DD160415A1 - PROCESS FOR THE PRODUCTION OF PEPTIDES - Google Patents

Abstract

The invention is a process for the preparation of peptides using activated esters of N-hydroxy compounds without isolation of intermediates. The amino components of dewachsenden peptide are in a halohydrocarbon with up to twice the moral excess of N "-Boc-protected Aminosaeureaffester of N-hydroxy compounds under Rueren with a alkaline-alkaline buffer o.Salzloesung coupled quentitatively u.racemisierungsfrei faster than without the use of this alkaline second phase, so that the resulting peptides in the organic phase are subsequently purified by conversion of the excess of carboxylic acid to an acidic amide by washing processes, deblocked by addition of a high acid organic acid with methanesulfonic acid on the N "-Box group, and by neutralization with sodium bicarbonate as amino components The process allows biologically active peptides or their intermediates to be prepared rapidly, with high purity and in good yields.

Description

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Process for the preparation of peptides

Gsgebiet applicatio of the invention

The invention relates to a process for the preparation of peptides in homogeneous solution without isolation of intermediates and thus enables a rational and economically advantageous presentation of biologically active peptide active ingredients or of their synthesis intermediates.

Character istik of the known technical solutions

Rapid peptide syntheses that allow 1-2 amino acid couplings per day are only possible . the isolation, purification and characterization steps required of conventional work can be eliminated after each amino acid coupling and every ^ amine group blocking. However, this is only possible if the two reactions mentioned can be carried out quantitatively, because otherwise, due to the lack of intermediate purification, faulty and core sequences of the peptides rapidly accumulate. This concern was accounted for by the plague-phase peptide synthesis introduced by LESRRIFrßLD (RB Merifield, Federation Proc..21, 412 (1962)). The amino component of the growing peptide chain

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anchored to a solid polymeric support and the peptide linkages are quantitatively effected as much as possible with a large excess of the activated carboxy components. Reagent excesses and soluble by-products can then be readily removed by filtration and etching, which is analogous to the more readily quantitative ones M ^ protection group blocking applies. However, the heterogeneous reaction procedure allows for numerous coupling

'· ·

despite the excesses used, no quantitative acylations, so that in general more or less heavily contaminated peptide mixtures are formed whose necessary intensive purification eliminates the advantages of rapid synthesis.

It is obvious that one can perform such quantitative couplings with an excess of activated Carboxylkomponentθ also in solution waiving the polymer bond of the amino component, if it is possible to remove the excess and possibly occurring coupling by-products. Several variants of a rapid peptide synthesis which can be carried out in solution are known from the literature (GRPettit, "Synthetic Peptides", Vol. 4,33 (1976)), which consist essentially in the activation of the carboxyl component and in the manner of the separation differentiate the reagent surpluses. The latter can be done in principle either by precipitation of the peptide from the reaction medium or by washing out the excess and by-products. With respect to the N -Deblockierungen you put with

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Advantage of the tert-butyloxycarbonyl protection, which is quantitatively and rapidly removable with sufficiently acidic acids, with only gaseous by-products formed and the deblocking can be easily washed out with aqueous alkaline solutions ·

The most well-known among the Sehnellsynthesen in solution is that developed by TILAK (DOS 2022032 and Tetrahedron Letters 849 (1970)) and later by BBYERRiANN (Rec.Trav.Chim.Pays-Bas 22,481 (1973)) as REMA technique (Repetitive Mixed Anhydride ) method using mixed anhydrides. Based on the amino component used, 1.5 equivalents of the mixed anhydride of an N-protected amino acid are required, and after the quantitative coupling (0.5 to 2 hours), the excess of the reagent is hydrolyzed by a bicarbonate solution. A water-miscible solvent is used for the coupling reaction, so that the peptide precipitates upon hydrolysis. This is washed, taken up again in the organic solvent, deprotected, precipitated and then used in the next synthesis oil. This technique is not suitable for the coupling of sterically demanding amino acids, such as isoleucine, valine, proline, etc., because it leads to partial malacylation by the mixed anhydride. This limitation is as disadvantageous as the frequent precipitation reactions of the peptide. · The misacylations can be avoided in principle if the peptide couplings are carried out with activated esters. However, since activated esters are generally less reactive than mixed anhydrides, one must on the one hand

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use larger excesses and on the other hand use as reactive as possible esters to come in reasonable times to quantitative couplings. H-hydroxysuccinimide ester (CH Schneider, W.Wirz, Helv.chim. Acta £ 5, 1062 (1972)), o-nitrophenyl ester (M.Bodanszky US 3944538, J.org.Chemistry, 8.3565 (1973)), and N-hydroxybenzotriazole esters (GTYoung, J.Chem.Soc., Perkin I 1979, 1901, H.Rolli et al., Int. J. Peptide Prot. Res. J. 41, 1980)) did not satisfy Er maintenance in this regard because too slow couplings required either high reagent excesses or too long reaction times. By contrast, the pentafluorophenyl esters (L. Kisfaludy, Tetrahedron Letters 1785 (1974)), which allow quantitative peptide coupling in 15-30 minutes when 2-3 equivalents of active ester are used, proved to be much more suitable for this purpose. The disadvantages are not so much the excess reagent but the high price of pentafluorophenol and its high toxicity. > In the active ester syntheses was usually

Coupled in a small volume of dimethylformamide, which ensures the necessary solubility of the growing peptides and favors the desired quantitative conversion by the high concentration of the reactants. The work-up of the reaction mixtures after the coupling pursues the aim of separating the excess of the Boc-amino acid active esters and the hydroxy compounds formed as coupling by-products from the IT ^ -Boc-protected coupling product. This could be done by adding the coupling solution with, for example, ethyl acetate (M.Bodanszky, J.org.Chemistry 3§, 3565 (1973)) or hexane (L.Kisfaludy, Tetra-

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hedron Letters 1785 (1974)) - depending on the solubility of the peptide - precipitate the coupling product. In most cases, the Boc-amino acid reactive ester and the hydroxy compound will remain in solution so that the following Boc cleavage step can be initiated after isolation of the peptide. It is more convenient, according to TILAIC (Tetrahedron Letters 849 (1970)), to use the excess activated carboxy Immediately after the coupling with a primary / tertiary diamine, for example 2-diethylaminoethylamine, the component is transferred to an amide which can be washed out clearly with aqueous acids. Either you give to the dimethylformamide coupling solution after amide formation, an excess of aqueous acid, in which case only the peptide precipitates and can be further processed accordingly (L »Kisfaludy, Tetrahedron Letters 1785 (1974))» or one does not take the coupling approach in one with water immiscible solvent and washed, the Boc-amino acid amide and the Hydroxyverbin- ^ _n dung with aqueous acid or base of (u H.Rolli. with., Int. J.Peptide Prot.Res. J £, 411 (1980)). Consequently, in the case of the last-mentioned work-up variant, the usual intermediate isolation of the peptide is obtained after the coupling step.

The latter also applies to a rapid synthesis, in which the coupling was carried out in an aqueous-organic two-phase system using a water-soluble carbodiimide (S.Hozaki, Chem.Letters 1977 »1057) .Dabei can be separated after the coupling, the aqueous phase and from the organic phase of the reagent excess and the urea formed with

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aqueous acids and bases are extracted while the resulting peptide remains in the organic phase. However, in order to achieve quantitative couplings in a time of about 2 hours, which is adequate for rapid synthesis, there were 4-fold excesses of the carboxy component, the carbodiiraid and the N-hydroxybenzotriazole added for racemization suppression. With regard to the simplicity and thus the economic viability of the method, the N -Boc deblocking step required after the couplings also deserves consideration. If the protected ipeptide is firmly isolated after coupling, it is expedient to cleave with trifluoroacetic acid by taking up the peptide therein and after approx. 15 minutes unblocking time trifluoroacetic acid distilled off, precipitated with ether and the peptide with free amino group again for the next coupling in DMP receives (HCBeyermann, Rec.Trav · Chim.Pays-Bas, 22, 481 (1973); M. Bodanszky, J.org. Chemistry ν ^ 8, 3565 (1973) et al.). The isolation of the Boc-group cleavage step may also be useful if the coupling product was not isolated after processing because relatively high concentrations of, for example, hydrochloric acid or trifluoroacetic acid are necessary in acidic deblocking in an organic solvent in the neutralization very much salt were (H.Rolli et al., Int.J.Peptide Prot.Res · 15,411 (1980)). Only NOZAKI (Chem.Letters 1977 .1057) has added HCl / dioxane to its organic phase in the last-mentioned manner and, after completion of the Boc elimination, the

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Acid neutralized with soda solution. Since the peptide is not isolated in this method either after coupling or after Boc cleavage, the authors also described the method as a "hold in solution method". However, the chosen Deblockierungsvariante also has disadvantages, because the necessary high addition of HCl / dioxane involves the risk that in the subsequent neutralization with the distribution of dioxane between the organic and aqueous phase, the amino component is partially washed out.

In conclusion, it should be noted that all of these methods require that the growing amino component be soluble in the organic coupling solvent. However, this can be affected in many cases by appropriate protective group selection at the terminal carboxyl group and at the amino acid side chains

Object of the invention

The object of the invention is to provide an economically advantageous preparation of pure biologically active peptides or peptide fragments of high molecular weight peptides as synthesis building blocks while avoiding long reaction times, the isolation of intermediates and a large excess of activated carboxyl and avoiding the activation of this component by relatively toxic compounds

Presentation of the. Essence of the invention

The object of the invention is to develop a process for the production of peptides which, by means of a rapid synthesis, allows the step-by-step construction of peptides without isolation.

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tion of intermediates allowed material-saving under quantitative coupling steps is feasible »does not require the use of toxic materials and in which the peptide to be synthesized is kept in solution in all stages.

Previously known technical solutions do not satisfy with respect to the coupling with the exception of the use of highly reactive pentafluorophenyl N-protected amino acids »because the coupling rates are too slow» so that quickly achievable quantitative reactions with reasonable excess reagents, especially sterically hindered amino acids can not be achieved are. Pentafluorophenyl esters of the carboxy components are also disadvantageous because of the high price and the considerable toxicity of the pentafluorophenol. The "keep in solution" of the growing peptide, which simplifies the synthesis, presents difficulties in the previously known processes, either because the separation of the reagent excesses and the coupling by-products can not be achieved without intermediate precipitation or because of the deblocking of the N-Boc group after each coupling step used, which must be relatively high doses and therefore can be neutralized subsequently only with the formation of large amounts of salt.

It has now surprisingly been found that active ester couplings between N-protected carboxylic and amino moieties in a water immiscible organic solvent can be effectively accelerated by stirring with a weakly alkaline (pH 7-9) aqueous phase without racemizing the carboxy moiety becomes. In this way

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According to the invention, in the two-phase coupling system using excess activated esters of N-hydroxy compounds, in particular the relatively hydrolysis-stable active ester of K-hydroxy-5-norbornene-2,3-dicarboximide (HONB) and N-hydroxysinecinimide (HOSu), quantitative reactions can easily be achieved. Depending on the type of amino acids present at the coupling site, the excess of the carboxyl component is selected to be twice equimolar and achieves quantitative conversions in a maximum of 2 hours, but often in a much shorter time. Greater excesses are required only for sterically hindered knots. It is advantageous to use halogenated as the coupling phase. Hydrocarbons such as dichloroethane, methylene chloride or chloroform and as the aqueous phase buffer or alkaline hydrolyzing salt solutions, preferably alkali metal bicarbonate solutions. Table 1 contains some peptide linking examples in which couplings of Η-protected amino acid OHB esters to amino acid esters in dichloroethane are compared in the presence and absence of an aqueous sodium bicarbonate phase.

The reaction acceleration apparently goes back to a base catalysis of the peptide coupling and the extraction of the acidic, water-soluble H-hydroxy compounds resulting from the reaction from the reaction medium. »

This coupling solution in the two-phase system now allows using the known acidic leaching of the excess carboxyl component after its conversion into an acid-soluble amide to dispense with an intermediate precipitation of the N-protected peptide.

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Table 1:

² "

Most Active Axin Component Solution M. Time ² "Analytical Da

^{t (h) d} ^

Z-Pro-ONB Il H-Gly-OBz1 1 0.2 64-66 (-54.6 -VaI-ONB Il 2 0.1 68-73 -55.61 Z-Ala-ONB η H-Leu-OMe 1 0.5 oil -34,4C il Il. ' Boc-Leu-ONB Il 2 0.3 oil -33.20 Z-Pro-ONB H-Leu-OMe 1 1.0 68-70 -66.50 ti ti 2 0.5 71-72 -68.60 ζ H-Gly-OBz1 1 5.0 129 -24V6 (] Il 2 2.0 133 -26.40 H-Leu-OMe 1 49.0 144 -53.6 (1 Il 2 24.0 144 -52.0 (1

Time to quantitative conversion Solvent system 1: dichloroethane Solvent system 2: dichloroethane / 1M NaHCOv

A sufficiently hydrophobic amino component is added in the halohydrocarbon phase with an up to double molar amount of an excess N ^ Boc-protected amino acid ONB ester (or a corresponding active ester of another N-hydroxy compound) and until quantitative acylation (TLC control ) is stirred with a 1M sodium bicarbonate solution, which usually takes less than 2 hours to complete. The bicarbonate phase is then separated off and the organic phase is treated with an amount of a primary and tertiary amino-bearing diamine, for example 4-picolylamine or N, N-dimethylethylenediamine, necessary for converting the excess active ester into an acid-soluble amide, and Stirred for minutes · Afterwards

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The organic phase is washed successively with a dilute acid, a bicarbonate solution and with water. Now take the splitting off of the Boc group with a strongly acidic organic acid, such as methanesulfonic acid (H.Yajima, Chem.Pharm.Bull, 2, 740 (FIG. 1977)) instead of HCl or trifluoroacetic acid, one comes with a rather low total acid ekofissentr at ion in order to split off the Boc group in 15 minutes quantitatively · This excess acid can be washed out very easily with a bicarbonate solution, so that the amino component in this reaction step does not have to be made between

The organic phase is added to the above-described washing processes for cleaving the Sn-Boc group of the peptide with the necessary for the preparation of a 0.5 M solution of methanesulfonic acid and stirred for 15 minutes. It is then neutralized with a saturated sodium bicarbonate solution and thus at the same time the two-phase system is restored with the amino-free amino component for the next peptide coupling.

The successful application of this synthesis scheme requires, as in the rapid syntheses described so far, sufficient solubility of the amino components of the growing peptide in the organic phase with correspondingly low solubility in the aqueous bicarbonate phase. If necessary, care must be taken by appropriate selection of the terminal and side chain protecting groups. The side-chain protecting group selection must further take into account that corresponding Boc-amino acid amides in aqueous acids

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have to be soluble.

Under these limitations, the method of the invention is particularly useful for preparing short chain biologically active peptides such as angiotensin, oxytocin, vasopressin, somatostatin, gonatropin releasing hormone (Gn-RH, LH-RH), enkephalin, substance P, pentagastrin, pancreocymin cholecystokinin, and the like. or to represent intermediates of larger bioactive peptides.

The good practicality of the synthetic scheme has been demonstrated on the peptides of the Examples, all of which have been prepared in very good yields and high purity. In general, a synthesis cycle for the coupling of an amino acid was carried out in 4-5 hours, the necessary solvent use is in contrast to the solid phase synthesis very kleinj because in the specified synthesis scheme 5-10 mmol of the growing Aminokomponentö in 100 ml of organic phase can handle well , The time savings over conventional peptide synthesis is significant as evidenced by the 2-day leu-enkephalin synthesis.

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embodiments

1 «General Synthesis Code

7 »5 mmol Boc-amino acid OHB ester (HOHB = N-hydroxy-5-norbornene-2,3-dicarboximide) and 5 mbar of the amino component (usually amino acid or peptide alkyl esters) in 100 ml of 1.2-dichloroethane in a mixture with 15 ml of 1 N sodium bicarbonate solution for 0.5 - 2 hours intensively stirred. Thereafter, the aqueous phase is separated off, the dichloroethane solution is mixed with 5 mmol of 3-dimethylamiopropylamine and stirred for a further 20 minutes. The reaction vessel is then washed 3 times each with 1 η HGl, 1 η NaHCOo and water, then the organic phase 3.25 ml of methanesulfonic acid and 2 ml of glacial acetic acid added and stirred again for 20 minutes. Finally, with sodium hydrogen; carbonate solution is neutralized and the Zv / Eiphasensystem restored for the next clutch.

2. Examples

2.1 Z-Gly-PhePhe-Tyr (BzI) -OMe (fragment B23-26 of the insulin B-chain)

Yield 2.74 g (71% based on the starting amino acid);

P. 161-164 ⁰ Cj WgO _ _g . ₀ o ( _{c = ljDFiF} ) _{; DC} uniform ^C 45 ^H 46V8 (770.8)

Ber. C 70.11 H 6.02 N 7.27

Gef. 70.00 6.07 7.07

Amino acid analysis : Gly -1.0 (1); Phe 2.0 (2); Tyr 0.7 (1)

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2.2 Z-Tyr (Bzl) -Gly-Gly-Ph e-Leu-OBzl (Leu-Enkephalin)

Yield 3.26 g (75% of theory); F. 151-153 ⁰ C; W ^ ⁰ -17.0 ° (c = 1, CHoOH); DC uniform according to KieseIge! Chromatography

C ₅₀ H ₅₅ N ₅ O ₉ (870.0)

Ber. C 69.03 H 6.37 N 8.05 Gef. 68.67 6.24 8.26

Amino acid ana Iy: jGljr 2.0 (2); Phe 1,1 (1); Leu 1.1 (1);

Tyr 1.0 (1)

2.3 Boc-Tyr (BaI) -Gly-Leu-OMe (Fragment 5-7 of Gn-RH)

Yield 2.31 g (83% of theory); F. 55-58 ⁰ C; Ώ0 uniform C ₃₀ H ₄₁ N ₃ O ₇ (555.7)

Ber. C 64.85 H 7.44 N 7.56 Vessel 65.36 7.55 7.47

Amino acid analysis: GOjr 1.0 (1); Leu 1.0 (1); Tyr 0.8 (1)

Claims (3)

229688 A Br estungs pr ouch 1. A process for the preparation of peptides, characterized in that an amino acid or peptide ester or amide with unprotected «st-amino group as the amino component and an ir 'Boc-protected amino acid reactive ester as carboxyl component' · using a slight excess of this carboxy component dissolved in a water-immiscible solvent, the two components with stirring the solution with an aqueous, weakly alkaline buffer or salt solution at a pH between 7 and 9 without racemization within a few minutes to 2 hours with each other coupled, then the aqueous phase separated, the organic, residual. Carboxyl component and · containing peptide containing phase for removing the carboxyl component with a primary and tertiary amino-carrying diamine treated with stirring, resulting acid amide washed with dilute acid, the organic phase washed with bicarbonate and water until neutral and then the remaining peptide in this phase with a strongly acidic organic acid, preferably methanesulfonic acid, unblocked on the nitrogen, the organic solution is washed neutral with the aqueous alkaline solution of the biphasic system and deblocked peptide in this organic phase is again subjected to further coupling with a N ^ -Boc-protected amino acid active in the two-phase system · ''; 2. The method according to item 1, characterized in that as
N ^ -Boc-protected arabic acid active ester preferably
Esters of II-hydroxy compounds, in particular relatively hydrolysis-stable esters of N-hydroxy-5-norbornene-2,3-c1-carboxylic acid imide or N-hydroxysuccinimide are used 3. The method according to item 1, characterized in that as
water-immiscible organic solvents, preferably halogenated hydrocarbons or ethyl acetate, and as the aqueous phase having the pH range of 7 to 9
either buffers, bases under pH stat conditions or
alkaline hydrolyzing salt solutions, preferably alkali metal bicarbonates find application «