Protected peptides calcitonin

 

(57) Abstract:

The invention relates to compounds of the formula X1-Leu-His-Lys(R1)-Leu-Gln-Thr-Tyr(R2)-Pro-Y, where X1- H-, A1O-CO-, H-Lys(R3)-Leu-Ser-Gln-Glu(B4)-, A1O-CO-Lys(R3)-Leu-Ser-Gln-Glu(B4)-; Y is-OH, -Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2;1there is tert-butyl; R1- protective group of the formula IN the1O-CO - Epsilon-amino group of the Lys residue, R2- protective group of the formula IN the2About-WITH - hydroxyl group of residue Taut, R3- protective group of the formula IN the3O-CO - Epsilon-amino group of the Lys residue IN4- protective group for the gamma-carboxyl group of Glu residue, and1IN2IN3and4may be the same or different and are selected from the range: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(substituted aryl)sulfonylated. The use of these protected peptides can facilitate acquisition of calcitonin salmon. table 2.

The invention relates to new original compounds used to produce calcitonin salmon and its analogues, namely to secure the peptides of General formula

X1-Leu-His-Lys(R1)-Leu-Gln-Thr-Tyr(R2)-Pro-Y

where X1- N AND1O-CO-, H-Lys(R3)-Leu-Se 1 tert-butyl;

R1- protective group of the formula IN the1O-CO - Epsilon-amino group of the Lys residue,

R2- protective group of the formula IN the2O-WITH - hydroxyl group of residue Tight,

R3- protective group of the formula IN the3O-CO - Epsilon-amino group of the Lys residue,

IN4- protective group for the gamma-carboxyl group of Glu residue,

and1IN2IN3and4may be the same or different and are selected from the range: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(substituted aryl)sulfonylated.

Gipokaltsiemicheskaya hormone calcitonin is the most important drug used for the treatment of diseases associated with impaired metabolism of calcium in the body: hypercalcemia of different origin, acute degeneration of the bones, osteoporosis, diseases of the page, slow splicing bones after fractures or surgical procedures. In medical practice the greatest application finds calcitonin salmon, biological activity is more than 40 times more than that of calcitonin of humans and mammals.

Calcitonin salmon is a polypeptide consisting of 32 amino acid residues, containing dial is/BR> The main source of calcitonin for medical purposes is chemical synthesis. For chemical synthesis of calcitonin are two main approaches that exist in the chemistry of peptides: solid phase, or the synthesis of insoluble polymer carrier, and a liquid-phase or synthesis in solution. Despite the significant progress made in recent years, solid-phase synthesis is still little adapted to obtain peptides in large quantities required for clinical trials and production of medicines. In this respect, the disadvantages of solid-phase synthesis include: a) the need to use full real-time protection three functional amino acids; b) the use of a large excess of protected amino acids, as a rule, not subject to recovery; C) limited ability to control the course of the synthesis, the inability purification of intermediate products; d) the difficulty scale; d) the risk of contamination of the target peptide similar in structure impurities, the complexity and the multi-stage purification of the final product; (e) high cost of raw materials - protected amino acids, reagents and polymers.

Therefore, the liquid-phase production of peptides as it provides tactical flexibility in the choice of protection schemes (from minimum to full) and methods of condensation, full control over the course of the synthesis, the possibility of purification of intermediate products, as well as the scalability of the whole process. However, the efficiency of the process, outputs and quality of final products significantly depend on the optimality of the tactical scheme of the synthesis.

Synthesis of calcitonin in solution is carried out by an Assembly of the full sequence of appropriately protected peptide segments, removal of the protective groups, the subsequent cleaning and selection of the target product. Protected peptide segments, in turn, are synthesized from the protected amino acids and/or of short peptide segments. Amino acid sequence of segments, nature and location of the structure of the protective groups, the methods and procedures of the Assembly of segments of the full sequence of calcitonin are determined by the tactical scheme of synthesis. That the scheme was effective and scalable, during the development, you must follow some General rules: a) splitting into segments should be performed so that the risk of racemization during condensation sledovateliach stages in the Assembly of molecules calcitonin; C) permanent and temporary protective group segments have, on the one hand, to provide the necessary level of protection from side reactions during the synthesis of the segments and their subsequent condensation into a single chain, on the other hand, is easily removed at the final stage of the process without damage to the structure of the target product; g) the protected peptide segments must be soluble in the solvents used for the condensation of the segments. These requirements are largely contradictory, so to achieve their full implementation in practice is very difficult.

Structural feature of the molecule calcitonin salmon is the presence of only three amino acid residues (in addition to the two Cys residues), requires real-time protection - Lys11, Lys18and Glu15high content of gidroksipiroxikam, and the presence of residues His and Arg, real-time protection which liquid-phase synthesis is not strictly necessary. This opens up the possibility for the synthesis of calcitonin using a variety of protection circuits - from minimal to complete.

Known methods for producing calcitonin salmon and its analogues of the peptide segments with full constant asiapaci patent 606816, Cl. C 07 K 001/06). Full protection helps to minimize possible side reactions in the Assembly sequence of calcitonin. However, the removal of a large number of protective groups with acidic reagents at the end of the synthesis itself is often a source of adverse reactions. In addition, the solubility of fully protected peptides in organic solvents decreases significantly with the increase of their length. This hampers the synthesis and makes it nearly impossible to clean and control the purity of the intermediates. Thereby lost one of the main advantages of liquid-phase synthesis of peptides.

Schematic of the synthesis of calcitonin and analogs of peptide segments with a minimum constant protection tert-Putilkovo of the type described, for example, article Guttman road S., Pless J., R. L. Huguenin, E. Sandrin, Bossert H., Zehnder, K. Helv. Chim. Acta, 1969, v. 52, p.1788-1795, in the patent application Germany 2025791, Cl. C 07 C, and intermediate (incomplete) options real-time protection (for example, Japan patent 0616694, Cl. C 07 K 7/06) allow to improve the solubility of the synthesized polypeptide. Cleavage under mild conditions a small number of protective groups at the final stage of the process gives a high yield of target product, which is not required the nogo type excludes the possibility of simultaneous use of the same type (for example, tert-butoxycarbonyl and tert-butilkoi) as a highly effective temporary protective groups for the amino and carboxyl groups are protected peptide segments. This greatly complicates synthetic approaches to obtain peptide segments that overlap the area from the 11th to the 23rd amino acid residue sequence of calcitonin salmon.

Thus, the development of efficient and scalable scheme liquid-phase synthesis of calcitonin is, essentially, the development of a set optimally protected peptide segments and their connections in a single amino acid sequence.

Object of the invention is to create new protected peptides that could be used as starting compounds for efficient and scalable synthesis of calcitonin salmon.

This object is achieved in that there are new compounds, namely, protected peptides calcitonin General formula

X1-Leu-His-Lys(R1)-Leu-Gln-Thr-Tyr(R2)-Pro-Y

where X1- N AND1O-CO-, H-Lys(R3)-Leu-Ser-Gln-Glu(B4)-, A1O-CO-Lys(R3)-Leu-Ser-Gln-Glu(B4)-;

Y-HE-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2;

AND1tert-butyl;2O-WITH - hydroxyl group of residue Tight,

R3- protective group of the formula IN the3O-CO - Epsilon-amino group of the Lys residue,

IN4- protective group for the gamma-carboxyl group of Glu residue,

and1IN2IN3and4may be the same or different and are selected from the range: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(substituted aryl)sulfonylated.

The subject of the invention, therefore, are peptide segments 16-23, 11-23, 16-32 and 11-32 (I-IV) sequence of calcitonin salmon having a protective group on the side radicals of amino acid residues Lys11, Lys18Glu15and Tight22(PL.1).

Segments I-IV can have a free-amino group (X2O - n) or blocked urethane protecting group (X2- AND1O-CO-), preferably tert-butoxycarbonyl (A1- tert-butyl). Side of the protective group R1-R3IN4are removed by reason group 2-alkyl - or 2-arylsulfonamides type: urethane (urethane) for residues Lys11and Lys18(R1IN1O-CO-, R3IN3O-CO-); carbonate for the remainder of Tight22(R2IN2O-CO-); clonaepam is Ocharovanie functional groups of amino acids described in the literature. 2-Alkyl(aryl)sulfonylation substituents IN1IN4in compounds I-IV can be present in various combinations, can be the same or different.

Peptide segments I-IV are new, not previously described substances.

For the synthesis of peptide segments I-IV can be used techniques and methods peptide synthesis described in the literature. Segment I (16-23) can be synthesized by the method of stepwise extension of the peptide chain from the C-end to N-with N-protected amino acids. As a temporary Nprotection in this case you can use the group to be removed by catalytic hydrogenolysis, for example carbobenzoxy or soft acidolysis, for example tert-butoxycarbonyl or 4-methoxybenzylideneamino. Permanent blocking of C-terminal carboxyl group of the synthesized peptide can be used ester protection, such as benzyl or 2,2,2-trihalogenmethanes ether. To activate the carboxyl groups of amino acid residues introduced into the peptide chain, you can use a variety of methods described in the literature, for example the method of activated esters, the method of mixed anhydrides, azide method, cinteza segment II (11-23) obtained with NC-protected segment 16-23 remove N-protective group, and increasing it further stepped up sequence 11-23, as described above, or condense it by known methods with pre-synthesized segment 11-15 General formula

X2-Lys(R3)-Leu-Ser-Gln-Glu(B4)-OH (V)

where X2there is a urethane protective group, preferably tert-butoxycarbonyl, and R3and4take the values specified above. Remove C-the protection obtained with NC-protected segment 11-23 gives target N-protected segment II.

For the synthesis of segment III (16-32) with NC-protected segment 16-23 remove C-protective group, and the N-protected segment I condense known methods described in the literature segment 24-32 formula

H-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2(VI).

Segment IV (11-32) can be synthesized in two ways: 1) N-easymany segment II condense with segment VI; 2) segment III with a free-amino groups condense with segment V

Obviously practicable receiving segments I-1V with any combination of substituents IN the1IN4when the above znachenie, to use a group selected from among those described in literature (table.2).

The above segments I-IV can be used as intermediates for obtaining calcitonin salmon. For the synthesis of the complete amino acid sequence of calcitonin salmon segment IV with a free-amino group derived from segment II or segment III, as described above, condensed by known methods with-protected segment of 1-10 amino acid sequence of calcitonin salmon, for example, described in the literature peptide formula

< / BR>
With the product of condensation is removed N-Sun-protection, for example, trifluoroacetic acid, and receive a peptide having the amino acid sequence of calcitonin salmon 1-32 and containing four amino acid residue is blocked by protective groups, 2-alkyl(aryl)sulfonylamino type:

< / BR>
For removal of the protective groups of the peptide VIII is subjected to short-term action of diluted basis, for example the processing of 0.1 N. aqueous sodium hydroxide solution within 3-20 minutes at a temperature of from 0 to 20oC, the mixture is neutralized, for example, by adding an excess of acetic acid, after which emit obtained from the raffia.

The invention is illustrated by examples. In the description of examples, the following abbreviations and symbols:

DMF - dimethylformamide,

DCGC - dicyclohexylcarbodiimide,

MBT - 1-hydroxybenzotriazole,

TFU - triperoxonane acid,

HPLC - high performance liquid chromatography,

Cpsc - 2-(4-chlorophenyl)sulfadimethoxine,

Msc - 2-methylsulfonylbenzoyl,

Psc - 2-vinylsulfonylacetamido,

Pse - 2-phenylsulfonyl,

Tse - 2-(4-tolyl)sulfonylated,

Tce - 2,2,2-trichlorethyl.

Abbreviations of amino acids and the protective groups used in accordance with the recommendations of the Commission on biochemical nomenclature at IUPAC-IUB, published in Eur. J. Biochem., 1984, v. 138, No. 1, pp. 9-37. Optically active amino acids listed in the descriptions of the examples, by default, have the L-configuration.

The values of the chromatographic mobility of Rfrefer to plates for thin-layer chromatography Alufolien Kieselgel 60 F254(Merck, Germany) in the system chloroform-methanol-acetic acid 95:5:3 (A) or 90:10:3 (B); ethyl acetate-pyridine-acetic acid-water 60: 5: 15:10 (). Detection of the spots on the plates was performed in UV-light, ninhydrin is metre MSBH-1 (NGOs "Electron", Ukraine) or mass spectrometer MALDI-TOF VISION 2000 (Thermo Bioanalysis, England). The analysis of amino acids was performed on the analyzer Biotronik LC5001 after acid hydrolysis of the samples of peptide material in sealed ampoules (6 N. model HC1, 24 h, 110oC).

Example 1. Obtain Boc-Leu-His-Lys(Psc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OH (segment A).

A. Vos-RHS-Use. 3.9 g of the BOC-RHS-IT was dissolved in 20 ml of dry ethyl acetate, add 50 mg of 4-dimethylaminopyridine and 2 ml of 2,2,2-trichloroethanol, cooled in an ice bath and added 4.2 g DCGK. Stirred 30 min at 0oC and 30 min at 20oC, the precipitate is filtered off, dicyclohexylamine, the filtrate is extracted with 50 ml water, 50 ml saturated NaHC3and 50 ml saturated NaCl, and then evaporated. Obtain 6.1 g of a colorless oil, Rf0.65-0.7 (A).

B. Boc-Tyr(Psc)-Pro-OTce. Oil Re-RHS-Use (example Ia) is dissolved in 30 ml of 2 M Hcl in acetic acid, over 30 min at 20oWith evaporated, the residue perevarivat with toluene, washed with ether and dissolved in 50 ml of DMF. To the solution was added 9.5 g of Boc-Tyr(Psc)-OH, 2.0 ml of N-methylmorpholine and 2.2 g of MBT, cooled in an ice bath and then added 3.5 g DCGK. Stirred for 1.5 h at 0oC and 2 h at 20oC, the precipitate is filtered off, dicyclohexylamine, to the filtrate, add 150 ml of Neorganicheskie layer evaporated to dryness and treat the residue with petroleum ether. Obtain 10.5 g of a colorless crystalline product, Rf0.7-0.75 (A); mass spectrum: (M+H)+724.2 (calculated: 723.08).

C. Boc-Thr-Tyr(Psc)-Pro-OTce. Boc-Tyr(Psc)-Pro-OTce (example 1B) are dissolved in 40 ml of 2 M Hcl in acetic acid, over 30 min at 20oWith evaporated, the residue perevarivat with toluene, washed with ether and dissolved in 70 ml of DMF. To the solution was added 4 g of Boc-Thr-OH, 1.8 ml of N-methylmorpholine and 2.4 g of MBT, cooled in an ice bath and then added 3.5 g DCGK. Stirred for 1.5 h at 0oC and 3 h at 20oC, then treated reaction mixture as described in example 1B. Obtain 11 g of a colorless amorphous substance, Rf0.40-0.45 (A); mass spectrum: (M+N)+823.5 (calculated: 823.19).

, Boc-Gln-Thr-Tyr(Psc)-Pro-OTce. Boc-Thr-Tyr(Psc)-Pro-OTce (example 1B) are dissolved in 50 ml of TFU, after 30 min at 20oWith evaporated, the residue is treated with ether and allocate 11 g trifenatate H-Thr-Tyr(Psc)-Pro-OTce. It is dissolved in 70 ml of DMF, the solution add 3.95 g of Boc-Gln-OH, 1.8 ml of N-methylmorpholine and 2.2 g of MBT, cooled in an ice bath and then added 3.3 g DCGK. Stirred for 1.5 h at 0oC and 3 h at 20oC, then treated reaction mixture as described in example 1B. The residue after evaporation of the extract is treated with ether and obtain 10.4 g of the target product, Rf0.15(example 1G) was dissolved in 50 ml of TFU, after 30 min at 20oWith evaporated, the residue is treated with ether and allocate 9.8 g trifenatate H-Gln-Thr-Tyr(Psc)-Pro-OTce. It is dissolved in 70 ml of DMF, the solution was added 3 g of the monohydrate of Boc-Leu-OH, 1.4 ml of N-methylmorpholine and 2 g of MBT, cooled in an ice bath and then added 2.8 g DCGK. Stirred for 1.5 h at 0oC and 8 h at 20oC, then treated reaction mixture as described in example g Obtain 9.6 g of the target Pentapeptide, Rf0.10-0.15 (A), 0.55-0.60 (B); mass spectrum: (M+N)+1067.3 (calculated: 1065.50).

that is, Boc-Lys(Psc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OTce. Boc-Leu-Gln-Thr-Tyr(Psc)-Pro-Otce (example 1D) was dissolved in 60 ml of TFU, after 30 min at 20oWith evaporated, the residue is treated with ether and allocate 9.5 g trifenatate H-Leu-Gln-Thr-Tyr(Psc)-RHS-Use. It is dissolved in 70 ml of DMF, the solution was added 6.5 g of 2,4,5-trichlorphenol ester Boc-Lys(Psc), 1.4 ml of N-methylmorpholine and 1.4 g of MBT. Stir the mixture for 8 h at 20oWith, then the solvent is distilled off under reduced pressure and treat the residue with ether. The precipitate is filtered off, washed with ether and obtain 11.4 g of the Hexapeptide, Rf0.35-0.40 (B); mass spectrum: (M+N)+1404.2 (calculated: 1405.91).

W. Boc-His(Boc)-Lys(Psc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OTce. Boc-Lys(Psc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OTce (example 1E) is dissolved in 60 ml of TFU, after 30 min at 20oWith uprima, to the solution was added 5.4 g of 2,4,5-trichlorphenol ester Boc-His(Boc), 1.4 ml of N-methylmorpholine and 1.35 g of MBT. Stir the mixture for 12 h at 20oWith, then the solvent is distilled off under reduced pressure and treat the residue with ether. The precipitate is filtered off, periostat from DMF-ether and obtain 12.3 g of heptapeptide,f0.30-0.40 (B); mass spectrum (after removal of the BOC-protection): (M+N)+1444.1 (calculated: 1442.94).

C. Boc-Leu-His-Lys(Psc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OTce. Boc-His(Boc)-Lys(Psc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OTce (example 1e) is dissolved in 60 ml of TFU, after 40 min at 20oWith evaporated, the residue is treated with ether and produce 12 g of bestreferat.ru H-His-Lys(Psc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OTce. It is dissolved in 70 ml of DMF, the solution was added 3 g of N-hydroxysuccinimide ester Boc-Leu and 2.2 ml of N-methylmorpholine. Stir the mixture for 18 h at 20oWith, then the solvent is distilled off under reduced pressure and treat the residue with ethyl acetate. The precipitate is filtered off, periostat from DMF-ether and obtain 11.6 g of oktapeptid,f0.10-0.15 (B); mass spectrum (after removal of the BOC-protection): (M+N)+1554.6 (calculated: 1556.11).

I. Boc-Leu-His-Lys(Psc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OH (segment Ia). To a solution of 6.5 g TSE-ether segment Ia (example 1H) in 60 ml of tetrahydrofuran, 15 ml of water and 5 ml of acetic acid at a fast peremeshivaya slurry and the filtrate evaporated to dryness. The residue is treated with 150 ml water, the precipitate was separated by filtration and dried in air. Product periostat of acetic acid ether and dried in vacuum. Obtain 5.3 g of the target segment Ia, Rf0.40-0.45 (In); mass spectrum: (M+N)+1524.1 (calculated: 1524.84); amino acid composition: Glx 0.93 (1); Thr 0.85 (1); Leu 2.00 (2); Pro 1.09 (1); Tight 0.91 (1); His 1.13 (1); Lys 1.02 (1).

Example 2. Obtain Boc-Leu-His-Lys(Msc)-Leu-G1n-Thr-Tyr(Psc)-Pro-OH (segment Ib).

a. Boc-Lys(Msc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OTce. 3.2 g of triptoreline H-Leu-Gln-Thr-Tyr(Psc)-Pro-OTce (example 1E) is dissolved in 15 ml of DMF, the solution was added 2 g of 2,4,5-trichlorphenol ester Boc-Lys(Msc), 0.5 ml of N-methylmorpholine and 450 mg OBT. Stirred mixture of 4 h at 20oWith, then the solvent is distilled off under reduced pressure and treat the residue with ether. The precipitate is filtered off, washed with ether and obtain 3.9 g of the Hexapeptide, Rf0.30-0.35 (B); mass spectrum: (M+N)+1342.2 (calculated: 1343.84).

B. Boc-His(Boc)-Lys(Msc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OTce. Boc-Lys(Msc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OTce (example 2A) is dissolved in 15 ml of TFU, after 30 min at 20oWith evaporated, the residue is treated with ether and produce 4.0 g trifenatate H-Lys(Msc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OTce. It was dissolved in 20 ml of DMF, the solution was added 1.8 g of 2,4,5-trichlorphenol ester Boc-His(Boc), 450 μl of N-methylmorpholine and 400 mg OBT. Paramashiva. The precipitate is filtered off, periostat of the acetic acid ester and obtain 4.0 g of heptapeptide, Rf0.30-0.40 (B); mass spectrum (after removal of the BOC-protection): (M+N)+1381.1 (calculated: 1380.87).

C. Boc-Leu-His-Lys(Msc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OTce. Boc-His(Boc)-Lys(Msc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OTce (example 2B) was dissolved in 15 ml of TFU, after 40 min at 20oWith evaporated, the residue is treated with ether and allocate 3.9 g of bis-trifenatate H-His-Lys(Msc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OTce. It was dissolved in 20 ml of DMF, the solution was added 900 mg of N-hydroxysuccinimide ester Boc-Leu and 0.82 ml of N-methylmorpholine. Stir the mixture for 24 h at 20oWith, then treat the mixture as described in example 1H and obtain 3.8 g of oktapeptid, Rf0.08-0.15 (B); mass spectrum (after removal of the BOC-protection): (M+N)+1495.1 (calculated: 1494.04).

, Boc-Leu-His-Lys(Msc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OH (segment Ib). To a solution of 1.8 g TSE-ether segment Ib (example 2B) in 20 ml of tetrahydrofuran, 5 ml of water and 2 ml of acetic acid with rapid stirring portions over 10 min to 1 g of activated zinc dust. The mixture is stirred for another 20 min, filter the slurry and the filtrate evaporated to dryness. The residue is treated with 50 ml water, the precipitate was separated by filtration and dried in air. Peptide periostat of acetic acid with ether, and dried in kislotnyi ingredients: Glx 1.04 (1); Thr 0.82 (1); Leu 2.00 (2); Pro 1.13 (1); Tight 0.95 (1); His 1.08 (1); Lys 1.01 (1).

Example 3. Obtain Boc-Lys(Psc)-Leu-Ser-Gln-Glu(OPse)-Leu-His-Lys(Psc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OH (segment II).

a. Boc-Glu(OPse)-OTce. 4.2 g of Boc-Glu(OPse)-OH was dissolved in 20 ml of dry ethyl acetate, add 20 mg of 4-dimethylaminopyridine and 1.2 ml of 2,2,2-trichloroethanol, cooled in an ice bath and add 2.3 g DCGK. Stirred 30 min at 0oC and 60 min at 20oC, the precipitate is filtered off, dicyclohexylamine, the filtrate is extracted with 20 ml of water, 30 ml of saturated NaHC3and 30 ml saturated NaCl, and then evaporated. The residue is crystallized from ether and obtain 5.0 g of a colorless crystalline product, Rf0.55-0.6 (A).

B. Boc-Gln-Glu(OPse)-OTce. Boc-Glu(OPse)-OTce (example 3A) was dissolved in 20 ml of TFU, after 30 min at 20oWith evaporated, the residue is crystallized from ether and obtain 4.8 g of triptoreline H-Glu(OPse)-OTce. It is dissolved in 15 ml of DMF, the solution was added 2.6 g of Boc-Gln-OH, 1.1 ml of N-methylmorpholine and 1.35 g of MBT, cooled in an ice bath and then added 2.3 g DCGK. Stirred for 1.5 h at 0oAnd 7 h at 20oC, the precipitate is filtered off, dicyclohexylamine, the filtrate is evaporated. The residue is washed with water, evaporated with ethanol and crystallized from ether. Obtain 5.4 g of the dipeptide, Rf0.15-0.20 (A), 0.40-0.45 (B); mass spectrum: ez 30 min at 20oWith evaporated, the residue is crystallized from ether and obtain 5.4 g of triptoreline H-Gln-Glu(OPse)-OTce. It is dissolved in 15 ml of DMF, the solution was added 3.4 g of pentafluorophenyl ester of Boc-Ser and 1.1 ml of N-methylmorpholine. Stirred for 1.5 h at 20oC and the reaction mixture is evaporated to an oil. The residue was washed with hexane, and then treated with ether and obtain 5.5 g of Tripeptide, Rf0.05-0.10 (A), 0.30-0.35 (B); mass spectrum: (M+N)+762.9 (calculated: 763.10).

, Boc-Leu-Ser-Gln-Glu(OPse)-OTcc. Boc-Ser-Gln-Glu(OPse)-OTce (example 3b) was dissolved in 20 ml of TFU, after 50 min at 20oWith evaporated, the residue is crystallized from ether and obtain 5.4 g of triptoreline H-Ser-Gln-Glu(OPse)-OTce. It is dissolved in 15 ml of DMF, the solution was added 3.4 g of 2,4,5-trichlorphenol ester BOC-Leu, 450 mg OBT and 1.1 ml of N-methylmorpholine. Stirred for 5 h at 20oC and the reaction mixture is evaporated to an oil. The residue is washed with water, evaporated with ethanol, then treated with ether and obtain 5.2 g of tetrapeptide, Rf0.05-0.10 (A), 0.40-0.45 (B); mass spectrum: (M+N)+877.4 (calculated: 876.28).

D. Boc-Lys(Psc)-Leu-Ser-Gln-Glu(OPse)-OTce. Boc-Leu-Ser-Gln-Glu(OPse)-OTce (example 3 g) was dissolved in 20 ml of TFU, after 40 min at 20oWith evaporated, the residue is crystallized from ether and obtain 5.3 g of triptoreline H-Leu-Ser-Gln-Glu(OPse)-Otce. It is dissolved in 25 ml is up to 2 h at 20oC and the reaction mixture is evaporated to an oil. The residue is washed with water, ether, evaporated with ethanol, then treated with ether and obtain 6.5 g of Pentapeptide, Rf0.05-0.10 (A), 0.30-0.35 (B); mass spectrum (after removal of the BOC-protection): (M+N)+1117.4 (calculated: 1116.56).

e. Boc-Lys(Psc)-Leu-Ser-Gln-Glu(OPse)-OH (segment Va). To a solution of 6.0 g TSE-ether segment Va (example 3D) in 50 ml of tetrahydrofuran, 10 ml of water and 2 ml of acetic acid with rapid stirring portions over 10 min to 2 g of activated zinc dust. The mixture is stirred for another 30 min, filter the slurry and the filtrate evaporated to dryness. The residue is treated with 100 ml water, the precipitate was separated by filtration and dried in air. Peptide periostat from a mixture of methanol and acetic acid ether and dried in vacuum. Obtain 4.2 g of the target segment Va, Rf0.55-0.60 (In); mass spectrum: (M+N)+1084.6 (calculated: 1085.30); amino acid composition: Glx 2.04 (2); Ser 0.85 (1); Leu 1.00 (1); Lys 1.06 (1).

W. Boc-Lys(Psc)-Leu-Ser-Gln-Glu(OPse)-Leu-His-Lys(Psc)-Leu-Gln-Thr-Tyr(Psc)-RHS-Use (TSE-aired segment IIA). 1.65 g TSE-ether segment Ia (example 1H) dissolved in 15 ml of TFU, after 40 min at 20oWith evaporated, the residue is treated with ether and produce 1.7 g of bis-trifenatate H-Leu-His-Lys(Psc)-Leu-Gln-Thr-Tyr(Psc)-Pro-OTce. It is dissolved in 10 ml of DMF, a solution of the Ledger. The mixture is stirred for 2 hours while cooling, then 18 h at 20oC. the precipitate is Filtered off, dicyclohexylamine, the filtrate evaporated to an oil and the residue is treated with 100 ml of ethyl acetate. The precipitate was separated by filtration, periostat from DMF-ethyl acetate, washed with ether and obtain 2.32 g of tridecapeptide; mass spectrum (after removal of the BOC-protection): (M+N)+2524.2 (calculated: 2522.26).

C. Segment IIa. To 2.3 g TSE-ester IIA (example g) in a mixture of 10 ml of DMF, 20 ml of tetrahydrofuran, 5 ml of water and 2 ml of acetic acid with rapid stirring portions over 10 min to 1 g of activated zinc dust. The mixture is stirred for 50 min, then treated as described in example 3E. Receive 2 g of segment II; mass spectrum (after removal of the BOC-protection): (M+N)+2389.6 (calculated: 2390.87); amino acid composition: Glx 2.73 (3); Ser 0.83 (1); Thr 0.87 (1); Leu 3.00 (3); Pro 1.11 (1); Tight 0.91 (l); His l.06 (l); Lys 2.02 (2).

Example 4. Obtain Boc-Leu-His-Lys(Psc)-Leu-Gln-Thr-Tyr(Psc)-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2(segment IIIa).

To a solution of 770 mg of the segment Ia (example 1i), 600 mg of bis-trifenatate segment VI (Helv. Chim. Acta, 1969, v. 52, 1788-1795), 135 mg OBT and 100 μl of N-methylmorpholine in 6 ml DMF added 140 mg DCGK and stirred the mixture for 24 h at 20oC. the Reaction mixture is treated as described is graperoot on column 415 cm with LiChroprep RP18 (Merck, Germany) in a gradient of acetonitrile from 10 to 50%) in water containing 0.2% TFU. The fractions containing pure product (>90% by HPLC), are combined and evaporated to dryness, the residue is treated with ether and obtain 0.94 g of triptoreline segment IIIA; mass spectrum: (M+N)+2394.9 (calculated: 2395.83); amino acid composition: Asx 1.01 (1); Glx 1.05 (1); Ser 0.81 (1); Thr 3.57 (4); Gly 1.94 (2); Leu 2.00 (2); Pro 2.08 (2); Tight 0.90 (1); His 1.06 (l); Lys 1.08 (l); Arg, 1.01 (1).

Example 5. Obtain Boc-Leu-His-Lys(Msc)-Leu-Gln-Thr-Tyr(Psc)-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2(segment IIIb)/

Get out of 750 mg segment Ib (example 2) and 600 mg of bis-trifenatate segment VI, as described for segment IIIa in example 4. After chromatographic purification allocate 0.86 g trifenatate segment IIIb; mass spectrum: (M+N)+2334.2 (calculated: 2333.76); amino acid composition: Asx 1.08 (1); Glx 1.00 (1); Ser 0.84 (1); Thr 3.50 (4); Gly 1.96 (2); Leu 2.00 (2); Pro 1.88 (2); Tight 0.95 (1); His 1.09 (1); Lys 1.03 (1); Arg 1.00 (1).

Example 6. Obtain Boc-Lys(Psc)-Leu-Ser-Gln-Glu(OPse)-Leu-His-Lys(Psc)-Leu-Gln-Thr-Tyr(Psc)-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2(segment IVa). Option 1.

To a solution of 1.25 g of the segment IIA (example 3), 600 mg of bis-trifenatate segment VI, 135 mg OBT and 120 μl of M-methylmorpholine in 10 ml of DMF added 160 mg DCGK and stirred the mixture for 40 h at 20oC. the Reaction mixture was treated as described in example 1G, the 15 cm with LiChroprep RP18 (Merck, Germany) in a gradient of acetonitrile from 10 to 70%) in water containing 0.2% TFU. The fractions containing pure product (>90% by HPLC), are combined and evaporated to dryness, the residue is treated with ether and obtain 1.15 g of triptoreline segment IVa; mass spectrum: (M+H)+3363.1 (calculated: 3361.97); amino acid composition: Asx 1.04 (1); Glx 3.15 (3); Ser 1.80 (2); Thr 3.48 (4); Gly 1.96 (2); Leu 3.00 (3); Pro 2.01 (2); Tight 0.88 (1); His 1.07 (1); Lys 2.10 (2); Arg 1.00 (1).

Example 7. Obtain Boc-Lys(Psc)-Leu-Ser-Gln-Glu(OPse)-Leu-His-Lys(Psc)-Leu-Gln-Thr-Tyr(Psc)-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2(segment IVa). Option 2.

With 1.30 g of the segment IIIa (example 4) remove the BOC-protecting action TFU, as described in example I, and obtain 1.33 g of Tris-trifenatate segment IIIA with a free-amino group, (M+N)+2295.3. It condense with 600 mg segment Va (example 3E), as described in example 6. After chromatographic purification allocate 1.28 g trifenatate segment IVa, identical obtained in example 6.

Example 8. Obtain Boc-Lys(Cpsc)-Leu-Ser-Gln-Glu(OTse)-Leu-His-Lys(Msc)-Leu-GIn-Thr-Tyr(Psc)-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2(segment IVb).

a. Boc-Glu(OTse)-OTce. 2.2 g of Boc-Glu(OTse)-OH was dissolved in 10 ml of dry ethyl acetate, add 10 mg of 4-dimethylaminopyridine and 0.65 ml of 2,2,2-trichloroethanol, cooled in an ice bath and add 1.2 g DCGK. Stirred for 30 min when the acetate and extracted with 20 ml of water, 30 ml saturated NaHCO3and 30 ml saturated NaCl, and then evaporated. The residue is crystallized from ether and obtain 2.5 g of a colorless crystalline product, Rf0.55-0.6 (A).

B. Boc-Gln-Glu(OTse)-OTce. Boc-Glu(OTse)-OTce (example 8A) was dissolved in 10 ml of TFU, after 30 min at 20oWith evaporated, the residue is crystallized from ether and obtain 2.5 g of triptoreline H-Glu(OTse)-OTce. It is dissolved in 10 ml of DMF, the solution was added 1.3 g of Boc-Gln-OH, 600 μl of N-methylmorpholine and 0.65 g of MBT, cooled in an ice bath and then add 1.2 g DCGK. Stirred for 1.5 h at 0oC and 10 h at 20oWith, then treat the mixture as described in example 3b. Obtain 2.8 g of the dipeptide, Rf0.15-0.20 (A), 0.40-0.45 (B); mass spectrum: (M+N)+689.4 (calculated: 690.05).

C. Boc-Ser-Gln-Glu(OTse)-OTce. With the dipeptide Boc-Gln-Glu(OTse)-OTce (example 8b) remove the BOC-protecting and condense it with 1.7 g of pentafluorophenyl ester of Boc-Ser, as described in example 3b. Obtain 2.8 g of Tripeptide, Rf0.05-0.10 (A), 0.30-0.35 (B); mass spectrum: (M+N)+777.9 (calculated: 777.11).

, Boc-Leu-Ser-Gln-Glu(OTse)-OTce. With peptide Boc-Ser-Gln-Glu(OTse)-OTce (example 8b) remove the BOC-protecting and condense it with 1.8 g of 2,4,5-trichlorphenol ester Boc-Leu, as described in example g Obtain 2.7 g of tetrapeptide, Rf0.05-0.10 (A), 0.30-0.35 (B); mass spectrum: (M+N)+892.0 (you the specifications and condense it with 2.3 g of 2,4,5-trichlorphenol ester Boc-Lys(Cpsc), as described in example 3D. Obtain 3.3 g of Pentapeptide (TSE-ether segment Vb), Rf0.05-0.10 (A), 0.30-0.35 (B); mass spectrum (after removal of the BOC-protection): (M+H)+1166.4 (calculated: 1165.04).

that is, Boc-Lys(Cpsc)-Leu-Ser-Gln-Glu(OTse)-OH (segment Vb). With 3 g TSE-ether segment Vb (example 8) remove Tce protection by treatment with zinc dust as described in example 3E. Obtain 2.3 g of the segment Vb, Rf0.55-0.60 (In); mass spectrum: (M+N)+1132.0 (calculated: 1133.78); amino acid composition: Glx 1.86 (2); Ser 0.89 (1); Leu 1.00 (l); Lys 1.03(1).

W. Segment IVb. With 600 mg of triptoreline segment IIIb (example 5) remove the BOC-protecting action TFU and obtain 620 mg of Tris-trifenatate segment IIIb with a free-amino group, (M+N)+2232.3. It condense with 330 mg segment Vb (example 8E), as described in example 6. After chromatographic purification allocate 630 mg trifenatate segment IVb; mass spectrum: (M+N)+3350.1 (calculated: 3348.39); amino acid composition: Asx 1.01 (1); Glx 3.20 (3); Ser 1.88 (2); Thr 3.63 (4); Gly 2.04 (2); Leu 3.00 (3); Pro 2.03 (2); Tight 0.94 (1); His 1.07 (1); Lys 2.01 (2); Arg 1.05 (1).

Example 9. Receiving calcitonin salmon (using segment IVa).

360 mg segment IVa dissolved in 3 ml of TFU, after 30 min at 20oWith evaporated to dryness and treat the residue with ether. Obtain 365 mg of Tris-trifenatate with the g segment VII (Helv. With him. Acta, 1969, v. 52, 1788-1795), 30 μl of N-methylmorpholine, 27 mg OBT and 40 mg DCGK, the mixture is stirred for 24 h at 20oC. the precipitate is Filtered off, dicyclohexylamine, to the filtrate add 40 ml of ethyl acetate, precipitated precipitate was separated, obtain 480 mg of a crude protected 32-peptide VIIIa. It is treated with TFU to remove the BOC-protection, as described above, then dissolved in 20 ml of a mixture of DMF-water (1:4). To the solution under strong stirring for 30 added dropwise 2 ml of 1 N. aqueous sodium hydroxide solution and stirred for further 5 min, then add 0.5 ml of acetic acid. The mixture is diluted with water to 50 ml and applied to a column 415 cm-cellulose CM-52 (Whatman, UK), equilibrated with 0.05 M ammonium acetate (pH 5.9). Spend the elution gradient from 0.1 to 0.7 M ammonium acetate (pH 5.9), the fractions containing the desired product are pooled and lyophilized. Obtain 182 mg of calcitonin salmon; chromatographic purity by HPLC 92%, the mass content of the peptide material is 76%. Mass spectrum: (M+N)+3432.8 (calculated: 3433.15); amino acid composition: Asx 2.01 (2); Glx 3.22 (3); Ser 3.58 (4); Thr 4.63 (5); Gly 3.04 (3); Val 0.94 (1); Leu 5.00 (5); Pro 2.11 (2); Tight 0.94 (1); His 1.07 (1); Lys 2.01 (2); Arg 1.05 (1); Cys is not defined.

Example 10. Receiving calcitonin salmon (using segment IVb).

36 is. Get 360 mg of Tris-trifenatate segment IVb with a free-amino group, (M+N)+3249.3. It is dissolved in 3 ml of dimethyl sulfoxide, add 130 mg of segment VII and then hold condensation and treating the reaction mixture as described in example 9. Obtain 510 mg of a crude protected 32-peptide VIIIb. It is treated with TFU to remove the BOC-protection, as described above, then this will release the sodium hydroxide as described in example 9, for 20 minutes Chromatography on cellulose CM-52 emit 131 mg of calcitonin salmon, identical obtained in example 9; chromatographic purity by HPLC 91%, the mass content of the peptide material is 78%.

Protected peptides of General formula

X1-Ley-His-Lys(R1)-Leu-Gln-Thr-Tyr(R2)-Pro-Y

where X1= H-, A1O-CO-, H-Lys(R3)-Leu-Ser-Gln-Glu(B4)-, A1O-CO-Lys(R3)-Leu-Ser-Gln-Glu(B4)-;

Y= -OH, -Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2;

AND1there is a third-butyl;

R1- protective group of the formula IN the1O-CO - Epsilon-amino group of the Lys residue;

R2- protective group IN2About-WITH - hydroxyl group of residue Tight;

R3- protective group of the formula IN the3O-CO - Epsilon-amino group of the Lys residue;

IN4- protective group for guymy or different and are selected from the range: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(substituted aryl)sulfonylated.

 

Same patents:

The invention relates to medicine, in particular to the medical industry, and can be used for treating immunodeficiencies involving violation of the process of differentiation of T-lymphocytes

Peptide derivatives // 2187511

Antitumor peptides // 2182911

The invention relates to oligopeptides derivative containing amino acid D-2-alkyltrimethyl, which is capable of releasing growth hormone (GH) from the somatotropic cells and active when administered orally

The invention relates to novel antagonists of LH-RF, and above all, peptidomimetics and peptide with a modified side chain, of the formula V, where D-Xxx, R1- R8described in the description, and their salts with pharmaceutically acceptable acids, as well as it describes their therapeutic use as analogue releasing factor, luteinizing hormone (LH-RF) with high antagonistic activity and not detecting unwanted side effects, especially no incentive edema

New peptides // 2162855
The invention relates to medicine, refers to the use of CGRP antagonists and/or for the preparation of cosmetic or pharmaceutical compositions, especially for topical application, for the treatment of ocular or palpebral of poonsuk (prurigo), ocular or palpebral pain and ocular or palpebral of dysesthesia

The invention relates to an improved process for the preparation of collagen from solubilizing and purified, may pasensyahan extract non-sterile native or telopeptide collagen, including: i) the stage of mixing and shearing of the extract in mixer with dual lateral incisors with the gradual increase of the initial rate of mixing at 500-1000 rpm without exceeding the speed of 10000 rpm and the gradual increase of temperature for 2-10oWith, preferably 3-5oWith, thus, to increase the initial ambient temperature of the extract to the maximum controlled temperature, component not exceeding 50oWith, and then (ii) the stage of sterilization in the liquid environment of the extract with obtaining sterile collagen in native or telopeptides, native or telopeptide collagen type I, obtained with the above method, with the following characteristics or properties: ratio2(I)1/1(I)2from 0.48 to 0.52; sterility in accordance with the standard of the European Pharmacopoeia; total nitrogen from 17,0 to 18.7%; hydrox

The invention relates to the field of biotechnology and biochemistry, and can be used in medicine

The invention relates to a method for cyclosporine And high purity by purification of the crude product containing cyclosporiasis complex by multi-step chromatography on silica gel at high load columns from 10 to 52%, using as eluent a mixture of toluene with acetone in an amount of from 10 to 30 vol.% or toluene with ethyl acetate in an amount of from 10 to 35 vol.%, cyclosporine And high purity with content cyclosporine L, U and D less than 0.05% and the content of cyclosporine and < 0,02% vol., industrial method of purification of cyclosporin a from a crude product containing complex cyclosporiasis

The invention relates to method modification, facilitating access to the treated (poly)peptides and to a method of cleaning using this method modifications

The invention relates to method modification, facilitating access to the treated (poly)peptides and to a method of cleaning using this method modifications

The invention relates to method modification, facilitating access to the treated (poly)peptides and to a method of cleaning using this method modifications

The invention relates to medicine, in particular to drugs, protein-containing, and methods for their preparation, namely natural cationic proteins from the tissues of the thymus gland of calves - histone

The invention relates to medicine, in particular to drugs, protein-containing, and methods for their preparation, namely natural cationic proteins from the tissues of the thymus gland of calves - histone

The invention relates to novel conjugates of affinity ligand - matrix containing the ligand associated with the matrix carrier, optionally via a spacer elements located between the matrix and ligand, and new conjugates affinity ligand - matrix, to their preparation and use for the purification of protein materials, such as, for example, immunoglobulins, insulin, factor VII, or human growth factor, or its analogs, derivatives and fragments, and their predecessors
Up!