DD218904A1 - PROCESS FOR THE PRODUCTION OF PEPTIDES - Google Patents

Abstract

The invention relates to a process for the preparation of peptides which, after complete or partial protection group blocking, can serve as peptide pharmaceuticals or as intermediates of active compounds. The aim of the invention is the preparation of chirally uniform peptides. The invention substantially restricts the reduction in solubility which otherwise occurs in the case of enzymatic segment linkages as the chain length of the reactants increases, as a result of which continuous process design is enabled when immobilized proteases are used. The peptides are prepared from a selectively protected amino acid or a selectively protected peptide in which the carboxy group which is in reaction is unblocked or carries an ester group, and a selectively protected amino acid or a selectively protected peptide including the corresponding amides in which the reacting Amino group is unprotected. In a protease-specific buffer system to which, if necessary, smaller amounts of organic solvents can be added, using solubilizing protecting groups in the presence of soluble or immobilized proteases. These peptides can be used after cleavage of the protecting groups as drugs or after partial deblocking as an intermediate for the synthesis of peptide drugs.

Description

Title of the invention

Process for the preparation of peptides

Field of application of the invention

The invention relates to a process for the preparation of peptides which, after complete or partial protection group blocking, can serve as peptide pharmaceuticals or as intermediates of active substances.

Characteristic of the known technical solution

For the preparation of peptides, various organic-chemical coupling methods (see Reviews: Wünsch, E. (1974) Synthesis of peptides, in Houben-Weyl, Bd · 15, 1/2, Methods of Organic Chemistry, Müller, B. (eds ·) Georg Thieme Verl., Stuttgart; Jakubke, H.-D. u. Jeschkeit, H. (1982) Amino Acids, Peptides, Proteins, Akademie Verl., Berlin), or suitable proteolytic agents are used to eliminate the racemization risk Enzymes as Biocatalysts for the Peptide-Joining Step (See Reviews: Jakubke, H.-D & u.-Kuhlry P. (1982) Pharmacia 21, 89; Chaiken, IM *, Komoriya, A *, Ohno, M, Widmer, F (1982) Appl. Biochenu Biotechnol, 2, 385)

In the known methods of enzymatic peptide synthesis, the formation of the peptide bond in the presence of proteases using suitable buffer systems and often with the addition of organic solvents to the partially protected reactants largely

to keep in solution © With increasing chain length, the solubility of the terminal partially blökkierten peptide segments decreases · The addition of high levels of organic solvents is generally not possible, as this proteases can be irreversibly damaged · In particular, when using immobilized proteases is an essential requirement for continuous process control that dissolves both starting products and the coupling product in the reaction medium ·

Object of the invention

The aim of the invention is the preparation of chirally uniform peptides by protease-catalyzed linking of the reactants using reversibly removable solubilizing protective groups.

Explanation of the essence of the invention

The object of the invention is to decisively reduce the solubility degradation of the reactants which has otherwise been detected in enzymatic peptide syntheses with increasing segment chain length and to enable a continuous process design when immobilized proteases are used by increasing the product solubility.

According to the invention, peptides are prepared from a selectively protected amino acid or a selectively protected peptide whose reactive carboxy group is unblocked or carries an ester moiety, and a selectively protected amino acid or a selectively protected peptide including the corresponding amides in which the amino acid in reaction is unblocked is, in a protease-specific buffer system, possibly low levels of organic solution j

can be added, using solubilizer

lisierender protecting groups in the presence of soluble or im j

mobilized proteases · After completion of the peptide synthesis, j

Product separation and work-up can the protective group I

In contrast to known enzymatic peptide syntheses with conventional protective groups, the solubility of the ester-grouping according to the invention for the Ν-protected oarboxy component increases its solubility, whereby segmental linkages with product precipitation occur when serine or thiole proteases are used be made possible «

An additional solubilizing protecting group at the C-terminus of the amino moiety allows segmental conformation without product precipitation under these conditions. Such kinetic controlled peptide linkage is a prerequisite for continuous process design using immobilized proteases. However, the invention is also suitable both for the segmental linkage in which either the cC-amino function of the carboxy component or the carboxy function of the amino component is substituted with a solubilizing moiety, as well as for the enzymatic condensation of ¬ segments whose protected puncta are completely blocked with solubilizing Sohutzgruppen ^

The invention will be explained in more detail below with reference to exemplary embodiments.

embodiments

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

Z. benzyloxycarbonyl

Boc tert -butyloxycarbonyl

OMe methyl ester

OBz / SO-Na 4-Sülfobenzylester

OCho choline ester

OEt (S ₂ O ₃ Na) 2-thiosulfatoethyl ester

example 1

Synthesis of Boc-Gln-Phe-Phe-Gly-Leu-Met-NH ^ 73.3 mg (0.1 mmol) of Boc-Gln-Phe-Phe-OBzl-SOJJa be ₃ ZNA ₂ CO ₃ (in 0.5 ml NaHCO 0.2 M, pH 10) and 0.3 ml of 33MP, mixed with 35.4 mg (0.1 mmol) of H-Gly ~ Leu-Met-NH ₂ and stirred for 30 min at room temperature after addition of 10 mg of chymotrypsin. After stopping the reaction with 2 ml of 1 N HCl, the precipitated product is fritted and washed successively with water, 1 N HCl, water, NaHCO ^ solution and twice more with water and dried. 55.5 mg (66 % d * Th) IV 235-238 ^° C

Example 2

Synthesis of Z-TyrtEzD-Pro-Phe-Leu-Gly-NH »

84.2 mg (0.1 mmol) of Z-Tyr (Bzl) -Pro-Phe-OBzl-S0 ₃ are dissolved in 0.6 ml of NaHCO ₃ ZNa ₂ CO ₃ (0.2 M, pH 10) and 0.4 ml of DMi ¹ , with 67 , 2 mg (0.3 mmol) of H-Leu-Gly-NH "added and after addition of 10 mg of chymotrypsin stirred for 10 min at room temperature and worked up as under 1).

Y. 52.1 mg (64 % of theory ) F. 97-99 ° C

Example 3

Synthesis of Z-Ala-Phe-Ala-Ala-NH ^.

53.3 mg (0.1 mmol) of Z-Ala-Phe-Oßt (S ₂ O ₃ Na) and 98 mg (0.5 mmol) of H-Ala-Ala-NH ₂ * HCl are dissolved in 1.5 ml of Michaelis buffer (pH 8) and 0 , 5 ml of 1N NaOH and stirred at room temperature for 20 min with 2.5 mg of chymotrypsin. The work-up is carried out as indicated under 1).

Y. 32.7 mg (64% of theory..) P. 266 - 27O ⁰ C

· ·; Y:; ί ..; , , ; ~ 5 -. '.

Example 4 Synthesis of Z-Pro-Phe-Leu-Gly-OPOE

41 mg (0.1 mmol) of Z-Pro-Phe-OMe are dissolved in 0.3 ml of methanol, plus 452 mg of LeU-Gly-OPOSg ₀₀₀ (equivalent to 0.1 mmol of leucine) and 2.5 mg of chymotrypsin in 2.5 ml of 0.2 M phosphate buffer pH 8 and stirred · After 60 min, saturated with NaCl and extracted several times with CH ₂ Cl ₂ , the extracts over Ua ₂ SO. dried, i. Concentrated and the residue crystallized from ethanol.

i. '

Y. 325 mg polymer peptide containing 52% Z-Pro-Phe-Leu-Gly (amino acid analysis) The use of 82 mg (0.2 mmol) leads to a tetrapeptide yield after 30 min of 89 %.

Example 5

Synthesis of Z-Phe-Leu-HH ^

a) 160 mg of Z-Phe-OPOSg _ooo (corresponding to o.o5mmol Phe) and 16.7 mg (0.1 mmol) of Leu-NH ₂ »HC1 are stirred in 5 ml of phosphate buffer with 5 mg of chymotrypsin for 20 min. The precipitate is filtered off with suction, washed with saturated NaHCO 3 solution and 1N HCl and dried.

Y. 8.7 mg (42 % of theory ) P. 189-191 ⁰ C

b) 42.1 mg (0.1 mmol) of Z-Phe-OCho and 83.5 mg (0.5 mmol) of H-LeO-HHg ^- HCl are dissolved in 1.5 ml of Michaelis buffer (pH 8) and 0.5 ml of 1N NaOH and stirred at room temperature for 20 min with 2.5 mg of chymotrypsin. The. Work-up is carried out as described under 1).

Y. 26.6 mg ((4.7% of theory) P. 189 -.. 191 ⁰ C

Claims (4)

ISrfindungsanspruche A process for the preparation of peptides in which an aminoacid blocked with an oC-araino protective group or a correspondingly protected peptide whose carboxy group which is in reaction is unblocked or carries a Sstergruppierung, with a protected at the G-terminal carboxy function or peptide including the corresponding amides in which the reacting amino group is unblocked, in a protease-specific buffer system, to which portions of organic solvents can be added, if necessary, is reacted with a protease, followed by completion of the coupling and described workup partial or total protection group blocking, thereby Geke η η eich η et that one or more solubilizing Schutzgruppelia be used for reversible blocking of the N-terminal amino function of the carboxy and the C-terminal functions of the carboxy and amino component · 2. Method according to item 1, characterized in that solubilizing ester-blowing groups are used on the carboxy component when using serine and thiol proteases. 3 · The method of item 1, characterized geke η _ä n characterized in that solubilizing Ssterschutzgruppen at the carboxyl and other solubilizing protecting groups on the K-terminus of the carboxyl component and / or at the C-terminus of the Aminokompönente in the presence of immobilized serine and Ihiolproteasen a peptide linkage in a homogeneous Enable phase 4 Method according to item 1, characterized in that the following are used as solubilizing protective groups: polyoxyethylene, thiosulfatoalkyl, sulfobenzyl, choline, carnitine esters