DD133231B1 - PROCESS FOR THE PRODUCTION OF PEPTIDES, PROTEINS AND PROTEIN SEQUENCES - Google Patents

Description

Procedure_for_the_product-2_УОП_Ре £ ^ д.аеп ^ _Рго ^ е1pеп_ипа._Рго ^ е1п-ee ^ uenzen

Scope of the invention

Chemical production of peptides and proteins, in particular of peptide hormones, by stepwise, repetitive extension with the corresponding protected amino acids or peptide fragments.

Characteristic of the known technical solutions

The methods for peptide synthesis described so far are as follows:

a) so-called conventional methods (E. WÜNSCH: Angew Chem, 85, 773 (1971):

All reactants are in the liquid phase, with the P.einisolierung taking place at each coupling stage of the bugged synthesis product difficulties.

b) solid support peptide syntheses (RB MERRIFIELD: Adv., Enzymol., 32, 221 (1969); RB IIERRIPIELD: J. Amer., Chem. Soc., 85, 2145 (1963); RB MERRIPIELD: Biochemistry, 3, 1385 (1964). JM STEWART and JD YOUNG: Solid Phase Peptide Synthesis Freeman, San Francisco (1969); G. LOSSE and K. NEUBERT: Z. Chem. , 48 (1970); G. LOSSE and R. ULBRICH: V Z. Techn. Univers. Dresden 22, 263 (1973); E. SCHRÖDER and K. LÜBKE: The peptides I, Academic Press, 1965) Stepwise peptide construction on a high molecular weight carrier, the transient peptide chain always on a polymeric

Carrier remains tied. This facilitates removal of the excess protected amino acids or peptide fragments or reagents used; However, the separation of the unreacted during a coupling peptides (trunk sequences) or formed Pehlsequenzen can only be done after completion of the synthesis, after detachment from the carrier. Isolating the desired product from the resulting peptide mixture is often a major problem.

c) Polymer Reagent Method (M.PRIDKIN et al .: J.Amer.Chem.Soc.90, 2953 (1968); Th. V / IELMD and Ch. BIRR: Chimia 22, 581 (1967); G LIMECKE and E. HAAKE: Naturwissenschaften 55, 343 (1968):

The use of an activating reagent bound to a polymer also facilitates the removal of the excess reactants. In contrast to the process mentioned under b), it is possible to separate off unreacted peptide at each coupling stage as in the conventional method mentioned under a). In this method, the growing peptide chain is not bound to the polymeric support.

d) The peptide product composition is significantly influenced by the properties of the carrier material. That Crosslinking, swellability, permeability to diffusing molecules, type and location of the anchor groups within the polymer as well as the mechanical properties (mechanical stability, pileability, grain size) play an essential role.

As the solid insoluble copolymer common for use in the peptide synthesis, the polystyrene resin proposed by LlERRIPIELD serves with 1 to 2 % divinylbenzene crosslinking and a grain size of 20 to 80 μm, which has a gel-like character. These are substituted with about 0.6 to 2.0 milliequivalents of chloromethyl groups or other groups per gram of resin to attach the growing peptide chain.

As a result of the unsatisfactory effect of the mentioned support structure on the course of the peptide syntheses carried out therewith, numerous variants of this basic type have been proposed in order to improve the physical or chemical properties of the support.

Thus, on the basis of polystyrene-divinylbenzene, also popcorn polymers (G. LOSSE and K. NEUBERT: Z. Chem. 22 »48 (197O)), shell resins (A. LOSSE: Tetrahedron Letters 1971, 4989; BAYER et al .: J. Jimer Chem. Soc. 92, 1735 (1970); DE-OS 2 109 027) and macroporous (macroreticular) polymers (M. TILAK and C. HOLLINDEH: Org. Prep, and Proceed. 3, 183 (1971); USP 1 950 969; S. SANO et al., Biochim., Biophys., Acta 1221, ² 44).

Of these, the macroreticular polymers have a larger surface area and easier accessibility of their pore framework to larger molecules, allowing for rapid in-diffusion of the reactants without the need to loosen the polymer structure beforehand, as in the case of gelatinous resins. This should result in an independence of the pore structure of solvent effects and thus a possibly more favorable synthesis process.

Comparative testing shows that once the diffusion of the reaction components in the solid phase has no essential importance and that, on the other hand, these polymers are indeed suitable for peptide syntheses, but can not reach the performance of the Merrifield polystyrene resins.

Conducting peptide synthesis on the solid support is inherently linked to a series of demands on the support material and the course of the synthesis itself, as successful synthesis can only be achieved if each reaction step is quantitative and selective. These conditions are in practice

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only partially realizable. Therefore, the pest phase method, despite its undisputed number of advantages, has certain disadvantages in terms of the performance of the synthesis, and in particular of the final product composition. These are caused by chemical conditions, such as incomplete reactions and side reactions, and by physical conditions, due to their carrier properties.

D _a s aim of the invention is to reduce the disadvantages of the known support materials such as rigidity of the backbone of the makroreticularen resins unfavorable behavior of gel polymers and the strong influence of solvent effects and thus to achieve a better course of the peptide synthesis.

Task:

The object of the present invention is to reduce the disadvantages of the known carrier materials used for peptide synthesis on the polymeric carrier in such a way that a better course of the peptide synthesis is achieved and thus the respective peptide is obtained in higher purity and in greater yield can / can ground.

Merkmale_der_Erfindung

According to the invention, polymeric carriers are used whose backbone consists of cross-linked polyvinyl hydrocarbons and a heterogeneously expanded G _e lstruktur has latent porosity, so that the to be established on the polymeric support polypeptides sterically can not interfere with and occur any unwanted reactions between the peptide chains. To form the active sites in the polymer, the copolymer is swollen in a solvent and chloromethylated with monochlorodimethyl ether or other suitable methods for creating active sites in the polymer are used (eg with benzhydrylamine).

The polymeric bodies thus prepared are linked to a protected amino acid and form the basis for the construction of polypeptides,

The synthesis of these polymeric support materials is carried out by copolymerization of ИопоѵіпуIverbindungen and a small proportion of PolyvinyIverbindungen in the simultaneous presence of not participating in the polymerization reaction substances, wherein the polymerization is carried out so that it comes to forming a heterogeneously expanded gel structure with latent porosity. The composition of the monomers and the additives is chosen so that a relatively heterogeneous but weitmaschiges polymer structure is formed. These resins, in the unswollen state, have no surface measurable by conventional methods and no macroporous structure. On the other hand, the swelling behavior and in particular the respiratory difference compared to the known gel resins is greatly changed, so that unfavorable effects on the diffusion and reaction behavior in the peptide synthesis can be avoided.

The preparation of the polymeric carrier is preferably carried out by the method of suspension polymerization, ie an oily polymerizable phase is distributed in an aqueous phase by stirring or shaking to small spherical droplets and initiated by 7ärmezufuhr the polymerization. During the polymerization, the droplets are solidified and small beads are formed. The diameter of the beads can be varied and depends on the stirring speed and other parameters. It is possible to use polymers with a diameter of from 10 to 1000 ₍ μ), preferably choosing a diameter of the balls between 40 and 300 μ .

The oily phase consists of a mixture of monomers, inert agents and polymerization initiators. The selection of the monomers must be such that the necessary active centers for binding the amino acids are already present in the matrix which forms or can be introduced by polymer-analogous reactions. Such

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Monomers are for example:! Compounds Halogenalkylviny such as chloromethyl styrene, vinyl aromatics such as styrene, vinyltoluene, Vinyläthylbenzol, vinyl benzophenone, Viny benzyl alcohol, acrylic and Ally compounds such as methyl methacrylate and allyl chloride, B _e i The monomers may therefore be one or Several of the alkenyl compounds which are copolymerized with a suitable crosslinking agent. Such are divinylbenzene, trivinylbenzene, glycol dimethacrylate, Divinyloxyäthan, butadiene, etc.

In order for the copolymer to obtain the steric structure which is suitable for the peptide synthesis, certain proportions of inert substances are added to the monomer mixture which themselves do not participate in the polymerization reaction but lead to an optimal matrix by interaction with the polymer chains. Such inert agents are substances which are miscible with the monomers, but the polymers swell only partially. As a result, partial separation of already formed polymers and inert agents occurs during the polymerization process, resulting in the formation of the heterogeneous structure. Aliphatic hydrocarbons are rbindungen for the copolymerization of vinylaromatic V _e is preferably used with a C-number of Cg to C .. as inert agent. For the copolymerization of AcryIverbindungen preferably aromatic hydrocarbons such as benzene, toluene, etc. are used. Of particular importance for the synthesis of the resins of the invention is the choice of the proportions of monomers, crosslinkers and inert agents. In this case, it should be avoided that the proportions used lead to the formation of macroreticular (porous) structures (DE-OS 1 950 969). A suitable range for producing polymeric materials according to the invention is 0.1 to 3% by weight, preferably 0 , 5 to 2 Ge \ v .-% crosslinker, for example divinylbenzene and at an Inertmittelanteil of 20 to 50 ^ S. The resulting resins are transparent and contain the inert medium in heterogeneous distribution. The removal takes place before or during the subsequent polymer-analogous reaction by extraction bzv /. Distillation.

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When using Halogenalky! Vinylaromaten as a monomer, the resins thus prepared without further. Treatment for peptide synthesis can be used. Polymers which have no reactive centers for binding with protected amino acids are prepared by methods known per se into the corresponding chloromethyl, oa, hydroxymethyl (M. BODANSZKY et al .: Chem. Ind. (London) 38, 1597 (1966 ), or benzhydrylamine derivatives (P. RIVAILLE et al .: HeIv.Chin Acta 54, 296 (1971); P. PIETTA et al .: Gazz., Chim. Italians 103, 483 (1973); P. PIETTA Chem. V. HRUBY et al .: J. Med Vb ₉ 624 (1973); Ind M. Bodanszky et al .: Chem (London et al .: J. Org Chem 39, 44 (1974)..... ) 38, 1597 (1966)).

Surprisingly, the support materials prepared in this way show substantially better properties with regard to the use for peptide synthesis than the hitherto known resins. Thus, in contrast to the macroreticular (porous) carriers, they have no porosity and inner surface. Furthermore, the resin framework of the new support materials, also in contrast to the mentioned macroreticular supports, a high flexibility and mobility and a lower cross-linking, which have an advantageous effect on the synthesis of peptides, preferably on the synthesis of very long-chain polypeptides.

Opposite the Gelpoylmeren, v / ie they z. B. proposed by 1! ERRIPIELD and are commonly used for peptide synthesis, the new resins are characterized by a smaller volume difference between swelling and non-diluting solvents and by improved kinetics, properties that prove to be carried out in the peptide synthesis very favorable because this once the solvent effects are reduced and on the other reactants can react better. As a consequence of the improved properties of the novel support materials, the peptide syntheses of various peptide syntheses result in a substantially higher purity and greater yield, based on the amount of polymer used,

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incurred when using previously known types of carriers

 In conjunction with the higher purity peptide Ausbeuto simultaneously decreases the number of lifting components, so that the cleaning effort decreases considerably.

For the preparation of peptides by the method according to the invention, the amino acid chain can be built up either stepwise from the various amino acid derivatives or using peptide fragments according to known methods. The peptide construction begins by binding the C-terminal amino acid to the reactive centers of the support material using known methods. Instead of the C-terminal amino acid and a peptide, for example, can be bound to the support a di- or tripeptide in the same Meise, whereupon the desired amino acid chain with amino acids or peptides, respectively, and is completed. The linking of the amino acids and / or peptide fragments is carried out by reacting an amino acid or a peptide having a protected cv-amino group and activated terminal carboxyl group with an amino acid bound to the carrier or a peptide having a free III-amino group.

The carboxyl group can be converted, for example, by conversion into an activated ester, such as p-nitrophenyl, N-hydroxysuccinimide, 2,4, 5-trichlorophenyl, pentachlorophenyl, 8-hydroxyquinoline (optionally with the addition of further activating additives) Acid anhydride, azide, imidazolide or by reaction by means of a carbodiimide (optionally with the addition of further activating additives), or Ν, Ν-carbonyldiimidazoles, activated.

Functionally free groups not involved in the reaction are suitably protected, in particular by means of radicals which are readily cleavable by hydrolysis or reduction or liquid hydrogen fluoride, using the groups known for peptide synthesis, e.g. the benzyl, p-chlorobenzyl, 2,6-dichlorobenzyl, p-nitrobenzyl, p-Lethoxybenzyl, benzhydryl, tertiary-butyl, tosyl, dinitrophenyl, nitro group.

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Examples of amino-protecting groups which may be mentioned are aliphatic, oxy-carbonyl groups, such as, for example, cyclohexyloxycarbonyl, adamantyloxycarbonyl and, in the first place, tert-butyl-oxycarbonyl- (B04-), or carbonic or thiocarbonic acid-derived groups, as appropriate aromatic radical-substituted carbobenzoxy groups (for example carbobenzoxy, p-bromo or p-chlorocarbobenzoxy, p-lethoxycarbobenzoxy), furthermore o-nitrophenylsulfenyl and optionally substituted aralkyl groups (diphenylmethyl or triphenylmethyl groups, by way of example)

Trifluoroacetic acid / dichloromethane, trifluoroacetic acid alone, HCl in glacial acetic acid or dioxane are preferred for cleavage of the ε-amino protecting groups. Other cleavage reagents and their cleavage conditions for the removal of specific amino-protecting groups are described in E. SCHRÖDER et al. K. LÜBKE: The peptides I, Academic Press, 1965, pages 72-75.

After the desired peptide has been built, the bond with the polymeric support material in a known manner, such as hydrogen bromide / glacial acetic acid, ammonolysis, alcoholysis, Hydrazino Iyse, but preferably with the aid of liquefied hydrogen fluoride at 0 C to room temperature, optionally with the addition of z. As anisole, Llercacptoäthanol be split.

When chloromethyl polymers are used, a C-terminal carboxyl function is obtained on detachment of the peptide from the carrier by means of liquid hydrogen fluoride, and a C-terminal amide function in the case of benzhydrylamine polymers. The crude peptide thus obtained is purified after liberation of remaining protective groups by using suitable purification methods (column chromatography and other purification operations such as reprecipitation and the like).

The inventive method will be explained in the following embodiments, without the invention being limited to these examples.

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^e · * · ^s 2 ^ ^e ^ - ^e

1. Synthesis of the polymeric backbone

In a 2 liter flat-bottomed beaker equipped with a reflux condenser and a heat control brush, a mixture of 313.7 g of styrene, 12.7 g of divinylbenzene of composition 51.2 ^c / o divinylbenzene and 48.8 % of ethylstyrene, 175 g Parex-Benain and 1.5 g of azodiisobutyric acid dinitrile in 1000 ml of Y / asoer, to which 5 g of Mg (OH) ₂ and 0.2 g of sapol were added, suspended by stirring. The stirring speed is adjusted so that the forming spherical droplets have a mean diameter of 60., U. The temperature is then increased to 70 C and kept constant until the gel point is over (about 5 hours), then heated to 95 ⁰ C and postpolymerized for 4 hours. The obtained polymer beads are filtered, washed free of 0.5 percent strength, sulfuric acid from the suspension stabilizer adhering and removing the Inertmittels treated with steam "Finally, the product at 100 ⁰ C is dried. Optionally, the polymer can be screened in the boundaries between 40 μ and 100 μ . It is in the form of gel-like transparent spheres of high strength. With the known methods, neither a measurable inner surface nor a pore volume can be detected. The advantageous structural properties of the polymers show up in a high swelling rate and a toluene swelling of 800 ml / 100 g of polymer. The yield is 305 g of resin.

2 «Chloromethylation of the polymeric backbone

a) In a heatable stirred vessel with reflux condenser, 100 g of polymer are swollen in 900 ml of dichloromethane for two hours. To this mixture are added 35 g monochlorodimethyl ether and 5 g tin IV chloride and heated to 40 C with stirring. At this temperature is chloromethylated for 3 hours. The resin is then filtered off from the liquid phase and on the filter first twice

200 ml of dioxane and then washed three times with 250 ml of methanol. The last running ethanol is free of chloride. Thereafter, the adhering methanol is suctioned off and dried in air for 25 hours. This gives 120 g of a chloromethylated styrene-divinylbenzene copolymer having a chlorine content of 5.5 % chlorine.

If necessary, the degree of chloromethylation can be controlled by varying the amount of chloro ether, reaction temperature, reaction time and amount of catalyst within the limits of 1 % to 23 % chlorine.

100 g of polymer are swollen at room temperature in 4-00 ml of anhydrous dichloromethane with stirring for one hour, then a mixture of 9.0 ml of tin IV is added at 18 C (internal temperature) within 12 minutes. chloride, 40 ml of chloromethyl methyl ether and 200 ml of anhydrous dichloromethane, and the mixture is stirred at 18 C (internal temperature) for one hour. / In is quickly sucked off and washed with small portions:

600 ml Dioxane - water 3: 1 600 ml Dioxane - 3N HCl 3: 1 360 ml Dioxane - water 1: 1 360 ml Dioxane - 7 / asser 1: 3 600 ml water 360 ml Dioxane - water 1: 3 360 ml Dioxane - water • 1: 1 360 ml Dioxane - water 3: 1 600 ml dioxane 360 ml Dioxane - methanol 3: 1 360 ml Dioxane - methanol 1: 1 360 ml Dioxane - !! ethanol 1: 3 $ 00 ml methanol

Instead of dioxane, acetone can also be used. The further work-up is described under a).

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3. Binding of the C-terminal amino acid or the corresponding di- or tripeptide to the reactive centers of the resin

a) 5 grams of chlorate methylate, prepared as described in Example 2, with a chlorine content of 1.5 milliequivalents per gram are added to a solution of 7 »5 milliequivalent Boc ~ alanine (or another H 'protected amino acid or II' - Protected di- or tripeptide) and 7.0 milliequiv alent triethylamine (or: diisopropylamine, diisopropylethylamine, cesium carbonate, etc.) in 20 ml of absolute ethanol after a ten-minute exposure time. Now the mixture is heated under reflux and shaking or vibrating with a Vibromischer to 75 ⁰ C for 24 hours. After cooling, it is necessary to suck off over a G3 glass filter frit and to wash the resin until the filtrate has no chloride, with 15 ml portions of the following solvents:

three times methanol, twice water, twice methanol, three times water, once methanol, twice water, twice methanol, twice water, five times methanol.

The product thus obtained is first dried in air, then over phosphorus pentoxide. The determination of the residual chlorine content of the polymer can be concluded to a Boc-alanine content of about 0.7 milliequivalent per gram of resin,! Lach cleavage of the Boc group using trifluoroacetic acid / dichloromethane (1: 1), as described in Example 4, after Reaction with pyridine hydrochloride, the content of free amino group according to DORIiAH (LC DORMAH: Tetrahedron Letters, 1,969, 2319) titrated. Thereafter, 0.57 milliequivalent Boc-alanine was esterified per gram of resin.

b) б gram of benzhydrylamine polymer prepared as described in (P. RIVAILLE et al .: HeIv.Chim.Acta 54, 296 (1971); P. PIETTA et al .: Gazz. Chim. Italians 103, 483 (1973);

P. PIETTA et al .: J. Org. Chem. 39, 44 (1974); HRUBT et al .: J. Med. Chem. 16, 624 (1973)), with a

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Reactive amino group content of 1 milliequivalent per gram is converted into a reaction vessel suitable for peptide synthesis (see, for example, JM STEWART and JD YOUNG: Solid Phase Peptide Synthesis Freeman, San Francisco (1969); E, SCHRODER and K. LUKE: The peptides I, Academic Press, 1965). The mechanical mixing of the reactants is carried out by means of a vibromixer. The amount of solvent and reagent is 60 milliliters.

After a three-time pre-wash with dichloromethane, a solution of 30 mmol Boc-glycine in dichloromethane is added, which was reacted with 15 mmol dicyclohexylcarbodiimide for one hour and freed from the excreted dicyclohesylurea and allowed to act the thus pre-activated amino acid for 60 minutes. After filtration, the resin is washed; 3 times 3 minutes each with: dichloromethane

Chloroform-methanol (2: 1)

dichloromethane,

Exposure of a pre-activated with 7 »5 mmol dicyclohexylcarbodiimide solution of 15 mmol Boc-glycine in dichloromethane (see above), repetition of the aforementioned Y / aschvorganges and a third reaction with a preactivated solution of 7.0 mmol Boc-glycine and 3.5 mmol dicyclohexylcarbodiimide , After carrying out the above-mentioned .Yaschvorganges a reaction with a mixture of 6 ml of acetic anhydride, 3 ml of pyridine and 31 ml of dichloromethane for 30 minutes to acetylate any remaining reactive amino groups of the carrier and thus to block for subsequent undesirable reactions. After a six-fold washing with dichloromethane, a reaction according to DORI-IAl · is carried out to determine the presence of basic groups. carried out with pyridine hydrochloride with subsequent titrimetric determination of the chloride content as triethylammonium chloride. This process, the subsequent Boc-Schutzgruppenabspaltung with determination of the bound to the carrier Boc-glycine amount is the S _c hema in Example 4 refer.

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- H

4. Scheme of Peptide Construction

No solvent or reagent

Einwir- number of kung (min)

60 1 3 3 3 3 3 3

1 dichloromethane

2 conversion with preactivated Boc-amino acid in dichloromethane-dimethylformamide

3 dichloromethane than Dime thy! Formamide

4 chloroform-ethanol (2: 1)

5 dichloromethane

6 trifluoroacetic acid-dichloromethane

(1: 1.2)

7 trifluoroacetic acid-dichloromethane

(1: 1.2)

8 trifluoroacetic acid-dichloromethane

(1: 1.2)

9 dichloromethane

10 chloroform

11 triethylamine-chloroform (1: 9)

12 chloroform

13 dichloromethane

14 0.3M pyridine.HCl in dichloromethane

15 dichloromethane

16 chloroform-methanol (2: 1)

17 chloroform

18 testing for chloride freedom

19 triethylamine-chloroform (1: 9)

20 chloroform

21 dichloromethane

20

20 3 3 5 3 3 5 3 3 3

5 3 3

Explanations of the peptide structure

The mechanical mixing of the reactants is generally carried out by rotation of the reaction vessel about its horizontal axis. It has now been found that it is much cheaper to carry out the mixing by means of vibromixing, since hereby the structure of the reaction vessel with its inlets and outlets can be carried out stationary. Furthermore, it turns out to be particularly advantageous if, instead of the usual alternating use of swelling and non-swelling solvents when using dec new carrier material in Peptidaufbau mainly only swelling v / irkende solvent (preferably: dichloromethane, dimethylformamide, chloroform) unf and intermediate chloroform-methanol Mixture »Approximately 20 milliliters of solvent or reagent are used per milliequivalent of reactive amino groups» Dicyclohexylcarbodiimide is used as a 1 molar solution in dichloromethane.

In the direct use of dicyclohexylcarbodiimide as the activation component is Ur. 2 of the scheme, instead of adding "pre-activated Вос-amino acid", add the solution of 3 milliequivalents of Boc-amino acid, which acts on the carrier for 3 minutes before adding the solution of 3 milliequivalents of dicyclohexylcarbodiimide. The reaction time is 60 to 180 minutes.

The "pre-activation" itself requires the one-hour reaction of б milliequivalents of protected amino acid with 3 equivalents of dicyclohexylcarbodiimide to be carried out separately and separation of the urea formed. When activated esters are used in Hr. 2 in the same manner as the "preactivated-amino acid" use, at reaction times of 180 to 300 minutes. All reactions may optionally be carried out with the addition of further activating substances. The course of the coupling of the amino acid and / or peptide fragments is determined by determining the unreacted free

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Follows amino groups, wherein preferably the determination method according to DORIiM (L.C. DORLIAN: Tetrahedron Letters, 2969, 2319) by means of pyridine hydrochloride application finds. For this purpose, steps no. 6 Ьіз no. 13 are omitted and the titration proceeds to steps no. 19 to no. 21. In case of incomplete reaction, the coupling process is repeated until the determination of the free amino groups gives a constant value. Only then takes place splitting off of the H ^ protective group, determination of the total content of free amino groups and coupling of the next amino acid or peptide fragment, wherein the steps listed in the scheme are used.

For example, to produce the luteinizing hormone releasing factor (LH-RH), tert-Boc amino acid derivatives are used for the peptide-forming reaction in the following order, and correspondingly prepared peptide fragments can be used:

N 1 -Boc-glycine, N 1 -Boc-L-proline, N 1 -Boc-N 1-nitro-tosyl) -L-arginine, N 2 -Boc-L-leucine, N ⁰ 1 -BoC-Gliquin, N ^ -Boc-O-BenzyKoder O-dichlorobenzyl) ~ L-tyrosine, N ⁰ ^ -. 0-benzyl (or O-tert-butyl) -L-serine, N ^ -Boc-L-tryptophan, N * -Boc -N ⁱⁿ -tosyl (or dinitrophenyl) -L-histidine, N ⁶ ^ -ZL-pyroglutamic acid (or free pyroglutamic acid). In the same way as the peptide structure is carried out on a benzhydrylamine resin, this can also be carried out on a chloromethylated carrier, wherein z. B. when LH-RH Boc-glycine according to Example 3a is bound to the carrier. The Boc-alanine bound to the carrier according to Example 3a is used, for example, to construct the insulin B-chain sequence B 24 B 30, using tert-Boc-amino acid derivatives or corresponding peptide fragments for the peptide-forming reaction in the following order: N * -Boc-L-alanine, K * -Boc-N ^ -Z- (or cyclohexyloxycarbonylo) - L-lysine, N * -Boc-L-proline, N * -Boc-O-benzyl- L-threonine, N ** · Boc-O-BenzyKoder O-dichlorobenzyl) -L-tyrosine, N ⁰⁶ -Boc-L-phenylalanine, N ** " -Boc-L-phenylalanine.

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This peptide construction may optionally be continued with the appropriate amino acids or peptide fragments until the insulin B chain has been formed. In the same way one synthesizes the insulin A chain or fragments of the chain, or biologically active peptides, such as thyrotropes releasing hormone (TRH), somatostatin, enkephalins

5. Peptide cleavage from the polymeric carrier

a) by means of hydrogen fluoride

The peptide-resin constructed according to the scheme should be thoroughly washed and dried. Approximately 2 grams of peptide polymer are treated with about 10 milliliters of redistilled, anhydrous hydrogen fluoride in the presence of redistilled anisole (about 0.3 to 1.5 milliliters) or mercaptoethanol for 30 to 60 minutes at 0 ^° C Peptide is removed from the remaining protecting groups and the resin.

Hydrogen fluoride and volatiles are removed by imposing high vacuum, the residue is treated with ether (to remove anisole and the like), the peptide is dissolved in 2 percent strength. Acetic acid and filtered from the residual polymer. The combined acetic acid solutions give after lyophilization a solid product consisting of the formed peptide. Optionally, the resulting peptide is still purified by use of suitable methods.

b) by means of ammonolysis

When the construction of a peptide containing a C-terminal acid amide group (e.g., LH-RH) on a chloromethylated polymer is carried out, the peptide detachment from the carrier is carried out by means of ammonolysis.

For this purpose, the peptide polymer, z. B. 6 grams, in a pressure bottle, adds 5 to 10 milliliters of dichlorides than and optionally catalytic amounts of a reaction accelerator, such as diethylene glycol, then 500 milliliters at -3 ⁰ C saturated with ammonia methanol.

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After the mixture has been at room temperature for about 24 to 72 hours, the excess ammonia is removed by applying a vacuum, aspirated through a glass filter frit, the residual resin thoroughly washed with methanol and the combined filtrates concentrated iV at 35 ^° C. To remove the scavenger groups _{, the} solid residue dried over P.0 _{o is} treated with liquid hydrogen fluoride (about 15 to 20 milliliters, addition of 0.3 milliliters of anisole) at 0 ^° C. for 60 minutes. Hydrogen fluoride is removed by applying pump vacuum, the residue is extracted with ether to give anisole and the like. Ä., Dissolves the peptide in 2 percent. Acetic acid, filtered from the residual polymer and the combined filtrates lyophilized. D _a s thus obtained, freed of all the protecting groups peptide is optionally purify.

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Claims (7)

Invention '- A process for the preparation of peptides, proteins, protein sequences with high purity and yield from the corresponding amino acid or peptide fragment derivatives by stepwise peptide construction on a polymeric support, characterized in that as a solid support active groups, heterogeneously expanded copolymers with latent porosity on the Basis of Mono- and Polyviny! Connections. 2. The method according to item 1, characterized in that the heterogeneously expanded gel structure with latent porosity of the polymeric support is achieved by polymerization of the monomers in the presence of inert agents. 3 «method according to item 1, characterized in that are used as MonovinyIverbindungen styrene or Acry! Acid derivatives. Method according to items 1 and 2, characterized in that 0.1 to 3% by weight, preferably 0.5 to 2% by weight, of polyvinyl compounds, such as divinylbenzene, trivinylbenzene or glycol diacrylates, are used. 5. The method according to item 1 to 3, characterized in that preferably 20 to 50 weight percent aliphatic hydrocarbons of the fractions Cg bio Cj. be used in styrene polymers, or low molecular weight hydrocarbons in acrylic acid derivative copolymers. 6. The method according to item 1 to 4, characterized in da3 preferably used as active groups chloromethyl or Benzhydrylaniin groups. 7. The method according to item 1 to 5, characterized in that in the peptide structure preferably only swelling acting solvent or a chloroform / I / methanol GemiGch used v / ground.