JPS6150998A - Manufacture of pentapeptide composition and intermediate polypeptide - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

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 organs of animals. However, until about 10 years ago, very little was known about the thymus gland, an organ that accounts for about 0.8% of a human's body weight at birth. The hypothesis that thymic hormones influence the expression of the immune system has been proposed.Despite intense interest and early speculation and experimentation regarding the function of the thymus, until recently little was known about its function. However, it is now known that the thymus is an accessory organ with epithelial (endocrine) and lymphatic (immunological) components, and therefore the thymus is involved in the body's immune function. It is well known that it is an accessory organ consisting of epithelial stroma derived from the third branchial arch and lymphocytes derived from stem cells generated in hematopoietic tissue.
rThe HumanTh such as go+dste1n
ymus J, Hcinscmann,
London, 1959), lymphocytes are differentiated within the thymus and are released from the thymus as mature thymus-derived III cells called T cells.
Circulates to the splenic lymph nodes. It seems that the induction of female cell differentiation within the thymus occurs mediated by the secretions of thymic epithelial cells, but due to the difficulties associated with biological tests, it is difficult to fully understand the hormones that are thought to be present. The reality up until now has been that it has not been possible to isolate or identify the structure.

Immediate F/I of the importance of the biochemical differentiation properties of the polypeptides of the present invention! Therefore, it should be noted that, broadly speaking, one of the functions of the thymus regarding immunity is to produce cells derived from the thymus called T cells, that is, lymphocytes. T cells form the majority of the recirculating small lymphocyte fluid reservoir. ■Cells have immunological specificity and are directly involved in cell-mediated immune responses (such as allograft reactions) as executive cells. However, ]-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 order for B cells to produce antibodies with many antigens, the presence of T cells with an appropriate reaction is essential. Many aspects of the process are still not completely understood.

From the above-mentioned point of view, the thymus gland is necessary for the expression of cellular immunity and humoral antibody reactions, and the above expression and reaction system can be achieved by differentiating hematopoietic stem cells and inducing T cells in the thymus gland. It can be said that the thymus gland has an influence on This inductive influence is caused by secretions of the epithelial cells of the thymus gland.

That is, it is carried out through the mediation of thymic hormones.

Furthermore, in order to understand the functioning of the thymus and lymphocyte cell lineages and the circulation of lymphocytes within the body, it is essential that 9↑ cells originate in the bone marrow and are carried by the blood to the thymus. The thymus subdivides into immunologically responsive T cells. These T cells migrate into the bloodstream and circulate between various tissue lymphatic systems and the bloodstream, including f3 MII Ill.

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 capsule of Fabricius, which is similar to the thymus gland. In mammals, no organ corresponding to Bricius has been found, and B cells are thought to differentiate within the bone marrow. The physiological substances that carry out this division are completely unknown.

Substances isolated from the thymus have been of great interest since it has been found that the thymus is associated with the immune properties of the body.In recent years, chemical studies on substances scattered in the bovine thymus have attracted much attention. 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, [Th
e L anCetJ, July 20, 1968 issue, l
), 119-122, rTriangle J,
Vol, 1 i, No, 1゜0, 7-
14. rAnnals of tl+e N
OW York ACad (3mV of 3ci
enccs J VOl, 183, D.

230-240.1971. [Cl1nical
andExperimental I n+mUn
oIOgy J, Vol. 4, N.

2, 1), 181-189. 19
67, rNature J, Vol, 247.
p, 1L14.1974, "p) r'oceedi
n(js 'or the National
ACademlof 3cicinces,
USA, Vol. 71. D, 1 474
-1478. 1974. rcell l, Vo
l.

5.0.36L365 and l), 367 -370°1
975, and rLancet J Vol, 2. t
), 256-259.19.75. GO1dSt13in and M anganaro
I'A nnals or the Ngw by
York Academy of 3Cien
ceSJ, Vof, 1g3, 0.230-240.
A 1971 article discloses the existence of thymic polypeptides that do not cause myasthenic neuromuscular disorders 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 types of polypeptides contained in the bovine thymus. One of these polypeptides, named thymotoxin, is thought to cause smoking cessation. However, this polypeptide has not been isolated;
It is believed to be a polypeptide with a molecular weight of approximately 7,000, and has been shown to have a strong net positive charge and be retained on CM-Sephadex (CM-S cphadex) at l-18,0. There is.

rNaturc J, 247, 11. January 4, 1975, describes products identified as Thymin I (Thymin I) and Thymine ■). These products are new polypeptides isolated from bovine thymus and have been found to have specific applications in various therapeutic areas. Similar names have been used for thymic substances isolated from the thymus in the past, so these thymine ■ and ■ substances are now referred to as thymine
opoietin I) and thymoboietin IT (
They are respectively called ``T hymopoictinl''. 1 These substances and their manufacturing methods were introduced in August 1975. 2
U.S. Patent Application No. 606,84 filed in the United States on 2nd.
It is described in the specification of No. 3.

U.S. Special Engineering'"No. 4,002,602 Mei 1st Period II F4
3 + col; 1, ubiquitous immunogenic polypeptides (U
biquitousImmunopoietic P
Disclosed are long chain polypeptides, referred to as Olypeptides.

This polypeptide is a 74-amino acid polypeptide that can be administered to bone marrow or 81-amino acid at nanogram concentrations in vitro.
I! It is characterized by its ability to induce T-cell and B-cell immune cell differentiation from precursors present in acupuncture. Thus, the polypeptides described above are useful in therapeutic areas involving thymic or immune defects or deletions, and the like.

U.S. Patent No. 4.0, issued January 11, 1977
Synthetic tridecapeptide compositions are disclosed in No. 02.740, Mei-I store. 3 These tridecapeptide compositions are not directed against B-lymphocytes, which are receptors for human bodies, but against T-lymphocytes. Can induce sphere differentiation. As such, this polypeptide is
It exhibits many of the properties of the long chain volibebtide isolated and named as thymovoyetin in No. 43.

The present invention provides synthetic polypeptides containing five amino acids (i.e., 5-amino acid polypeptides) having certain active moiety structural units; 1911 at sunrise
The long chain polypeptide isolated in U.S. Pat. It was found that the characteristics of

Therefore, it is an object of the present invention to provide novel polypeptides of biological importance.

Another aspect of the present invention is that nanograms of 8) J degrees increase the ability of bone marrow cells to differentiate into T cells and give rise to thymus-derived lymphocytes, thus improving human and animal immunity. The object of the present invention is to provide novel polypeptides that are highly effective in this system.

Yet another object of the present invention is to provide compositions and methods for use in biochemical operations as well as new intermediate products and methods for synthesizing new polypeptides.

The present invention will be explained in more detail below.

In order to satisfy the above objects and advantages, the present invention provides novel polypeptides having the following channel unit as an active site.

-ARG-LYS-ASP-VAITYR- Furthermore, novel peptide-resin intermediates formed in the process of manufacturing the polypeptides of the present invention are also provided, these intermediates having structural units of the following formula: It is.

RI R2R3 In this case, peptide intermediates that do not contain resins and other protecting groups are also within the scope of the invention. In the above formula, R+, Rz, R3, RJ and R5 are protective groups bonded to each of the indicated amino acids, if necessary, and the resin is a solid polymeric substance that acts as a reaction support. The present invention further provides a method for producing the polypeptides of the present invention by solid-phase peptide synthesis,
The present invention also provides therapeutic compositions and methods for administering polypeptides to humans and animals so that their biochemical effects do not extend to humans and animals.

As mentioned above, the present invention relates to novel polypeptides having therapeutic effects in various fields, unique intermediates produced in the process of producing polypeptides, therapeutic compositions, uses utilizing the polypeptides, and This article relates to a method for producing polypeptides.

According to a main embodiment of the present invention, polypeptides having the following amino acid structural unit as an active site are provided.

1. ARG-LYS-ASP-VAL-TYR In yet another practical IM embodiment, pentapeptides containing an F amino acid structure as a sensitive site, which can be expressed by the following general formula, are provided. .

In the above formula, R and R' are each a substituent bonded to the pentapeptide, and these substituents have a substantial effect on the biochemical activity and sex of the amino acid glue carving activity (7?1 structure). In other words, these two terminal amino acids are provided with functional groups or derivatives (i.e., R and R') that do not substantially affect the biochemical activity of the pentapeptide molecule. The formula ■ means that the terminal amino acids of this pentapeptide chain can be modified when attached without departing from the scope of the invention.Thus, the terminal amino 11021 and carboxylic acid groups are This means that it is not essential for imparting biochemical activity as in the case of pentapeptides with unsubstituted terminal amino acid groups and the biochemical activity referred to in the present invention. Both pentapeptides in which the terminal group is substituted with one or more functional groups that do not affect the scope of the present invention are included within the scope of the present invention.

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 polypeptides with other amino acids. From these points of view, the pentapeptides of the present invention appear to be unique. Because,
This is because these pentapeptides exhibit the same biochemical activity as long-chain natural peptides having the above-mentioned peptide structural unit as a part thereof. The biochemical activity of peptide molecules is believed to be due to the stereochemical characteristics of the molecule, ie, its unique folded structure. Note that the bonds of polypeptides are not rigid but flexible and have a sheet-like, spiral-like, etc. shape.As a result, the molecule as a whole becomes flexible and folds in a certain state. We have discovered that pentapeptides are similar to long-chain natural polypeptides and therefore exhibit the same biochemical properties. However, the pentapeptide can be substituted with various functional groups as long as the substituents do not interfere with the natural folded structure of the L pentapeptide molecule.

Biochemical activity of pentapeptide molecules and natural superposition MIJ
The holding power of the structure is clear from the following facts. That is, the pentapeptide structures of the present invention are described in U.S. Patent Application No. 606. a43g light■0) and rNat
It shows the same biochemical properties as the natural 49-amino acid named zymo yetin described in ureJ247', 11.1975. Furthermore, the pentapeptide structure of the present invention can be obtained from rcellJ.

Vol. 5.36.7-370. August 1975 issue and US Patent! '! It has the same biochemical properties as the synthetic tridecapeptide disclosed in No. 4,002,740. In both of these long-chain polypeptides, the pentapeptide of the invention can be identified, albeit in a form bound within its molecule to other amines rIi described in the above-mentioned literature. However, the biochemical activities of the pentapeptide of the present invention, thymovoyetin, and tridecapeptide are the same, and the other amino acids and terminal amino acids are different from each other. The above publication directly proves that the pentapeptide of the present invention is an active site since the i-transformed peptide chain does not affect the biochemical properties of the basic pentapeptide pathology construct.

From this, it can be seen that R and R' in formula ① have basic form activity (
It will be understood that any substituent can be used so long as it does not substantially affect the biochemical activity of the 14-construct.

R and R' may be, for example, any of the following substituents.

Me:':+-+ Rdan' Hydrogen 0H CI=C7 alkyl N +-12C5-CI
27 Reel N HR7C6-020 Alkaryl C6-C20 Aralkyl OR7CI-C7 Alkanoyl C2-C7 Alkenyl C2-C7 Alkynyl Where R7 is CI-07 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, C6- Alkaryl R and R2 of C2 may also be a neutral amino acid group or the remainder of a polypeptide chain having L20 carbon atoms.

These are shown below.

R R' GLN VANGLU
GLNGLY
LEUGLtJ-GLN TY
RGLY-GLU VAL-GLNGL
Y-GLU-GLN VAL-LEtJVAL-
D YR GLN-LEU GLN-TYR GLN=VΔL L-eu-TY+Comb EU-LEU TYI or-LE For example, a novel pentapeptide having the following formula has been provided.

In the above formula, R is hydrogen, (+-07 alkyl such as methyl, ethyl, 05-CI2 aryl such as phenyl, or CI-C7 alkanoyl such as acetyl, butionyl, etc.), and R' is OH , NHz.

NHR7 or N(R7)2. The most preferred polypeptides are those of the above formula in which R is hydrogen and R' is 1.

Also included within the scope of this invention are pharmacologically satisfactory salts of pentapeptides.

Acids that can form salts with pentapeptides include hydrochloric acid,
Inorganic acids such as hydrobromic acid, perchloric acid, nitric acid, diocyanic acid, sulfuric acid, phosphoric acid, formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid , anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid, etc.
I can do something.

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 Abbreviation Glycine G-LY Shiichi Glutamine Fl! 2 GLU glutamine GLNL L-arginine AR, GL-lysine YS L-aspartic acid A, 5P L-valine VAL cy-dyrosine °rYR Lo ysine EU active site of the polypeptide construct of the present invention is a five amino acid peptide, and this peptide is described in U.S. Patent Application No. 606
．． It has been found to exhibit properties similar to the 49-amino acid polypeptide isolated from bovine thymus as disclosed in US Pat. The peptides of the present invention are 1 ng
It is unique in that it can induce selective differentiation of Thy-1'' L cells (rather than OR'' BI3 cells) at a concentration of -10 tig/-yl. Here, T
hV-1 is a differentiation antigen (dif[ereMUIanion all
oantioen), and CR is not T cells but S cells.
It is a complement receptor present on S+ cells.

When these synthetic peptides were studied in vitro by inductive analysis, they were found to be similar to thymopoietin I [(T
It is found that it has IG specificity similar to that of hymopoietin (①). That is, these peptides differentiated Thy-1' cells and induced them to become Thy-1+1 cells, but did not differentiate OR-' cells into CR+1 cells. In vitro, it behaves similar to thymoboietin and produces intracellular cyclic muscle adenyl M (AMP).
Although many substances have been identified that are capable of inducing cell differentiation, most substances are inactive at such low concentrations and the peptides of the present invention do not exhibit TIIA cell differentiation versus cR"B1[ID2La differentiation. It should be noted that the synthetic peptides of the present invention also have the same effect on neuromuscular transmission as thymovoyedin. For example, 24 hours after a single injection of 10-100 μ9 per mouse,
Impaired neuromuscular transmission occurred, detectable by electromyography.

Because of the above-mentioned properties of the polypeptides of the present invention, these polypeptides are useful for therapeutic treatment of humans and animals. This is because polypeptides can MS differentiate lymphoid stem cells originating from hematopoietic tissues into thymus-derived cells, or TtIll 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, since the polypeptide compounds of the present invention can perform any one of the functions of the thymus, these compounds have applications in various thymic (multiple and immunological) fields. The field is Dige, a condition of congenital lack of thymus.
orge ) syndrome treatment. 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 at low temperatures, polypeptides can increase or promote therapeutic stimulation of cellular immunity and thus inhibit bacterial or mycoplasmal infections, tuberculosis. Polypeptides are effective in the treatment of chronic infectious diseases in living organisms such as leprosy, acute and chronic viral infectious diseases, and are effective in supporting the overall immunity of the body. I think that the. Furthermore, the polypeptide compounds are also believed to be useful in fields where 111 cell immunity is a problem, particularly in fields where there is a deficiency in immunity, such as in the aforementioned Gougeo syndrome group. In addition, when the production of antibodies becomes excessive due to an imbalance between (1) cells and B cells, the polypeptide compound can correct this excessive state by stimulating the production of T cells. Peptide compounds are effective in the treatment of certain autoimmune diseases in which 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 a variety of antibodies. The polypeptides are also effective in inhibiting the unfettered proliferation of thymoboietin-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. The polypeptides of the present invention affect neuromuscular transmission, and administration of the polypeptides to insects is effective in treating diseases associated with convulsive excessive neuromuscular transmission, for example.

Yet another important property of the polypeptides of the present invention is that they are highly active at very low concentrations of about 111 g/yf or more, and their viscosity reaches its maximum at concentrations of about 1000 g/yf or more. This means that As a carrier for this polypeptide, any known carrier that is commonly used for such purposes can be used, such as the usual "F1 (aqueous solution)", and in this case, protein dilutions such as serum albumin can be used. It is desirable to include an agent to prevent the polypeptide used at the above-mentioned low temperature from being adsorbed and lost to the glassware. The polypeptides of the present invention are active in a range of about 1 m9 or more per kilogram of body weight.

For the treatment of DiGeorge syndrome, polypeptides can be administered at a rate of approximately 1.0-0-1 O per 1 KO of body weight.J:
stomach. 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△
...erican Chemical 5ocietyJ
85. p, 2149-2154.1963.
It was synthesized using a concept similar to the yield method. The synthesis was performed by stepwise addition of several protected amino acids to a growing peptide chain that was covalently attached to a solid resin particle. In this synthesis method, the drug and by-products are 'a''
A and intermediate id crystals were removed. This method generally involves covalently bonding the first amino acid of the peptide chain to a solid polymer, and then sequentially adding amino acids one at a time.
This is a method in which a desired structural unit is constructed by adding types step by step. After 1 hour, the bead is separated from the solid support and the protecting groups are likewise removed. According to this method, the growing peptide chain is completely attached to the insoluble solid particles, making it difficult to filter and wash to remove drugs and nv1 organisms.

The amino acids can be attached to a suitable polymer, but in this case said polymer must be insoluble in the solvent used, and must also 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 bond the first protected amino acid. Various polymers can be used for this, such as cellulose, polyvinyl alcohol, polymethacrylates, sulfonated polystyrene, but in the synthesis of the present invention chloromethylated copolymers of styrene and divinylbenpine are used. was 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. Fluoresamine (“1 uo
The point of using (Fe1i
Analyt by x etal, Bioche
Polypeptides were synthesized by an improved solid-state synthesis method in 1973).

If the coupling did not indicate completion, the coupling was repeated using the same protected amino acid before α-amino deprotection.

A general method of construction is to first esterify an amino acid and a hydroxyl-protected mono-D-sine into a resin in anhydrous alcohol containing an aminoamine.

The bound amino acid resin was washed with 8 filtration, alcohol and then water, and dried. The protecting group attached to the amino group of the protected tyrosine (for example, t-benzyloxycarbonyl, abbreviated as t-BOC) was then 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 was bonded to the amino acid-resin by reacting with L-valine. Then the protected form of L-aspartic acid,
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. Other neutral amino acid residues at the end of the molecular chain
The addition can be carried out using known reactions.

A series of reactions for synthesizing an active 414 construct consisting of five amino acids can be performed as follows.

R3Ra α-BOC-As p-Val--ryr-resin↓
R3Ra excluding α-amino protecting group R2N-ASp-Va LTyr-resin ↓ α-B
OC-Lys-COOHR2R3Ra α-800-1j/5-Asp-Va I-Tyr-resin↓ Hz N Lys-ASI)-ValTyr-resin↓ excluding α-amino protecting group
a-AOC-Aro-COOH RI R2R3R4 α-AOC-ArGI-LVS-ASI)-VaI-T
Vr-resin ↓ HEN excluding all protective groups ArOLys-Asp-vat-Tyr-c
ool-1 In the above series of reactions, Ra, R2, R
3 and R4 are protecting groups for various reactive groups on the side chains of amino acids, and these protecting groups are not affected or removed when the growth reaction is interrupted, except for the α-amino protecting group. I don't do it. In the above pentapeptide resin intermediate, Ra is tosyl (Tos), R2 is benzyloxycarbonyl (Z), R3 is benzyl (Bzl),
RJ is O-2,6-dichlorobenzyl (Bzl (CI
2>) and α-Rs are preferably t-amyloxycarbonyl. The resin may be any of the aforementioned resins as long as it is effective in the method of the present invention.

The bond in the Pevdi resin is split and the peptide is attached to the resin at the same time as the protecting group R+. Rz. Rz. The final product, peptide, is also released from R+ and α-Rs. Protecting groups and resin separation are treated with anhydrous fluoride. 1751 15
! TZZ・2.

As described in No. 1/J, a method using 8 insoluble solid molecules is preferred for packaging the polypeptides of the present invention, but the polypeptides may be prepared using other methods of production. It is also possible to produce similar products. Other types of solid supports, such as N-methyl-benzhydrylamine resins or benzhydrylamine resins, can also be used and are advantageous for the production of polypeptides under certain conditions.

In this case, the C-terminal amino acid is attached directly to the resin, and the final peptide is split or cleaved from the resin substrate in II F to yield the C-terminal amino acid. The method using N-methyl-benzhydrylamine resin is "3i.

chemical and biophysical
Research Communications
, 48. 1 100-1105 (1972)
” is described by Monahan et al. The method of using benzhydrylamine resin is “J
Oural of lvl edicinal
Chemistry, 1973, vol.
, P. 545-549J by J.
, Rivier) et al. Derivatives of scale can be prepared from the basic form pentabebubu using known methods. To prepare the derivatives, it is necessary to block functional groups that might inhibit the reaction, resulting in -C of the desired product. For example, the α-carboxylic acid group of aspartonic acid or the α-amino group of ricin is
This is a group that should be L-blocked during the production process of the above derivative.

As mentioned above, in carrying out the method of the present invention, it is necessary to protect or monochain the amino group, which allows the reaction to be controlled and the desired product to be obtained. Suitable amine protecting groups that can be used include those capable of forming J crystals that protect strongly basic amino groups, or urethane protecting substituents such as benzyloxycarbonyl and t-butyloxycarbonyl.

In the case of amino acids in which the reaction is carried out at the carboxyl end of the molecule, it is preferable to use tert-butyloxycarbonyl or °C-amyloxycarbonyl to protect the α-amino group. After the reaction, these BOC and AOC protecting groups can be easily removed by the relatively mild action of an acid (for example, trifluoroacetic acid) in the next step (in which the α-amino-7 base itself reacts). can be excluded. This acid treatment has no particular effect on the protective groups used in the peptide chain. Therefore, the α-7 amino group is protected against subsequent reactions and the α-amino group is protected by reaction with any substance that can be removed after said reaction under conditions that do not specifically impart F g to the peptide molecule. This means that it is possible to do so. Examples of such substances are derivatives of specific carboxylic acids capable of acylating amino groups.

Generally, an amine group can be protected by reaction with a compound containing a group represented by the following formula.

I -0-C- In the above formula, R may be any group as long as it prevents the amine group from participating in the next horn bubbling reaction and can be removed without destroying the molecule. Such Ht R is a straight chain or branched alkyl which may be unsaturated, preferably one having L10 carbon atoms, aryl, preferably one having 6 carbon atoms.
-15, aralkyl, preferably carbon number 7-
18, cycloalkyl, preferably carbon number 5-8
heterocyclic groups such as alkaryl, preferably having 7 to 18 carbon atoms, or isonicodinyl. Aryl, aralkyl and alkaryl may be further substituted with one or more alkyl having about 4 carbon atoms. Preferred groups for R include tert-butyl, tert-amyl,
These are phenyltolyl, xylyl, and benzyl groups. Particularly preferred specific amine protecting groups include benzyloxycarbonyl, Cp in which one or more phenyl rings are present;
or [substituted benzyloxycarbonyl substituted with a halogen such as 3r, lower alkoxy such as 21-, me-L-xy, lower alkyl, tart-butyloxycarbonylmiloxycarbonyl, cyclohexyloxycarbonyl, vinyloxycarbonyl, Examples include adamantyloxycarbonyl and biphenylisopropoxycarbonyl. Other FUNs that can be used include isonicotinyloxycarbonyl, phthaloyl, o-tolylsulfonyl,
Formil et al.

According to the method of the invention, peptides are obtained by reacting free 1α-amino groups with compounds containing blocked α-amino groups. In order to carry out the coupling from the reaction sign, the carboxyl component, ie, the carboxyl group, of the compound that is receptive to the reaction is activated. As a result, the carboxyl group can react with the free amino group attached to the peptide chain. Activation can be achieved by changing the carboxyl group to a reactive group such as ester, anhydride, azide, or acid chloride. Anonymous contains an amino group and a carboxyl group, and during these reactions, usually one group participates in the reaction and the other is protected. Prior to coupling, the protecting group attached to the α- or terminal amino group of the peptide bound to the resin is
It is removed under conditions that do not substantially affect other protecting groups, such as those attached to the ε-amino group of the sine molecule.

A preferred method for carrying out this removal step is mild hydrolysis with M, such as by reaction with trifluoro-6 at air temperature.

A specific pentapeptide represented by the following formula is produced through the series of steps described above.

82N-Atma G-LYS-ASP-VAL-T Y
R-COOH This pentapeptide represents the basic active drawing unit of the polypeptide of the invention. Of course, the terminal amino acid groups of AGR and TYR in the second substituted pentapeptide may be further substituted as described above, but this substituted pentapeptide can be prepared by reacting this basic pentapeptide with an appropriate drug to produce the desired derivative. You can use it. These types of reactions such as acylation, esterification, amidation, etc. are well known reactions.

Additionally, other amino acids, ie, amino acid groups that do not affect the biochemical activity of the wood-shaped pentapeptide molecule, may be added to the peptide chain. This addition reaction can be carried out in the same continuous reaction as the polypeptide synthesis.

EXAMPLES The present invention will be described below with reference to Examples, but the present invention is not limited to these Examples. In the examples and the main text of Suishi Ma, unless there is a specific indication of 1, the part does not refer to the part II.

[Example 1] In order to synthesize the polypeptide of the present invention, the following materials were purchased.

α-AOC-NQ-Tos-L-arginine α-BO
C-2-Chlor-benzyloxycarbonyl-synthin α-BOC-
0-benzyl-mono-aspartate α-800-L-valine α-BOC-2.6-cyclolbenzyl-L-tyrosine In the above drug, BOC is t-butyloxycarbonyl and AOC is t-amyloxycarbonyl. , Tos represents Toshir, respectively. Sample mulberry, dicyclohexylcarposiimide, and fluorescein amine used for structure determination during sequential reactions of amino acids.

And the resin was sold by 4 people per 1 set. The resin used was a polystyrene divinylbenpine resin having a particle size of 200-400 mesh and containing 1% divinylbenzene and 0.75 mmol chloride/g resin.

When synthesizing the polypeptide, 2 mmol of α-BO
C-0-2.6-cyclobenzyl-tyrosine 1
8 in absolute alcohol containing mmol of triethylamine
The mixture was treated at 0° C. for 24 hours to esterify to 2 mmol of chlormethylated resin.

The resulting bound amino acid resin was filtered 8 times, washed with absolute alcohol and dried. Then α-130G or α-A
The OC amino acids were similarly sequentially coupled to the deprotected α-amino groups of the peptide resin using the appropriate dicyclohexylcarposiimide to form the bebudid resin of the present invention. After each conjugation reaction, nine replicates of the resin were tested with fluores7mine, and if positive fluorescence, the conjugation was assumed incomplete and the reaction was repeated with the same protected amino acid used. Ta. As a result of several coupling reactions, a pentapeptide-resin represented by the following formula was obtained.

TO3Z BZl In the above formula, AOC represents amyloxycarbonyl, Tos represents tosyl, Z represents benzyloxycarbonyl, 3zl represents benzyl, and BZI (C12) represents 0-2.8-dichlorobenzyl.

The resolution of this peptide-resin conjugate and the removal of the protecting groups were performed using a Ken resolution device (Peninsula L).
ab, lnc, 1fj) using anhydrous hydrogen fluoride for 60 minutes at O'C, in this case using 1.2xl of anisole per gram of Bevdid resin as scavenger.

The peptide mixture was lyophilized in vacuo, washed with anhydrous ether, and the peptides were extracted with glacial acetic acid and water. , 1N
Peptides were subjected to Cloa 1-Graph analysis on P-6 Biogel (13i0-Get) in acetic acid.

As a result, it was found that the purified peptide had a purity of 94% and was suitable for the following supplementary selection.

Example 2 To investigate the activity and properties of the polypeptide of Example 1, tests were conducted on healthy 1i1 5-6 week old nu7 nu mice of both sexes. The mouse has BALB/C backing (
Thymocytes displaying T11y-1,2 surface antigen) and maintained under normal conditions. Regarding antisera, anti-Thy-'1,2 sera were made in Thy-1 homologous mice.

For in vitro induction of differentiation of T by-1+ T cells or CR+8 cells, prothymocytes (P r
Komuro & Boys induced the differentiation of thymocytes from thymocytc.
e) “The method described by I-ancet, 1
, 740.19'73). This method uses the harvest of Thy-1, 2 as a guideline for 711]11a differentiation.

On the other hand, CR+ from CR-B1 cell precursor in a test tube
Induction of differentiation into B cells was carried out under the same conditions as for thymocytes, but in this case, 3J, IL, etc. were used for analysis using rabbit-tailed rabbit antibodies and non-degradable complement, which may aggregate to some extent. The ability of CR+8 cells to bind sheep red blood cells coated with A population of healthy nu/nu mouse spleen cells sorted on a discrete gradient of bovine blood sperm albumin was used as a source of both precursors (Thy-L and CR-). This is because the cell population contains few or all<"rhy-i cells and the number of CR cells is small.

As a result, this polypeptide exhibited a similar selectivity to that of thymovoietin (III) in inducing the differentiation of T lymphocytes rather than T lymphocytes, which are complement receptors (CR). This pentapeptide is I nu 10
Differentiation of Thy-1'' T cells was induced in the v:i degree range of t1g/xi. Differentiation of CRFF3.flu cells was not induced in the concentration range of O, O'1 ng/10 μg/11.

[Example 3] In order to examine the effect of the peptide of Example 1 on neuromuscular transmission, 10-100 μQ of the peptide was included in a normal salt solution. injected into the membrane.

24 hours later, l”1ancct J, Vol, 1
2. Neuromuscular transmission was investigated using electromyography according to the method described in J. O., 256-259.1975.

Neuromuscular disorders were observed in mice given peptides.
It was.

[Example 4] rJourn, Med, Che'm, Vol.
1'6°No, 5. p, 545-8 (1973)
The pentapeptide of Example 1 was prepared on a benzhydrylamine resin substrate using the method described by J. I built A. In this production, dicyclohexylcarposiimide was used as a force bubbling agent, and the coupling reaction was performed using CH2'''C'
(Carried out in 12. The obtained peptide was separated from the substrate using hydrogen fluoride to obtain the peptide)! ! ? After separation, the protecting group was removed to obtain the following centering body.Identification was performed by thin layer chromatography and electrophoresis, and the following results were obtained.

Thin layer chromatography sample: 30μg silica gel (Brinkmann glass plate, 5x'20cm,
Jj7 0.25mm) R>, n-butanol:pyridine:acetic acid (10A
c): Water 30:15:3:12 R: Ethyl acetate (EtOAc): Pyridine: Acetic acid: Water 5:5:1:3 Water 1:1:1:1 Spray reagent: Pauli assay, ninhydride 1 ), Sakaro reagent and I2 compound Δrg-Ly, 5-A5p-Val-Tyr-NHz descendant chroma L graph I1 1J proof 0.53 0.68 0.26 electrophoresis method electrophoresis method 3mm filter fi (11.5cmX56cm)
Sample: 100 μ9 pl-15,6, pyridine acetate L buffer solution.

1000V, 1 hour Spray Reagents: Ninhydrin and Sakaguchi test sample This amidated pentapebd is 89% towmy (7 Womey)
When used at a concentration of 1 μg/y1 in solution, it exhibited a maximum activity of 1'05% and a minimum activity of 70% compared to the basic pentapeptide of Example 1.

[Example 5] The basic pentapeptide obtained in Example 1 was esterified to produce an ethyl alcohol derivative (q). To produce the ester derivative, the M group of aspartic acid was replaced with a mild acid-sensitive blocking group. In this case, the t-butyl group is preferred as the protecting group.The protecting group for the α-amino group is one sensitive to mild bases, preferably a fluorine group. Nylmethoxycarbonyl group.If such α-amino protecting group is used, the effect on the acid-sensitive protecting group of aspartic acid can be removed by adding each amino acid residue.Protected pentapeptide resin After formation, the two protecting groups are removed by treatment with a mild base followed by a mild acid.The pentapeptide is cleaved from the resin by transesterification with ethyl formate using sulfuryl chloride as a catalyst. Separation selectively yields the C-terminal ester.Esterification does not occur with the free acid group of the aspartic acid residue.All remaining protecting groups are removed, and the peptide of the following formula is recovered.

(Example 7) In this example, r Biocbc + n1cal an
d 31ophysical Re5carcl+
Communications vol, 4 F3
.

1100-1105 (1972)
The pentapeptide of Example 1 was prepared using N-methyl-benzhydrylamine resin as a solid support according to the method of Onahan et al. I built A. The protected pentapeptide was separated from the resin using hydrogen fluoride to yield the amine-substituted irN dJ form 111 below.

Example 8 In this example, instead of the protected L-arginine,
The basic form of pentapebutide of Example 1 was prepared using the method of Example 1 except that N-methyl-arginine of
-Methyl-substituted rA FN form was synthesized. 45f of the peptide of the following formula was obtained.

Example 9 A metal amine polypeptide similar to that obtained in Example 8 was synthesized using benzylhydrylamine resin as the solid resin in the same manner as described in Example 4. The pentapeptide indicated on the resin was separated from the resin with hydrogen fluoride and all protecting groups were removed to yield an amide polypeptide of the formula below.

[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.

[Example 12-21] Using the reaction techniques described above for lengthening polypeptides, the following various polypeptides were obtained. The terminal amino group and carboxyl group are each substituted with R and R', and the basic amino acid has the following formula.

Here, R and R' are the amino acids or OH shown in the table below.

The polypeptide derivatives obtained in Examples 5-23 retain biochemical activities similar to those described herein for the basic amino acid structural unit.

The present invention provides novel polypeptides as described above, and this polypeptide is a complement receptor (CR").
It is effective in many therapeutic fields because it can induce differentiation of T lymphocytes rather than B lymphocytes. Further provided by the present invention are novel intermediate polypeptides, methods for producing the peptides, therapeutic compositions and uses of the compositions, derivatives of the above pentapeptides. Although the present invention has been described with respect to several preferred embodiments, it is of course not limited to only these embodiments.

Claims (5)

[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 the α-amino protected form. L-valine is bonded to L-tyrosine-resin by reaction with L-valine, the α-amino protecting group is removed from the L-valine moiety, and L-asparagine is obtained by reaction with α-amino protected L-aspartic acid. The acid is combined with L-valine-L-tyrosine-resin and the α-amino protecting group is removed from the L-aspartic acid moiety to form the α-amino protected form L-
React with lysine to convert L-lysine to L-aspartic acid L
-valine-L-tyrosine- is bonded to the resin, and the α-amino protecting group is removed from the L-lysine moiety to form the α-amino protected L-
React with arginine to convert L-arginine to L-lysine.
A method for producing a polypeptide represented by the following formula and its salts, which comprises binding L-aspartic acid-L-valine-L-tyrosine to a resin, and then removing all protecting groups from the peptide, the method comprising: NH-ARG-LY
S-ASP-VALTYR-COR' In the above formula, R and R' are terminal groups of the polypeptide that do not substantially affect the biochemical ability of the polypeptide, and R and R' are the following groups: It is selected from the group consisting of ¥R¥ ¥R¥ Hydrogen OH Alkyl of C_1-C_7 NH_2 Aryl of C_5-C_1_2 NHR_7C_6-C
Alkaril of _2_0 N(R_7)_2C_6-C
Aralkyl of _2_0 OR_7C_1-C_7 alkanoyl VAL C_2-C_7 alkenyl GLN C_2-C_7 alkynyl LEU GLN TYR GLU VAL-GLN GLY VAL-LEU GLU-GLN VAL-TYR GLY-G1-N GLN- LEU GLY-GLU GLN-TYR GLY-GLU-GLU GLN-VAL LEU-TYR LEU-LEU TYR_-LEU VAL-GLN-LEU VAL-GLN-LEU-TYR_ Here, R_7 is the alkyl of C_1-C_7, C_2-
C_7 alkenyl, C_2-C_7 alkynyl, C
Aralkyl of _6-C_2_0, or C_6-C_2
A method for producing a pentapeptide composition, characterized in that it is an alkaryl of _0. (2) The method according to claim 1, wherein the reactive side chains of the amino acids to be reacted are protected during the reaction period. (3) The method according to claim 1, wherein the polymer resin is a resin selected from the group consisting of cellulose, polyvinyl alcohol, polymethacrylate, sulfonated polystyrene, and chloromethylated polymers of styrene and divinylbenzene. . (4) An intermediate polypeptide having the following formula, ▲There are mathematical formulas, chemical formulas, tables, etc.▼ In the above formula, R_1, R_2, R_3, R_4 and R_5 are protecting groups, and R_1, R_2, R_3 and R_4 is a protecting group carried on a reactive side chain, R_5 is a protecting group carried on an amino acid, R_5 is R_1, R
It can be removed under conditions that do not affect the _2, R_3 and R_4 groups. and an intermediate polypeptide characterized in that the resin is an insoluble polymeric substance having a stable physical shape and covalently bonded to adjacent amino acids. (5) R_1 is tosyl, R_2 is ε-2-chlorobenzyloxycarbonyl, R_3 is benzyl, R_4 is O-
5. The intermediate polypeptide according to claim 4, wherein 2,6-dichlorobenzyl, R_5 is amyloxycarbonyl.