leu). In parallel, control mixtures prepared in the same composition but without Cu / Zn-SOD, resp. test compounds. After 15 min incubation at 37 ° C, the mixtures were cooled in an ice bath, centrifuged (10 min) at 78,500 ms.

From the difference in absorbance at A '= 550 nm of supernatants, reaction mixtures containing Cu / Zn-SOD, or test compounds, resp. without these components, the amounts formed O · were calculated using the molar absorption coefficient of cytochrome c (reduced-oxidized) ε = 9.6 * -2 nmol.cm.

The formation of Oj radicals, determined in the analyzed mixtures according to the mentioned method, results in the dismutase activity of Cu / Zn-SOD and the tested aqua-N-salicylideneglutamate mandates K / L- and D, L-forms / summarized in tab. 1.

TABLE 1

Scavenger pmoly 0.7 activity (%) Cu / Zn-SOD 7.7 100 Ki Ru / sal-L-Glu // _H2O / ^ .2 HJO 9.5 123.4 K £ Cu / sal-D, L-glu // H ₂ O / 7 6.9 89.6

Claims (1)

OBJECT OF THE INVENTION Process for influencing the catalytic activity of Cu (II) for the dismutant elimination of superoxide Ot radicals, characterized in that the substrate is treated with its coordination compounds in which it is chelated by a ligand of the general formula C oc ₆ h ₄ -ch = n-ch / coo ^- / -ch ₂ - ch ₂ -coorJ ² “where R is H, K ⁺ , or Na ⁺ , derived from salicylaldehyde and L-glutamic, respectively. D, L-glutamic acid.