CN113413456A

CN113413456A - Method for producing recombinant peptides and resulting peptides

Info

Publication number: CN113413456A
Application number: CN202110591854.1A
Authority: CN
Inventors: N·F·麦索多夫; L·A·安德烈耶娃; D·V·格里科夫
Original assignee: OVB Ireland Ltd
Current assignee: OVB Ireland Ltd
Priority date: 2013-05-28
Filing date: 2013-05-28
Publication date: 2021-09-21
Also published as: JP2015515855A; CN104662034B; JP6552960B2; KR102093096B1; KR20160001591A; CN104662034A

Abstract

The present invention relates to a method for producing recombinant peptides and the resulting peptides. The present invention relates to biochemistry and biotechnology and to the production of peptides with sexual and reproductive function stimulating activity, said peptides having the general formula: A-Thr-Lys-Pro-B-C-D-X, wherein A is 0, Met (O), Thr, Ala, His, Phe, Lys, Gly, B is 0, Gly, Asp, Trp, Gln, Asn, Tyr, Pro, Arg, C is 0, Arg, Phe, Tyr, Gly, His, Pro, Lys, D is 0, Val, Gly, Tyr, Trp, Phe, His, X is OH, OCH₃、NH₂Wherein 0 is the absence of an amino acid residue, with the proviso that if A.noteq.0, then B and/or C and/or D.noteq.0, and if B.noteq.0, then C and/or D.noteq.0, excluding the peptides Phe-Thr-Lys-Pro-Gly, Thr-Lys-Pro-Pro-Arg, Thr-Lys-Pro-Arg-Gly. The invention also relates to the use of the gene engineeringMethods of producing the peptides.

Description

Method for producing recombinant peptides and resulting peptides

The present application is a divisional application of the 'method for producing recombinant peptide and resulting peptide' patent application No. 201380028491.4, filed 2013, month 5, 28.

Technical Field

The present invention relates to the field of biochemistry and relates to recombinant methods for producing peptides and the peptides produced. In particular, the invention relates to peptides having the following general formula:

A-Thr-Lys-Pro-B-C-D-X, wherein:

A-0、Met、Met(O)、Thr、Ala、His、Phe、Lys、Gly

B-0、Gly、Asp、Trp、Gln、Asn、Tyr、Pro、Arg

C-0、Arg、Phe、Tyr、Gly、His、Pro、Lys

D-0、Val、Gly、Tyr、Trp、Phe、His

X-OH、OCH₃、NH₂,

wherein 0 is the absence of an amino acid residue, with the proviso that if A.noteq.0, then B and/or C and/or D.noteq.0, and if B.noteq.0, then C and/or D.noteq.0, 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).

Background

Small peptides are known to exhibit extremely high activity, stimulating self-healing in disordered organs. It is well known that smaller peptides are initially derived from animal tissue and subsequently one learns to produce them under laboratory conditions.

Peptide-containing pharmaceutical products are capable of regenerating damaged somatic cells, restoring lost function to affected organs, and revitalizing them. Peptide-containing pharmaceutical products have been developed more than thirty years ago; since then, several hundred trials have been conducted, demonstrating the efficacy of peptides in the treatment and prevention of various systemic diseases and single organ-and systemic-rejuvenation.

In vivo, the peptide serves as an information messenger: they transfer information from one cell to another, enabling all events to be completed well and in time. If a cell is functioning properly, the corresponding organ functions well. If a disorder occurs, the entire organ is affected, resulting in disease. Clearly, disease can be treated by introducing a deletion into the body, but this approach will "pet" the cells completely and the cells will appropriately terminate normal functioning. Thus, it is desirable that peptide messengers (peptides) be delivered to cells to function so that the body will self-heal. Each organ has a supply of reserve stem cells. If the supply pool is completely depleted, the person survives 100-110 years. The peptide is the same for all mammals. Thus, if bovine peptide (calf peptide) is introduced into a human, the human body will treat it as a natural molecule. The main problem is to find out how to isolate peptides from animal organs. This technique was invented in Military Medical academic as early as 1971 by professor Vladimir Morozov and professor Vyacheslave Havinson. Pharmaceutical products were developed and then, based on this, dietary supplements were manufactured because of the convenience of use of dietary supplements. In their studies of the aging process and its method of influence, staff at the St.Petersburg Institute of Gerontology concluded that: peptides were introduced into the mouse experimental group diet to increase their life expectancy (life expectance) by 30-38%. Subsequently, peptide studies for the elderly were performed at the Kiev Institute of Gerontology and St.Peterburg, the senior. This shows that the mortality rate is almost 2-fold reduced, indicating a high age-protective (geroprotective) activity of the peptide. Long-term studies and use of peptide medical products have shown their efficacy in patients of different age groups; however, particular efficacy was observed in the elderly (50 years old). The absolute advantage of peptide bioregulators is the absence of any adverse reactions. Over 1500 million people with various pathologies received the product over 26 years. Their efficacy averaged 75-95%. Peptide deficiency dramatically accelerates tissue consumption and body aging with age and pathological changes. In fact, sufficient amounts of peptides are required for cells and tissues to function adequately, which in turn, supports optimal functioning of genes. Peptides that function in cells specific for them are synthesized there. Thus, upon pathological changes and aging, cellular function is disrupted; thus, the regeneration of the peptide is likewise influenced. Accordingly, cell function is in turn affected. Thus, the tissue process is degraded and eventually expressed clinically. Therefore, the use of small peptides in medicine is one of the major innovations, and by stimulating cell proliferation and tissue regeneration, the rate of aging can be significantly slowed, and cell life extended. Another important advantage of peptides is their antitumor effect. At present, the use of peptides in rehabilitation (physical rejuvenation) and cancer prevention is the best choice, since it rejuvenates cells and tissues not only by regulating and synchronizing all the cycle processes, but also by increasing the ability of cells to divide, without allotypes (atypia is an abnormal cell structure, or abnormality).

Of course, in the current state of the art, the production of small peptides from animal tissue is impractical because the process is very expensive, not to mention the humanity of the production process.

Modern advanced production methods include the use of recombinant microorganisms. The most convenient microorganism is escherichia coli (e. Commercial strains of E.coli suitable for laboratory conditions are known in the art, e.g.E.coli K12, E.coli 0104. These and similar known strains of E.coli can be used to obtain strains that produce the claimed small peptides. The nucleic acid encoding the corresponding target small peptide can be integrated into any known strain suitable for laboratory conditions. Vectors, such as bacterial plasmids, viruses, virosomes, hybrid vectors containing phage DNA, and plasmids, are used to insert DNA into a host cell. These vectors include, for example, cosmids and phagemids.

In addition, the peptides can be produced by conventional chemical synthesis.

The proposed peptides solve the problem of extending the scope of tools for stimulating reproductive and sexual function and treating reproductive and sexual dysfunction, which is still applicable at present. Currently, psychotherapy, antidepressants, anxiolytics, Adaptogens (Adaptogens) and vitamins, general plant stimulants and dietary supplements are used for the pathogenesis of this pathology. This treatment is long-term, inefficient and associated with a number of side effects.

One of the most effective types of physiologically active substances suitable for the production of stimulators of reproductive and sexual functions is peptides which are endogenous substances and which have virtually no negative side effects.

Our own studies showed that the heptapeptide Selank (a synthetic analogue of the endogenously produced peptide Tuftsin) having the general formula Thr-Lys-Pro-Arg-Pro-Gly-Pro (SEQ ID NO:2680) can be used as a tool for the prevention and treatment of reproductive and sexual dysfunction (russian federal patent No. 2404793). However, the synthesis of the heptapeptide selak is multistage, which greatly increases the cost of pharmaceutical products based thereon; in addition, it is subject to strong proteolysis in vivo, which reduces its stimulatory effects on the prevention and treatment of reproductive and sexual dysfunction.

Disclosure of Invention

The object of the invention is to expand the scope of tools with stimulating activity of reproductive and sexual functions.

The technical result achieved when implementing the present invention is to enhance the efficacy of prevention and treatment of reproductive and sexual dysfunction, to reduce the duration of treatment course and to reduce the cost of medication, wherein peptides having the following general formula are proposed as pharmaceutical products:

A-Thr-Lys-Pro-B-C-D-X, wherein:

A -0、Met、Met(O)、Thr、Ala、His、Phe、Lys、Gly

B-0、Gly、Asp、Trp、Gln、Asn、Tyr、Pro、Arg

C-0、Arg、Phe、Tyr、Gly、His、Pro、Lys

D-0、Val、Gly、Tyr、Trp、Phe、His

X-OH、OCH₃、NH₂，

wherein 0 means no amino acid residue.

Provided that if A ≠ 0, then B and/or C and/or D ≠ 0; if B.noteq.0, C and/or D.noteq.0, 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).

The amino acid residues at positions A, B, C, D and X were selected based on bioinformatic analysis of the frequencies of amino acid residues at the corresponding positions of the N-terminal and C-terminal amino acid residues in the database [ EROP-Moscow (http:// op. inbi. ras. ru /) Zamylatin A.A ]. The selection of these amino acid residues is made based on the criterion that the occurrence of amino acid residues at this position is greater than 50%. The amino acid residue samples were experimentally confirmed by synthesis of individual peptides and their stimulation of reproductive and sexual functional activity in an in vivo model. The location of the pharmacophore within the peptide is determined experimentally. It is known that any peptide is exposed to peptidases and degraded into specific fragments. For this reason, in the synthesis of the seven peptide fragment of Selank: Thr-Lys (SEQ ID NO:2678), Thr-Lys-Pro (SEQ ID NO:5), Pro-Gly-Pro (SEQ ID NO:2677), Arg-Pro-Gly-Pro (SEQ ID NO:2676), Pro-Arg-Pro-Gly-Pro (SEQ ID NO:2675) and their activities were investigated. The results are provided in table 4. In this analysis, the pharmacophore is determined: it is Thr-Lys-Pro (SEQ ID NO: 5). As shown in the study (table 5, example 10), it was shown that the addition of a separate tentatively identified amino acid residue to the C-terminus maintained peptide activity (stimulation of reproductive and sexual functions), assuming that the number of amino acid residues in the peptide was 3 or more than 4. Tetrapeptides do not have reproductive and sexual stimulation activity.

The mentioned technical results are achieved by the directed synthesis of various peptides (excluding tetrapeptides) having the general formula a-Thr-Lys-Pro-B-C-D-X and by the use of these peptides as stimulators of reproductive and sexual function for the prevention and treatment of reproductive and sexual dysfunction. Our own studies have shown that synthetic peptides, i.e., the Thr-Lys-Pro (SEQ ID NO:5) tripeptide corresponding to the general formula A-Thr-Lys-Pro-B-C-D-X, the Thr-Lys-Pro-Arg-Pro (SEQ ID NO:6) pentapeptide and the Thr-Lys-Pro-Arg-Pro-Phe (SEQ ID NO:7) hexapeptide, can be recommended as reproductive and sexual function stimulators.

All peptides (excluding tetrapeptides) having the general formula A-Thr-Lys-Pro-B-C-D-X are recommended as reproductive and sexual function stimulators, with a common pattern, i.e., the presence of a Thr-Lys-Pro tripeptide molecule in their molecular structure.

Thr-Lys-Pro-Arg-Pro-Gly-Pro (SEQ ID NO:2680) heptapeptide (Selank) was used as a control.

The results of the research conducted show that peptides (excluding tetrapeptides) having the general formula A-Thr-Lys-Pro-B-C-D-X have reproductive function and sexual function stimulating activity and are useful as pharmaceutical products for the prevention and treatment of reproductive and sexual dysfunction.

Certain peptides of the general formula A-Thr-Lys-Pro-B-C-D-X are shown in Table 1.

TABLE 1

Drawings

FIG. 1 shows a diagram of Arg-Pro-Gly-Pro (SEQ ID NO:2676) tetrapeptide synthesis;

FIG. 2 shows a diagram of Pro-Arg-Pro-Gly-Pro (SEQ ID NO:2675) pentapeptide synthesis;

FIG. 3 shows a diagram of Pro-Gly-Pro (SEQ ID NO:2677) tripeptide synthesis;

FIG. 4 shows a diagram of Thr-Lys-Pro-Arg-Pro-Phe (SEQ ID NO:7) hexapeptide synthesis;

FIG. 5 shows a diagram of the synthesis of Thr-Lys-Pro-Arg-Pro (SEQ ID NO:6) pentapeptide;

FIG. 6 shows a diagram of Thr-Lys (SEQ ID NO:2678) dipeptide synthesis;

FIG. 7 shows a diagram of the synthesis of the Thr-Lys-Pro-Phe (SEQ ID NO:2679) tetrapeptide;

Detailed Description

The following are examples illustrating the present invention.

Peptide synthesis with the general formula A-Thr-Lys-Pro-B-C-D-X was performed by peptide chemistry in solution using L-amino acids. Peptide synthesis was performed by stepwise extension of peptide chains, as well as fragment condensation using the mixed anhydride method, the carbodiimide method, the activated ester method, and the mixed anhydride method with 1-hydroxybenzotriazole added as an auxiliary nucleophile. All intermediates and best products were isolated and characterized. The evaporation of the solution was carried out at 40 ℃ using a vacuum evaporator. The melting points determined with a Boetui apparatus (Boethius appaatus) are given without correction. The obtained compounds were tested for properties by TLC on a silica gel coated plate (Czech Republic). Substances were detected by UV light using ninhydrin, Barton's reagent, paul reagent, lyde-Hoppe reagent and o-tolidine in a chlorine environment. The optical rotation was measured by an AI-EPO polarimeter. Peptide homology was tested by High Performance Liquid Chromatography (HPLC) and peptide structure was determined by mass spectrometry. All solvents were accordingly anhydrous. The melting point was not corrected.

The peptides were also obtained by genetic engineering techniques using host cells engineered from known laboratory strains of e.coli, transformed with known commercially available plasmids containing nucleic acids encoding the target peptides.

The examples describe peptide synthesis.

Example 1 Synthesis of Arg-Pro-Gly-Pro tetrapeptide

The synthesis of the tetrapeptides was performed according to the scheme shown in FIG. 1.

For the synthesis, a mixed acid anhydride method, an azide method and a carbodiimide method are used. Derivatives of L-amino acids were used for the synthesis. The evaporation of the solution was carried out at 40 ℃ using a rotor evaporator. The melting points determined with a Bottuyus apparatus are given without correction. The obtained compounds were tested for properties by TLC on a silica gel coated plate (Czech Republic). The substance was detected by spraying the plate with a solution of ninhydrin and/or o-tolidine. Providing chromatographic mobility (Rf) values in the following solvent systems: 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).

The optical rotation was measured by an AI-EPO polarimeter.

Elemental analysis using a Carlo-Erba model 1106 analyzer.

I.Boc-Pro-Gly-OEt.8.3g (3.45mmol) Boc-Pro-OH was dissolved in 50ml CH₂Cl₂Cooling to 5 deg.C; then, 38.45mmol (5.38ml) of TEA was added. The reaction mixture was cooled down to-25 to-30 ℃. At this temperature, 38.45mmol (4.84ml) of isobutyl chloroformate were added using a pipette. The temperature of the reaction mixture was maintained in the range of-18 to-20 ℃ for 20 minutes. At the same time, a solution of 5.9g (42.3mmol) of HCl.H-Gly-Oet in 75ml of chloroform in a 1.1-fold excess contains 5.92ml of TEA. The solution was cooled down to-25 ℃ and the contents were poured into the ether solution immediately after the mixed anhydride had formed in the first flask. The reaction mixture was incubated at-10 ℃ for 1 hour and then stirred on a magnetic stirrer at 4 ℃ for 12 hours. The reaction mixture was evaporated and then 250ml of ethyl acetate were added; the ethyl acetate solution was then washed 3 times with 25ml0.1N HCl and 25ml H₂Wash 3 times with O and once with saturated NaCl solution. Over MgSO₄The organic layer was dried, filtered and evaporated. Under vacuum over P₂O₅KOH and paraffin dried residue.

Yield: 10.2g (30.3mmol) 78.83%

Rf-0.5(7)；0.862(8)

Melting point 68-70 ℃.

₂ ₃II.Boc-Pro-Gly-NH.10.2g Boc-Pro-Gly-OEt (30.3mmol) was dissolved in 80ml of anhydrous methanol and a 4-fold excess of hydrazine hydrate, i.e. 5.88ml (121.2mmol), was added. The solution was stirred at room temperature on a magnetic stirrer for 12 hours. The reaction mixture was evaporated and then twice with ether; then, ether (. about.5 ml) was poured into the residue and placed in the refrigerator overnight (seeding agent) for better crystallization. The precipitated crystals were filtered, washed with ether using a filter and dried in a desiccator.

Yield: 6.9g (20.79mmol) 68.62%

Rf-0.284(7)；0.474(8)；0.189(9)

The melting point is 98-100 ℃.

III.Boc-Pro-Gly-Pro-OBzl.58.4mmol (4-fold excess) of hydrogen chloride in ethyl acetate were added to 4.7g (14.6mmol) of Boc-Pro-Gly-N in 40ml of DMF₂H₃Cooling down to-20 ℃ and, immediately, 1.73ml (14.6mmol) of freshly distilled tert-butyl nitrite; the reaction mixture was then stirred at-5 ℃ for 30 minutes. The reaction mixture was cooled down to-40 ℃ and a solution of 8.2ml (58.4mmol) of TEA in 4ml of DMF cooled down to-10 ℃ was added; when the temperature of the reaction mixture rose to-20 deg.C, 3.7g (15.3mmol) of 1.05 fold excess HCl.H-Pro-OBzl were added to 20ml DMF and 2.14ml TEA. Then, it was stirred on a magnetic stirrer at 4 ℃ for 24 hours. The reaction mixture was evaporated and the residue was dissolved in 200mL ethyl acetate with 20mL H₂O2 times, 20ml 10% KHSO₄The solution was washed 3 times with 20ml H₂O3 washes with 20mL 5% NaHCO₃Washed 3 times with 20ml H₂Wash 3 times with O. Over MgSO₄The ethyl acetate solution was dried. Then evaporating it and addingA small amount of ether (. about.10 mL) to the residue. Then, it was placed in a refrigerator for crystallization. For better product crystallization, seed crystals were added. The precipitated crystals were filtered and washed with a small amount of ether using a filter. Then, it is dried in a dryer.

Yield: 5.358g (11.65mmol) 79.92%

Rf-0.326(7)；0.947(8)；0.390(9)

Melting point 125-.

[α]_D ²²＝-101.18°(с＝0.85；СН₃ОН).

Elemental analysis: c62.89 (62.73); n9.21 (9.14); h7.52 (7.24).

IV.TFA·H-Pro-Gly-Pro-OBzl.5.358g (11.65mmol) Boc-Pro-Gly-Pro-OBzl was dissolved in 29.13ml dichloromethane; then, 29.13mL TFA was added, incubated at room temperature for 45 minutes, evaporated 2 times with absolute ethanol, 2 times with benzene, 2 times with ether, and dissolved in benzene; then, hexane was poured. Decant hexane and dry in a desiccator under vacuum over KOH, P₂O₅And paraffin drying the resulting material while replacing the drying agent several times.

Yield: 4.63g (9.7mmol) 98%

Rf-0.043(7)；0.247(8)；0.018(9)。

₂V.Boc-Arg(NO)-Pro-Gly-Pro-OBzl.3.09g of Boc-Arg (NO)₂) -OH (9.7mmol) was dissolved in 50mL THF and 10mL DMF, 2.07g (10.67mmol) DCC was added, cooled to 0 deg.C and stirred for 40 min; then, the TFA-H-Pro-Gly-Pro-OBzl solution was added to 50ml of THF and 4.46ml (9.7mmol) of TEA. The reaction mixture was stirred for three days. The DCM precipitate was filtered off; evaporating the solution under vacuum; then, 200ml of hexane was added to the residue. Thus, the desired product was separated as an oil, dissolved in 500mL ethyl acetate and washed 3 times with 25mL0.1N HCl, with 25mL H₂O washes were three times and once with NaCl saturated solution. Over MgSO₄The organic layer was dried, filtered and evaporated. The residue was dissolved in ethyl acetate and precipitated with dry ether. The precipitate was filtered and passed through P under vacuum₂O₅KOH and paraffin drying with simultaneous desiccant replacementSeveral times.

Yield: 4.76g (7.9mmol) 85%

Rf-0.44(1)；0.8(11)

Melting point 108-

[α]_D ²²＝-77.8°(с＝0.5；СН₃СООН)。

VI.Boc-Arg-Pro-Gly-Pro.4.76g (6.5mmol) of Boc-Arg (NO)₂) -Pro-Gly-Pro-OBzl in 100ml methanol; 1ml of 1N hydrochloric acid and 4.67g of a catalyst, i.e. 10% palladium oxide on neutral alumina, are then added and the hydrogenation is carried out at room temperature under 1atm. in a stream of dry hydrogen for 6 hours. Then, the catalyst was filtered off and washed with methanol on the filter. The collected (pooled) filtrate was evaporated to dryness. The residue was precipitated from dry methanol with ether. Then, it was dried under vacuum while the desiccant was changed several times.

Yield: 4.23g (5.8mmol) 89%

Rf-0.125(4)；0.57(6)；0.37(5)

Melting point 123-.

VII.Arg-Pro-Gly-Pro.4.23g (5.8mmol) Boc-Arg-Pro-Gly-Pro-OH was suspended in 10ml 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. Reprecipitation from anhydrous methanol with ether. The resulting precipitate was dissolved in 7.5ml of 30% ethanol and applied to Amberlyst A-21 (AcO)^-Form) column for acetate/hydrochloride exchange. The peptide was eluted with 200ml of 30% ethanol, evaporated to dryness under vacuum and precipitated from methanol with anhydrous ether. Yield: 3.69g (4.93mmol) 85%

Rf-0.287(4)；0.145(5)；0.338(14)

Melting point 120 ℃ 122 DEG C

HPLC results: column: supercosil ABZ Plus, size 4.6X 250 mm; the flow rate is 1 mL/min; eluent A: NH (NH)₄H₂PO₄+Н₃РО₄(50m M, pH 2.8); eluent B: MeOH

Gradient: 0-20min (0-40% B); retention time 21.21 min.

Rf-0.24(6)

Melting point 151 deg.C

[α]_D ²²＝-65.0°(с＝0.5СН₃СООН)

HPLC results: column: zorbax ODS d 4.6 mm; t 35 deg.C

The flow rate is 1 mL/min; a50 mM NH₄H₂PO₄(pH 2.5); b ═ a + MeOH (1: 1); 10-60% B (within 25 min). Retention time 16.5 min.

Example 2 Synthesis of Pro-Arg-Pro-Gly-Pro pentapeptide

Peptide synthesis was performed by the classical method of peptide chemistry using natural L-amino acids according to the scheme shown in figure 2.

Firstly, producing Pro-Gly-Pro tripeptide; then, the pentapeptide is obtained by stepwise assembling peptide chains from the N-terminus. For the synthesis, a mixed acid anhydride method, an azide method and a carbodiimide method are used.

Chromatographic mobility (Rf) values for 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).

I.Boc-Pro-Gly-OEt.8.3g (38.45mmol) Boc-Pro was dissolved in 50ml CH₂Cl₂Cooling to +5 deg.C; and 38.45mmol (5.38ml) TEA was added. The reaction mixture was cooled down to-25 to-30 ℃. At this temperature, 38.45mmol (4.84ml) of isobutyl chloroformate were added using a pipette. The temperature of the reaction mixture was maintained in the range of-18 to-20 ℃ for 20 minutes. At the same time, a solution of 5.9g (42.3mmol) of HCl.H-Gly-Oet in 75ml of chloroform in a 1.1-fold excess contains 5.92ml of TEA. The solution was cooled down to-25 ℃ and the contents were poured into the ether solution immediately after the mixed anhydride had formed in the first flask. The reaction mixture was incubated at-10 ℃ for 1 hour and then stirred on a magnetic stirrer at 4 ℃ for 12 hours. The reaction mixture was evaporated, and 250ml of ethyl acetate was added thereto; the ethyl acetate solution was washed 3 times with 25ml0.1N HCl and 25ml H₂Wash 3 times with O and once with saturated NaCl solution. Over MgSO₄The organic layer was dried, filtered and evaporated. In trueSpace meridian P₂O₅KOH and paraffin dried residue.

Yield: 10.2g (30.3mmol) 78.83%

Rf-0.5(3)；0.862(4)。

II.Boc-Pro-Gly-N ₂ H ₃ .10.2g Boc-Pro-Gly-OEt (30.3mmol) was dissolved in 80ml of anhydrous methanol and a 4-fold excess of hydrazine hydrate, i.e. 5.88ml (121.2mmol), was added. The solution was stirred at room temperature on a magnetic stirrer for 12 hours. The reaction mixture was evaporated and then twice with ether; then, ether (. about.5 ml) was poured into the residue and placed in the refrigerator overnight (seeds were added for better crystallization). The precipitated crystals were filtered, washed with ether using a filter and dried in a desiccator.

Yield: 6.9g (20.79mmol) 68.62%

Melting point of 98-100 deg.C

Rf-0.284(3)；0.474(4)；0.189(5)。

III.Boc-Pro-Gly-Pro-OBzl.

58.4mmol (4-fold excess) of hydrogen chloride in ethyl acetate were added to 4.7g (14.6mmol) of Boc-Pro-Gly-N in 40ml of DMF₂H₃Cooling down to-20 ℃ and, immediately, 1.73mL (14.6mmol) of freshly distilled tert-butyl nitrite; the reaction mixture was then stirred at-5 ℃ for 30 minutes. The reaction mixture was cooled down to-40 ℃ and a solution of 8.2ml (58.4mmol) of TEA in 4ml of DMF cooled down to-10 ℃ was added; when the temperature of the reaction mixture rose to-20 deg.C, 3.7g (15.3mmol) of 1.05 fold excess HCl.H-Pro-OBzl were added to 20ml DMF and 2.14ml TEA. Then, it was stirred on a magnetic stirrer at 4 ℃ for 24 hours. The reaction mixture is evaporated and the residue is dissolved in 200ml of ethyl acetate and washed with 20ml of H₂O2 washes with 20mL 10% KHSO₄The solution was washed 3 times with 20ml H₂O3 washes with 20ml 5% NaHCO₃Washed 3 times with 3ml of H₂Wash 3 times with O. Over MgSO₄The ethyl acetate solution was dried. It was then evaporated and a small amount of ether (. about.10 mL) was added to the residue. Then, the sample was placed in a refrigerator. For better crystallization of the product, additionAnd (4) seed crystals. The precipitated crystals were filtered and washed with a small amount of ether using a filter. Then, it is dried in a dryer.

Yield: 5.358g (11.65mmol) 79.92%

Rf-0.326(3)；0.947(4)；0.390(5)

Melting point 125-

[α]_D ²²＝-101.2°(с＝0.85；СН₃ОН)

Elemental analysis: c62.89 (62.73); n9.21 (9.14); h7.52 (7.24).

IV.TFA·H-Pro-Gly-Pro-OBzl.5.358g (11.65mmol) Boc-Pro-Gly-Pro-OBzl was dissolved in 29.13ml dichloromethane; adding 29.13ml TFA, incubating at room temperature for 45 min, evaporating 2 times with absolute ethanol, 2 times with benzene, 2 times with ether, and dissolving in benzene; then, hexane was poured. Decant the hexane and pass through P in a desiccator under vacuum₂O₅The resulting material was dried with KOH and paraffin. Yield: 4.63g (9.7mmol) 98%

Rf-0.043(3)；0.247(4)；0.018(5)。

V.Boc-Arg(NO2)-Pro-Gly-Pro-OBzl.3.09g (9.7mmol) of Boc-Arg (NO)₂) Dissolved in 50ml THF and 10ml DMF; then, 2.07g (10.67mmol) of DCC was added, cooled to 0 ℃ and stirred for 40 minutes; a solution of TFA. H-Pro-Gly-Pro-OBzl was added to 50ml THF and 4.46ml (9.7mmol) TEA. The reaction mixture was stirred for three days. The DCM precipitate was filtered off; evaporating the solution under vacuum; then, 200ml of hexane was added to the residue. The desired product was then separated as an oil, dissolved in 500ml of ethyl acetate and washed 3 times with 25ml of 0.1N HCl and 25ml of H₂O washes were three times and once with NaCl saturated solution. Over MgSO₄The organic layer was dried, filtered and evaporated. Under vacuum over P₂O₅The dry residue of KOH and paraffin,

yield: 4.76g (7.9mmol) 85%

Rf-0.44(1)；0.8(6)

Melting point 108-.

₂VI.TFA-Arg(NO)-Pro-Gly-Pro-OBzl.4.76g (7.9mmol) of Boc-Arg(NO₂) -Pro-Gly-Pro-OBzl in 20ml dichloromethane; adding 20ml TFA, incubating at room temperature for 45 min, evaporating 2 times with absolute ethanol, 2 times with benzene, 2 times with ether, and dissolving in benzene; then, hexane was poured. Decant the hexane and pass through P in a desiccator under vacuum₂O₅The resulting material was dried with KOH and paraffin.

Quantitative yield.

Rf-0.16(1)；0.27(6)。

₂VII.Boc-Pro-Arg(NO)-Pro-Gly-Pro-OBzl.1.7g (7.9mmol) Boc-Pro was dissolved in 20mL THF and 1.07g (7.9mmol) BT was added and cooled down to 0 deg.C; 1.8g of DCC in 50mL of THF was added. TFA-Arg (NO) was added during 40min₂) A solution of-Pro-Gly-Pro-OBzl (7.9mmol) in 50mL THF and 1.1mL (7.9mmol) TEA were added to the reaction mixture. Stirring at 0 ℃ for 2 hours and at room temperature for 2 days; then, DCM was filtered off, evaporated in vacuo, dissolved in 500mL ethyl acetate and reacted with Boc-Pro-Arg (NO)₂) Similar treatment with-Pro-Gly-Pro-OBzl.

Yield: 4.07g (67.8%)

Rf-0.42(1)；0.72(6)；0.31(7)

Melting point 147-.

VIII.Boc-Pro-Arg-Pro-Gly-Pro.4.07g (6.5mmol) were dissolved in 100ml of methanol; 1ml of 1N hydrochloric acid and 0.85g of catalyst, i.e. 10% palladium oxide on neutral alumina, are added and the hydrogenation in dry hydrogen vapour is carried out at room temperature for 6 hours at 1atm. Then, the catalyst was filtered off and washed with methanol on the filter. The collected filtrate was evaporated to dryness. The residue was precipitated from anhydrous methanol with ether. Yield: 3.02g (5.8mmol) 89%

Rf-0.125(1),0.57(2),0.37(6)。

IX.Pro-Arg-Pro-Gly-Pro.3.02g (5.8mmol) Boc-Pro-Arg-Pro-Gly-OH was suspended in 10ml 2N hydrochloric acid in dioxane and incubated for 45 min at room temperature. Then, dry ether was added and the precipitate was washed by decantation with dry ether. Reprecipitation from anhydrous methanol with ether. The resulting precipitate was dissolved in 7.5ml of 30% ethanol and applied to Amberlyst A-21(AcO^-Form) column for acetate/hydrochloride exchange. The peptide was eluted with 200ml of 30% ethanol, evaporated to dryness under vacuum and precipitated from methanol with anhydrous ether.

Yield: 2.27g (75%)

Rf-0.2(2)；0.1(6)

Melting point of 180 ℃ and 185 DEG C

[α]_D ²⁰＝-105°(с＝0.4；СН₃СООН)。

Amino acid composition relative to arginine: pro 2.78 (3); gly 1.1 (1).

Gradient: 0-20min (0-40% B); retention time 10.13 min.

Example 3 Synthesis of Pro-Gly-Pro tripeptide.

The synthesis of the Pro-Gly-Pro tripeptide was performed according to the diagram shown in FIG. 3. The synthesis of Pro-Gly-Pro tripeptide is carried out by using a novel protective group and a peptide bond formation method in solution. The mixed anhydride method using PivCl was used for peptide bond formation. Tert-butoxycarbonyl protection (Boc) was used for the protection of the amino group and benzyl ester (OBzl) was used to protect the carboxyl group. Peptide chain extension was performed using a stepwise approach.

Derivatives of L-amino acids are used for the synthesis. The evaporation of the solution was carried out at 40 ℃ using a vacuum evaporator. Melting points determined with a Boetui apparatus (Boethius appaatus) are given without correction.

The obtained compounds were tested for properties by TLC on a silica gel-coated plate (Czech Republic). The substance was detected by spraying the plate with a solution of ninhydrin and/or o-tolidine. Providing chromatographic mobility (Rf) values in the following solvent systems: (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. Elements were analyzed using a Carlo-Erba model 1106 analyzer.

I.e. Boc-Pro-Gly-OH production.

1. 10.75g (50mmol) Boc-Pro was dissolved in 150ml acetonitrile and cooled down to-5 ℃; then, 7.7ml (50mmol) Triethylamine (TEA) were added to the solution and while stirring on a magnetic stirrer, it was cooled down to-20 ℃. 6.8ml (55mmol) of pivaloyl chloride (PivCl) were added to the cooled solution, stirred on a magnetic stirrer at-10 ℃ for 20 minutes, then cooled to-30 ℃; then, a pre-cooled solution of Gly was added. Simultaneously, a Gly solution was prepared.

2. 4.5g Gly (60mmol, 1.2 fold excess) were dissolved in 35ml water and 60ml acetonitrile, and 8.4ml (60mmol) triethylamine was added. The mixture was cooled down to-10 ℃ and added to the solution in the first flask after 20 minutes. The reaction mixture was incubated at-10 ℃ for 1 hour and stirred on a magnetic stirrer at 18-20 ℃ for 2 hours. The reaction mixture was evaporated on a rotary evaporator. About 50ml of water was added to the residue. With a 3-fold excess of NaHSO₄(24.84g) the aqueous solution was acidified to pH 3 and extracted 5 times with 100ml ethyl acetate. By H₂O(50ml)、10％KHSO₄Solution (50ml), H₂The collected ethyl acetate solution was washed with O (50ml) and saturated NaCl (50 ml). Over MgSO₄The ethyl acetate solution was dried. The dried ethyl acetate was filtered and evaporated. Dry ether was added to the residue. Upon addition of ether to the flask, the product precipitated, filtered with dry ether using a filter and washed. Passing KOH, P in a desiccator under vacuum₂O₅And paraffin drying the resulting material while replacing the drying agent several times.

Product M.W.272.3

Yield: 5.97g (21.74 mmol); (43.5%)

Melting point 70 ℃.

Rf-0.863 (acetone-phenyl-acetic acid) (50:100: 1);

0.746 (benzene-ethanol) (8: 2); 0.903 (chloroform: methanol) (9: 1);

0.847 (ethyl acetate: acetone: 50% acetic acid: water) (2:1: 1).

Production of Boc-Pro-Gly-Pro-OBzl.

1. 5.97g (21.74mmol) Boc-Pro-Gly-OH was dissolved in 100ml acetonitrile and cooled down to-5 ℃; then, a 1.1-fold excess (3.35ml, 23.9mmol) of Triethylamine (TEA) was added to the solution and it was cooled down to-20 ℃ while stirring on a magnetic stirrer. A 1.1-fold excess (2.34ml, 23.9mmol) of pivaloyl chloride (PivCl) was added to the cooled solution, stirred on a magnetic stirrer at-10 ℃ for 20 minutes, then cooled to-30 ℃; then, a pre-cooled solution of HCl Pro-OBzl was added. Simultaneously, HCl Pro OBzl is prepared.

2. 6.3g of HCl Pro-OBzl (26.1mmol, 1.2 excess) are dissolved in 50ml of acetonitrile, and 4.0ml (28.71 mmol; 1.1-fold excess) of triethylamine are added. The mixture was cooled down to-10 ℃ and added to the solution in the first flask after 20 minutes. The reaction mixture was incubated at-10 ℃ for 1 hour and stirred on a magnetic stirrer at 18-20 ℃ for 2 hours. The reaction mixture was evaporated. 300ml of ethyl acetate were added to the evaporation residue. By H₂O (3 times in 25 mL), 10% KHSO₄Solution (3 times with 25 ml) H₂O (3 times with 25 ml), 5% NaHCO₃(with 25ml 3 times) H₂The ethyl acetate solution was washed with O (25ml 3 times) and saturated NaCl solution (25ml once). Over MgSO₄The ethyl acetate solution was dried. The dried ethyl acetate was filtered and evaporated. About 100ml of dry ether was added to the residue. Upon addition of ether to the flask, the product precipitated, filtered with dry ether using a filter and washed. Passing KOH, P in a desiccator under vacuum₂O₅And paraffin drying the resulting material while replacing the drying agent several times.

Product M.W.459.82

Yield: 8.12g (17.66 mmol); (81.23%)

Melting point 125-

Rf-0.326 (acetone: benzene: acetic acid) (50:100:1)

0.390 (Hexane: acetone) (3:2)

0.947 (chloroform: methanol) (9:1)

0.716 (methanol)

0.620 (benzene: ethanol) (8:2)

Production of Boc-Pro-Gly-Pro-OH.

8g (17.4mmol) of Boc-Pro-Gly-Pro-OBzl was dissolved in 100ml of methanol, and 0.5ml of CH was added₃COOH and palladium black were stirred simultaneously on a magnetic stirrer and hydrogen was passed through over 2 hours. The solution was filtered, evaporated, 3 times with benzene and 2 times with ethyl acetate. It was then precipitated from acetone with ethereal hexane and passed through P in a desiccator₂O₅The precipitate formed in the flask was dried with KOH and paraffin.

Product M.W.369.39

Yield: 6.3g (17.05mmol) 98%

Melting point of 99-100 deg.C

Rf:0.560 (chloroform-methanol) (9:1)

0.812 (chloroform-methanol-acetic acid) (42:7:1)

0.187 (acetone: benzene: acetic acid) (50:100:1)

0.164 (Hexane: acetone) (3:2)

Production of Pro-Gly-Pro.

40.6ml dichloromethane and 40.6ml TFA were added to 6.0g (16.24mmol) Boc-Pro-Gly-Pro-OH and incubated for 45 min at room temperature; after removal of the protecting group, the solution was evaporated 2 times with anhydrous methanol, 2 times with benzene and 2 times with ether. It was precipitated from acetone with ether. Under vacuum over P₂O₅KOH and paraffin dried residue. The dried product was reprecipitated from anhydrous methanol with dry diethyl ether.

Product M.W.381.39

Yield: 5.6g (14.72mmol) (90.62%)

Dissolving the obtained TFA Pro-Gly-Pro-OH precipitate in 10ml 30% ethanol; then, 25mL Amberlyst A-21 (CH) was added₃COO^-) Ion exchange resin to exchange trifluoroacetate for acetate and stir on magnetic stirrer for 45 minutes; then, it was washed with 150ml of 30% ethanol, evaporated to dryness under vacuum and precipitated from dry methanol with dry diethyl ether. The precipitate obtained is filtered off and passed over P under vacuum₂O₅The KOH and paraffin were dried in a desiccator while the desiccant was changed several times.

Product M.W.328.34

Yield: 4.54g (13.84mmol) (94%)

Melting point 143-

[α]_D ²²-31.5°(с1,МеОН)

Rf:0.59 (chloroform: methanol: ammonia) (6:4:1)

0.52 (chloroform: methanol: ammonia) (4:4.5:1.5)

0.524 (ethanol: ammonia) (7:3)

Example 4 Synthesis of Thr-Lys-Pro-Arg-Pro-Phe hexapeptide.

The synthesis of Thr-Lys-Pro-Arg-Pro-Phe hexapeptide was performed according to the scheme shown in FIG. 4.

The synthesis of Thr-Lys-Pro-Arg-Pro-Phe hexapeptide was performed using a novel protecting group and peptide bond formation in solution. The TBA salt method, the activated ester method, the carbodiimide method, and the mixed anhydride method are used to form peptide bonds. Both stepwise (stepwise approach) and block-wise approach (blockwise approach) are used.

Example 5 Synthesis of Thr-Lys-Pro-Arg-Pro pentapeptide.

The synthesis of Thr-Lys-Pro-Arg-Pro pentapeptide was performed according to the scheme shown in FIG. 5.

The synthesis of Thr-Lys-Pro-Arg-Pro pentapeptide is carried out using a novel protecting group and peptide bond formation in solution. The TBA salt method, the activated ester method, and the carbodiimide method are used to form peptide bonds. Both step-wise and block processes are used.

Example 6 Synthesis of Thr-Lys dipeptide.

The synthesis of Thr-Lys dipeptide was performed according to the scheme shown in FIG. 6. The synthesis of Thr-Lys dipeptide is performed using novel protecting groups and peptide bond formation in solution. Peptide bonds were formed using TBA salt method. The peptides described in examples 7-9 were synthesized using derivatives of both protected and free L-amino acids. The solvents used in peptide synthesis are accordingly anhydrous. Peptides were tested for homogeneity (homogeneity) by High Performance Liquid Chromatography (HPLC) using a Millichrome A-02 mini-column liquid chromatography system. (ii) by Mass Spectrometry Using a Bruker Mass spectrometer (

Daltonik GmbH).

Example 7 Synthesis of Thr-Lys-Pro-Phe tetrapeptide.

The synthesis of Thr-Lys-Pro-Phe tetrapeptide was performed according to the scheme shown in FIG. 7.

The synthesis of Thr-Lys-Pro-Phe tetrapeptides was performed using novel protecting groups and peptide bond formation in solution. The TBA salt method and the activated ester method are used to form peptide bonds. The peptide chain is extended using a stepwise method. Using derivatives of both protected and free L-amino acids. The evaporation of the solution was carried out at 40 ℃ using a vacuum evaporator. The melting point determined with a Bottuyus apparatus is given without correction. The obtained compounds were tested for properties by TLC on a silica gel coated plate (Czech Republic). The substance was detected by spraying the plate with a solution of ninhydrin and/or o-tolidine. Providing chromatographic mobility (Rf) values in the following solvent systems: (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-phenyl-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).

I.Z production of Lys (Boc) -Pro-OH.

A13% solution of TBA (98ml) was added to 46.32mmol (5.34g) Pro and evaporated twice with ethanol, twice with an ethanol/benzene mixture and 2 times with benzene. 300ml of anhydrous ethyl acetate was added; the reaction mixture was cooled down to 0 ℃ and 23.16mmol (12.66g) of the previously synthesized Z-Lys (Boc) -OPfp were added and stirred on a magnetic stirrer for 1 hour. The reaction mass was evaporated and 40ml of water was added to the evaporation residue. The aqueous solution was washed 3 times with 80ml of ether. After washing with ether, the aqueous solution was acidified with citric acid to pH 3. After acidification, the aqueous solution was extracted 3 times with 40ml aliquots of ethyl acetate. Washing the extracted ethyl acetate with 20ml water for 3 times, and adding 20ml 10% KHSO₄The solution was washed 3 times and 3 times with 20ml water. Over MgSO₄DryingEthyl acetate solution. Then, it was filtered and evaporated on a rotary evaporator. The resulting material was precipitated from ethyl acetate with hexane. Decant hexane and dry in a desiccator under vacuum over KOH, P₂O₅And paraffin drying the resulting material, and repeatedly replacing the drying agent. Yield: 9.68g (20.26 mmol); (87.51%).

Melting point of 76-77 deg.C

Rf-0.705 (butanol: acetic acid: water) (4:1: 1);

0.560 (benzene-ethanol) (8: 2);

0.476 (chloroform: methanol) (9: 1);

0.813 (isopropanol: formic acid: water) (20:5: 1);

0.297 (acetone-benzene-acetic acid) (50:100:1).

Production of H-Lys (Boc) -Pro-OH.

300ml of anhydrous methanol, 2ml of acetic acid and palladium black were added to 9.68g (20.26mmol) of Z-Lys (Boc) -Pro-OH and hydrogenated in dry hydrogen vapor at room temperature under 1atm for 8 hours. Then, the catalyst was filtered off and washed with methanol in a filter. The collected filtrate was evaporated to dryness. The residue was precipitated from anhydrous methanol with ether. Then, it was dried under vacuum while the desiccant was replaced several times. Yield: 6.38g (18.58 mmol); (91.87%)

Melting point of 98-99 deg.C

Rf-0.110 (chloroform-methanol-acetic acid) (42:7: 1);

0.166 (butanol: acetic acid: water) (4:1: 1);

0.235 (chloroform: methanol: ammonia) (8:1.75: 0.25);

0.494 (isopropanol: formic acid: water) (20:5:1) (2:1: 1).

Production of Boc-Thr-Lys- (Boc) -Pro-OH.

A solution of 13% TBA (39.4ml) was added to 18.58mmol (6.38g) Lys (Boc) -Pro-OH and evaporated 2 times with ethanol, 2 times with ethanol/benzene mixture and 2 times with benzene. 250ml of anhydrous ethyl acetate are added; the reaction mixture was cooled down to 0 ℃ and 9.2mmol (3.74g) of the previously synthesized Boc-Thr-OPfp was added and stirred on a magnetic stirrer for 1 h. The reaction mass is evaporated and 40 is addedml water to evaporate the residue. The aqueous solution was washed 3 times with 80ml of ether. After washing with ether, the aqueous solution was acidified to pH 3 with 18.58mmol (3.95g) of citric acid. After acidification, the aqueous solution was extracted 3 times with 40ml aliquots of ethyl acetate. The ethyl acetate collected after extraction was washed 3 times with 20ml of water and 20ml of 10% KHSO₄The solution was washed 3 times and 3 times with 20ml water. Over MgSO₄The ethyl acetate solution was dried.

Then, it was filtered and evaporated on a rotary evaporator. The resulting material was precipitated from ethyl acetate with hexane. Decant hexane and dry in a desiccator under vacuum over KOH, P₂O₅And paraffin drying the resulting material while replacing the drying agent several times.

Yield: 3.32g (6.1 mmol); (66.26%) melting Point 105 ℃ C. and 107 ℃ C

Rf-0.297 (acetone-phenyl-acetic acid) (50:100: 1);

0.234 (chloroform: methanol) (9: 1); 0.560 (benzene-ethanol) (8:2).

Production of Boc-Thr-Lys (Boc) -Pro-Phe-OH.

Production of Boc-Thr-Lys (Boc) -Pro-OSu.

3.53mmol (0.38g) of hydroxysuccinimide was added to 1.66g (3.05mmol) of Boc-Thr-Lys (Boc) -Pro in 50ml of anhydrous ethyl acetate, and the resulting solution was cooled down to 0 ℃ while stirring on a magnetic stirrer; then, 0.76g (3.53mmol) of DCC (dicyclohexylcarbodiimide) was added and stirred on a magnetic stirrer at room temperature for 2 hours. After the reaction had stopped, the resulting reaction mixture was filtered off and the precipitate was discarded. To the resulting solution was added 200ml of anhydrous ethyl acetate. The collected ethyl acetate was washed 2 times with 20ml of NaCl saturated solution and with 20ml of 10% KHSO₄The solution was washed twice with 20ml of saturated NaCl solution 2 times and with 20ml of 5% NaHCO₃Washed 2 times and 2 times with 20ml of saturated solution of NaCl. Over MgSO₄The ethyl acetate solution was dried. Then, it was filtered and evaporated on a rotary evaporator. The resulting material was precipitated from ethyl acetate with ether and hexane. Passing through KOH and P under vacuum₂O₅And paraffin filtration and drying of the precipitate while replacing the desiccant several times.

Yield: 1.52g (2.37mmol) 77.74%.

Rf-0.560 (benzene-ethanol) (8: 2); 0.457 (chloroform: methanol) (9: 1).

2. After preparation, 1.52g (2.37mmol) Boc-Thr-Lys (Boc) -Pro-OSu was dissolved in 25ml dimethylformamide and added to a preparation containing 0.392g (2.37mmol) L-Phe in 25ml dimethylformamide. The solution was stirred at room temperature on a magnetic stirrer. The reaction mixture was evaporated on a rotary evaporator and precipitated from benzene with ether. The precipitate was filtered and passed through P under vacuum₂O₅KOH and paraffin were dried while the desiccant was changed several times.

Yield: 1.03g (1.64mmol) 69.0%.

Rf-0.063 (isopropanol: formic acid: water) (20:5:1) (2:1: 1).

0.745 (chloroform: methanol: ammonia) (8:1.75: 0.25);

V.H-Thr-Lys-Pro-Phe-OH.

1.03g (1.64mmol) Boc-Thr-Lys (Boc) -Pro-Phe-OH was added to 8.2mL dichloromethane and 8.2mL TFA, the mixture was then incubated at room temperature for 45 min, and after removal of the protecting group, the solution was evaporated 2 times with anhydrous methanol, 2 times with benzene and 2 times with ether. It was precipitated from methanol with ether. Under vacuum over P₂O₅KOH and paraffin dried residue. The resulting precipitate was dissolved in 5ml of 30% ethanol and applied to Amberlyst A-21 (AcO)^-Form) column for acetate/hydrochloride exchange. The peptide was eluted with 100ml of 30% ethanol, evaporated to dryness under vacuum and precipitated from anhydrous methanol with anhydrous ether. The resulting precipitate was filtered and passed over P under vacuum₂O₅The KOH and paraffin were dried in a desiccator while the desiccant was changed several times.

Yield: 0.7g (1.43mmol) (87%).

Melting point 129-131 deg.C

Rf: -0.201 (butanol: acetic acid: pyridine: water) (30:6:20: 24);

0.156 (chloroform: methanol: ammonia) (4:4.5: 1.5).

Chromatographic and mass spectrometric analysis of the peptide sequences described in examples 1-7 are shown in table 2.

Table 2.

Note:

corresponding to [ M + nN]⁺Molecular peak of (1)

The strongest ions formed in fragmentation of the molecular ion peak at the energy of collision with a 35eV helium atom.

Table 2 shows data of High Performance Liquid Chromatography (HPLC) using a millihrome a-02 mini column liquid chromatography system and characteristics of synthetic peptides obtained using a thermo electron LCQ Advantage MAX mass spectrometer.

Developing chromatographic conditions allows for a chromatographically homogeneous product to be readily obtained.

Chromatographic conditions for peptide analysis.

And (3) chromatography: Milichrom-A02

Column: prontosil 120-5C18aq,2 x 75mm

Eluent A: 0.2M LiClO₄+5mM HClO₄

Eluent B: methanol.

Table 3 shows the gradient shape of the synthetic peptide separation.

TABLE 3

Time	％В
		0	5
16.5	80

Flow rate: 150 mul/min

Setting of wavelength: 210,220,230,240nm

Conditions of Mass Spectrometry

Equipment: ThermoElectron LCQ Advantage MAX

An ion source: performing electrospray; molecular ion fragments were introduced directly by ion collision (He) at 35eV at a flow rate of 5. mu.l/min into a 10. mu.g/ml peptide solution in 0.1% acetic acid

Source temperature: at 250 ℃ to obtain a mixture.

Ionization potential of 3.5kV

Example 8 identification of pharmacophore location.

To identify the pharmacophore, fragments of the parent peptide selak were synthesized: Thr-Lys; Thr-Lys-Pro; Pro-Gly-Pro; Arg-Pro-Gly-Pro; Pro-Arg-Pro-Gly-Pro; and efficacy testing was performed in vivo using a relevant preclinical model (Lordosis test).

We studied the efficacy of the following groups of peptides on the sexual behaviour of female rats at a dose of 100 μ g/rat: Thr-Lys; Thr-Lys-Pro; Pro-Gly-Pro; Arg-Pro-Gly-Pro; Pro-Arg-Pro-Gly-Pro. Hormone stimulated females following ovariectomy were recorded for sexual activity when they were in direct contact with sexually active males, or when such contact was not possible. It was found that the Thr-Lys-Pro peptide increased the female pre-sexual acceptance behavior (productive behavior) from 14 ± 4 to 29 ± 6 times during the monitoring period (p ═ 0.028, Wilcoxon test). The effect on lordotic response in females has the same trend (p ═ 0.09): the number of lordosis increased from 0.73. + -. 0.12 to 0.97. + -. 0.12 by the Thr-Lys-Pro peptide. These results indicate a sexual motivation to be enhanced in the context of the action of Thr-Lys-Pro peptide. The effect is particularly pronounced in the case of sufficient behavior. The results of the efficacy study of the following peptides in the lordotic model are shown in table 4: Thr-Lys; Thr-Lys-Pro; Pro-Gly-Pro; Arg-Pro-Gly-Pro; and Pro-Arg-Pro-Gly-Pro.

Table 4.

Table 4 demonstrates that the Thr-Lys-Pro tripeptide is a pharmacophore; furthermore, as shown by the results in Table 4, the smaller sequence, Thr-Lys dipeptide, did not work.

Example 9 pharmacophore assay

To test the pharmacophore, peptides based thereon, i.e., the Thr-Lys-Pro (SEQ ID NO:5) tripeptides corresponding to the general formula A-Thr-Lys-Pro-B-C-D-X, 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) pentapeptides and Thr-Lys-Pro-Arg-Pro-Phe (SEQ ID NO:7) hexapeptides were synthesized and potency tests were performed in vivo using the relevant pre-diagnostic model (lordosis test).

We investigated the efficacy of the following groups of peptides on female rat sexual behaviour at a dose of 100 μ g/rat: 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). The sex behavior of ovariectomized, hormone-stimulated females in direct contact with sexually active males, or when such contact is not possible, is recorded. It was found that peptides from the group comprising 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 pre-neutral acceptance behavior in females from 14 ± 4 to 26 ± 4 to 36 ± 6 behaviors during the monitoring period (p ═ 0.028, wilkokson test). Meanwhile, Thr-Lys-Pro-Arg (SEQ ID NO:4) and Thr-Lys-Pro-Phe (SEQ ID NO:2679) do not affect the intensity of female pre-sexual acceptance behavior and do not increase the number of lordosis, and the basic parameters of Thr-Lys-Pro-Arg (SEQ ID NO:4) and Thr-Lys-Pro-Phe (SEQ ID NO:2679) tetrapeptides were maintained at the level of the negative control. The effect on the female lordotic response has the same trend (p ═ 0.09). The peptide functions significantly in the absence of direct contact with the partner. The results indicate that the motivation for action in the context of the 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) peptides is enhanced and the motivation for effect is absent in the Thr-Lys-Pro-Arg (SEQ ID NO:4) and Thr-Lys-Pro-Phe (SEQ ID NO:2679) tetrapeptides. The effect is specific and evident in the case of sufficient behaviour. The results of the efficacy study of the following peptides in the lordotic model are shown in table 5: Thr-Lys-Pro (SEQ ID NO: 5); 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).

Table 5.

Industrial applicability

The present invention relates to the field of biochemistry and, in particular, to a method for producing peptides that exhibit high activity and are capable of stimulating self-healing of disordered organs. In particular, the present invention expands the scope of tools for stimulating sexual function and treating sexual dysfunction, while reducing the duration of treatment sessions and the cost of drug therapy.

Claims

1.A pharmaceutical composition comprising a peptide of formula Thr-Lys-Pro-Arg-Pro-X, wherein X is carboxy-terminal OH, OCH₃Or NH₂And (4) a base.

2. The pharmaceutical composition of claim 1, wherein the peptide is present in the composition in an amount effective for stimulatory or reproductive function.

3. The pharmaceutical composition of claim 1, wherein the peptide is produced by expression in a recombinant microorganism.

4. The pharmaceutical composition of claim 1, wherein the peptide is produced by chemical synthesis.