SU487066A1 - Method for producing human insulin β-sulfonate - Google Patents

Description

one

The invention relates to the improvement of the method of producing human insulin used in medicine.

A known method for producing human insulin S-S-sulfonate B-valued by condensing lower peptides, such as the protected peptide B1-9, with the peptide Bi-zo by a known method, removing the protective groups by oxidative sulfitolysis and isolating the target product in a known manner.

The disadvantages of this method are the complexity of the process and the rather low yield of the target product.

In order to simplify the process technology and increase the yield of the target product, a protected peptide of sequence 1-8 is proposed to be condensed with a protected peptide of sequence 9-14 by the method of shifted anhydrides, the resulting protected peptide of sequence 1-14 is converted into hydrazide by treatment with trifluoroacetic acid; the protected sequence of sequences 15–20 condense with the protected peptide sequences 21–30 by the method of activated esters; from the obtained protected peptide of sequence 15–30, remove the N-terminal protecting group by acidolysis; Peptide hydrazide of the sequence 1-14 to be fused in the azide method with a taper of peptide sequence 15-

2

30 and from the obtained protected peptide, sequences 1-30, remove the protecting groups by the action of hydrogen bromide in trifluoroacetic acid.

Example 1. Bis- (carbobenzoxy-phenyl-npl-valyl-asparaginyl-glutaminyl-histidylleucyl) -cistinyl-bis- (glycpl-O-greg-butyl ether-seryl-histidyl-lecyl-valyl) - ftreg-butyl ether-glutamyl alanyl gretbutyloxycarbonyl hydrazide.

0.120 g of carbobenzoxy-O-greg-butyl ether-seryl-histidyl-leucyl-valyl--) - tert-butyl ether-glutamyl-alanyl-trig-butyloxycarbonl-hydrazide is suspended in 5 ml of methanol and hydrogenated in the presence of palladium on carbon for two days The catalyst is filtered off, washed on the filter with water and methanol, the filtrate is evaporated to dryness, the residue is dissolved in 2 ml of hexametapol, and 0.120 g of bis (carbobenzoxp- (|) e-ilalapyl-valcyl-lysyl) gistpdil-leucyl) is added to the solution. - cystappl-bis-glntsin and 0,031 g of dptsiklogeksnlkarbodiimida, maintain two days prn 0 ° C n two days at room temperature. Dicyclohexylurea is filtered off, the filtrate is diluted with ether, the precipitate is filtered off, washed on the filter with a mixture of methanol-ether, dried, and 0.125 g of a product containing 1.2 (1) cysteine, 1.8 (2) histoid, 0.7 (1 ) acnapan-iiiOBoii acid, 0.4 (1) serine, 1.0 (1) glycine, 1.5 (2) glutamic acid, 1.7 (2) valine, 0.6 () phenylalanium, 1.8 ( 2) leucine, 0.7 (1) alanine.

Hereinafter, the theoretical amount of amino acids is indicated in parentheses.

Example 2. Bis- (o-nitrofeylsulfenyllein-tyrno-leuc-valyl) -cystynl-bns (gl-n-y-trig-butyl ester-glutamyl-arginyl-glycyl-phenylalanyl-phenylalanyl-tyrosyl-threonyl-isonyl-glynyl-aronyl-glycyl-phenylalanyl-phenylalanyl-tyrosyl-threonyl-trionyl-glycyl-glycyl-phenyl-yl-tri-butyl-aryl-tri-butyl-yl-aryl-valyl). -butyloxycarbonyl-lnzil-threonnn).

To a solution of 1.0 g of bis- (o-nitrophenylsulfenyl-leucyl-tyrnoznol-leucyl-valyl) -cystinylbns-glycine and 0.2 g / g-nitrophenol in 15 ml of DMF at -10 ° C is added 0.3 g of dicyclohexylcarbodnnmide , left overnight at 0 ° C, dicyclohexyl urea was filtered off, the filtrate was diluted with water, extracted with ethyl acetate, dried and the extract was evaporated to dryness. After crystallization of the residue from a mixture of isonropyl alcohol-hexane, 0.6 g (50%) of the product is obtained, mp. 270 ° C (decomp.); -22 ° (, DMF).

Found,%: C 54.39; H 6.73.

C86HiloNi6O24S4.

Calculated,%: C 54.39; H 6.00.

0.9 g of carbobenzoxy-7-G; og-butnlovy ester-glutamyl-arginyl-glycyl-feiyl-alanyl-phenyl-anilyl-tyrrocon-threonyl-stil-e-G; oetbutyloxycarboyl-lysn-threo-iine is dissolved in 10 ml of methanol, 5,36 ml Oh, Yun. sulfuric acid and hydrogenated in the presence of palladium on carbon for three days. The catalyst is filtered off, the filtrate is evaporated to dryness, the residue is dissolved in 50% methanol and treated with Amberlite IRA-410 ion exchange resin (OH — form). The resin is filtered off, washed on the filter with 50% methanol, the filtrate is evaporated to dryness, the residue is dried under vacuum over phosphorus pentoxide and dissolved in 4 ml of DMF. To the resulting solution, 0.4 g of the product obtained above is added, the mixture is kept at room temperature for one day, diluted with ether, the precipitate is separated by centrifugation, and crystallized from methanol to obtain 0.7 g (77%) of product, m.p. 174-178 ° C; -26 ° (, DMF).

Found,%: C 56.89; H 7.33.

С212НзоО 4205284 2Н2О.

Calculated,%: C 56.92; H 6.85.

Amnnoslot composition: cystenn 1.1 (1), lysnn 0.9 (1), arginine 0.6 (1), glycine 1.7 (2), glutamic acid 1.0 (1), tyrosine 1.0 (2 ), leucine 2.7 (2), phenylalanine and barn 3.5 (3), threonine 1.9 (2).

Example 3. 5,5-Dsulfonate b-chain.

50 mg of the product obtained in Example 1 are dissolved in 10 ml of trfluoroacetic acid, kept at room temperature for 1 hour, evaporated to dryness, the residue is diluted with water and again evaporated to dryness. This operation is repeated three more times. Residue after

the evaporated is dissolved in 10 ml of DMF, 0.07 ml of 2.00 N. of HCl solution in tetrahydrofuran is added, 0.1 ml of a solution containing 1.9 mg of nitrite IATRIM is added at -20 ° C, and the mixture is incubated for 30 min. 20 ° C, cool the resulting azide to -10 ° C and neutralize the triethn;:; amine.

62 mg of the product obtained in Example 2 are dissolved with 3 5 ml of methanol, cooled to

0 ° C and add 0.1 ml of acetic acid and 0.1 ml of 0.3 p. Solution of HCl in methanol. After 10 min, the mixture is evaporated to dryness, the residue is dissolved in water and treated with Amberlite IRA-410 ion exchange resin (OH — form).

The resin is filtered off, washed with water on the filter, the filtrate is evaporated to dryness, the residue is dissolved in a minimum amount of DMF, and the nr -40 ° C is added to the solution containing aznd. The mixture was kept for three days at -5 ° C and at room temperature for two days, evaporated to dryness, dissolved with 30 ml of trifluoroacetic acid and dried hydrogen bromide passed through the solution for 2 hours. Solution

It is evaporated to dryness at room temperature, the residue is dried in vacuum over solid alkali with phosphorus oxide and dissolved in 10 ml of dioxane and 6 ml of 0.2 and. Tris buffer (pH 8.1). To the resulting solution is added

110 mg of sodium sulfite and 60 mg of sodium tetratanate, incubated for 12 hours at room temperature and 2 hours at 37 ° C, dialyzed for 6 hours against several changes of distilled water, the solution after dialysis is evaporated

up to a small amount of liquidized. 12 mg (13%) of the product are obtained, and, -95 + 5 ° (with 0.1, 0.5 N. acetic acid).

Amino acid analysis: lysine 1.15 (1), histidine 1.92 (2), arginine 0.92 (1), aspartic acid 1.05 (I), threonine 1.94 (2), series 0.97 (1 ), glutamic acid 3.18 (3), nrolin 1.10 (1), glincine 3.0 (3), alanine 1.0 (1), cstein 1.59 (2), valnn 2.85 (3) , leucine 3.77 (4), tyrosine 1.85 (2), phenylalanine 2.72 (3).

Electrophoresis was carried out on paper with a nanoparticle of 1000–1200 in an exposure time of 50–60 min in 2% acetic acid, nro was benzidine and chlorine, and n in a buffer solution containing 4M urea, formic acid – iy j acetic acid (13 : 3: 2), with a Pauli reagent. In all cases, the product was individual, its mobility was in conjunction with the mobility of B-tsepn insulin bis-sulfonate of a bull isolated from natural insulin and used as a witness.

Subject invention

A method for producing S-S-sulphonate of human B-chain insulin by condensation of lower nanthones by a known method, followed by removal of the protective primer by oxidative sulfitolysis and release of the desired product by a known method, characterized in that

and increasing the yield of the target product, the protected peptide of sequence 1-8 is condensed with a protected peptide of sequence 9-14 by the method of shifted anhydrides and the resulting protected peptide of sequence 1-14 is converted into a hydrazide by treatment with trifluoroacetic acid; the peptide sequence 15–20 is condensed with a protected peptide sequence 21–30 by the method of activators; p (o) ()) and the K-terminal protective group of acidool is removed from the obtained peptide sequence 15–30; The peptide hydrazide of the sequence 1-14 is condensed by the azide method with the protected peptide of 15-30 sequence and the protective primer obtained from the obtained peptide of sequence 1-30 is removed by the action of hydrogen bromide in trifluoroacetic acid.