JPH0137399B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel polypeptides, novel methods for producing polypeptides, and uses of polypeptides. It is well known that many polypeptides have been isolated from various animal organs. However, until about 10 years ago, about the same weight as a human being at birth,
Little is known about the thoracic gland, an organ that accounts for 0.8% of the population, and the hypothesis has been that a small amount of neuromuscular inhibitory substances exist in the thoracic gland and that thoracic hormones affect the expression of the immune system. It's just a thing. Despite intense interest and early speculation and experimentation regarding the function of the thoracic gland, until recently little was known about the function of the thoracic gland. However, it is now known that the thymus is an accessory organ with epithelial (endocrine) and lymphatic (immunological) components and thus is involved in the immune function of the body. That is, it is known that the thoracic gland is an accessory organ consisting of epithelial stroma originating from the third branchial arch and lymphocytes derived from stem cells generated in hematopoietic tissue (Goldstein et al., "The Human
Lymphocytes differentiate within the thymus and leave the thymus as mature thymus-derived cells called T cells, circulating to the blood, lymph, and splenic lymph nodes. .
The induction of stem cell differentiation within the thymus seems to occur mediated by the secretions of the epithelial cells of the thymus, but due to various difficulties associated with biological testing, it has not been possible to completely isolate the hormones that are thought to be present. The reality up until now was that the structure could not be identified. To understand the importance of the biochemical differentiation properties of the polypeptides of the present invention, it is important to understand that, broadly speaking, the function of the thymus in relation to immunity is to produce cells derived from the thymus called T cells, namely lymphocytes. It is important to remember that this is something that should be done. T cells form the majority of the recirculating small lymphocyte fluid reservoir. T
The cells have immunological specificity and are directly involved in cell-mediated immune responses (such as allograft reactions) as executive cells. However, T cells do not secrete liquid antibodies. These antibodies are secreted by cells derived directly from the bone marrow, independent of the thymus. These bone marrow-derived cells are called B cells. However, in contrast to many antigens, the presence of suitably reactive T cells is essential for B cells to produce antibodies. The mechanism of this generation process through cooperation between T cells and B cells is still not completely understood. From the above-mentioned point of view, the thymus is necessary for the expression of cellular immunity and humoral antibody response, and is necessary for the differentiation of hematopoietic stem cells.
It can be said that the thymus influences the above expression and reaction system by inducing cells through the thymus. This inductive effect is due to secretions of the epithelial cells of the thymus,
That is, it is carried out through the use of thymus hormones. Furthermore, in order to understand the functioning of the thymus and lymphocyte cell system and the circulation of lymphocytes within the body, it is essential to understand that stem cells originate in the bone marrow and are carried in the blood to reach the thymus. The thymus subdivides into immunologically responsive T cells. These T cells migrate into the bloodstream and circulate with B cells between various tissue lymphatic systems and the bloodstream. The body's cells that secrete antibodies also originate from hematopoietic stem cells, but the differentiation of somatic cells is not determined by the thymus. Therefore, the somatic cells are named bone marrow-derived cells or B cells. In birds, somatic cells are differentiated in an organ called the bursa of Fabricius, which resembles the thymus. In mammals, no organ corresponding to the bursa of Fabricius has been found, and B.
The cells are believed to differentiate within the bone marrow. The physiological substances that drive this division are completely unknown. In recent years, much interest has been focused on substances isolated from the thoracic gland, as it has been found that the thoracic gland is associated with the immune properties of the body.In recent years, chemical studies on substances scattered in the thoracic gland of cattle have been conducted. A relatively large number of collections of papers based on this have been published. In fact, the present applicant and others have published numerous papers regarding research in the above field. Related publications include, for example, “The
Lancet”, July 20, 1968, p.119-122,
"Triangle", Vol, 11, No. 1, p.7-14,
“Annals of the New York Academy of
Sciences” Vol. 183, p. 230-240, 1971,
[Clinical and Experimental Immunology]
Vol.4, No2.p.181-189, 1967, [Nature],
Vol.247.p.11-14, 1974, “Proceedings of the
National Academy of Sciences, USA,
Vol.71, p.1474-1478, 1974, "Cell", Vol.5,
p.361-365 and p.367-370, 1975, and "Lancet" Vol. 2, p.256-259, 1975. Goldstein and Manganaro, “Annals of the New York Academy of
183, p. 230-240, 1971, discloses the existence of a thoracic polypeptide that causes myasthenic neuromuscular disorder in animals. This neuromuscular disorder is similar to the human disease myasthenia gravis. Furthermore, the above article found that there are two effects caused by different polypeptides contained in the bovine thorax. One of these polypeptides, named thymotoxin, is thought to cause smoking cessation. However, although this polypeptide has not been isolated, it appears to be a polypeptide with a molecular weight of approximately 7000, has a strong net positive charge, and is retained in CM-Sephadex at a pH of 8.0. It is shown. Nature, 247, 11. January 4, 1975, describes a product identified as Thymin and thymine. These products are new polypeptides isolated from bovine thorax,
It has been found that it has particular application in various therapeutic areas. Since similar names have been used in the past for other substances isolated from the thorax of cattle, these thymine and substances are now called Thymopoietin and Thymopoietin, respectively. . These substances and their manufacturing methods were introduced on August 22, 1975.
No. 606,843, filed in the United States on D.A. US Pat. No. 4,002,602 describes ubiquitous immunogenic polypeptides.
Long chain polypeptides, referred to as immunopoietic polypeptides, are disclosed. This polypeptide is a 74-amino acid polypeptide characterized by its ability to induce T-cell and B-cell immune cell differentiation from precursors present in bone marrow or spleen at nanogram concentrations in vitro. It is. Thus, the polypeptides described above are useful in therapeutic areas involving thymus or immune defects or deletions, and the like. U.S. Patent No. 1, issued January 11, 1977.
No. 4002740 discloses synthetic tridecapeptide compositions. These tridecapeptide compositions can induce differentiation of T-lymphocytes, but not B-lymphocytes, which are complement receptors. As such, this polypeptide exhibits many of the properties of the long chain polypeptide isolated and designated as thymopoietin in the aforementioned US patent application Ser. No. 606,843. The present invention provides synthetic polypeptides containing five amino acids (i.e., 5-
A method for producing amino acid polypeptide),
This polypeptide was published in the above-mentioned publication and on August 22, 1975.
Many of the properties of the long chain polypeptide isolated and termed thymopoietin in U.S. Patent Application No. 606,843 filed on D.A. and the tridecapeptide described in U.S. Pat. No. 4,002,740 It turns out that it has. Therefore, it is an object of the present invention to provide a method for producing novel biologically important polypeptides. Another object of the present invention is that it has the ability to differentiate bone marrow cells into T cells and give rise to thymus-derived lymphocytes at nanogram concentrations, and is therefore highly effective for human and animal immune systems. An object of the present invention is to provide a method for producing certain novel polypeptides. The present invention will be explained in more detail below. In order to satisfy the above objects and advantages, the present invention provides a method for producing novel polypeptides having the following structural unit as an active site. -ARG-LYS-ASP-VAL-TYR- Furthermore, novel peptide-resin intermediates formed during the production process of the polypeptides of the present invention are also obtained, and these intermediates have the structural unit of the following formula. It is. In the above formula, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are protecting groups bonded to each amino acid as required, and the resin is a solid polymer that acts as a reaction support. It is a molecular substance. The present invention is a method for producing polypeptides by solid-phase peptide synthesis. As mentioned above, the present invention relates to novel polypeptides having therapeutic effects in various fields, various intermediates generated in the process of producing polypeptides, therapeutic compositions,
The present invention relates to uses of the polypeptides and methods for producing the polypeptides. According to a reference example of the present invention, polypeptides having the following amino acid structural unit as an active site can be obtained. ARG-LYS-ASP-VAL-TYR In yet another reference example, a pentapeptide containing the above amino acid structure as an active site and can be represented by the following general formula is obtained. In the above formula, R and R' are each a substituent bonded to the pentapeptide, and these substituents do not substantially affect the biochemical activity of the basic active structure of the amino acid. . In other words, when attaching to these two terminal amino acids a functional group or derivative (i.e., R and R') that does not substantially affect the biochemical activity of the pentapeptide molecule, the terminal amino acids of this pentapeptide chain The formula means that can be modified. Therefore, terminal amino acid and alkoxylic acid groups are not essential to confer biochemical activity, as is the case with certain polypeptides. Substitutions of these functional groups include conventional substitutions such as acylation of free amino groups and amidation of free carboxylic acid groups, and substitution of other amino acids with polypeptides. From these points of view, the pentapeptides of the present invention appear to be unique. This is because these pentapeptides exhibit the same biochemical activity as long-chain natural peptides of which the peptide structural unit is a part. The biochemical activity of peptide molecules is believed to be due to the stereochemical characteristics of the molecule, ie, the unique folding structure of the molecule. Note that the bonds of polypeptides are not rigid but flexible, and are sheet-like.
It has a spiral shape. As a result, the molecule as a whole becomes flexible and folds in a certain manner. The inventors have found that pentapeptides fold similarly to long chain natural polypeptides and therefore exhibit the same biochemical properties. For this reason, pentapeptides can be substituted with a variety of functional groups as long as the substituents do not substantially affect the biochemical activity or interfere with the natural folded structure of the pentapeptide molecule. can. The biochemical activity and retention of the natural folded structure of the pentapeptide molecule are evident from the following facts. That is, the pentapeptide structure of the present invention
It exhibits the same biochemical properties as the naturally occurring 49-amino acid named thymopoietin as described in US Patent Application No. 606,843 and Nature 247 , 11, 1975. Furthermore, the pentapeptide structure of the present invention is described in "Cell", Vol. 5, 367-370,
It has the same biochemical properties as the synthetic tridecapeptide disclosed in August 1975 and US Pat. No. 4,002,740. In both of these long polypeptides, the pentapeptide of the invention can be identified, albeit in a form linked within the molecule to other amino acids described in the above-mentioned literature. However, the biochemical activities of the pentapeptide of the present invention and thymopoietin and tridecapeptide are the same, and the peptide chain in which other amino acids and terminal amino acids are substituted does not affect the biochemical properties of the basic pentapeptide structure. The above publication directly proves that the pentapeptide of the present invention is an active site. From this it will be understood that any substitution definition can be used for R and R' in the formula as long as it does not substantially affect the biochemical activity of the basic active structure. It is presumed that R and R' may be, for example, any of the following substituents. R R ' Hydrogen OH C ₁ -C ₇ alkyl NH ₂ C ₅ -C ₁₂ aryl NHR ₇ C ₆ -C ₂₀ alkaryl N(R ₇ ) ₂ C ₆ -C ₂₀ aralkyl OR ₇ C ₁ -C ₇ alkanoyl C ₂ - _C7 alkenyl _C2 - _C7 alkynyl where _R7 is _C1 - _C7 alkyl, _C2 - _C7 alkenyl, _C2 - _C7 alkynyl, _C6 - _C20 alkaryl or It is a _C6 - _C20 aralkyl group. R and _R2 may also be neutral amino acid groups or residues of polypeptides containing 1-20 carbon atoms.
These are shown below. R R ' GLN VAN GLU GLN GLY LEU GLU−GLN TYR GLY−GLU VAL−GLN GLY−GLU−GLN VAL−LEU VAL−TYR GLN−LEU GLN−TYR GLN−VAL LEU−TYR LEU−LEU TYR−LEU VAL− GLN-LEU VAL-GLN-LEU-TYR According to another reference example of the present invention, a novel pentapeptide having the following formula can be obtained. In the above formula, R is C ₁ − such as hydrogen, methyl, ethyl, etc.
C ₇ alkyl, C ₅ -C ₁₂ aryl such as phenyl, or C ₁ -C ₇ alkanoyl such as acetyl, propionyl, and R′ is OH, NH ₂ ,
NHR ₇ or N(R ₇ ) ₂ . The most preferred polypeptides are those in the above formula where R is hydrogen and R' is H. Acids that can form salts with pentapeptides include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, as well as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, and pyruvic acid. ,
Organic acids such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, and sulfanilic acid can be mentioned. In order to show the structure of the above pentapeptide, its amino acid components are shown by abbreviations for convenience, but these abbreviations indicate the following amino acids. Amino acid abbreviations Glycine GLY L-glutamic acid GLU L-glutamine GLN L-arginine ARG L-lysine LYS L-aspartic acid ASP L-valine VAL L-tyrosine TYR L-leucine LEU The polypeptide structure of the present invention has five active sites. This peptide has been found to exhibit properties similar to the 49-amino acid polypeptide isolated from bovine thorax as disclosed in U.S. Patent Application No. 606,843. . The peptides of the present invention are characterized in that they can induce selective differentiation of Thy-1 ⁺ T cells (rather than CR ⁺ B cells) at concentrations of 1 ng-10 μg/ml.
Here, Thy-1 is a differentiation antigen present on T cells but not on B cells.
CR is a complement receptor that exists on B cells, not T cells. When these synthetic peptides were studied by induction assay in vitro, they were found to have induction specificity similar to Thymopoietin. That is, these peptides differentiated Thy-1 ^-1 cells and induced them to become Thy-1 ⁺¹ cells, but did not differentiate CR ^-1 cells into CR ⁺¹ cells. Although many substances have been identified in vitro that behave similarly to thymopoietin and are capable of generating intracellular cyclic muscle adenylate (AMP) and inducing T cell differentiation, most substances do not It should be noted that the peptides of the invention are selective for inducing T cell differentiation but not for CR ⁺ B cell differentiation. Experiments have shown that the synthetic peptides of the present invention also affect neuromuscular transmission similar to thymopoietin. For example, 10− per mouse
Twenty-four hours after a single 100 μg injection, impaired neuromuscular transmission occurred, detectable by electromyography. Because of the above-mentioned properties of the polypeptides of the invention, these polypeptides are useful for therapeutic treatment of humans and animals. This is because polypeptides can induce lymphoid stem cells originating from hematopoietic tissues to differentiate into thymus-derived cells, or T cells, which are responsible for the body's immune response. As a result, the substances of the invention are likely to have a wide variety of therapeutic uses. First, because the polypeptide compounds of the present invention can perform certain functions within the thymus function, these compounds have a variety of thymus function and immunological applications. The main field of use is the treatment of Digeorge syndrome, a condition of congenital absence of the thymus. This deletion can be overcome by injection of polypeptides of the invention. Because of the biochemical properties of polypeptides that are highly active even when used in low concentrations, polypeptides increase or promote therapeutic stimulation of cellular immunity and thus are effective against bacterial or mycoplasmal infections, tuberculosis, leprosy, leprosy, etc. Polypeptides appear to be effective in the treatment of chronic infectious diseases in living organisms, such as acute and chronic viral infections, and in supporting the body's overall immunity. . Furthermore, polypeptide compounds are also believed to be useful in areas where cellular immunity is a problem, particularly in areas where there is an immune deficiency, such as in the aforementioned disease group. Additionally, if an imbalance of T cells and B cells results in excessive antibody production, polypeptide compounds can stimulate T cell production to correct this excessive condition. Peptide compounds are effective in treating certain autoimmune diseases where damaging antibodies are present, such as systemic lupus erythema. Furthermore, because of the aforementioned properties of polypeptides, they are effective in vitro in developing T cell surface antigens and in developing functional capacity to respond to cell division substances and antigens. In addition, cells can cooperate to enhance the ability of B cells to produce various antibodies. The polypeptides are also effective in inhibiting the free expansion of thymopoietin-responsive lymphocytes. Another important property of polypeptides is their ability to be used in living organisms to restore cells using the properties of T cells. Therefore, the polypeptides of the present invention can be said to be substances that have activity in many fields because they have the effect of enhancing immune responses in individuals. Since the polypeptides of the present invention affect neuromuscular transmission, administration of the polypeptides in large amounts is effective in treating diseases associated with, for example, convulsive excessive neuromuscular transmission. Yet another important property of the polypeptides of the present invention is that they are highly active at very low concentrations of about 1 ng/ml or more, and their activity is maximum at concentrations of about 100 ng/ml or more. That's what it means. As a carrier for this polypeptide, any known carrier commonly used for such purposes can be used, such as a normal saline solution containing a protein diluent such as bovine serum albumin. It is desirable to prevent the polypeptide used at the above-mentioned low concentration from being adsorbed and lost to glassware. The polypeptides of the present invention are active in a range of about 1 mg or more per 1 kg of body weight. For the treatment of diabetic syndrome, polypeptides may be administered at a rate of about 1.0-10 mg/kg body weight. Generally, the above dosages can also be used for the treatment of other conditions or diseases mentioned above. The polypeptides of the present invention are described in "Journal Of
American Chemical Society” 85, p.2149−
It was synthesized using a concept similar to the Merrifield method as reported in 2154, 1963. The synthesis was carried out by stepwise addition of several protected amino acids to a growing peptide chain covalently attached to a solid resin particle. In this synthetic method, the drug and by-products were removed by filtration and crystallization of the intermediate. Generally speaking, this method involves covalently bonding the first amino acid of a peptide chain to a solid polymer, followed by stepwise addition of amino acids one type at a time to construct the desired structural unit. . Finally, the peptide is separated from the solid support and the protecting groups are removed as well. According to this method, the growing peptide chain is attached to completely insoluble solid particles, making it convenient to filter and wash to remove drugs and by-products. The amino acids can be attached to a suitable polymer, which must be insoluble in the solvent used and must be in a physically stable form to permit easy filtration. The polymer carrying the amino acid must contain a functional group that can reliably covalently attach the first protected amino acid. Various polymers can be used for this purpose, such as cellulose, polyvinyl alcohol, polymethacrylate, and sulfonated polystyrene; however, in the synthesis of the present invention, a chloromethylated copolymer of styrene and divinylbenzene is used. used. Various functional groups of the amino acids, which are active but should not participate in the reaction, were protected throughout the synthesis reaction with known protecting groups commonly used in the case of polypeptides. Functional groups such as side chains on arginine, lysine, aspartic acid, and tyrosine were protected with protecting groups that could be removed upon completion of the stepwise addition without adversely affecting the final polypeptide product. The use of fluorescamine (Felix
et al., Analyt.Biochem., 52, 377, 1973), polypeptides were synthesized by an improved solid-state synthesis method. If the coupling was not indicated to be complete, the coupling was repeated using the same protected amino acid before α-amino deprotection. As a general method of construction, first, an amino acid and hydroxy-protected L-tyrosine were esterified into a resin in absolute alcohol containing an aminoamine.
The bound amino acid resin was filtered, washed with alcohol and then water, and dried. Next, the protecting group attached to the amino group of the protected tyrosine (for example, t-benzyloxycarbonyl, abbreviated as t-BOC) was removed without affecting other protecting groups. The bound amino acid-resin with a free amino group is a protected form of L-valine, preferably alpha t-BOC-L-valine,
L-valine was bonded to the amino acid-resin by reacting with L-valine. Then the protected form L-aspartic acid, L-
The complete peptide molecule was synthesized by reacting with lysine and L-arginine, respectively, in the same manner as above. An active structure consisting of five basic amino acids was formed by the above method. Addition of other neutral amino acid residues to the ends of the molecular chain can be carried out using known reactions. A series of reactions to synthesize the active structure of five amino acids can be performed as follows.

[Example 1]

In order to synthesize the polypeptide of the present invention, the following materials were purchased. α-AOC-Ng-Tos-L-arginine α-BOC-2-chloro-benzyloxycarbonyl-L-lysine α-BOC-O-benzyl-L-aspartic acid α-BOC-L-valine α-BOC-2 , 6-dichlorobenzyl-L-tyrosine In the above drug, BOC represents t-butyloxycarbonyl, AOC represents t-amyloxycarbonyl, and Tos represents tosyl. Reagents, dicyclohexyl carbodiimide, fluorescein amine, and resin used for structure determination during the sequential reactions of amino acids were purchased from commercial sources. The resin used was a polystyrene divinylbenzene resin having a particle size of 200-400 mesh and containing 1% divinylbenzene and 0.75 mmol chloride/g resin. In synthesizing the polypeptide, 2 mmol of α-BOC-O-2,6-dichlorobenzyl-L
-Tyrosine was esterified to 2 mmol of chloromethylated resin by treatment in absolute alcohol containing 1 mmol of triethylamine at 80 DEG C. for 24 hours. The resulting bound amino acid resin was filtered, washed with absolute alcohol and dried. α-BOC or α-AOC amino acids were then similarly sequentially coupled to the deprotected α-amino groups of the peptide resin using an equivalent amount of dicyclohexyl carbodiimide to form the peptide-resin of the invention. After each coupling reaction, a small amount of the resin was tested with fluorescein amine, and if positive fluorescence, the coupling was assumed to be incomplete and the reaction was repeated with the same protected amino acid used. As a result of several coupling reactions, a pentapeptide-resin represented by the following formula was obtained. In the above formula, AOC is amyloxycarbonyl,
Tos is tosyl, Z is benzyloxycarbonyl,
Bzl represents benzyl, and Bzl (Cl ₂ ) represents O-2,6-dichlorobenzyl. The resolution of this peptide-resin conjugate and the removal of the protecting groups were carried out using a Kelf resolution device (Peninsula Lab).
(manufactured by NC.) using anhydrous hydrogen fluoride at 0°C for 60 minutes. In this case, 1.2 ml of anisole per gram of peptide resin was used as a scavenger. The peptide mixture was lyophilized in vacuo, washed with anhydrous ether, and the peptides were extracted with glacial acetic acid and water. P-6 biogel in 1N acetic acid (Bio-
Peptides were subjected to chromatographic analysis on Gel). As a result, it was found that the obtained peptide had a purity of 94% and had the following structure. [Pharmacological Test Example] In order to investigate the activity and properties of the polypeptide of Example 1, tests were conducted on healthy 5-6 week old nu/nu mice of both sexes. The mouse is
They were reared on BALB/C backing (thymocytes displaying Thy-1.2 surface antigen) and maintained under normal conditions. Regarding antiserum, anti-Thy−1.2 serum
-1 homologous mouse. Regarding the in vitro induction of Thy−1 ⁺ T cell or CR ⁺ B cell differentiation, Komuro & Boyse induced differentiation of thymocytes from prothymocytes in vitro. It was carried out according to the method described (Lancet, 1, 740, 1973). This method uses the acquisition of Thy-1.2 as a measure of T cell differentiation. On the other hand, ^CR from in vitro B cell precursors
Induction of differentiation into CR ⁺ B cells was performed under the same conditions as for thymocytes, but in this case, the criteria for analysis were rabbit antibodies and non-degradable complement in an amount that caused some agglutination. The ability of CR ⁺ B cells to bind coated sheep red blood cells was used. Healthy nu/nu fractionated on discontinuous gradients of bovine serum albumin
Mouse spleen cell populations were cultured with both precursors (Thy-1 ^- and
^CR- ) was used as a source. This is because the cell population contains little or no Thy-1 ⁺ cells and only a small number of CR ⁺ cells. As a result, this polypeptide showed similar selectivity to thymopoietin in inducing the differentiation of T lymphocytes rather than B lymphocytes, which are complement receptors (CR ⁺ ). This pentapeptide has a concentration range of 1ng-10μg/ml.
Differentiation of Thy−1 ⁺ T cells was induced. 0.01ng/10μ
It did not induce differentiation of CRFB cells in the concentration range of g/ml. [Reference Example 3] In order to examine the effect of the peptide of Example 1 on neuromuscular transmission, 10-100 μg of the peptide was added in a normal salt solution.
This peptide was injected intraperitoneally. twenty four
After some time, "Lancet", Vol, 12, p.256-259, 1975
Neuromuscular transmission was investigated using electromyography using the method described in . Mice injected with the peptide had neuromuscular damage. [Reference example 4] “Journ.Med.Chem.Vol.16, No.5, p.545-8
The pentapeptide of Example 1 was prepared on a benzhydrylamine resin substrate using the method by J. Rivier described in J. Rivier (1973). In this production, dicyclohexylcarbodiimide was used as a coupling agent, and the coupling reaction was
Performed in CH ₂ Cl ₂ . The obtained peptide is separated from the substrate using hydrogen fluoride to liberate the peptide, and the protecting group is removed to obtain the following derivative. It was identified by thin layer chromatography and electrophoresis, and the following results were obtained. Thin layer chromatography Sample: 30μg silica gel (Brinkmann glass plate, 5x
20cm, thickness 0.25mm) R ¹ _f : n-butanol: pyridine: acetic acid (HOAc): water 30:15:3:12 R ² _f : ethyl acetate (EtOAc): pyridine: acetic acid: water 5:5:1 :3 R ³ _f : Ethyl acetate: n-butanol: acetic acid: water 1:1:1:1 Spray reagent: Pauli reagent, ninhydrin, Sakaguchi reagent and ₂ compounds Arg-Lys-Asp-Val-Tyr-NH ₂ thin layer Chromatography R ¹ _f R ² _f R ³ _f 0.53 0.68 0.26 Electrophoresis Peptide transferred to cathode 7.5cm Electrophoresis Watmann 3mm filter paper (11.5 x 56cm) Sample: 100μg PH5.6, pyridine acetate buffer solution, 1000V ,
1 hour Spray Reagents: Ninhydrin and Sakaguchi Reagent This amidated pentapeptide has a maximum activity of 105% and It showed a minimum activity of 70%. [Reference Example 5] The basic pentapeptide obtained in Example 1 was esterified to obtain an ethyl alcohol derivative. To produce ester derivatives, it is necessary to block or protect the acid group of aspartic acid with a mild acid-sensitive blocking group;
A t-butyl group is preferred as a protecting group. The protecting group for the α-amino group is sensitive to mild bases, and is preferably a fluorenylmethoxycarbonyl group. If such a protecting group for the α-amino group is used, the effect on the acid-sensitive protecting group of aspartic acid can be removed by adding each amino acid residue. After formation of the protected pentapeptide resin, treatment with a mild base followed by a mild acid removes the two protecting groups. The C-terminal ester is selectively obtained when the pentapeptide is cleaved from the resin by transesterification with ethyl formate using sulfuryl chloride as a catalyst.
Esterification does not occur at the free acid group of the aspartic acid residue. All remaining protecting groups are removed and a peptide of the following formula is recovered. [Reference Example 7] In this reference example, “Biochemical and
Biophysical Research Communications
Described in Vol.48, 1100-1105 (1972)
N- as a solid support according to the method of Monahan et al.
The pentapeptide of Example 1 was prepared using methyl-benzhydrylamine resin. The protected pentapeptide was separated from the resin using hydrogen fluoride to obtain the amino-substituted derivative shown below. [Reference Example 8] In this reference example, the method of Example 1 was used except that an equivalent amount of N-methyl-L-arginine was used instead of the protected L-arginine, and the N of the basic pentapeptide of Example 1 was -Methyl-substituted derivatives were synthesized. A peptide of the following formula was obtained. [Reference Example 9] The same metal amine polypeptide obtained in Reference Example 8 was synthesized in the same manner as described in Reference Example 4 using benzylhydrylamine resin as a solid resin. The obtained pentapeptide having a protecting group and directed to the resin was separated from the resin with hydrogen fluoride, and all protecting groups were removed to obtain an amide polypeptide of the following formula. [Reference Example 11] The following esterified polypeptide was synthesized according to the method of Example 5, except that an equivalent amount of N-methyl-L-arginine was used in place of the protected L-arginine. The polypeptide derivative obtained in Reference Example 5-23 retains the same biochemical activity as described in the main text of the present specification for the basic amino acid structural unit. As mentioned above, the present invention provides novel polypeptides, which can induce the differentiation of complement receptor (CR ⁺ ) T lymphocytes rather than B lymphocytes, and are therefore useful in many cases. Effective in the therapeutic field. Further provided by the present invention are derivatives of the above pentapeptides, novel intermediate polypeptides, methods for producing the peptides, therapeutic compositions and uses of the compositions. Although the invention has been described with respect to certain preferred embodiments, it is of course not limited to these embodiments.

Claims (1)

[Claims] 1. L-tyrosine whose amino and hydroxyl groups are protected is esterified and bonded to a water-insoluble polymer resin through a covalent bond, and the α-amino protecting group is removed from the L-tyrosine moiety to form α. - React with amino-protected L-valine to bond L-valine to L-tyrosine-resin, remove the α-amino protecting group from the L-valine moiety, and react with α-amino-protected L-aspartic acid. to convert L-aspartic acid to L-valine-L.
-tyrosine-resin, remove the α-amino protecting group from the L-aspartic acid moiety, and react with α-amino protected L-lysine to convert L-lysine into L-aspartic acid-L-valine-tyrosine-resin. and remove the α-amino protecting group from the L-lysine moiety to form the α-amino protected L-arginine into L-lysine-L-L-aspartic acid-L-valine-L-
A peptide intermediate bound to a tyrosine resin and having the formula: In the above formula, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ form a peptide intermediate which is a protecting group supported on an amino acid, and all the protecting groups are removed from the peptide intermediate and the polymer is After removing from the resin, the following formula is obtained: A production method characterized by producing a pentapeptide represented by: 2. The method according to claim 1, characterized in that the reactive side chains of the reactive amino acids are protected during the reaction period. 3 Claims characterized in that R ₁ is tosyl, R ₂ is benzineoxycarbonyl, R ₃ is benzyl, R ₄ is 0-2,6-dichlorobenzyl, and R ₅ is t-amyloxycarbonyl. The method described in Section 2. 4. Claim 1, wherein the polymeric resin is a resin selected from the group consisting of cellulose, polyvinyl alcohol, polymethacrylate, sulfonated polystyrene, and a chloromethyl copolymer of styrene and divinylbenzene. Method described.