METHOD FOR PRODUCING A RECOMBINANT PEPTIDE AND RESULTANT PEPTIDE
20170158735 ยท 2017-06-08
Assignee
Inventors
- Nikolay Fedorovich MYASOEDOV (Moscow, RU)
- Lyudmila Alexandrovna Andreeva (Moscow, RU)
- Dmitriy Viktorovich Golikov (Moscow, RU)
Cpc classification
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61P15/00
HUMAN NECESSITIES
C07K5/1013
CHEMISTRY; METALLURGY
International classification
Abstract
The invention relates to peptides with the following general formula:
A-Thr-Lys-Pro-B-C-D-X, where:
A0, Met, Met (O), Thr, Ala, His, Phe, Lys, Gly
B0, Gly, Asp, Trp, Gln, Asn, Tyr, Pro, Arg
C0, Arg, Phe, Tyr, Gly, His, Pro, Lys
D0, Val, Gly, Tyr, Trp, Phe, His
XOH, OCH.sub.3, NH.sub.2,
where 0 is no amino acid residue, provided that if A0, then B and/or C and/or D0, if B0, then C and/or D0,
excluding the peptides Phe-Thr-Lys-Pro-Gly (SEQ ID NO: 1), Thr-Lys-Pro-Pro-Arg (SEQ ID NO: 2), Thr-Lys-Pro-Arg-Gly (SEQ ID NO: 3).
Claims
1-2. (canceled)
3. A pharmaceutical composition comprising a peptide of formula:
A-Thr-Lys-Pro-B-C-D-X, wherein: A is absent, Met, Met(O), Thr, Ala, His, Phe, Lys; B is absent, Gly, Asp, Trp, Gln, Asn, Tyr, Pro, Arg; C is absent, Arg, Phe, Tyr, Gly, His, Pro; D is absent, Val, Tyr, Trp, Phe, His; X is a carboxy terminal OH, OCH.sub.3, or NH.sub.2 group, provided that (i) when A is present, at least one of B, C, or D is also present, or when B is present, then C and/or D is also present and (ii) the peptide is not a tetrapeptide and is not Thr-Lys-Pro-X (SEQ ID NO: 5), Phe-Thr-Lys-Pro-Gly-X (SEQ ID NO: 1), Thr-Lys-Pro-Pro-Arg-X (SEQ ID NO: 2), Thr-Thr-Lys-Pro-Arg-X (SEQ ID NO: 607), Lys-Thr-Lys-Pro-Arg-X (SEQ ID NO: 772), Thr-Lys-Pro-Gly-Pro-X (SEQ ID NO: 460), Thr-Lys-Pro-Gly-Arg-X (SEQ ID NO: 456), Thr-Lys-Pro-Arg-Tyr (SEQ ID NO: 494), or Thr-Lys-Pro-Arg-Gly-X (SEQ ID NO: 3), wherein said peptide is present in the composition in an amount effective for stimulating sexual or reproductive function.
4. The pharmaceutical composition of claim 3, wherein said peptide is present in the composition in the form of a salt.
5. The pharmaceutical composition of claim 4, wherein said peptide salt is produced by chemical synthesis.
6. The pharmaceutical composition of claim 5, wherein said chemical synthesis includes a step of ion-exchange resin replacing a counter-ion.
7. A method for stimulating sexual or reproductive function in a mammal in need thereof comprising administering to said mammal the pharmaceutical composition of claim 3.
8. The method of claim 7, wherein the mammal has a reproductive or sexual dysfunction.
9. A method for treating a reproductive or sexual dysfunction in a mammal in need of such treatment comprising administering to said mammal the pharmaceutical composition of claim 3.
10. A method for stimulating sexual or reproductive function in a mammal in need thereof comprising administering to said mammal the pharmaceutical composition of claim 4.
11. The method of claim 10, wherein the mammal has a reproductive or sexual dysfunction.
12. A method for treating a reproductive or sexual dysfunction in a mammal in need of such treatment comprising administering to said mammal the pharmaceutical composition of claim 4.
13. A method for stimulating sexual or reproductive function in a mammal in need thereof comprising administering to said mammal a pharmaceutical composition comprising a peptide of formula A-Thr-Lys-Pro-B-C-D-X, wherein: A is absent, Met, Met(O), Thr, Ala, His, Phe, Lys, Gly; B is absent, Gly, Asp, Trp, Gln, Asn, Tyr, Pro, Arg; C is absent, Arg, Phe, Tyr, Gly, His, Pro, Lys; D is absent, Val, Gly, Tyr, Trp, Phe, His; X is a carboxy terminal OH, OCH.sub.3, or NH.sub.2 group, provided that (i) when A is present, at least one of B, C, or D is also present, or when B is present, then C and/or D is also present and (ii) the peptide is not a tetrapeptide.
14. The method of claim 13, wherein said peptide is present in the composition in the form of a salt.
15. The method of claim 13, wherein the mammal has a reproductive or sexual dysfunction.
16. A method for treating a reproductive or sexual dysfunction in a mammal in need of such treatment comprising administering to said mammal a pharmaceutical composition comprising a peptide of formula A-Thr-Lys-Pro-B-C-D-X, wherein: A is absent, Met, Met(O), Thr, Ala, His, Phe, Lys, Gly; B is absent, Gly, Asp, Trp, Gln, Asn, Tyr, Pro, Arg; C is absent, Arg, Phe, Tyr, Gly, His, Pro, Lys; D is absent, Val, Gly, Tyr, Trp, Phe, His; X is a carboxy terminal OH, OCH.sub.3, or NH.sub.2 group, provided that (i) when A is present, at least one of B, C, or D is also present, or when B is present, then C and/or D is also present and (ii) the peptide is not a tetrapeptide.
17. The method of claim 16, wherein said peptide is present in the composition in the form of a salt.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0022]
[0023]
[0024]
[0025]
[0026]
[0027]
[0028]
BEST EXAMPLE OF THE INVENTION
[0029] The following are the examples illustrating the invention.
[0030] Synthesis of peptides with the general formula A-Thr-Lys-Pro-B-C-D-X was performed by methods of peptide chemistry in solution using L-amino acids. Peptide synthesis was carried out by stepwise elongation of the peptide chain, as well as fragment condensation using mixed anhydride method, carbodiimide method with the addition of 1-hydroxybenzotriazole as an auxiliary nucleophile, activated ester method, and mixed anhydride method. All intermediate and final products were isolated and characterized. Evaporation of solutions was conducted using vacuum evaporator at 40 C. Melting points, determined with Boethius apparatus, are given without correction. Identity of the obtained compounds was tested by TLC on Silufol silica gel-coated plates (Czech Republic). Substances were detected by UV light using ninhydrin, Barton's reagent, Pauly reagent, Reindel-Hoppe reagent, and o-tolidine in chlorine environment. Specific rotation was determined by AI-EPO polarimeter. Peptide homogeneity was tested by high performance liquid chromatography (HPLC), and peptide structure was confirmed by mass spectrometric methods. All solvents were correspondingly absolutized. Melting points were not corrected.
[0031] Peptides were also obtained by genetic engineering techniques using host cells engineered by known laboratory strains of E. coli, transformed with known commercially available plasmids containing nucleic acid encoding the target peptide.
[0032] The examples describe peptide synthesis.
Example 1. Synthesis of Arg-Pro-Gly-Pro Tetrapeptide
[0033] Synthesis of the tetrapeptide was performed according to the diagram shown in
[0034] In synthesis, mixed anhydride method, azide method and carbodiimide method were used. Derivatives of L-amino acids were used for synthesis. Evaporation of solutions was conducted using rotor evaporator at 40 C. Melting points, determined with Boethius apparatus, are given without correction. Identity of the obtained compounds was tested by TLC on Silufol silica gel-coated plates (Czech Republic). Substances were detected by spraying the plate with a solution of ninhydrin and (or) o-tolidine. Substances were detected by spraying the plate with a solution of ninhydrin and (or) o-tolidine. Chromatographic mobility (Rf) values in the following solvent systems are provided: acetone:benzene:acetic acid (50:100:1)(1); chloroform:methanol (9:1)(2); hexane:acetone (3:2)(3); butanol:acetic acid:water (4:1:1)(4); butanol:acetic acid:pyridine:water (30:6:20:24)(5); chloroform:methanol:ammonia (6:4:1)(6); benzene:ethanol (8:2)(7); Ethyl acetate:acetone:50% acetic acid:water (2:1:1)(8); chloroform:methanol (14:1)(9); chloroform:methanol:ammonia (8:1.75:0.25)(10); chloroform:methanol:ammonia (6.5:3.0:0.5)(11).
[0035] Specific rotation was determined by AI-EPO polarimeter.
[0036] Elemental analysis using Carlo-Erba model 1106 analyzer.
[0037] I. Boc-Pro-Gly-OEt.
[0038] 8.3 g (3.45 mmol) of Boc-Pro-OH were dissolved in 50 ml of CH.sub.2Cl.sub.2, cooled down to 5 C.; then, 38.45 mmol (5.38 ml) of TEA were added. The reaction mixture was cooled down to 2530 C. At this temperature, 38.45 mmol (4.84 ml) of isobutyl chloroformate were added using a pipette. The reaction mixture temperature was kept in the range of 1820 C. for 20 minutes. Simultaneously, a solution of 5.9 g (42.3 mmol) of 1.1-fold excess of HCl.H-Gly-OEt in 75 ml of chloroform, containing 5.92 ml of TEA. The solution was cooled down to 25 C. and, after formation of mixed anhydride in the first flask, its contents were poured to the ether solution right away. The reaction mixture was incubated for 1 hour at 10 C. and then stirred for 12 hours at 4 C. on a magnetic stirrer. The reaction mixture was evaporated, then 250 ml of ethyl acetate were added; then, ethyl acetate solution was washed 3 times with 25 ml of 0.1N HCl, 3 times with 25 ml H.sub.2O, and once with saturated solution of NaCl. The organic layer was dried over MgSO.sub.4, filtered and evaporated. The residue was dried under vacuum over P.sub.2O.sub.5/KOH and paraffin.
[0039] Yield: 10.2 g (30.3 mmol) 78.83%
[0040] Rf0.5 (7); 0.862 (8)
[0041] Melting point 68-70 C.
[0042] II. Boc-Pro-Gly-N.sub.2H.sub.3.
[0043] 10.2 g of Boc-Pro-Gly-OEt (30.3 mmol) were dissolved in 80 ml of absolute methanol, and 4-fold excess of hydrazine hydrate, viz. 5.88 ml (121.2 mmol), was added. The solution was stirred for 12 hours on a magnetic stirrer at room temperature. The reaction mixture was evaporated, then two times evaporated with ether; then, ether (5 ml) was poured over the residue and left in a refrigerator overnight (for better crystallization, seeding agent was added). The precipitated crystals were filtered, washed with ether using a filter and dried in a desiccator.
[0044] Yield: 6.9 g (20.79 mmol) 68.62%
[0045] Rf0.284 (7); 0.474 (8); 0.189 (9)
[0046] Melting point 98-100 C.
[0047] III. Boc-Pro-Gly-Pro-OBzl.
[0048] 58.4 mmol (4-fold excess) of hydrogen chloride in ethyl acetate were added to the solution of Boc-Pro-Gly-N.sub.2H.sub.3, containing 4.7 g (14.6 mmol) in 40 ml of DMF, cooled down to 20 C., and, immediately, 1.73 mL (14.6 mmol) of freshly distilled tert-butyl nitrite; then, the reaction mixture was stirred for 30 minutes at 5 C. The reaction mixture was cooled down to 40 C., and solution of 8.2 mL (58.4 mmol) of TEA in 4 mL of DMF, cooled down to 10 C., was added; when the temperature of the reaction mixture had risen to 20 C., 3.7 g (15.3 mmol) of 1.05-fold excess of HCl.H-Pro-OBzl were added to 20 ml of DMF and 2.14 ml of TEA. Then, it was stirred at 4 C. for 24 hours on a magnetic stirrer. The reaction mixture was evaporated, and the residue was dissolved in 200 mL of ethyl acetate and washed 2 times with 20 ml of H.sub.2O, 3 times with 20 ml of 10% sodium KHSO.sub.4, 3 times with 20 ml of H.sub.2O, 3 times with 20 ml of 5% NaHCO3, and 3 times with 20 ml of H.sub.2O. Ethyl acetate solution was dried over MgSO.sub.4. Then it was evaporated, and a small amount of ether (10 mL) was added to the residue. Then, it was left in a refrigerator for crystallization. For better product crystallization, seeding agent was added. The precipitated crystals were filtered and washed with a small amount of ether using a filter. Then, they were dried in a desiccator.
[0049] Yield: 5.358 g (11.65 mmol) 79.92%
[0050] Rf0.326 (7); 0.947 (8); 0.390 (9)
[0051] Melting point 125-126 C.
[0052] [].sub.D.sup.22=101.18 (c=0.85; CH.sub.3OH).
[0053] Elemental analysis: C, 62.89 (62.73); N, 9.21 (9.14); H, 7.52 (7.24).
[0054] IV. TFA.H-Pro-Gly-Pro-OBzl.
[0055] 5.358 g (11.65 mmol) of Boc-Pro-Gly-Pro-OBzl were dissolved in 29.13 ml of methylene chloride; then, 29.13 mL of TFA were added, incubated for 45 minutes at room temperature, evaporated 2 times with absolute ethanol, 2 times with benzene, 2 times with ether and dissolved in benzene; then, hexane was poured over. Hexane was decanted, and the resulted substance was dried under vacuum in a desiccator over KOH, P.sub.2O.sub.5 and paraffin, while desiccant was changed several times.
[0056] Yield: 4.63 g (9.7 mmol) 98%
[0057] Rf0.043 (7); 0.247 (8); 0.018 (9).
[0058] V. Boc-Arg(NO2)-Pro-Gly-Pro-OBzl.
[0059] 3.09 g of Boc-Arg (NO.sub.2)OH (9.7 mmol) were dissolved in 50 mL of THF and 10 ml DMF, 2.07 g (10.67 mmol) of DCC were added, cooled down to 0 C., stirred for 40 minutes; then, a solution of TFA.H-Pro-Gly-Pro-OBzl was added to 50 ml of THF and 4.46 ml (9.7 mmol) of TEA. The reaction mixture was stirred for three days. DCM precipitate was filtered off; the solution was evaporated under vacuum; then, 200 ml of hexane were added to the residue. With this, the desired product was separated as an oil, which was dissolved in 500 mL of ethyl acetate and washed 3 times with 25 ml of 0.1N HCl, three times with 25 ml of H.sub.2O, and once with saturated solution of NaCl. The organic layer was dried over MgSO.sub.4, filtered and evaporated. The residue was dissolved in ethyl acetate and precipitated with dry ether. The precipitate was filtered and dried under vacuum over P.sub.2O.sub.5/KOH and paraffin, while desiccant was changed several times.
[0060] Yield: 4.76 g (7.9 mmol) 85%
[0061] Rf0.44 (1); 0.8 (11)
[0062] Melting point 108-110 C.
[0063] [].sub.D.sup.22=77.8 (c=0.5; CH.sub.3COOH).
[0064] VI. Boc-Arg-Pro-Gly-Pro.
[0065] 4.76 g (6.5 mmol) of Boc-Arg(NO.sub.2)Pro-Gly-Pro-OBzl were dissolved in 100 ml of methanol; then 1 ml 1N hydrochloric acid and 4.67 g of catalyst, viz. 10% palladium oxide on neutral aluminum oxide, were added, and hydrogenation in the stream of dry hydrogen at room temperature under 1 atm. was conducted for 6 hours. Then, the catalyst was filtered off and washed on the filter with methanol. Pooled filtrate was evaporated to dryness. The residue was precipitated with ether from the absolute methanol. Then, it was dried under vacuum, while desiccant was changed several times.
[0066] Yield: 4.23 g (5.8 mmol) 89%
[0067] Rf0.125 (4); 0.57 (6); 0.37 (5)
[0068] Melting point 123-125 C.
[0069] VII. Arg-Pro-Gly-Pro.
[0070] 4.23 g (5.8 mmol) of Boc-Arg-Pro-Gly-Pro-OH were suspended in 10 ml of 2N hydrochloric acid in dioxane and incubated at room temperature for 45 min. Then, dry ether was added, and the precipitate was washed by decantation with dry ether. It was reprecipitated with ether from absolute methanol. The resulting precipitate was dissolved in 7.5 ml of 30% ethanol and applied to Amberlyst A-21 (AcO.sup. form) column for acetate/hydrochloride salt exchange. Peptide was eluted with 200 ml of 30% ethanol, evaporated to dryness under vacuum and precipitated with absolute ether from methanol.
[0071] Yield: 3.69 g (4.93 mmol) 85%
[0072] Rf0.287 (4); 0.145 (5); 0.338 (14)
[0073] Melting point 120-122 C.
[0074] HPLC results: Column: Supercosil ABZ Plus, size 4.6250 mm; flow rate 1 mL/min; Eluent A:
[0075] NH.sub.4H.sub.2PO.sub.4+H.sub.3PO.sub.4 (50 mM, pH 2.8); eluent B: MeOH
[0076] Gradient: 0-20 min (0-40% B); retention time 21.21 min.
[0077] Rf0.24 (6)
[0078] Melting point=151 C.
[0079] [].sub.D.sup.22=65.0 (c=0.5 CH.sub.3COOH)
[0080] HPLC results: Column: Zorbax ODS d=4.6 mm; t=35 C.
[0081] Flow rate 1 mL/min; A=50 mM NH.sub.4H.sub.2PO.sub.4 (pH 2.5); B=A+MeOH (1:1); 10-60% B (in 25 min). Retention time 16.5 min.
Example 2. Synthesis of Pro-Arg-Pro-Gly-Pro Pentapeptide
[0082] Peptide synthesis was performed by classical methods of peptide chemistry using natural L-amino acids according to the diagram shown in
[0083] First, Pro-Gly-Pro tripeptide was produced; then, pentapeptide was obtained by stepwise buildup of the peptide chain from the N-terminus. In synthesis, mixed anhydride method, azide method and carbodiimide method were used.
[0084] Chromatographic mobility (Rf) values in the following solvent systems are provided: butanol: acetic acid: water (4:1:1)(1); chloroform:methanol:ammonia (6:4:1)(2); acetone: benzene: acetic acid (50:100:1)(3); chloroform:methanol (9:1)(4); hexane:acetone (3:2)(5); butanol:acetic acid:pyridine:water (30:6:20:24)(6); chloroform:methanol (14:1)(7).
[0085] I. Boc-Pro-Gly-OEt.
[0086] 8.3 g of Boc-Pro (38.45 mmol) were dissolved in 50 ml of CH.sub.2Cl.sub.2, cooled down to +5 C., and 38.45 mmol (5.38 ml) of TEA were added. The reaction mixture was cooled down to 2530 C. At this temperature, 38.45 mmol (4.84 ml) of isobutyl chloroformate were added using a pipette. The reaction mixture temperature was kept in the range of 1820 C. for 20 minutes. Simultaneously, a solution of 5.9 g (42.3 mmol) of 1.1-fold excess of HCl.H-Gly-OEt in 75 ml of chloroform, containing 5.92 ml of TEA. The solution was cooled down to 25 C. and, after formation of mixed anhydride in the first flask, its contents were poured to the ether solution right away. The reaction mixture was incubated for 1 hour at 10 C. and then stirred for 12 hours at 4 C. on a magnetic stirrer. The reaction mixture was evaporated and 250 ml of ethyl acetate were added to it; the ethyl acetate solution was washed 3 times with 25 ml of 0.1N HCl, 3 times with 25 ml H.sub.2O, and once with saturated solution of NaCl. The organic layer was dried over MgSO4, filtered and evaporated. The residue was dried under vacuum over P.sub.2O.sub.5/KOH and paraffin.
[0087] Yield: 10.2 g (30.3 mmol) 78.83%
[0088] Rf0.5 (3); 0.862 (4).
[0089] II. Boc-Pro-Gly-N.sub.2H.sub.3.
[0090] 10.2 g of Boc-Pro-Gly-OEt (30.3 mmol) were dissolved in 80 ml of absolute methanol, and 4-fold excess of hydrazine hydrate, viz. 5.88 ml (121.2 mmol), was added. The solution was stirred for 12 hours on a magnetic stirrer at room temperature. The reaction mixture was evaporated, then two times evaporated with ether; then, ether (5 ml) was poured over the residue and left in a refrigerator overnight (for better crystallization, seeding agent was added). The precipitated crystals were filtered, washed with ether using a filter and dried in a desiccator.
[0091] Yield: 6.9 g (20.79 mmol) 68.62%
[0092] Melting point 98-100 C.
[0093] Rf0.284 (3); 0.474 (4); 0.189 (5).
[0094] III. Boc-Pro-Gly-Pro-OBzl.
[0095] 58.4 mmol (4-fold excess) of hydrogen chloride in ethyl acetate were added to the solution of Boc-Pro-Gly-N.sub.2H.sub.3, containing 4.7 g (14.6 mmol) in 40 ml of DMF, cooled down to 20 C., and, immediately, 1.73 mL (14.6 mmol) of freshly distilled tert-butyl nitrite; then, the reaction mixture was stirred for 30 minutes at 5 C. The reaction mixture was cooled down to 40 C., and solution of 8.2 mL (58.4 mmol) of TEA in 4 mL of DMF, cooled down to 10 C., was added; when the temperature of the reaction mixture had risen to 20 C., 3.7 g (15.3 mmol) of 1.05-fold excess of HCl.H-Pro-OBzl were added to 20 ml of DMF and 2.14 ml of TEA. Then, it was stirred at 4 C. for 24 hours on a magnetic stirrer. The reaction mixture was evaporated, and the residue was dissolved in 200 mL of ethyl acetate and washed 2 times with 20 ml of H.sub.2O, 3 times with 20 ml of 10% sodium KHSO.sub.4, 3 times with 20 ml of H.sub.2O, 3 times with 20 ml of 5% NaHCO.sub.3, and 3 times with 3 ml of H.sub.2O. Ethyl acetate solution was dried over MgSO.sub.4. Then it was evaporated, and a small amount of ether HO mL) was added to the residue. Then, it was left in a refrigerator. For better product crystallization, seeding agent was added. The precipitated crystals were filtered and washed with a small amount of ether using a filter. Then, they were dried in a desiccator.
[0096] Yield: 5.358 g (11.65 mmol) 79.92%
[0097] Rf0.326 (3); 0.947 (4); 0.390 (5)
[0098] Melting point 125-126 C.
[0099] [].sub.D.sup.22=101.2 (c=0.85; CH.sub.3OH)
[0100] Elemental analysis: C, 62.89 (62.73); N, 9.21 (9.14); H, 7.52 (7.24).
[0101] IV. TFA.H-Pro-Gly-Pro-OBzl.
[0102] 5.358 g (11.65 mmol) of Boc-Pro-Gly-Pro-OBzl were dissolved in 29.13 ml of methylene chloride; 29.13 mL of TFA were added, incubated for 45 minutes at room temperature, evaporated 2 times with absolute ethanol, 2 times with benzene, 2 times with ether and dissolved in benzene; then, hexane was poured over. Hexane was decanted, and the resulted substance was dried under vacuum in a desiccator over P.sub.2O.sub.5/KOH and paraffin.
[0103] Yield: 4.63 g (9.7 mmol) 98%
[0104] Rf0.043 (3); 0.247 (4); 0.018 (5).
[0105] V. Boc-Arg(NO.sub.2)Pro-Gly-Pro-OBzl.
[0106] 3.09 g (9.7 mmol) of Boc-Arg (NO.sub.2) were dissolved in 50 ml of THF and 10 ml DMF; then, 2.07 g (10.67 mmol) of DCC were added, cooled down to 0 C. and stirred for 40 minutes; a solution of TFA.H-Pro-Gly-Pro-OBzl was added to 50 ml of THF and 4.46 ml (9.7 mmol) of TEA. The reaction mixture was stirred for three days. DCM precipitate was filtered off; the solution was evaporated under vacuum; then, 200 ml of hexane were added to the residue. With this, the desired product was separated as an oil, which was dissolved in 500 mL of ethyl acetate and washed 3 times with 25 ml of 0.1N HCl, three times with ml of H.sub.2O, and once with saturated solution of NaCl. The organic layer was dried over MgSO4, filtered and evaporated. The residue was dried under vacuum over P.sub.2O.sub.5/KOH and paraffin.
[0107] Yield: 4.76 g (7.9 mmol) 85%
[0108] Rf0.44 (1); 0.8 (6)
[0109] Melting point 108-110 C.
[0110] VI. TFA-Arg(NO.sub.2)Pro-Gly-Pro-OBzl.
[0111] 4.76 g (7.9 mmol) of Boc-Arg (NO.sub.2)Pro-Gly-Pro-OBzl were dissolved in 20 ml of methylene chloride, 20 mL of TFA were added, incubated for 45 minutes at room temperature, evaporated 2 times with absolute ethanol, 2 times with benzene, 2 times with ether, then dissolved in benzene; then, hexane was poured over. Hexane was decanted, and the resulted substance was dried under vacuum in a desiccator over P.sub.2O.sub.5/KOH and paraffin.
[0112] The yield is quantitative.
[0113] Rf0.16 (1); 0.27 (6).
[0114] VII. Boc-Pro-Arg(NO.sub.2)Pro-Gly-Pro-OBzl.
[0115] 1.7 g (7.9 mmol) of Boc-Pro were dissolved in 20 mL of THF, and 1.07 g (7.9 mmol) of BT were added, cooled down to 0 C.; 1.8 g of DCC in 50 ml of THF were added. In 40 min, a solution of TFA-Arg(NO.sub.2)Pro-Gly-Pro-OBzl (7.9 mmol) in 50 mL of THF and 1.1 mL (7.9 mmol) of TEA were added to the reaction mixture. It was stirred for 2 hours at 0 C. and for 2 days at room temperature; then, DCM was filtered off, evaporated under vacuum, dissolved in 500 mL of ethyl acetate and treated similarly to Boc-Pro-Arg(NO.sub.2)Pro-Gly-Pro-OBzl.
[0116] Yield: 4.07 g (67.8%)
[0117] Rf0.42 (1); 0.72 (6); 0.31 (7)
[0118] Melting point 147-148 C.
[0119] VIII. Boc-Pro-Arg-Pro-Gly-Pro.
[0120] 4.07 g (6.5 mmol) were dissolved in 100 ml of methanol; 1 ml of 1N hydrochloric acid and 0.85 g of catalyst, viz. 10% palladium oxide on neutral aluminum oxide, were added, and hydrogenation in the stream of dry hydrogen at room temperature under 1 atm. was conducted for 6 hours. Then, the catalyst was filtered off and washed on the filter with methanol. Pooled filtrate was evaporated to dryness. The residue was precipitated with ether from the absolute methanol.
[0121] Yield: 3.02 g (5.8 mmol) 89%
[0122] Rf0.125 (1), 0.57 (2), 0.37 (6).
[0123] IX. Pro-Arg-Pro-Gly-Pro.
[0124] 3.02 g (5.8 mmol) of Boc-Pro-Arg-Pro-Gly-Pro were suspended in 10 ml of 2N hydrochloric acid in dioxane and were incubated at room temperature for 45 min. Then, dry ether was added, and the precipitate was washed by decantation with dry ether. It was reprecipitated with ether from absolute methanol. The resulting precipitate was dissolved in 7.5 ml of 30% ethanol and applied to Amberlyst A-21 (AcO.sup.-form) column for acetate/hydrochloride salt exchange. Peptide was eluted with 200 ml of 30% ethanol, evaporated to dryness under vacuum and precipitated with absolute ether from methanol.
[0125] Yield: 2.27 g (75%)
[0126] Rf0.2 (2); 0.1 (6)
[0127] Melting point 180-185 C.
[0128] [].sub.D.sup.20=105 (c=0.4; CH.sub.3COOH).
[0129] Amino acid composition versus arginine: Pro 2.78 (3); Gly 1.1 (1).
[0130] HPLC results: Column: Supercosil ABZ Plus, size 4.6250 mm; flow rate 1 mL/min; Eluent A:
[0131] NH.sub.4H.sub.2PO.sub.4+H.sub.3PO.sub.4 (50 mM, pH 2.8); eluent B: MeOH
[0132] Gradient: 0-20 min (0-40% B); retention time 10.13 min.
Example 3. Synthesis of Pro-Gly-Pro Tripeptide
[0133] The synthesis of Pro-Gly-Pro tripeptide was carried out according to the diagram shown in
[0134] Synthesis of Pro-Gly-Pro tripeptide was carried out using modern protecting groups and methods of peptide bond formation in solution. Mixed anhydride method with PivCl was used for peptide bond formation. tert-butyloxycarbonyl protection (Boc) was used for protection of amino groups, and benzyl ester (OBzl) was recruited for protection of carboxyl group. Stepwise approach to peptide chain elongation was used.
[0135] Derivatives of L-amino acids were used for synthesis. Evaporation of solutions was conducted using vacuum evaporator at 40 C. Melting points, determined with Boethius apparatus, are given without correction.
[0136] Identity of the obtained compounds was tested by TLC on Silufol silica gel-coated plates (Czech Republic). Substances were detected by spraying the plate with a solution of ninhydrin and (or) o-tolidine. Chromatographic mobility (Rf) values in the following solvent systems are provided: (ethyl acetate:acetone:50% acetic acid:water (2:1:1); benzene:ethanol (8:2); chloroform:methanol:ammonia (6:4:1); chloroform:methanol:acetic acid (42:7:1); acetone:benzene:acetic acid (50:100:1); chloroform:methanol (9:1); hexane:acetone (3:2); butanol:acetic acid:water (4:1:1); butanol:acetic acid:pyridine:water (30:6:20:24); hexane:ethyl acetate (4:1); chloroform:methanol:ammonia (8:1.75:0.25); (isopropanol:formic acid:water) (20:5:1); (chloroform:methanol:ammonia) (7:2.5:0.5); methanol. Specific rotation was determined by AI-EPO polarimeter). Elemental analysis using Carlo-Erba model 1106 analyzer.
[0137] I. Production of Boc-Pro-Gly-OH.
[0138] 1. 10.75 g (50 mmol) of Boc-Pro were dissolved in 150 ml of acetonitrile, cooled down to 5 C.; then, 7.7 ml (50 mmol) of triethylamine (TEA) were added to the solution, and it was cooled down to 20 C., while stirring on a magnetic stirrer. 6.8 ml (55 mmol) of pivaloyl chloride (PivCl) were added to the cooled solution, stirred on a magnetic stirrer for 20 minutes at 10 C. and then cooled down to 30 C.; then, precooled solution of Gly was added. Simultaneously, Gly solution was prepared
[0139] 2. 4.5 g of Gly (60 mmol, 1.2-fold excess) were dissolved in 35 ml of water and 60 ml of acetonitrile, 8.4 ml (60 mmol) of triethylamine were added. The mixture was cooled down to 10 C. and added to the solution in the first flask after 20 minutes. The reaction mixture was incubated for 1 hour at 10 C. and stirred for 2 hours at 18-20 C. on a magnetic stirrer. The reaction mixture was evaporated on a rotary evaporator. About 50 mL of water were added to the residue. The aqueous solution was acidified with a 3-fold excess of NaHSO.sub.4 (24.84 g) to pH=3 and extracted 5 times with 100 ml of ethyl acetate. Pooled ethyl acetate solution was washed with H.sub.2O (50 mL), 10% solution of KHSO.sub.4 (50 mL), H.sub.2O (50 mL), and saturated NaCl (50 mL). Ethyl acetate solution was dried over MgSO4. Dried ethyl acetate was filtered and evaporated. Dry ether was added to the residue. Upon the addition of ether to the flask, it precipitated the product, which was filtered and washed with dry ether using a filter. The resulted substance was dried under vacuum in a desiccator over KOH, P2O5 and paraffin, while desiccant was changed several times.
[0140] Product M.W. 272.3
[0141] Yield: 5.97 g (21.74 mmol); (43.5%)
[0142] Melting point 70 C.
[0143] Rf0.863 (acetone-benzene-acetic acid) (50:100:1);
[0144] 0.746 (benzene-ethanol) (8:2); 0.903 (chloroform:methanol) (9:1);
[0145] 0.847 (Ethyl acetate:acetone:50% acetic acid:water) (2:1:1).
[0146] II. Production of Boc-Pro-Gly-Pro-OBzl.
[0147] 1. 5.97 g (21.74 mmol) of Boc-Pro-Gly-OH were dissolved in 100 ml of acetonitrile, cooled down to 5 C.; then, 1.1-fold excess (3.35 ml, 23.9 mmol) of triethylamine (TEA) was added to the solution, and it was cooled down to 20 C., while stirring on a magnetic stirrer. 1.1-fold excess (2.34 ml, 23.9 mmol) of pivaloyl chloride (PivCl) was added to the cooled solution, stirred on a magnetic stirrer for 20 minutes at 10 C. and then cooled down to 30 C.; then, precooled solution of HCl.Pro-OBzl was added.
[0148] Simultaneously, HCl.Pro-OBzl was prepared.
[0149] 2. 6.3 g of HCl.Pro-OBzl (26.1 mmol, 1.2 excess) were dissolved in 50 ml of acetonitrile, 4.0 ml (28.71 mmol; 1.1-fold excess) of triethylamine were added. The mixture was cooled down to 10 C. and added to the solution in the first flask after 20 minutes. The reaction mixture was incubated for 1 hour at 10 C. and stirred for 2 hours at 18-20 C. on a magnetic stirrer. The reaction mixture was evaporated. 300 ml of ethyl acetate were added to the evaporated residue. Ethyl acetate solution was washed with H.sub.2O (3 times with 25 mL), 10% solution KHSO.sub.4(3 times with 25 ml) H.sub.2O (3 times with 25 ml), 5% NaHCO.sub.3(3 times with 25 ml) H.sub.2O (3 times 25 mL), and saturated solution of NaCl (once with 25 mL). Ethyl acetate solution was dried over MgSO.sub.4. Dried ethyl acetate was filtered and evaporated. About 100 ml of dry ether were added to the residue. Upon the addition of ether to the flask, it precipitated the product, which was filtered and washed with dry ether using a filter. The resulted substance was dried under vacuum in a desiccator over KOH, P.sub.2O.sub.5 and paraffin, while desiccant was changed several times.
[0150] Product M.W. 459.82
[0151] Yield: 8.12 g (17.66 mmol); (81.23%)
[0152] Melting point 125-126 C.
[0153] Rf0.326 (acetone:benzene:acetic acid) (50:100:1)
[0154] 0.390 (hexane: acetone) (3:2)
[0155] 0.947 (chloroform:methanol) (9:1)
[0156] 0.716 (methanol)
[0157] 0.620 (benzene: ethanol) (8:2)
[0158] III. Production of Boc-Pro-Gly-Pro-OH.
[0159] 8 g (17.4 mmol) of Boc-Pro-Gly-Pro-OBzl were dissolved in 100 ml of methanol, 0.5 ml of CH.sub.3COOH and palladium black were added, while stirring on a magnetic stirrer, and hydrogen was passed through for 2 hours. The solution was filtered, evaporated, evaporated 3 times with benzene and 2 times with ethyl acetate. Then, it was precipitated with ether hexane from acetone, and the precipitate formed in the flask was dried in a desiccator over P.sub.2O.sub.5/KOH and paraffin.
[0160] Product M.W. 369.39
[0161] Yield: 6.3 g (17.05 mmol) 98%
[0162] Melting point 99-100 C.
[0163] Rf: 0.560 (chloroform-methanol) (9:1)
[0164] 0.812 (chloroform-methanol-acetic acid) (42:7:1)
[0165] 0.187 (acetone:benzene:acetic acid) (50:100:1)
[0166] 0.164 (hexane:acetone) (3:2)
[0167] IV. Production of Pro-Gly-Pro.
[0168] 40.6 ml of methylene chloride and 40.6 ml of TFA were added to 6.0 g (16.24 mmol) of Boc-Pro-Gly-Pro-OH and incubated for 45 minutes at room temperature; after removal of the protecting group, the solution was evaporated 2 times with absolute methanol, 2 times with benzene and 2 times with ether. It was precipitated with ether from acetone. The residue was dried under vacuum over P.sub.2O.sub.5, KOH and paraffin. The dried product was reprecipitated with dry diethyl ether from absolute MeOH.
[0169] Product M.W. 381.39
[0170] Yield: 5.6 g (14.72 mmol) (90.62%)
[0171] The resulting precipitate of TFA.Pro-Gly-Pro-OH was dissolved in 10 ml of 30% ethanol; then, 25 mL of Amberlyst A-21 (CH.sub.3COO.sup.) ion-exchange resin was added for exchange of trifluoroacetate to acetate salt and stirred on a magnetic stirrer for 45 minutes; then, it was washed with 150 ml of 30% ethanol, evaporated to dryness under vacuum and precipitated with dry diethyl ether from absolute methanol. The resulting precipitate was filtered off and dried in a desiccator under vacuum over P.sub.2O.sub.5, KOH and paraffin, while desiccant was changed several times.
[0172] Product M.W. 328.34
[0173] Yield: 4.54 g (13.84 mmol) (94%)
[0174] Melting point 143-145 C.
[0175] [].sub.D.sup.22=31.5 (c 1, MeOH)
[0176] Rf: 0.59 (chloroform:methanol:ammonia) (6:4:1)
[0177] 0.52 (chloroform:methanol:ammonia) (4:4.5:1.5)
[0178] 0.524 (ethanol: ammonia) (7:3)
Example 4. Synthesis of Thr-Lys-Pro-Arg-Pro-Phe Hexapeptide
[0179] Synthesis of Thr-Lys-Pro-Arg-Pro-Phe hexapeptide was performed according to the diagram shown in
[0180] Synthesis of Thr-Lys-Pro-Arg-Pro-Phe hexapeptide was carried out using modern protecting groups and methods of peptide bond formation in solution. TBA salt method, activated ester method, carbodiimide method and mixed anhydride method were used for peptide bond formation. Both stepwise and blockwise approaches were used.
Example 5. Synthesis of Thr-Lys-Pro-Arg-Pro Pentapeptide
[0181] Synthesis of Thr-Lys-Pro-Arg-Pro pentapeptide was performed according to the diagram shown in
[0182] Synthesis of Thr-Lys-Pro-Arg-Pro pentapeptide was carried out using modern protecting groups and methods of peptide bond formation in solution. TBA salt method, activated ester method and carbodiimide method were used for peptide bond formation. Both stepwise and blockwise approaches were used.
Example 6. Synthesis of Thr-Lys Dipeptide
[0183] Synthesis of Thr-Lys dipeptide was performed according to the diagram shown in
Example 7. Synthesis of Thr-Lys-Pro-Phe Tetrapeptide
[0184] The synthesis of Thr-Lys-Pro-Phe tetrapeptide was carried out according to the diagram shown in
[0185] Synthesis of Thr-Lys-Pro-Phe tetrapeptide was carried out using modern protecting groups and methods of peptide bond formation in solution. TBA salt method and activated ester method were used for peptide bond formation. Stepwise approach to peptide chain elongation was used. Derivatives of both protected and free L-amino acids were used for synthesis. Evaporation of solutions was conducted using vacuum evaporator at 40 C. Melting points, determined with Boethius apparatus, are given without correction. Identity of the obtained compounds was tested by TLC on Silufol silica gel-coated plates (Czech Republic). Substances were detected by spraying the plate with a solution of ninhydrin and (or) o-tolidine. Chromatographic mobility (Rf) values in the following solvent systems are provided: (butanol:acetic acid:water) (4:1:1); (benzene-ethanol) (8:2); (chloroform:methanol) (9:1); (isopropanol:formic acid:water) (20:5:1); (chloroform-methanol-acetic acid) (42:7:1); (chloroform:methanol:ammonia) (8:1.75:0.25); (acetone-benzene-acetic acid) (50:100:1); (chloroform:methanol:ammonia) (6:4:1); (chloroform:methanol:ammonia) (44.5:1.5); (butanol:acetic acid:pyridine:water) (30:6:20:24).
[0186] I. Production of Z-Lys(Boc)-Pro-OH.
[0187] 13% solution of TBA (98 ml) was added to 46.32 mmol (5.34 g) of Pro and evaporated two times with ethanol, two times with ethanol/benzene mixture, and 2 times with benzene. 300 ml of absolute ethyl acetate were added; the reaction mixture was cooled down to 0 C., and 23.16 mmol (12.66 g) of previously synthesized Z-Lys (Boc)-OPfp were added and stirred on a magnetic stirrer for 1 hour. The reaction mass was evaporated, and 40 ml of water were added to the evaporation residue. The aqueous solution was washed 3 times with 80 ml of ether. After washing with ether, the aqueous solution was acidified with citric acid to pH 3. After acidification, aqueous solution was extracted with ethyl acetate 3 times in 40 ml aliquots. Pooled ethyl acetate after extraction was washed 3 times with 20 ml of water, 3 times with 20 ml of 10% solution of KHSO.sub.4, and 3 times with 20 ml of water. Ethyl acetate solution was dried over MgSO.sub.4. Then, it was filtered and evaporated on a rotary evaporator. The resulted substance was precipitated with hexane from ethyl acetate. Hexane was decanted, and the resulted substance was dried under vacuum in a desiccator over KOH, P.sub.2O.sub.5 and paraffin, repeatedly changing desiccant.
[0188] Yield: 9.68 g (20.26 mmol); (87.51%).
[0189] Melting point 76-77 C.
[0190] Rf0.705 (butanol:acetic acid:water) (4:1:1);
[0191] 0.560 (benzene-ethanol) (8:2);
[0192] 0.476 (chloroform:methanol) (9:1);
[0193] 0.813 (isopropanol:formic acid:water) (20:5:1);
[0194] 0.297 (acetone-benzene-acetic acid) (50:100:1).
[0195] I. Production of H-Lys(Boc)-Pro-OH.
[0196] 300 ml of absolute methanol, 2 ml of acetic acid, and palladium black were added to 9.68 g (20.26 mmol) of Z-Lys(Boc)-Pro-OH, and hydrogenation was performed in the stream of dry hydrogen at room temperature under 1 atm for 8 hours. Then, the catalyst was filtered off and washed on the filter with methanol. Pooled filtrate was evaporated to dryness. The residue was precipitated with ether from the absolute methanol. Then, it was dried under vacuum, while desiccant was changed several times.
[0197] Yield: 6.38 g (18.58 mmol); (91.87%)
[0198] Melting point 98-99 C.
[0199] Rf0.110 (chloroform-methanol-acetic acid) (42:7:1);
[0200] 0.166 (butanol:acetic acid:water) (4:1:1);
[0201] 0.235 (chloroform:methanol:ammonia) (8:1.75:0.25);
[0202] 0.494 (isopropanol:formic acid:water) (20:5:1) (2:1:1).
[0203] III. Production of Boc-Thr-Lys-Boc)-Pro-OH.
[0204] 13% solution of TBA (39.4 ml) was added to 18.58 mmol (6.38 g) of Lys (Boc)-Pro-OH and evaporated 2 times with ethanol, 2 times with ethanol/benzene mixture and 2 times with benzene. 250 ml of absolute ethyl acetate were added; the reaction mixture was cooled down to 0 C., and 9.2 mmol (3.74 g) of previously synthesized Boc-Thr-OPfp were added and stirred on a magnetic stirrer for 1 hour. The reaction mass was evaporated, and 40 ml of water were added to the evaporation residue. The aqueous solution was washed 3 times with 80 ml of ether. After washing with ether, the aqueous solution was acidified with 18.58 mmol (3.95 g) of citric acid to pH 3. After acidification, aqueous solution was extracted with ethyl acetate 3 times in 40 ml aliquots. Pooled ethyl acetate after extraction was washed 3 times with 20 ml of water, 3 times with 20 ml of 10% solution of KHSO.sub.4, and 3 times with 20 ml of water. Ethyl acetate solution was dried over MgSO.sub.4.
[0205] Then, it was filtered and evaporated on a rotary evaporator. The resulted substance was precipitated with hexane from ethyl acetate. Hexane was decanted, and the resulted substance was dried under vacuum in a desiccator over KOH, P.sub.2O.sub.5 and paraffin, while desiccant was changed several times.
[0206] Yield: 3.32 g (6.1 mmol); (66.26%) Melting point 105-107 C.
[0207] Rf0.297 (acetone-benzene-acetic acid) (50:100:1);
[0208] 0.234 (chloroform:methanol) (9:1); 0.560 (benzene-ethanol) (8:2).
[0209] IV. Production of Boc-Thr-Lys(Boc)-Pro-Phe-OH.
[0210] 1. Production of Boc-Thr-Lys(Boc)-Pro-OSu.
[0211] 3.53 mmol (0.38 g) of hydroxysuccinimide were added to 1.66 g (3.05 mmol) of Boc-Thr-Lys(Boc)-Pro in 50 ml of absolute ethyl acetate, and the resulting solution was cooled down to 0 C., while stirring on a magnetic stirrer; then, 0.76 g (3.53 mmol) DCC (dicyclohexylcarbodiimide) were added and stirred on a magnetic stirrer at room temperature for 2 hours. After reaction stopped, the resulting reaction mixture was filtered off and the precipitate was discarded. 200 ml of absolute ethyl acetate were added to the resulting solution. Pooled ethyl acetate was washed 2 times with 20 ml of saturated solution of NaCl, two times with 20 ml of 10% sodium KHSO.sub.4, 2 times with 20 ml of saturated solution of NaCl, 2 times with 20 ml of 5% NaHCO.sub.3, and 2 times with 20 ml of saturated solution of NaCl. Ethyl acetate solution was dried over MgSO.sub.4. Then, it was filtered and evaporated on a rotary evaporator. The resulted substance was precipitated with ether and hexane from ethyl acetate. The precipitate was filtered and dried under vacuum over KOH, P.sub.2O.sub.5 and paraffin, while desiccant was changed several times.
[0212] Yield: 1.52 g (2.37 mmol) 77.74%.
[0213] Rf0.560 (benzene-ethanol) (8:2); 0.457 (chloroform:methanol) (9:1).
[0214] 2. After preparation, 1.52 g (2.37 mmol) of Boc-Thr-Lys (Boc)-Pro-OSu were dissolved in 25 ml of dimethylformamide and added to the prepared solution, containing 0.392 g (2.37 mmol) of L-Phe in 25 ml of dimethylformamide. The solution was stirred on a magnetic stirrer at room temperature. The reaction mixture was evaporated on a rotary evaporator and precipitated with ether from benzene. The precipitate was filtered and dried under vacuum over P.sub.2O.sub.5/KOH and paraffin, while desiccant was changed several times.
[0215] Yield: 1.03 g (1.64 mmol) 69.0%.
[0216] Rf0.063 (isopropanol:formic acid:water) (20:5:1) (2:1:1).
[0217] 0.745 (chloroform:methanol:ammonia) (8:1.75:0.25);
[0218] V. Production of H-Thr-Lys-Pro-Phe-OH.
[0219] 1.03 g (1.64 mmol) of Boc-Thr-Lys (Boc)-Pro-Phe-OH were added to 8.2 mL of methylene chloride and 8.2 ml of TFA, then the mixture was incubated for 45 minutes at room temperature, and, after removal of protecting groups, the solution was evaporated 2 times with absolute methanol, 2 times with benzene, and 2 times with ether. It was precipitated with ether from methanol. The residue was dried under vacuum over P.sub.2O.sub.5KOH and paraffin. The resulting precipitate was dissolved in 5 ml of 30% ethanol and applied to Amberlyst A-21 (AcO.sup.-form) column for acetate/hydrochloride salt exchange. Peptide was eluted with 100 ml of 30% ethanol, evaporated to dryness under vacuum and precipitated with absolute ether from absolute methanol. The resulting precipitate was filtered and dried in a desiccator under vacuum over P.sub.2O.sub.5, KOH and paraffin, while desiccant was changed several times.
[0220] Yield: 0.7 g (1.43 mmol) (87%).
[0221] Melting point 129-131 C.
[0222] Rf:0.201 (butanol:acetic acid:pyridine:water) (30:6:20:24);
[0223] 0.156 (chloroform:methanol:ammonia) (4:4.5:1.5).
[0224] Chromatographic and mass spectrometric analysis of peptide sequences, described in Examples 1-7, is shown in Table 2.
TABLE-US-00002 TABLE 2 Mass spectrometric characteristics Chromatographic Fragmenta- SEQ characteristics tion of the ID Tr, Purity, *[M + molecular No Peptide NO MW min % H].sup.+ ion peak** 1 Thr-Lys- 6 597 8.01 97 598 369(100), Pro-Arg- 272(31), Pro 580(29) 2 Thr-Lys- 7 744 14.8 96 745 516(100) Pro-Arg- 0.263(59), Pro-Phe 327(38) 3 Pro-Arg- 2675 522 9.14 89 523 25(100) Pro-Gly- 0.425(27), Pro 407(11) 4 Thr-Lys- 4 500 5.72 94 501 457(100) Pro-Arg 0.272(55), 484(47), 5 Arg-Pro- 2676 425 6.82 85 426 408(100), Gly-Pro 293(54), 254(24) 6 Pro-Gly- 2677 269 5.42 >98 270 173(100), Pro 155(98), 116(75) 7 Thr-Lys- 5 344 4.15 93 345 230(100), Pro 129(19), 212(16) 8 Thr-Lys 2678 247 1.77 95 248 230(100), 129(78), 84(30) 9 Thr-Lys- 2679 500 5.72 94 501 457(100) Pro-Phe 0.272(55), 484(47) Note. *Molecular peak corresponding to [M + H].sup.+ **Most intense ions formed in the fragmentation of the molecular ion peak at the energy of collisions with helium atoms of 35 eV.
[0225] Table 2 shows the data of high performance liquid chromatography (HPLC) using Millichrome A-02 microcolumn liquid chromatographic system and mass spectrometric characteristics of synthesized peptides obtained using ThermoElectron LCQ Advantage MAX mass spectrometer.
[0226] Developed chromatographic conditions allow to easily obtain chromatographically homogeneous product.
[0227] Chromatographic conditions for analysis of peptides.
[0228] Chromatograph: Milichrom-A02
[0229] Column: Prontosil 120-5C18aq, 2*75 mm
[0230] Eluent A: 0.2M LiClO4+5 mM HClO.sub.4
[0231] eluent B: methanol.
[0232] Table 3 presents gradient shape for separation of the synthesized peptides.
TABLE-US-00003 TABLE 3 Time % B 0 5 16.5 80
[0233] Flow rate: 150 l/min
[0234] Set of wavelengths: 210, 220, 230, 240 nm
[0235] Mass spectrometry conditions
[0236] Equipment: ThermoElectron LCQ Advantage MAX
[0237] Ion source: electrospray; direct introduction of peptide solution with a concentration of 10 g/ml in 0.1% acetic acid at a flow rate of 5 l/min Molecular ion fragmentation at 35 eV by ion collisions (He)
[0238] Source temperature: 250 C.
[0239] Ionization potential 3.5 kV
Example 8. Identification of Pharmacophore Position
[0240] To identify pharmacophore, fragments of the parent peptide Selank were synthesized:
[0241] Thr-Lys; Thr-Lys-Pro; Pro-Gly-Pro; Arg-Pro-Gly-Pro; Pro-Arg-Pro-Gly-Pro; and efficacy tests were conducted in vivo using relevant pre-clinical model (Lordosis test).
[0242] We studied the efficacy of the following group of peptides: Thr-Lys; Thr-Lys-Pro; Pro-Gly-Pro; Arg-Pro-Gly-Pro; Pro-Arg-Pro-Gly-Pro in the dose of 100 g/rat in relation to sexual behavior of female rats. Sexual behavior was recorded in ovariectomized hormonally stimulated females in direct contact with the sexually active male, or when such a contact was impossible. It was found that Thr-Lys-Pro peptide increased the intensity of proceptive behavior in females from 144 to 296 acts during monitoring (p=0.028, Wilcoxon test). The effect on the lordosis reaction in females had the same trend (p=0.09): the number of lordoses under the action of Thr-Lys-Pro peptide increased from 0.730.12 to 0.970.12. These results indicate the intensification of sexual motivation on the background of Thr-Lys-Pro peptide action. The effect is specific and manifested in an adequate behavioral situation. Results of efficacy studies of the following peptides: Thr-Lys; Thr-Lys-Pro; Pro-Gly-Pro; Arg-Pro-Gly-Pro; and Pro-Arg-Pro-Gly-Pro; in the model of lordosis are shown in Table 4
TABLE-US-00004 TABLE 4 Test Number of acts SEQ ID Progesterone, product, of proceptive Percentage Group NO mg/rat g/rat behavior of lordoses Negative Control 0.5 0 14 4 0.73 0.12 Thr-Lys 2678 0.5 100 13 4 0.70 0.02 Thr-Lys-Pro 5 0.5 100 29 6 0.97 0.12 Pro-Gly-Pro 2677 0.5 100 13 5 0.74 0.09 Arg-Pro-Gly-Pro 2676 0.5 100 15 4 0.69 0.14 Pro-Arg-Pro-Gly-Pro 2675 0.5 100 14 6 0.73 0.14 Positive Control 1.0 0 22 11 0.98 0.09
[0243] Table 4 proves that Thr-Lys-Pro tripeptide is the pharmacophore; furthermore, the smaller sequence, i.e. Thr-Lys dipeptide, does not function, as results from the Table 4.
Example 9. Pharmacophore Test
[0244] To test the pharmacophore, peptides based on it were synthesized, viz. Thr-Lys-Pro tripeptide, Thr-Lys-Pro-Arg (SEQ ID NO: 4) and Thr-Lys-Pro-Phe (SEQ ID NO: 2679) tetrapeptides, Thr-Lys-Pro-Arg-Pro (SEQ ID NO: 6) pentapeptide, and Thr-Lys-Pro-Arg-Pro-Phe (SEQ ID NO: 7) hexapeptide, corresponding to the general formula A-Thr-Lys-Pro-B-C-D-X, efficacy test were conducted on the in vivo, using relevant pre-clinical models (lordosis test).
[0245] We studied the efficacy of the following group of peptides: Thr-Lys-Pro (SEQ ID NO: 5); Thr-Lys-Pro-Arg (SEQ ID NO: 4); Thr-Lys-Pro-Arg-Pro (SEQ ID NO: 6); and Thr-Lys-Pro-Arg-Pro-Phe (SEQ ID NO: 7); at a dose of 100 g/rat in relation to sexual behavior of female rats. Sexual behavior was recorded in ovariectomized hormonally stimulated females in direct contact with the sexually active male, or when such a contact was impossible. It was found that peptides from the group, including Thr-Lys-Pro (SEQ ID NO: 5), Thr-Lys-Pro-Arg-Pro (SEQ ID NO: 6), and Thr-Lys-Pro-Arg-Pro-Phe (SEQ ID NO: 7), increased the intensity of proceptive behavior in females from 144 to 264-366 acts during monitoring (p=0.028, Wilcoxon test). At the same time, Thr-Lys-Pro-Arg (SEQ ID NO: 4) and Thr-Lys-Pro-Phe (SEQ ID NO: 2679) do not affect the intensity of proceptive behavior of females and do not increase the number of lordoses, and the basic parameters of Thr-Lys-Pro-Arg (SEQ ID NO: 4) and Thr-Lys-Pro-Phe (SEQ ID NO: 2679) tetrapeptides remain at the level of negative control. The effect on the lordosis reaction in females had the same trend (p=0.09). In the absence of direct contact of partners, peptide action was manifested. The results indicate the intensification of sexual motivation on the background of Thr-Lys-Pro (SEQ ID NO: 5), Thr-Lys-Pro-Arg-Pro (SEQ ID NO: 6), Thr-Lys-Pro-Arg-Pro-Phe (SEQ ID NO: 7) peptide action and lack of sexual motivation effect in Thr-Lys-Pro-Arg (SEQ ID NO: 4) and Thr-Lys-Pro-Phe (SEQ ID NO: 2679) tetrapeptides. The effect is specific and manifested in an adequate behavioral situation. Results of efficacy studies of the following peptides: Thr-Lys-Pro; Thr-Lys-Pro-Arg (SEQ ID NO: 4); Thr-Lys-Pro-Phe (SEQ ID NO: 2679); Thr-Lys-Pro-Arg-Pro (SEQ ID NO: 6); Thr-Lys-Pro-Arg-Pro-Phe (SEQ ID NO: 7); in the model of lordosis are shown in Table 5.
TABLE-US-00005 TABLE 5 Number of Test acts of Progesterone product, proceptive Percentage Group SEQ ID NO mg/rat g/rat behavior of lordoses Negative Control 0.5 0 14 4 0.73 0.12 Thr-Lys-Pro 5 0.5 100 28 4 0.96 0.12 Thr-Lys-Pro-Arg 4 0.5 100 14 4 0.72 0.02 Thr-Lys-Pro-Phe 2679 0.5 100 13 5 0.73 0.09 Thr-Lys-Pro-Arg-Pro 6 0.5 100 36 6 0.99 0.09 Thr-Lys-Pro-Arg-Pro- 7 0.5 100 26 4 0.94 0.14 Phe Positive Control 1.0 0 22 11 0.98 + 0.09
INDUSTRIAL APPLICABILITY
[0246] The invention relates to the field of biochemistry, in particular, to a method for producing peptides exhibiting high activity and capable of stimulating self-healing in the organs where a disturbance occurred. In particular, the invention allows to expand the range of tools for stimulation of sexual function and treatment of sexual dysfunction, while reducing the duration of course therapy and the costs of medications.