DD144538A1 - PROCESS FOR THE PRODUCTION OF PEPTIDES - Google Patents

Abstract

The invention relates to a process for the preparation of Peptides} after complete or partial cleavage of the Protecting groups as active substances or, as intermediates of active substances can serve. The aim of the invention is the production of steric uniform peptides, by the invention is otherwise at Enzymatic Peptidsynthesen in buffer systems with the addition of Water-miscible organic solvents occurring Enzyraschädiguxig pushed back decisively and by easy separation of the protease from the reaction mixture a multiple reuse of the Biocatalyst allows. The peptides are made from a selectively protected amino acid or a selectively protected Peptide in which, the unreacted Carbcxygruppe in the reaction or bearing an ester group, and a selectively protected one Amino acid or a selectively protected peptide including the corresponding amides in which the reacting amino group unprotected, in a protease-specific buffer system with a soluble or immobilized protease with the addition of 'Wa'sser immiscible organic solvents or mixtures thereof Solvent. These peptides can cleave off the protecting groups as pharmaceuticals or after partial deblocking. as intermediates, used for the synthesis of other therapeutically useful peptide active ingredients become.

Description

~ ^Λ ~ 2 1-38 83

VEEPIHREI FOR THE PREPARATION OF PEPTIDEH

The invention relates to a process for the preparation of peptides which, after complete separation of the protective groups, may serve as active substances or as intermediates of active compounds,

Characteristic of the known technical Jjösuneen

For the preparation of peptides various organic chemical Verföiren, such as the azide method, the anhydride method, the Bicyclohex.ylcarbodiiraid method, the variant-rich method of the active esters and many others are used (see * Overview: WÜHBCH, - E * (1974 ) Synthesis of Peptides, in Houben-Y / eyl, Ed ₀ 15 * 1/2, Methods of Organic Chemistry, MÜLLER, ((Hrsg) Georg Thieme Verlag, Stuttgart) In the production of peptides by means of these mentioned methods There are various difficulties, which are characterized by racemization, side reactions, complex temperature control, complicated purification processes, etc. A particularly serious disadvantage of the conventional processes is the still unresolved problem of avoiding racemization, especially in the case of pragment condensation. long-chain biologically active Peptidwirkstof ™ fe is a Rafseraisierungsfreier course of the coupling reaction

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Since the optical purity of the synthesis products is a prerequisite for the full biological activity of the peptide active ingredients, there are eminent disadvantages in the industrial application of said organic-chemical processes.

In addition to the chemosynthetic peptide synthesis methods, procedures were also described in which proteases were used as catalysts for the formation of the peptide bond (BERGMÄHN, M ₄₅ and KUMKBL-CQMAT, H * (1937); 'J Biol. Chern, JLlä »707) * The use of proteolytic enzymes as catalysts in the peptide-coupling reaction eliminates racemization and other side reactions, as well as allowing easy process control«. In the known methods of enzymatic peptide synthesis (reviewed by ISOWA, Y. (1978) "Yuki Gosei Kagaku Kyokaishi," 26, 195-250), the peptide bond formation takes place in the presence of proteolytic enzymes using suitable buffer systems with addition of Water-miscible organic solvents have been used to keep the reactants largely in solution. It has also been shown that water-miscible organic cosolvents shift the equilibrium of the protease-catalyzed reaction in the direction of the synthesis (G.A. "HOMMDBSRG _} J" A . * MATT IS u M "Laskowski, Sr. (1978) ν Biochemistry, Η, 5220; K. 'HSfOUYB _e et al (I979) e J * * Amer Chem Soc _e,.. J01, 751)" The However, use of water-miscible organic solvents has the disadvantage that a) by the addition of high levels of such solvents, such as methanol, dimethylformamide, thylsulfoxid, tetrahydrofuran u «a. the protease used is damaged and b) a recovery of the biocatalyst for the purpose of repeated use of such reaction mixtures in industrial process management virtually impossible. is. In addition, alcohols can be used as cosolvents in the. enzymatic peptide synthesis, when added in very high proportions, can also function as a reactant partner (E.G, BiGALLS et al. (1975) "Biotech").

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Bioeng., 22, 1627-1637) and contaminate the end product with the resulting Ester ™ derivatives *

OF INVENTION The aim of the dung. ' ^::

The aim of the invention is the production of optically uniform peptides through the use of soluble and immobi ». lysed proteases under reaction conditions which ensure multiple reuse of the biocatalysts.

The invention is intended to substantially reduce the possible damage to the enzyme and the consequent reduction in catalytic activity of enzymatic peptide syntheses with the addition of water-miscible organic co-solvents, while at the same time allowing multiple reuse of the biocatalyst. are prepared from a selectively protected amino acid or a selectively protected peptide whose reacting carboxy group is unblocked or carries an ester moiety, and a selectively protected amino acid or a selectively protected peptide including the corresponding imides Reactive imino group is unblocked, in a protease-specific buffer system with the addition of water-immiscible organic solvents or mixtures of such solvents in the presence of '. Soluble or immobilized .Proteases · Sfach terminated .- Peptide synthesis, product from separation and workup, the protective groups can be partially or completely cleaved if necessary by known methods. In contrast to known enzymatic peptide syntheses in buffer systems with the addition of water-miscible organic solvents, the use of water with miscible cosolvents according to the invention means that the phase in the buffer phase is present. Enzyme is not exposed to damaging influences, and at the same time it can. Enzyme recovered after the coupling reaction by phase separation · By the

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If inert, water-immiscible organic solvents are used, undesired side reactions, as are possible, for example, in the case of the use of alcohols known from the literature, are completely excluded. The use of immobilized proteases also allows enzymatic peptide synthesis without product precipitation. The proven multiple reuse of both the soluble and the immobilized enzyme has high economic relevance. The invention is suitable for the stepwise construction of a peptide as well as for fragment condensations, thereby blocking those functional Groups to be selectively protected "may be used in peptide chemistry common protecting groups including polymeric resins with suitable anchor groups. As an enzyme, all known proteases can be used, wherein the selection of the enzyme used as a catalyst must be carried out depending on the object of synthesis due to the substrate specificity • Interesaanterweise could H + tert-butyloxy-carbonyl-protected Car ™ boxykomponenten with chymotrypsin as a catalyst with the aid of the inventive method be successfully linked, although in the literature such conversions do not arrive.

The invention will be explained in more detail below on 6 exemplary embodiments. «

In the examples, the amino acids are abbreviated according to the internationally valid rules »In addition, the following abbreviations are used;

Ac acetyl

Z · benzyloxycarbonyl

Boc tert.-butyloxycarbonyl

OMe methyl ester

DCC dicyclohexylcarbodiimide

HOBs I-hydroxybenaotriazoi

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·. , :. , ...; ;. , , - 5 -

Example 1

It does not mean that Ac _n -Le uP fce -Le u-

a) with dissolved ,, chymotrypsin

66.9 mg (Ο ,, 2 inraol) of Ac-Leu-Piie-OMe and 26 mg (0.2 mmol) of Leu-IHp are dissolved in 1.5 ml of 1,1,2-trichloroethene, while stirring with 0, 5 ml of a 0.1 mil solution of chymotrypsin in HaHCO ₃ ZNa ₂ CO ₃ -Puffer (0.2 M ₅ pH 10) and stirred for 20 min at room temperature. The precipitated product is fritted and washed successively with water, 1'N HGl , water, HaHGOo solution and washed twice with water '·· Subsequently, the product at 110 ⁰ G 1 hour »dried · Ausbo 72.8 mg (84% d, Th ·) P _e 284-285 ⁰ G

To a suspension of 100 mg of gyiüotrypsin fixed on silica gel (corresponding to about 2 mg chymotrypsin) in 1 ml of 0.2 liter carbonate buffer (pH 9.7), 66.9 mg (0.2 mmol) of Ac-Leu-Phe are added -OMe and 26 mg (0.2 mmol) of leu-lHp dissolved in 1.5 ml of dichloromethane, added and stirred vigorously for 1.5 hours at room temperature. The reaction mixture is placed on a glass frit and the estimated peptide is 7 times T ml Removed methanol from immobilized enzyme. The filtrate is placed in an irrigated container, transferred to a syringe, and processed as described under a). 55 mg (64 %). '/ P * 284 - 285 ⁰ C

Example 2

 - 6 -,. :. , .,

213883

Z- Ala-Phe, -Le u-HEU

a) with,

76.9 mg (0.2 mmol) of Z-Ala-Phe-OMe and 26 mg (0.2 mmol) of LeU-HH ₉ are mixed with 0.5 ml of carbon tetrachloride and 0.5 ml of a 0.6 mM solution of chymotrypsin in NaHCOo / lTapCOo buffer (0.2 M, pH 10) and stirred at room temperature for 1.5 hours »The precipitated product is filtered off and washed successively with water, 1 U HGl, water, UaHCOo-Lö-Bung and twice with The product is then dried at 110 ^° C. for 1 hour, yielding 69.8 mg (72 $ Sd × th ^-1 ) I ¹ . 224 - 226 ⁰ G

b)

76.9 mg (0.2 mmol) of Z- Ala-Phe-OMe are dissolved in 1.5 ml of carbon tetrachloride and 26 mg (0.2 mmol) of Leu-ϊΤΗ dissolved in 1.2 ml of NaIlGO ₃ A ^T a ₂ CO ₃ buffer (0.2 M, - pH 10), and 250 mg of chymotrypsin bound to silica gel (corresponding to about 5 mg chymotrypsin) added, after 2stdg _β stirring at room temperature Y ^ the precipitate is transferred to a glass frit, the product with 8 times 1 ml-methanol separated from the immobilized enzyme and worked up as under 1b)

Y. 67.6 mg (70% d "Th") P. 224-226 ⁰ C.

Synthes

a) with ge Io _i 6 _i tem _{ii <} Chymp _r tr ^ jDgi _; n

78.5 mg (0.2 mmol) Boc-Leu-Pne-OMe and 260 mg (2 mmol). LeU-HH ₂ are in relation with 1.5 sil of a mixture of 1,1,2-di-chloroethers and petroleum ether (30 - 50 ⁰ G)

1: 2 and 1 ml of a 0.6 ml solution of chymotrypsin in lTäHCO ₃ / Ha ₂ CO ₃ buffer (0.2 M, pH 10) and stirred for 2 hrs. At room temperature. The precipitated pro duct is a frit and washed successively with water, 1 HCl, water, NaHCO ^ solution as well as the deluded tion for the reac organic Lösimgsmitte! Mix ge wash · The product is? P ^ 5 ^ ^m desiccator * Yield _e 84.2 mg (86 % a.T.) F. 205-208 ⁰ G

b) lit i

IP - 48 ° (c = 0.5, ethanol)

78.5 g (0 _s 2 mmol) of Boc-Leu-Phe-OHe are dissolved in 1 ml of carbon tetrachloride and thereto 109.7 mg (0.845 mmol) of LeU-UH ₂ dissolved in 1 ml of carbonate buffer (0.2 M, pH 10) and 250 rag of chymotrypsin fixed to silica gel (corresponding to about 5 Big Bnsssym) and stirred for 4 hrs. At room temperature. After separation of the reaction product from the immobilized enzyme with 5 times 1 nil of methanol and concentration of the filtrate is worked up as usual 35.3 mg (36 ld "Th _e ) F. 203-208 ⁰ C

| ³ - 49 ° Cc = 0.45, ethanol)

75.6 mg (0.2 mmol) of Boc-Leu-Phe-OH and 26 mg (0.2 mmol) of LeU = KH ₂ are dissolved in 0.5 ml of carbon tetrachloride, under stirring with 1.5 l of a 1.5 mM solution of papain (12 IS / mg) in McIlvaine »buffer added and 24 hours» - at '.' 30 ⁰ C: stirred "The precipitated product is fritted and successively with water, 0.5 IT. HCl, 7pro7 _The ammonia "solution and. Water .gewaschen · The peptide is about ₁ PPO -getrocknet * '''-...;

Y. 76 mg (78% of theory..) P. 205-207 ⁰ C ^5-48.8 ° (c 0.5, ethanol)

A comparative peptide obtained chemosynthetically by the DCC / HOBt method was synthesized in 6iproz. Y. receive:

P 205-207 ⁰ G [ ⁰⁴ Jd ⁵ - 44.3 ° (c = 0.5, ethanol)

Synthesis of Boc-Ala-Phe- »Leu-IT £ U

Analogously to Example 3a) are 70.1 mg (0.2 mmol) Boc-Ala

Phe-OMe and 260 mg (2 mmol) Leu-HHp reacted with chymotrypein as catalyst »Ausb. 68 _S 6 mg (76.5% of th _e.) P. 186 -188 ⁰ C.

Synthesis of, BpC-Gly ₁ -Gly-Phe ^

Analogously to Example 3a), 78.7 mg (0.2 mmol) of Boc-Gly-.gly-Phe-OMe and 260 mg (2 mmol) of Leu-KHo are reacted with chymotrypsin as catalyst (reaction time: 1 h _e ) _e y. 59.1 mg (60 ύ fr Th..) P. 163-165 ⁰ C.

Bjlg ^ JjXJj

an enzyme for the synthesis of

a) with dissolved chymotrypsin

200.7 mg (0.6 mmol) of Ac-Leu-Phe-OMe and 78 rag (0.6 mmol) of Leu-ϊίΗ ^ are mixed with 0.5 ml of dichloromethane and 1.5 ml of a 0.1 mM solution of chymotrypsin in carbonate buffer (0.2 M, pH 10) and stirred for 20 min. At room temperature «The precipitated product is fritted and worked up analogously to Example 1a).

Ausbe 202.0 mg (78% d "Th.) · P 284 - 285 ⁰ C iltrat obtained after the separation of the product 3 is taken from the aqueous phase and this for converting 133V ⁸ mg (0.4 mmol) Ac - »Leu-Phe» OMe with 52 mg (0.4 mmol) of Leu-ML in 0.25 ml of dichloromethane used Ausbo 131.2 mg (76 % of theory ) F. 284 - 285 ⁰ C.

The aqueous filtrate obtained in the separation of the product is subjected to a third batch of reaction of 66, S mg (0.2 mmol) Ac-Leu-Phe-OMe and 26 mg (0.2 mmol) of LeU-UH ₂ in the presence of 0.2 ml dichloromethane added. 64.6 mg (74 % of Th ₉ ) 'F .; 284.- 285 ⁰ C

The immobilized chymotrypsin resulting from the product separation in Example Ib) is used to react 66.9 mg (0.2 mmol) of Ac-Leu-Phe-OMe with 26 mg (0.2 mmol) of Leu-lHo Ib) worked up. 53 ₅ 6 Big (62% of th _e.) P _8284-285 C ⁰

Claims (3)

A method of producing peptides in which a selectively protected amino acid or a selectively protected peptide, the carboxy group of which is reactive, is unblocked or carries an ester moiety, with a selectively protected amino acid or a selectively protected peptide including the corresponding amides, wherein the reacting amino group is unblocked, is reacted in a protease-specific buffer system with a proteolytic enzyme, wherein after completion of the coupling and described workup, if necessary, the Schutsgruppen partially or completely cleaved by known methods, characterized geke η ζ eich η et that the reaction under Addition of water-immiscible organic solvents or mixtures of such solvents is carried out 2 · Method according to item 1, characterized in that immobilized proteases are used. - "- 2 13883 E rf in d -u η g s a n d r e c h 3 "method according to item 1 and 2, characterized geke η η - · ' ζ I η et that dissolved or immobilized proteases are reused several times» 4c method according to point 1 to 3 »ge ge k. e η η draw t _s that as water-immiscible solvents preferably the following solvents are used; Dichloromethane, carbon tetrachloride s 1, t, 2-Triehlor- = ethen, petroleum ether ·; .