Divalent l-glutamic acid diether-based neoglycoconjugates and production method thereof

FIELD: chemistry.

SUBSTANCE: use of two carbohydrate residues in the structure of a divalent neoglycoconjugate enables sixfold increase in binding efficiency compared to the monovalent derivative of monolactosyl-L-glutamate-succinate-dihexadecyl-L-glutamic acid, which can be used to produce high-efficiency directed systems for delivering medicinal agents. EFFECT: high affinity of carbohydrate residues to proteins by forming new divalent ligands based on L-glutamic acid diether, which are specific to corresponding receptors which contain residues of different carbohydrates, via a 1,3-dipolar cycloaddition reaction.

2 cl, 2 ex

 

The invention relates to the field of Bioorganic chemistry, in particular derivatives of amino acids and peptides belonging to the class of aliphatic diesters containing two carbohydrate residue.

The use of bivalent derivative of carbohydrate as a ligand asialoglycoprotein receptors, galactinol and other receptors depending on the selected glycoside is a promising direction to increase the affinity of binding transport systems with the corresponding proteins.

Structural components bivalent neoglycoconjugates based diapir L-glutamic acid are carbohydrate residues and fragments of two diesters L-glutamic acid.

Known monovalent derivative of lactose, in which the spacer elements of the molecule presents a derivative of diethylene glycol.

The presence of the carbohydrate residue and the use of the spacer facilitates the binding of the conjugate with the corresponding receptors. However, the disadvantage of this compound is its relatively high cytotoxicity (due to the presence of disulphide bonds) and low efficiency of binding to the lectin RCA.

The closest technical solution to the claimed invention is monoacetyl-L-glutamate-succinate-dihexadecyl-L-glutamic acid.

In this connection the presence of diapir L-glutamic acid contributes to commit derived in the lipid bilayer, and ectothermy the remainder serves as a ligand galactopoietic receptors for giving nanosystems address function. However, there is only one carbohydrate residue in the molecule provides a relatively low efficiency of interaction with specific receptors or lectins.

The technical result of the invention is to increase the affinity of carbohydrate residues to proteins through the creation of a new bivalent ligands based on diapir L-glutamic acid, specific to the corresponding receptors, contain the remains of various carbohydrates (e.g. lactose, mannose, and other), by the reaction of 1,3-dipolar cycloaddition.

Dihexadecyl ester of L-glutamic acid is derived from natural amino acids, so the connection based on it are of low toxicity and high Biodegradability. The application of the principle of "click-chemistry", based on the reaction of 1,3-dipolar cycloaddition, allows you to quickly and effectively synthesize the proposed conjugate. The presence of two carbohydrate residues increases the efficiency of interaction and the binding constant of the derivative with a specific what ecapture the cell surface.

To achieve the technical result developed a method of producing compound, comprising the following stages: synthesis Dobropolyeugol ether succinate dihexadecyl ester of L-glutamic acid, synthesis azidoaniline derived carbohydrate and conjugation of these components with the formation of 1,3-triazole connecting ring.

The implementation of the present invention is confirmed by the examples.

Example 1. Synthesis galactosyl-L-glutamate-succinate-dihexadecyl-L-glutamic acid.

To 0.5 g dihexadecyl ester of L-glutamic acid is added 0.126 g of succinic acid anhydride. After stirring for 10 h carboxyl group of the compounds activate N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide and add 0,065 g Dobropolyeugol ester of L-glutamic acid. Dobreprogramy ether succinate dihexadecyl ester of L-glutamic acid allocate column chromatography in the system chloroform-methanol, 10:1. Output 0,345 g (45%), Rf to 0.73 (chloroform-methanol, 5:1).

The mass spectrum of the [M]+: 901,661 (M+).

To a solution of 2,003 g 1,2,3,6,2',3',4',6'-OCTA-O-acetyl-β-D-lactoside gain of 1.18 ml of 2-bromoethanol in the presence piratage complex of boron TRIFLUORIDE and incubated for 6 h at room temperature. The reaction mass is neutralized to a 25%ammonia solution to pH 7. The solution industry is with 3×50 ml of water.

Received 2,3,6,2',3',4',6'-hepta-O-acetyl-1-O-(2-bromacil)-β-D-lactoside allocate column chromatography in the system hexane-ethyl acetate, 5:1. Output 2,034 g (86%), Rf of 0.43 (hexane-ethyl acetate, 3:1).

1H-NMR spectrum (δ, ppm): 1.96, 2.01, 2.02, 2.03, 2.05, 2.07, 2.09 (s, 24N, COCH3), 3.25-3.42 (t, 4H, OCH2CH2), 3.60-3.63 (m, 2H, H-5, H-5'), 3.80-4.0 (m, 2H, H-6), 4.10-4.20 (m, 2H, H-6'), 4.30-4.35 (d, 1H, H-4), 4.50-4.55 (d, 1H, H-1, J1,2 7.8 Hz), 4.53-4.58 (d, 1H, H-1', J1,2 7,7 Hz), 4.90-5.0 (d, 1H, H-3'), 5.20-5.34 (m, 2H, H-2, H-2'), 5.18-5.26 (m, 2H, H-4', H-3).

To 200 mg of the obtained compound added to 85.8 mg of sodium azide and stirred at 65°C for 20 hours the Reaction mass is filtered, poured into 20 ml of water and extracted with 3×30 ml of anhydrous chloroform. The organic layer is separated, the solvent is removed in vacuum.

Get 2,3,6,2',3',4',6'-hepta-O-acetyl-1-O-(2-azidoethyl)-β-D-lactoside. Yield 170 mg (78%), Rf of 0.54 (hexane-ethyl acetate, 3:1).

1H-NMR spectrum (δ, ppm): 1.96, 2.01, 2.02, 2.03, 2.05, 2.07, 2.09 (s, 24N, COCH3), 3.25-3.42 (t, 4H, och2CH2), 3.60-3.63 (m, 2H, H-5, H-5'), 3.80-4.0 (m, 2H, H-6), 4.10-4.20 (m, 2H, H-6'), 4.30-4.35 (d, 1H, H-4), 4.50-4.55 (d, 1H, H-1, J1,27,8 Hz), 4.53-4.58 (d, 1H, H-1', J1,27,7 Hz), 4.90-5.0 (d, 1H, H-3'), 5.20-5.34 (m, 2H, H-2, H-2'), 5.18-5.26 (m, 2H, H-4', H-3).

To a solution of 200 mg 2,3,6,2',3',4',6'-hepta-O-acetyl-1-O-(2-azidoethyl)-β-D-lactoside in dry methanol under stirring at room temperature was added 0.2 ml of freshly prepared 0.1 M solution of sodium methylate to achieve pH abesolut solution ion-exchange resin KU-2 (H +-form), filtered and removed solvent in vacuo.

Get 1-O-(2-azidoethyl)-β-D-lactoside. Output to 0.108 g (93%), Rf 0,06 (hexane-ethyl acetate, 3:1).

IR spectrum (νmaxcm-1): 3300 (O-H), 2900, 1435, 1340 (C-H), 1215 (C-O), 1140-1030 (C-O, 4 bands, carbohydrate skeleton).

To 50 mg of 1-O-(2-azidoethyl)-β-D-lactoside add to 35.8 mg Dobropolyeugol ether succinate dihexadecyl ester of L-glutamic acid. The reaction of lead in acetonitrile in the presence of catalytic amounts of CuI and DIPEA. Get galactosyl-L-glutamate-succinate-dihexadecyl-L-glutamic acid. Output 0,048 g (57,4%), Rf of 0.23 (chloroform-methanol, 3:1).

The mass spectrum of the [M]+: 1723,461 (M+).

Example 2. Synthesis dimensia-L-glutamate-succinate-dihexadecyl-L-glutamic acid.

Dobreprogramy ether succinate dihexadecyl ester of L-glutamic acid get analogously to example 1.

To a solution of 5.12 in city of 1,2,3,4,6-Penta-O-acetyl-α-D-mannopyranoside add to 1.14 ml of 2-bromoethanol in the presence piratage complex of boron TRIFLUORIDE and incubated for 12 hours at 20°C. the Reaction mass is neutralized to a 25%ammonia solution to pH 7. The solution was washed with 3×100 ml of water.

Received 2,3,4,6-Tetra-O-acetyl-1-O-(2-bromacil)-α-D-mannopyranoside allocate column chromatography in the system hexane-ethyl acetate, 5:1. Output 3,342 g (59%), Rfto 0.45 (hexane-ethyl acetate, 3:1).

1H-NMR spectrum (CDCl3/sub> , δ, ppm): 1.99, 2.05, 2.10, 2.16 (s, M, COCH3), 3.50-3.53 (t, 2H, OCH2CH2), 3.86-4.0 (m, 2H, OCH2CH2), 4.10-4.16 (m, 2H, H-6), 4.24-4.30 (DD, 1H, H-5), 4.88-4.87 (d, 1H, H-1, J121,78), 5.26-5.31 (m, 2H, H-2, H-4), 5.34-5.37 (DD, 1H, H-3).

To 200 mg of the obtained compound added to 85.8 mg of sodium azide and stirred at 65°C for 20 hours the Reaction mass is filtered, poured into 30 ml of water and extracted with 3×30 ml of anhydrous chloroform. The organic layer is separated, the solvent is removed in vacuum.

Get 2,3,4,6-Tetra-O-acetyl-1-O-(2-azidoethyl)-α-D-mannopyranoside. Output 0,170 g (78%), Rfof 0.65 (hexane-ethyl acetate, 3:1).

1H-NMR spectrum (CDCl3, δ, ppm): 1.99, 2.05, 2.10, 2.16 (s, M, COCH3), 3.50-3.53 (t, 2H, OCH2CH2), 3.86-4.0 (m, 2H, OCH2CH2), 4.10-4.15 (m, 2H, H-6), 4.24-4.29 (DD, 1H, H-5), 4.88-4.87 (d, 1H, H-1, J121,78), 5.26-5.31 (m, 2H, H-2, H-4), 5.34-5.37 (DD, 1H, H-3).

To 200 mg of 2,3,4,6-Tetra-O-acetyl-1-O-(2-azidoethyl)-α-D-mannopyranoside in anhydrous methanol with stirring at room temperature was added 0.1 ml of freshly prepared 0.1 M solution of sodium methylate in methanol to achieve a pH of 8. Absoluut solution ion-exchange resin KU-2 (H+-form), filtered and removed solvent in vacuo.

Get 1-O-(2-azidoethyl)-α-D-mannopyranoside. The output of 0.125 g (82%) Rfof 0.15 (hexane-ethyl acetate, 3:1).

IR spectrum (νmaxcm-1): 3300 (O-H), 2900, 1435, 1340 (C-H), 1215 (C-O), 1140-1030 (C-O, 4 bands,carbohydrate skeleton).

To 50 mg of 1-O-(2-azidoethyl)-α-D-mannopyranoside add 28,8 mg Dobropolyeugol ether succinate dihexadecyl ester of L-glutamic acid. The reaction of lead in acetonitrile in the presence of catalytic amounts of CuI and DIPEA.

Get dmannose-L-glutamate-succinate-dihexadecyl-L-glutamic acid. Output 0,044 g (45,4%), Rf is 0.19 (chloroform-methanol, 3:1).

The mass spectrum of the [M]+: 1399,691 (M+).

Using synthesized bivalent neoglycoconjugates get mixed liposomes and investigate their interaction with lectins.

The synthesized compound is inserted into liposomes based on dihexadecyl-N-(L-Omnitel)-L-glutamate in an amount of 5%. The particles have a size 45-61 nm, which is optimal for transport systems. Prepared colloidal solutions remain stable for weeks when stored at room temperature.

To 2 ml of a dispersion of liposomes containing the corresponding bivalent neoglycoconjugate (2 mg/ml), add 10 µl of 1% solution of lectin and record the binding kinetics by measuring the change in optical density of the solution at 400 nm after a certain period of time.

The use of two carbohydrate residues in the structure of bivalent neoglycoconjugate increases 6 times the binding efficacy compared with monovalent derivative of MES is lactose-L-glutamate-succinate-dihexadecyl-L-glutamic acid, that can be used to create efficient targeted delivery systems of drugs.

1. Bivalent neoglycoconjugate based diapir L-glutamic acid

as ligands uglevodistymi receptors.

2. A method of obtaining a bivalent neoglycoconjugates based diapir L-glutamic acid, comprising the following stages: synthesis Dobropolyeugol ether succinate dihexadecyl ester of L-glutamic acid, synthesis azidoaniline derived carbohydrate and conjugation of these components with the formation of 1,3-triazole connecting ring.



 

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