Method of obtaining fluorescent derivatives of catecholamines and their metabolites by method of derivatisation

FIELD: chemistry.

SUBSTANCE: invention relates to a novel method of obtaining fluorescent catecholamines selected from dopamine and adrenalin, and their metabolites, selected from homovanillic and vanillin-mandelic acids, by a method of derivation. The compounds can be used as highly sensitive and selective markers for the determination of various diseases. The method of derivation includes oxidation of the initial compounds and their interaction with amines that form condensed structures in a medium of the CAPS-buffer solution or glycin - KOH 0.2 mM hydrogen peroxide in the presence of horseradish peroxidase as a catalyst. The process in preferably carried out in a 0.1 M buffer solution with the concentration of horseradish peroxidase 0.01-1 mcM; concentration of hydrogen peroxide - 100 mcM, amine concentration - 0.1-33 mM; concentration of catecholamines and metabolites - 0.03-1 mcM.

EFFECT: method is simple and producible as it does not require higher temperature and is realised in a water solution.

2 cl, 2 dwg, 3 ex

 

The invention relates to medicine and can be used for highly sensitive and selective detection of markers of various diseases.

The problem with this definition is an important task of modern chemical analysis. Currently the most common markers of various diseases are physiologically active substances, such as catecholamines (CA) (dopamine (DA), adrenaline (AD)), and their main metabolites (gomovanilinovoi and vanillylmandelic acid (GVK and CMC, respectively)). The possibility of determination of these compounds in biological fluids and human tissues at levels mu concentrations and lower (according to studies in related biochemical analysis fields) allows you to diagnose various diseases in its earliest stages.

A significant problem in the determination of trace quantities of CA and their metabolites in real biological objects occurs when they contain components absorbing in the visible region of the spectrum and thereby interfering matrix effects. To improve not only the sensitivity of the method to determine these compounds (at the level of mu concentrations), but also selectivity and promising technique, which consists in the translation of the definition of short wavelength in the visible region of the spectrum (λex 340-355 and λem470-485 nm), where there are no matrix effects of biological objects by derivatization of SPACECRAFT and their metabolites organic molecules. Furthermore, the resulting compounds have significantly more intense fluorescence than the original-defined compounds, which increases not only the selectivity but also the sensitivity analysis.

Known method for the derivatization of SPACECRAFT and their metabolites 0,1 M diphenylethylenediamine (DEAD) in the presence of potassium ferrocyanide, catalyzing the oxidation of the dissolved oxygen molecules KA up to KA-o-quinones, which in turn interact with derivatization reagent (DEAD) by Michael's reaction (N. Nohta, A. Mitsui, Y. Ohkura. High-performance liquid chromatographic determination of urinary catecholamines by direct pre-column fluorescence derivatization with 1,2-diphenylethylenediamine. J. Chromatogr. 380 (1986) 229-231.). The end product derivatization with KA are DEAD 2-phenyl(4,5-dihydropyrrolo)[2,3-f]benzoxazole derivatives, having a more intense fluorescence than the KA and their metabolites in the visible region of the spectrum. The process takes place in a mixture of buffer solution and methanol: 0.3 M CAPS - CON with a pH of 10 as methanol (30:70 vol.%). Oxidation with air oxygen in the presence as catalyst of a 20 mm solution of ferrocyanide K3[Fe(CN)6], at temperature T=50°C for 20 min.

However, the low adaptability of this process is due to�the selection of toxic solvent 70%. methanol, the need for temperature control of the reaction mixture at an elevated temperature of 50°C, the duration time of obtaining derivatives of catecholamines and their metabolites necessary intensity, which is 100 Rel. units.

Known adopted as a prototype (K. V. of Contributors. New aspects of native and immobilized horseradish peroxidase for the determination of inhibitors and substrates. Author's abstract on competition of degree of candidate of chemical Sciences, majoring in Analytical chemistry 02.00.02. Moscow. 2010. 22 p) derivatization method of SPACECRAFT and their metabolites 0.5 M benzylamine (BA) or diphenylethylenediamine (DEAD) in micellar and aqueous-organic medium in the presence of peroxidase from horseradish roots, catalyzing the oxidation by hydrogen peroxide molecules to KA up to KA-o-quinones, which in turn interact with derivatization reagent, which was used benzylamine (BA) and diphenylethylenediamine (DEAD) by Michael's reaction.

The process takes place in 5 mm solution of CTMA in 0.1 M CAPS - KOH pH 11 (derivateservlet agent - BA), or 0.5 M buffer solution glycine - KOH, DMSO (70:30 vol.%, derivateservlet agent - DED), the hydrogen peroxide concentration of 1 mm, t=30 min (less time 5 min derivatization was achieved only for adrenaline), T=37°C.

The end product derivatization KA BA or DEAD are 2-phenyl(4,5-DIH�droprole)[2,3-f]benzoxazole derivatives, having in the visible region of the spectrum more intense fluorescence than the KA and their metabolites.

However, this method has the same drawbacks as the above analog: low manufacturability of this process due to the presence of toxic solvent 30 vol.% DMSO and incubation of the reaction mixture at an elevated temperature of 37°C.

The present invention solves the problem of increasing the efficiency of formation of fluorescent derivatives of catecholamine while improving the manufacturability of the process.

The problem is solved by a method of producing a fluorescent derivatives of catecholamines and their metabolites derivatization method, comprising the oxidation of parent compounds and their processing forming a condensed structure of amines, in the presence of a catalyst - peroxidase from horseradish roots. The novelty in this case is that the oxidation of parent compounds catalyzed by horseradish peroxidase, carried out in the presence of 0.1 M CAPS buffer solution (pH 11) and 100 μm solution of hydrogen peroxide. The increase in 5 times the concentration of the used buffer solution and a 10 times reduction in the concentration of hydrogen peroxide in comparison with the prototype leads to the stabilization of the biocatalyst - horseradish peroxidase and reduced denaturing step n� oxidant enzyme - peroxide moderadores carried out in aqueous solution at room temperature, i.e. increases the efficiency of peroxidase in the claimed process derivatization of catecholamines and their metabolites by means of amines, which allows to obtain fluorescent derivatives of the same intensity (100 Rel. units), but room temperature (without temperature control) and in the aquatic environment (in the absence of organic solvents and surfactants, which reduce the durability of devices and consumables to the emergence of environmental problems).

The most intense signal is obtained when carrying out the process in 0.1 M CAPS buffer solution at a concentration of horseradish peroxidase is 0.01 - 1 μm; the concentration of hydrogen peroxide is 100 μm, the concentration derivatizing agent amine is 0.1 to 33 mm; the concentration of catecholamines and metabolites - 0.03 to 1 micron.

The highest intensity of the fluorescence signal obtained using as amines benzylamine, diphenylethylenediamine or o-phenylenediamine. Last for derivatization stated catecholamines and metabolites were not applied.

The technical result of the invention thus consists not only in reducing the reaction time from 30 to 5 min, but also the improvement of the technological process due to exception of prolonged incubation of the reaction mixture at a value� temperature in the presence of toxic solvents (methanol, DMSO).

Selection oxidative systems by varying the nature of the biocatalyst and oxidizer (horseradish peroxidase - hydrogen peroxide, mushroom tyrosinase is the oxygen of the air, laccase - oxygen) showed that only the selected authors of the present invention in the inventive system conditions combines high technology process with high yield obtained by the derivatization products.

Analysis of the known technical solutions allows to make a conclusion that the invention is not known from the prior art that demonstrates its compliance with the criterion of "novelty."

The essence of the present invention for professionals not obvious from the prior art, which allows to make a conclusion about its compliance with the criterion of "inventive step".

The possibility of carrying out of the method on the traditional equipment with the achievement of the objectives testifies to the invention, the criterion of "industrial applicability".

Fig. 1 shows absorption spectra of HELL (1), HELL-o-quinone product of the peroxidase oxidation of HELL (2) and 2-phenyl-4,5-dihydropyrrole-2-hydroxy-3-methyl-benzoxadiazole - product derivatization HELL with BA (3), (garden - 30 μm, the reaction time is 5 min).

Fig. 2 shows the mass spectrum of the products of derivatization of adrenaline with a BA in optimum conditions of the reaction.

These examples suggest, but do not limit the claimed invention.

Example 1. The method of obtaining 2-phenyl-4,5-dihydropyrrole-2-hydroxy-3-methyl-benzoxazole, a fluorescent derivative of catecholamines by enzymatic derivatization HELL with BA

As a derivative of catecholamines was taken adrenaline (AD), as derivatizing agent benzylamine (BA).

The reaction derivatization HELL with asthma was performed by the following method: in the glass vial was sequentially introduced to 2.7 ml of 0.1 M CAPS buffer solution-KOH (pH of 11.0), 0.100 ml of a 0.1 M solution of BA (33 mm), 0.100 ml of a 30 μm solution of peroxidase (1 μm), a solution of AD (30-300 nm) and 0.100 ml of 3 mm solution of H2O2(0.1 mm). The reaction mixture was mixed thoroughly, transferred to a quartz cuvette and measured the intensity of fluorescence fluorimetry method. After 5 minutes of the start of the reaction (λex=350 nm, λem=495 nm). These results indicate that the increase in the fluorescence intensity at about 1.5 times through one and the same time after start of the reaction compared to the ways derivatization of catecholamines and their metabolites in the presence of an inorganic catalyst.

The identity of composition of the products of derivatization of catecholamines on the example of adrenaline (AD) in the claimed conditions and according to the method prototype (used with�Itanium as a catalyst of potassium ferrocyanide) was monitored by the methods of spectrophotometry(Fig. 1+ and liquid chromatography / mass spectrometry.)

From the absorption spectra it is seen that in the case of peroxidase oxidation of adrenaline that belongs to a class of catecholamines, there is a maximum absorption at 300 nm corresponding to the products of its oxidation, the molecules of which contain o-heneidy fragment. In the spectrum of absorption of the products of derivatization HELL in the presence of potassium ferrocyanide has a maximum at 350 nm corresponding to the wavelength of fluorescence excitation of derivatized HELL. The obtained spectral data provide indirect evidence about the identity of the products of derivatization HELL, obtained in the presence of HRP and potassium ferrocyanide.

For a more rigorous proof of identity of the reaction products derivatization KA, obtained in the presence of HRP and potassium ferrocyanide, studied the mass spectra of the products of derivatization of adrenaline with BA (Fig. 2). The data obtained are strictly in line with literature data (Justin P. Pennington, Christian Schoneich, John F. Stobaugh. Selective Fluorogenic Derivatization with Isotopic Coding of Catechols and 2-Amino Phenols with Benzylamine: A Chemical Basis for the Relative Determination of 3-Hydroxy-tyrosine and 3-Nitro-tyrosine Peptides.Chromatographia November 2007, Volume 66, Issue 9-10, pp. 649-659, K. B. Of Contributors. New aspects of native and immobilized horseradish peroxidase for the determination of inhibitors and substrates. Author's abstract on competition of degree of candidate of chemical Sciences, majoring in Analytical�th chemistry 02.00.02. Moscow. 2010. 22 p).

On the mass spectrum shows peaks of molecular ions, confirming the identity of the final products of enzymatic and non-enzymatic (method prototype) derivatization (last described in the literature).

Example 2. The method of obtaining 2-phenyl-4,5-dihydropyrrolo-benzoxazole, a fluorescent derivative of catecholamines on the example of enzymatic derivatization YES with DEAD.

As a derivative of catecholamines was taken dopamine (DA), as derivatizing agent - diphenylethylenediamine (DEAD).

The derivatization reaction with DEAD was performed by the following method: in the glass vial was sequentially injected with 2.7 ml of 0.5 M buffer solution, glycine-KOH (pH 8.0), 0,010 ml of a 0.1 M solution of DEAD (33 mm), 0.100 ml of a 30 μm solution of peroxidase (1 μm), the solution is YES (30-300 nm) and 0.100 ml of 3 mm solution of H2O2(100 μm). The reaction mixture was mixed thoroughly, transferred to a quartz cuvette and measured the fluorescence intensity after 5 min from the beginning of reaction (λex=350 nm, λem=495 nm).

Example 3. The method of obtaining 2-phenyl-benzoxazol-6-acetic acid, a derivative of catecholamine metabolites, for example, enzymatic derivatization with GVK BA.

As a derivative of catecholamine metabolites was taken gomovanilinovoi acid (GVK), as derive�tiirough agent benzylamine (BA).

The derivatization reaction with GVK BA conducted by the following method: in the glass vial was sequentially introduced to 2.7 ml of 0.1 M CAPS buffer solution-KOH (pH of 11.0), 0.100 ml of a 0.1 M solution of BA (33 mm), 0.100 ml of a 30 μm solution of peroxidase (1 μm), the solution GVK (0.1 to 10 μm in the system) and 0.100 ml of 3 mm solution of H2O2(100 μm). The reaction mixture was mixed thoroughly, transferred to a quartz cuvette and measured the fluorescence intensity after 5 min from the beginning of reaction (λex=350 nm, λem=430 nm).

As can be seen from the above examples, the present invention solves the problem of obtaining fluorescent derivatives of catecholamines and their metabolites, as well as more intensely fluorescent derivative in less time while improving the manufacturability of the process by reducing the reaction time (from 30 to 5 min), non-heating and temperature control, the use of toxic or corrosive organic solvents.

1. A method of producing a fluorescent derivative of a catecholamine selected from dopamine and epinephrine, and their metabolites selected from homovanillic and vanillylmandelic acids, derivatization method, comprising the oxidation of parent compounds and their interaction with forming a condensed structure of amines in the presence of a catalyst, ex�featuring the what derivatization compounds is carried out in the presence of CAPS buffer solution or glycine-KOH 0.1 mm hydrogen peroxide using the catalyst of horseradish peroxidase.

2. A method according to claim 1, characterized in that the process is carried out in a 0.1 M buffer solution at a concentration of horseradish peroxidase is 0.01 - 1 μm; the concentration of hydrogen peroxide is 100 μm, the concentration of amine is 0.1 to 33 mm; the concentration of catecholamines and metabolites of 0.03-1 μm.



 

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