RU2425051C1 - Method of producing d-glucose labelled with carbon isotope in position 1 - Google Patents

Abstract

FIELD: chemistry. ^ SUBSTANCE: invention relates to a method of producing D-glucose which is labelled with carbon isotopes 13C or 14C in position 1, involving condensation of D-arabinose with labelled nitromethane, taken in equimolar ratio, in dimethylsulphoxide in the presence of a base, followed by benzene extraction of excess dimethylsulphoxide, freeze-drying from benzene, cooling the reaction mixture of 1-desoxy-1-nitroalditol salts to liquid nitrogen temperature, adding water, followed by slow thawing while stirring and decomposition of 1-desoxy-1-nitroalditol salts contained in the obtained aqueous solution, and extraction of D-glucose via chromatography. Said method increases output and improves the process. ^ EFFECT: obtaining products used in medicine as isotopic diagnostic agents, as well as in scientific research and other fields. ^ 3 cl, 4 ex

Description

The invention relates to organic chemistry, in particular to methods for producing hexoses containing carbon isotopes ¹³ C or ¹⁴ C, in particular D-glucose labeled with a carbon isotope in position 1. The resulting products can be used in medicine as isotopic diagnostic tools, and in scientific research and other fields. For example, 1- ¹³ CD-glucose can be used to diagnose cancer.

In [JCSowden, HOFischer, J. Am. Chem. Soc. 69, 1963 (1947)] a method for producing D-glucose by condensing D-arabinose with nitromethane in methanol in the presence of sodium methylate and subsequent decomposition of 1-deoxy-1-nitroaldite salts by the action of sulfuric acid at low temperature is disclosed. The disadvantage of the proposed method is the low yield of the target product due to the large loss of salts of 1-deoxy-1-nitroaldites due to their retrodecay upon separation from the reaction medium by treatment with cation exchange resin and subsequent crystallization. Another serious drawback of this technique is the need to use a large excess of nitromethane (11 equiv.), Which makes this approach not applicable for the synthesis of isotope-labeled glucose. Later this same method was applied for the synthesis of 1- ¹⁴ C-glucose, on the basis of ¹⁴ C-labeled nitromethane [JCSowden, J. Biol. Chem. 180, 55 (1949)]. The yield of glucose per nitromethane in both cases does not exceed 6%.

A similar approach was used in [K.-O.Schoeps, B. Langstrom, S. Stone-Elander, C. Halldin, Appl. Radiat. Isot, 42, 877 (1991)] for the synthesis of 1- ¹¹ C-glucose, but dimethyl sulfoxide (DMSO) was used instead of methanol as solvent and as base - NaOH aqueous solution. Sulfuric acid was also used in this work to decompose salts of 1-deoxy-1-nitroaldites. The reaction mixture was neutralized on a column with anion exchange resin, DMSO was separated on a column with an amine phase, and the monosaccharide was isolated by HPLC. For the synthesis used a lack of nitromethane (0.7 equiv.); while the yield of glucose per nitromethane was 10%. The proposed method is not suitable for the synthesis of large quantities of glucose, because scaling this process requires expensive preparative HPLC columns and is not cost effective. In addition, a feature of both of the above methods is the acid decomposition of salts of 1-deoxy-1-nitroaldites, which requires the neutralization of the acid with a large amount of anion exchange resin or barium hydroxide, which is extremely inconvenient from a preparative point of view when conducting synthesis on a large scale, although acceptable for small-scale syntheses .

Therefore, another variant of the nitromethane method for producing L-glucose from L-arabinose was proposed, where the decomposition of 1-deoxy-1-nitroaldite salts to free monosaccharides was carried out by ozone [Patent 278067 SK, C07H 3/02, 02/19/1992] (prototype), which practically does not limit the scaling of the synthesis. In this work, the condensation of nitromethane with arabinose was carried out in the presence of sodium methylate in DMSO, the excess of which was removed by filtering the precipitate of 1-deoxy-1-nitroaldite salts, followed by their washing with alcohols. After treating the obtained salts with ozone, the resulting D-glucose was isolated from the reaction mixture by crystallization in the form of phenylhydrazone. The disadvantage of this method is the need for filtering salts of 1-deoxy-1-nitroaldite, followed by washing with n-butanol and a mixture of n-butanol and methanol in air in order to separate dimethyl sulfoxide, during which partial decomposition of these salts and formation of side impurities proceeds, which led the authors purify L-glucose in the form of phenylhydrazone and only then isolate it in free form using ligand exchange chromatography. In addition, a significant excess of nitromethane (2.8 eq.) Is used in this method, which greatly increases the cost of this approach to the synthesis of isotope-labeled glucose. The yield of glucose per nitromethane was 7%.

The objective of the invention was to develop an effective method for producing labeled D-glucose labeled in position 1, providing increased yield and process improvement. The problem is solved in that the condensation of D-arabinose with labeled nitromethane, taken in an equimolar ratio, is carried out in a solution in dimethyl sulfoxide in the presence of a base. Sodium methylate or sodium hydroxide is used as the base. To separate the excess dimethyl sulfoxide, extraction of the reaction mixture with benzene is used at room temperature, after lyophilization of the benzene residues, sodium salts of 1-deoxy-1-nitroaldite are obtained in the form of a dry powder or dry foam. The resulting mixture of salts of 1-deoxy-1-nitroaldite is cooled to the temperature of liquid nitrogen and water is added. Subsequent slow thawing with stirring allows you to get an aqueous solution of sodium salts of 1-deoxy-1-nitroaldite under mild conditions. The decomposition of the salts of 1-deoxy-1-nitroaldites contained in the resulting aqueous solution by the action of sulfuric acid or ozone results in a mixture of D-glucose, D-mannose and D-arabinose, from which labeled D-glucose is isolated by ligand exchange chromatography.

A feature and one of the advantages of the proposed method is the use of an equimolar (1: 1) ratio of reagents (arabinose and labeled nitromethane), which allows to increase the efficiency of the use of isotopic raw materials, minimizing its consumption, and thereby increase the yield of target glucose based on the nitromethane introduced into the reaction . Using this technique, synthetic glucose was first obtained with a yield of 14% based on nitromethane introduced into the reaction.

The invention is illustrated by the following examples of its implementation.

Example 1

1 g of D-arabinose (6.66 mmol) was dissolved in 6 ml of DMSO, 360 μl (6.66 mmol) of labeled nitromethane ( ¹³ CH ₃ NO ₂ ) was added with stirring, the flask was filled with argon, 2 ml of MeONa solution was added with stirring. MeOH (obtained from 212 mg or 8.8 mmol Na) and stirred at room temperature for 16 hours. Methanol was distilled off in vacuo without heating, 40 ml of benzene was added to the resulting solution with vigorous stirring, the resulting suspension was centrifuged, and the supernatant was discarded. The extraction with benzene was repeated two more times until the complete absence of DMSO in the extract (control - the absence of non-volatile residue after evaporation of benzene). The resulting yellow precipitate was frozen and lyophilized. Received 2 g of a yellow powder, which was frozen in liquid nitrogen, added 10 ml of water, allowed to thaw the formed ice and ozone was blown into the resulting solution at + 10 ° C until the color of the reaction mixture substantially disappeared. The remainder of the ozone was blown off, sparging argon through the resulting solution. The reaction mixture was deionized by sequential treatment with ion exchange resins Amberlyst 15 (H ⁺ ) and Dowex 1 × 8 (HCO ₃ ^- ), the solution was lyophilized. The resulting syrup was fractionated on a Dowex 50 × 8 (Ca ²⁺ ) column, eluting with water. The yield of D-glucose labeled with ¹³ C isotope was 170 mg (14.0%).

Example 2

The synthesis was carried out analogously to example 1, however, after adding labeled nitromethane ( ¹³ CH ₃ NO ₂ ) to the flask, 670 μl of a 10 N solution of NaOH in water (6.7 mmol) was added to the flask with stirring and further stirred at 40 ° C for 30 min. The solution was cooled to room temperature and 40 ml of benzene were added with vigorous stirring and further treated as described in Example 1 until DMSO was completely removed. The resulting yellow syrup was frozen and lyophilized. The resulting yellow foam was subjected to ozonolysis, as described in example 1, blowing ozone to a pH of the reaction mixture of 7.5-8.0 (5.5 min). The yield of D-glucose labeled with ¹³ C isotope was 125 mg (10.3%).

Example 3

The synthesis was carried out analogously to example 2, but used a half load: 500 mg of D-arabinose (3.33 mmol), 3 ml of DMSO, 180 μl of labeled nitromethane ( ¹³ CH ₃ NO ₂ ) (3.33 mmol) and 350 μl of 10 N solution NaOH (3.5 mmol). The yellow foam obtained after separation of DMSO by extraction with benzene was frozen in liquid nitrogen, 10 ml of water was added, and the ice formed was thawed. The resulting solution was added with vigorous stirring to a mixture of 1.1 ml conc. H ₂ SO ₄ and 1.4 ml of water at -20 ° C. Allowed to warm to + 10 ° C with stirring. The solution was diluted with water to a volume of 30 ml, the acid was neutralized with Dowex 1 × 8 anion exchanger (HCO ₃ ^- ) to pH 7, the resin was filtered. The filtrate was desalted by sequential treatment with Dowex 50 × 8 (H ⁺ ) cation exchanger and Dowex 1 × 8 anion exchanger (HCO ₃ ^- ). The filtrate was frozen and lyophilized. The resulting syrup was fractionated on a Dowex 50 × 8 (Ca ²⁺ ) column, eluting with water. The yield of D-glucose labeled with isotope C was 71 mg (11.5%).

Example 4

2.5 g of D-arabinose (16.65 mmol) was dissolved in 15 ml of DMSO, 900 μl (16.65 mmol) of labeled nitromethane ( ¹³ CH ₃ NO ₂ ) was added with stirring, the flask was filled with argon, 670 mg of NaOH powder was added with stirring ( 16.65 mmol) and stirred at 40 ° С h. 40 ml of benzene were added to the resulting solution with vigorous stirring, the resulting suspension was centrifuged, the supernatant was discarded. The extraction with benzene was repeated two more times until the complete absence of DMSO in the extract (control - the absence of non-volatile residue after evaporation of benzene). The resulting yellow precipitate was frozen and lyophilized. The resulting yellow powder was frozen in liquid nitrogen, 15 ml of water was added, the ice formed was thawed, and ozone was blown into the resulting solution at + 10 ° C to pH≈7. The remainder of the ozone was blown off, sparging argon through the resulting solution. The reaction mixture was deionized by sequential treatment with ion exchange resins Amberiyst 15 (H ⁺ ) and Dowex 1 × 8 (HCO ₃ ^- ), the solution was lyophilized. The resulting syrup was fractionated on a Dowex 50 × 8 (Ca ²⁺ ) column, eluting with water. The yield of D-glucose labeled with ¹³ C isotope was 378 mg (12.5%).

Similarly, when using ¹⁴ C-labeled nitromethane ( ¹⁴ CH ₃ NO ₂ ), D-glucose labeled with the ¹⁴ C isotope can be obtained. The advantage of this method is that the benzene extraction of DMSO at room temperature allows you to quickly get sodium salts of 1-deoxy-1-nitroaldites in dry form without noticeable decomposition of the latter. This increases the storage stability of sodium salts of 1-deoxy-1-nitroaldite, increases the yield of target D-glucose calculated on the basis of nitromethane introduced into the reaction, and also sharply reduces the amount of dimethyl sulfoxide impurity, which greatly facilitates the control of the completeness of salt conversion during ozonolysis and the isolation of the target product. The proposed benzene extraction of dimethyl sulfoxide from the reaction mixture greatly simplifies the method for producing D-glucose and has not previously been used in such a process.

To reduce the decomposition of sodium salts of 1-deoxy-1-nitroaldite when used in water, an original technique was used, which consisted of pre-cooling the vessel containing dry sodium salts of 1-deoxy-1-nitroaldite to the temperature of liquid nitrogen and quickly adding the required amount of water, which at the same time immediately freezes. Thawing the resulting salt mixture with stirring allows one to obtain an aqueous solution of sodium salts of 1-deoxy-1-nitroaldite under mild conditions. This procedure also increases the yield of the target product, D-glucose, based on the nitromethane introduced into the reaction.

The separation of monosaccharide is carried out using ligand exchange chromatography in water, which allows you to confidently clean gram quantities of mixtures and greatly simplifies the procedure for the isolation of the final product.

One of the advantages of the proposed method is also the use of an equimolar (1: 1) ratio of reagents (arabinose and nitromethane), which allows to increase the efficiency of the use of isotopic raw materials, minimizing its consumption.

Comparison of various methods for the synthesis of glucose (Glc) from arabinose (Aga) by the nitromethane method Way Ara, mmol CH ₃ NO ₂ , mmol CH ₃ NO ₂ / Ara, mol / mol Solvent removal Method for the decomposition of salts of 1-deoxy-1-nitroaldites The output of Glc calculated on CH ₃ NO ₂ ,% Glc extraction method (Patent 278067 SK) 66.6 185 2.78 Filtration O ₃ 7 The formation of phenylhydrazone, Prototype crystallization, then ligand-exchange chromatography Example 1 6.66 6.66 1,0 Benzene extraction O ₃ fourteen ligand exchange chromatography Example 2 6.66 6.66 1,0 Benzene extraction O ₃ 10 ligand exchange chromatography Example 3 3.33 3.33 1,0 Benzene extraction H ₂ SO ₄ 12 ligand exchange chromatography Example 4 16.65 16.65 1,0 Benzene extraction O ₃ 12 ligand exchange chromatography

Claims (3)

1. The method of producing D-glucose labeled with a carbon isotope in position 1, which consists in the condensation of D-arabinose with labeled nitromethane taken in an equimolar ratio in dimethyl sulfoxide in the presence of a base, subsequent benzene extraction of excess dimethyl sulfoxide, lyophilization from benzene, cooling the resulting mixture of salts 1-deoxy-1-nitroaldites to the temperature of liquid nitrogen, adding water, followed by slow thawing with stirring and decomposition of the salts of 1-deoxy-1-nitroaldites contained in the resulting aqueous solution, and isolation of D-glucose by chromatography. 2. The method according to claim 1, characterized in that the base used is sodium methylate or sodium hydroxide. 3. The method according to claim 1, characterized in that sulfuric acid or ozone is used to break down the sodium salts of 1-deoxy-1-nitroaldites.