RU2768707C1 - Method for producing fluorescent derivatives of arabinogalactan - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology and a method of producing novel luminescent-labeled biopolymers based on arabinogalactan. In particular, the described method of producing fluorescent arabinogalactan derivatives involves mixing a rhodamine dye with a cationite in NH4+form at room temperature for 1 hour, separation of aqueous solution and multiple washing of cationite with distilled water to colorless washing water; adding sulphated arabinogalactan to the cationite in the form of an ammonium salt and stirring at room temperature for 30 minutes; filtration; evaporation of water at 50–60 °C; washing solid residue with ethanol.EFFECT: high efficiency and environmental friendliness, as well as reduced duration of synthesis of fluorescent water-soluble derivatives of biopolymers.1 cl, 1 tbl, 3 ex

Description

The invention relates to the field of biotechnology and relates to a method for obtaining new luminescent-labeled biopolymers based on arabinogalactan, which can be used to visualize cell membranes, as well as to create drug delivery systems [S.A. Kuznetsova et al. Sulfated Derivatives of Arabinogalactan and Their Anticoagulant Activity. // Russ J Bioorg Chem 46, 1323-1329].

The most widely used fluorescent labels today are fluorescein and rhodamine derivatives because they can be appropriately excited by commonly used light sources such as an argon laser (488 nm and 514 nm lines) and a mercury arc lamp (546 nm line). However, they have some undesirable properties that may prevent their use. For example, fluorescein derivatives can be photobleached at a relatively high rate and exhibit a pH-dependent uptake that results in a significant decrease in fluorescence at physiological pH 7 or below. Rhodamine derivatives are relatively photostable and exhibit pH-insensitive fluorescence compared to fluorescein derivatives. They penetrate cells more easily than fluorescein derivatives. As a result, rhodamines are widely used in fluorescence microscopy, as fluorescent probes and molecular markers in chemistry and biochemistry, in flow cytometry and enzyme immunoassay [Yuan-Qiang Sun et al. Rhodamine-Inspired Far-Red to Near-Infrared Dyes and Their Application as Fluorescence Probes // Angew. Chem. Int. Ed. 2012, 51, 7634-7636].

Currently, fluorescent natural polymers are of interest due to their promising applications in biomedical labeling, tracing, imaging, and diagnostics, especially in drug delivery systems. A unique feature of natural polymers compared to synthetic ones is their ability to biodegrade. The combination of polysaccharides or polysaccharide derivatives with fluorescence methods leads to promising applications in the biomedical field, experimental medicine and biology.

Among polysaccharides, dextran, chitosan, heparin, etc. have been used to create their fluorescent derivatives. Organic fluorophores can be attached to polymer chains through covalent or non-covalent bonds and are capable of fluorescing in a wide range of wavelengths, depending on their nature. The synthesis of fluorescent polymers is usually carried out using two established methods. The first is through the polymerization of fluorescent monomers, and the second is through the attachment of fluorescent fragments to the polymer base due to covalent binding [V.N. Serova et al. Spectral and fluorescent characteristics of rhodamine 6 G in modified (co)polymethacrylate matrices. // Bulletin of the Kazan Technological University. 2012 15(6):111-113]. To preserve the properties of the natural polymer, the latter method is often more useful [Hun Min Lee et al. Simple Synthesis of Fluorophore Using Water-Soluble Chitosan Oligomer. // International Conference on Applied Mathematics, Simulation and Modeling (AMSM 2016). P 435-436].

However, most fluorescently labeled polymers are characterized by low fluorescence quantum yields, which may be due to changes in the structure of dye molecules caused by their covalent attachment to polysaccharide macromolecules. At the same time, fluorescent molecules used as probes have quantum yields starting from 5%. This makes it important to obtain fluorescent polymers by non-covalent interaction of the fluorophore with biomacromolecules, for example, due to electrostatic attraction [SA. Sealing et al. Influence of medium acidity on the spectral-fluorescent properties of solutions of poly-Schiff bases based on diamine-fluorophores. // Macromolecular compounds. 1996 Series A, vol. 38, no. 7, p. 1222-1227], [T.M. Akimkin et al. Spectral study of the non-covalent interaction of thiacarbocyanine dyes with hyaluronic acid. // High Energy Chemistry, 2011, Volume 45, No. 6, p. 553-558].

A general approach to the production of fluorescent polysaccharides based on fluorescein has been described [Charles G. Glabe et al. // Analytical Biochemistry. Volume 130, Issue 2, 15 April 1983, Pages 287-294]. When obtaining fluorescent polysaccharides, it is proposed to use cyanogen bromide for their activation. However, this reagent is highly toxic. Cyanogen bromide is volatile and readily absorbed through the skin or gastrointestinal tract. Therefore, toxic effects can occur through inhalation, physical contact, or ingestion.

A known method for producing labeled oxidized dextran, which includes the reaction of a fluorescent dye with activated oxidized dextran, followed by purification of the final product from impurities by ultrafiltration [RU 2426545. Published: 20.08.2011 Bull. No. 23. Method for obtaining fluorescent derivatives of dextrans]. Fluorescein is used as a fluorescent dye, and 1,1'-carbonyldiimidazole is used as an activating reagent. Purification of the final product from impurities is carried out by ultrafiltration on Vivaflow 200 membrane filters with a separation parameter of 10 kDa for dextran and PAD M _r =60 kDa, and 5 kDa for dextran and PAD Mr=35 kDa. The process includes 27 full concentration cycles. The degree of purification is controlled spectrophotometrically. Purity is checked by capillary electrophoresis.

The disadvantages of the method: the duration and multi-stage process, the use of expensive activating reagent - 1,1'-carbonyldiimidazole. Since the relative molecular weight of native dextran reaches hundreds of millions of daltons, it becomes necessary to carry out the hydrolysis of native dextran to obtain a drug with a given molecular weight distribution.

Recently, various fluorescent derivatives of chitosan (a natural polymer consisting of 1,4-linked N-acetyl-D-glucosamine and D-glucosamine subunits) have been synthesized. Most of the approaches for the synthesis of a dye-labeled chitosan molecular system due to its low solubility have been developed using chemical reagents and by covalent addition of fluorophores to the amino group of the biopolymer, such as fluorescein isothiocyanate, rhodamine B isothiocyanate.

In a method for producing chitosan labeled with rhodamine B isothiocyanate [Odette Ma et al. Precise derivatization of structurally distinct chitosans with rhodamine B isothiocyanate. // Carbohydrate Polymers 72 (2008) 616-624] chitosan is dissolved in acetic acid for 18 hours, an equal volume of anhydrous methanol is added to the chitosan solution, followed by stirring for 3 hours, then at room temperature for 18 hours in the dark, the process is carried out addition of rhodamine B isothiocyanate to the biopolymer. At the end of the labeling period with rhodamine B isothiocyanate - chitosans are precipitated by adding sodium hydroxide solutions, washed many times with deionized water and sublimated.

Disadvantages of the method: technological complexity, which consists in the duration of both the process of dissolving chitosan in acetic acid and attaching a fluorophore to it, the need to control the degree of protonation of chitosan, the use of toxic methanol, the process without access to light.

It is known to obtain fluorescent chitosan by chemical modification of chitosan with fluorescein isothiocyanate in dimethyl sulfoxide. The method is laborious and lengthy; toxic triethylamine is used in the synthesis. To purify the reaction product, dialysis is used, fractionation by chromatography on a biogel column [V.I. Gorbach, I.M. Yermak. Liposomes as carriers of sulfated polysaccharides from seaweed for their delivery to the body. // Health. Medical ecology. Science 3 (70) - 2017].

Described is a method for producing a chitosan oligomer labeled with fluorescein isothiocyanate, which consists of β-(1,4)-2-amido-2-deoxy-D-glucan and β-(1,4)-2-acetoamido-2-deoxy-D- glucan (acetylglucosamine). The derivatives have been synthesized by reacting the amine groups in the oligomer with the isothiocyanate groups of fluorescein isothiocyanate in ethanol [Hun Min Lee et al. Simple Synthesis of Fluorophore Using Water-Soluble Chitosan Oligomer. // International Conference on Applied Mathematics, Simulation and Modeling (AMSM 2016). R.435-436].

Due to the low solubility of chitosan, to convert it into a water-soluble state, this method includes an additional operation - obtaining a water-soluble chitosan oligomer by acidic or enzymatic hydrolysis of the original chitosan. This increases the duration of the production process labeled with chitosan isothiocyanate fluorescein, which is a significant disadvantage of this method.

A known method of obtaining fluorescent heparin. Fluorescent labeled heparin was prepared to study the effect of heparin on the interaction between biomacromolecules or cells using a fluorescent probe. Fluorescent heparin is obtained by reacting partially N-desulfurized heparin and 5-isothiocyanatofluorescein in carbonate buffer with stirring for 3 hours. Fluorescent heparin is isolated from the reaction mass on a Sephadex column. Dialysis and lyophilization are used to isolate and purify the fluoresceinylthiocarbamoyl derivative of partially N-desulfated heparin.

The disadvantages of the method: multi-stage and duration of the process; the use of a large number of reagents and methods for the purification of the target fluorescent heparin; the need for fractionation of heparin and the preliminary operation of partial N-desulfation in a toxic pyridine environment [Hideki Uchiyama and Kinzo Nagasawa. Preparation of Biologically Active Fluorescent Heparin Composed of Fluorescein-Labeled Species and Its Behavior to Antithrombin III. //1 Biochem. 89, 185-192 (1981)].

Closest to the proposed method is the "Method for obtaining chitosan derivatives for visualizing cell membranes and creating drug delivery systems with increased mucoadhesion" [RU 2697872, 21.08.2019]. To do this, chitosan is first acylated with caproic anhydride, then alkylated in the presence of glycidyltrimethylammonium chloride, followed by dialysis and freeze drying of the product, then fluorescein isothiocyanate is added. The resulting chitosan derivative has a polycation structure. Fluorescein isothiocyanate is a negatively charged fluorescent dye that is a fluorescent probe for biological experiments. The disadvantages of this method are the duration and multi-stage process of synthesis and purification of the fluorescent derivative of chitosan, the use of toxic methanol, in addition, chitosan has a low solubility in water.

The objective of the invention is to develop a simple, convenient and environmentally friendly method for attaching a fluorescent dye to a biopolymer, as well as to increase the efficiency and environmental friendliness of the synthesis of fluorescent water-soluble derivatives of biopolymers, increase the reliability of visualization, and reduce the duration of the synthesis of fluorescent water-soluble derivatives of biopolymers.

The technical result is achieved due to the fact that when implementing the method for obtaining fluorescent derivatives of arabinogalactan in the first stage, the rhodamine dye is mixed with cation exchanger in NF ₄ ⁺ -form at room temperature for 1 hour. Then the aqueous solution is separated and the cation exchange resin is repeatedly washed with distilled water until colorless washings are obtained. Next, sulfated arabinogalactan in the form of an ammonium salt is added to the cation exchanger and stirred at room temperature for 30 minutes, filtered, and water is evaporated at 50-60°C. The solid residue is washed with ethanol.

The proposed method differs from the prototype:

1) the nature of the polysaccharide: in the prototype - chitosan, which is slightly soluble in water, in the claimed method, the ammonium salt of sulfated arabinogalactan isolated from Siberian larch was used to obtain a fluorescent derivative. Polyfunctionality, water solubility, and membranotropism of arabinogalactan are clear advantages over other synthons (chitosan, dextran, heparin) used for the synthesis of biologically active fluorescent conjugates. Sulfated arabinogalactan has anticoagulant and hypolipidemic activity, is a potential heparinoid, and also has great prospects as an independent antimicrobial agent compared to other matrices due to the fact that this modified biopolymer is practically non-toxic, highly soluble in water, and the inheritance of the nanostructured morphology of the original arabinogalactan makes it possible to preserve its immunomodulatory properties. and transmembrane properties in the synthesis of new physiologically active polymers, including pharmaceutical and medical preparations [21-28];

2) simplification of the method by reducing the number of stages and using a fluorescent dye by ion exchange to introduce a fluorescent dye into the structure of the polysaccharide (sulfated arabinogalactan);

3) the nature of the fluorescent label (in the prototype - fluorescein isothiocyanate, in the claimed method - rhodamine B - a fluorescent dye, the advantages of which over fluorescein derivatives are that it is relatively photostable, exhibits pH-insensitive fluorescence and penetrates into cells more easily than fluorescein derivatives) .

Thus, thanks to these distinctive features, by reducing the stages and using a simple ion exchange method, it was possible to achieve the efficiency of the process of attaching a fluorescent dye to a biopolymer, to obtain fluorescent water-soluble, low-toxic derivatives of arabinogalactan biopolymer, which have a quantum yield sufficient for visualization due to non-covalent binding of a fluorophore to a macromolecule polysaccharide.

The method is carried out as follows.

The process includes three stages.

The first stage - sulfation of arabinogalactan is carried out according to the method described in the patent [RU 2521649, publ. 07/10/2014].

The second stage is the fixation on the KU-2-8 cation exchanger in the NH ₄ ⁺ form of the fluorescent dye rhodamine B.

The third stage is the introduction of a fluorophore, rhodamine B, into the structure of the sulfated polysaccharide.

The method is confirmed by specific examples. Example 1

Arabinogalactan produced by Chemistry of Wood LLC (Irkutsk, Russia) under the name of the drug "FibrolarS" isolated from Siberian larch wood (Larix sibirica Ledeb.) is sulfated according to the method described in the patent [RU 2521649, publ. 07/10/2014]. The sulfation process is carried out at a temperature of 80°C for 2 hours. At the end of the sulfation process, a product containing 8.2% sulfur is isolated.

To fix the rhodamine dye (rhodamine B) on the cation exchanger KU-2-8, which is in the NH ₄ ⁺ -form, 0.1 g of the corresponding dye in 30 ml of water is added to 5 ml of the swollen cation exchanger and the mixture is stirred at room temperature for 1 hour . An aqueous solution is separated from the cation exchanger, the modified cation exchanger is washed repeatedly with portions of water, 20–30 ml each, until colorless washings are obtained, then the cation exchanger modified with rhodamine dye is mixed with an aqueous solution of 0.3 g of sulfated arabinogalactan (sulfur content 8.2% (mass.)) in 20 ml of water at room temperature for 30 min. An aqueous solution of sulfated arabinogalactan with rhodamine B attached to it is separated from the cation exchange resin by filtration, evaporated at a temperature of 50-60°C, the resulting solid residue is washed with ethanol to remove the dye not attached to the polysaccharide to colorless washing ethanol solutions. Get 0.25 g of water-soluble target product with a quantum yield of 17±2%.

Examples 2.3. Analogously to example 1, the difference in the sulfur content in sulfated arabinogalactan (see table). Depending on the conditions of the sulfation process, sulfated arabinogalactan is obtained containing 10.1÷12.8% (wt.) sulfur. Sulfated arabinogalactan containing 10.1% sulfur (example 2) is obtained by sulfation of arabinogalactan at a temperature of 80°C for 3 hours; containing 12.8% sulfur (example 3) - at 90°C for 2 hours. During the sulfation of arabinogalactan, with an increase in both the duration of the process and the temperature greater than 90-100°C, the sulfur content in sulfated arabinogalactan increases slightly, but the process is accompanied by a greater degree of depolymerization of the original and sulfated arabinogalactan. With an increase in the sulfur content, a slight increase in the quantum yield occurs.

Thus, by adding a fluorescent dye to sulfated arabinogalactan using a simple ion exchange method, fluorescent water-soluble derivatives of arabinogalactan have been synthesized with the quantum yield necessary for visualization. All samples obtained as a result of synthesis fulfill the task in view of the studied range of changes in the sulfur content in the initial sulfated arabinogalactan.

Claims (1)

The way to obtain fluorescent derivatives of arabinogalactan, including mixing rhodamine dye with cation exchanger in NH ₄ ⁺ -form at room temperature for 1 hour; separation of the aqueous solution and repeated washing of the cation exchanger with distilled water to colorless washings; adding sulfated arabinogalactan in the form of ammonium salt to the cation exchanger and stirring at room temperature for 30 minutes; filtration; evaporation of water at 50-60°C; washing the solid residue with ethanol.