DE1493658B2 - PROCESS FOR THE MANUFACTURING OF HYDROXY-AMINO ACID, BUTYLAETHERS AND THEIR N-CARBON BENZOXY COMPOUNDS - Google Patents

Description

The synthesis of peptides of the hydroxyamino acids has hitherto been unprotected in many cases Hydroxyl function carried out. Acetyl, benzoyl, tosyl, introduced to protect the OH group and carbobenzoxy radicals caused side reactions under the conditions of peptide synthesis, such as z. B. Oxazoline ring closures (serine, threonine), nitrosation (azide method for tyrosine), partial racemization by acid-base catalysis (serine, threonine) and N, O-acyl changes. These difficulties could by using the O-tetrahydropyranyl or the O-benzyl ethers of the hydroxyamino acids be circumvented, the latter being the best. However, O-benzyl ether could only from Tyrosine and serine, the latter only by total synthesis, starting from acrylonitrile and subsequent resolution. A direct O-benzylation of serine, threonine, and hydroxypyroline has so far been unsuccessful.

With regard to the requirements of peptide natural product synthesis, recently for masking the amino and carboxyl groups selectively and under mild conditions cleavable tert-butoxycarbonyl and tert-butyl ester groups are used (HeIv. Chim. Acta, 45, p. 2473 [1962]), it seemed expedient to also remove the hydroxyl function, which can be selectively cleaved under analogous conditions Protect tert-butyl ether grouping. A first step was with the representation and the Use of hydroxyamino acid tert-butyl ether tert-butyl esters done (Recueil Trav. Chim., 81, p. 1 [1962], and H. Am. Soc, 85, p. 201 [1963]).

These derivatives of hydroxyamino acids are suitable because of the blocked carboxyl group of peptide synthesis only for use as a terminal component. Subject of the present Registration is a process for the preparation of the free hydroxyamino acid tert-butyl ether as a starting material for the synthesis of peptides from hydroxyamino acids.

It has been found that the desired hydroxyamino acid - tert. - butyl ether as well as theirs N-α-carbobenzoxy compounds are obtained by treating the Ν-Λ-carbobenzoxyhydroxyamino acids with p-nitrobenzyl bromide and triethylamine esterified, then with isobutene in the presence of catalysts, preferably concentrated sulfuric acid, and finally by catalytic reduction both splits off the carbobenzoxy radical as well as the p-nitrobenzyl radical. By treating with alkali it is too possible to split off only the ester residue while maintaining the N-α-carbobenzoxy residue.

In the process according to the invention, the O-butyl ether group remains during the entire process the peptide synthesis and is only in one of the last stages simultaneously with the protective groups split off other side chain functions. A particular advantage of the method is that all intermediate products in well crystallizing

ίο shape and high purity can be isolated. The preservation of the optical purity is through the conversion into optically pure starting material secured. This is a considerable advance compared to the previously known methods that lead to lead to a more or less strong racemization of the amino acids.

example 1
a) N-carbobenzoxy-threonine

238.2 g threonine are dissolved in 1000 ml of 2N sodium hydroxide solution and treated portionwise at 0 to 3 ⁰ C with 1500 ml of 2N sodium carbonate solution and 340 g of carbobenzoxy chloride. The alkaline solution will

shaken out with ether and then with 6N hydrochloric acid acidified to the Congo red envelope. The precipitated oil is extracted three times with ethyl acetate. Finally, the aqueous phase is saturated with sodium chloride and extracted with ethyl acetate. The ethyl acetate phases are combined and washed with water and brine, finally dried and im Reduced vacuum. After recrystallization from ethyl acetate / petroleum ether the product melts at 98.5 to 99.5 ° C; Yield 480 g (95% of theory).

b) N-carbobenzoxy-threonine-p-nitrobenzyl ester

Dissolve 50.6 g of N-carbobenzoxy-threonine and 42 ml of triethylamine in 200 ml Essigester, added 64.8 g of p-nitrobenzyl bromide are added and heated 8 to 9 hours at 8O ⁰ C. After cooling, is filtered off with suction and washed with amine hydrobromide Essigester . The combined Essigesterphasen are washed with 2N hydrochloric acid, 10 ° / ₀ sodium bicarbonate solution and finally with brine until neutral. After drying, it is concentrated, dissolved in a little ethyl acetate and petroleum ether is added until it becomes cloudy. The precipitated product is washed with ethyl acetate / petroleum ether. Melting point:

111.5 to 112.5 ° C, yield: 75.4 g (97 ° / ₀ of theory). To determine the optical purity of this compound, the protective groups were _{split off by hydrogenation with H 2} / Pd. This resulted in optically pure L-threonine: [ec] ff : -27.96 ± 0.5 °; [Oi] ISe : -33.67 ± 0.5 ° (c = 2.0; water). Starting material: [cc] f : -27.85 ± 0.5 °; [oc] |, " ₆ : -33.64 (c-a; water).

c) N-carbobenzoxy-threonine tert-butyl ether p-nitrobenzyl ester

4OgN-carbobenzoxy-threonine-ρ-nitrobenzyl esters are dissolved in a thick-walled round-bottomed flask ir 400 ml of methylene chloride and ^{cooled to 0} ° C. Now 360 ml of isobutene and 5 ml of concentrated sulfuric acid are slowly added. The flask is hermetically sealed and stored for 4 days at room temperature. Then it is cooled to 5 ° C

and carefully opened. The solution is then shaken three times with ice-cold 5% sodium carbonate solution. The combined sodium carbonate solutions are washed twice with methylene chloride and the methylene chloride phases are washed twice with water until neutral. After drying, the temperature is concentrated in vacuo ^{at 35 ° C. water bath.} A light yellow crystalline product remains, which is dissolved in a little ethyl acetate and petroleum ether is added until a dark brown oil has separated out. It is separated off and further quantities of petroleum ether are carefully added to the solution, after which fine white crystals precipitate (quickly by inoculation), which are easily soluble in ethyl acetate and ether and insoluble in petroleum ether. Yield 37 g (81% of theory); Melting point 55 to 56.5 ° C.

d) Threonine tert-butyl ether

29 g of N - carbobenzoxy - ρ - nitrobenzyl - threonintert.butyläther are dissolved in aqueous methanol and hydrogenated with a Pd catalyst in the presence of 4.5 ml of acetic acid. After evaporation of the solvent in vacuo, the residue crystallizes. It is triturated with ethanol to remove p-toluidine and reprecipitated from methanol / acetone; Melting point 259 to 26O ⁰ C (Z). Yield 9.7 g (85% of theory).

e) N-carbobenzoxy-threonine-tert-butyl ether-dicyclohexylamine salt

A solution of 9.4 g of N-carbobenzoxy-threonintert.butylether-p-nitrobenzyl ester in dioxane-water is saponified with 10.6 ml of 2N NaOH with the addition of phenolphthalein as an indicator by stirring for 2 hours. The solution, adjusted to pH 7 with 1N HCl, is largely freed from dioxane on a rotary evaporator in vacuo, and the oil which has separated out is redissolved with water. This is covered with ether and acidified with citric acid. From the separated, washed and dried ethereal phase, N-carbobenzoxy-threonine-tert-butyl ether is extracted exhaustively with potassium bicarbonate _{solution, the combined KHCO 3} solutions are acidified again with citric acid and the separated oil is taken up in ether. After washing, drying and evaporation in vacuo, a white crystalline residue remains, which was dissolved in ether-petroleum ether and converted into the corresponding salt with 3.84 g of dicyclohexylamine. Recrystallization from ethanol / ether / petroleum ether gives colorless needles with a melting point of 146 ° to 147 ° C. Yield 8.3 g (80% of theory).

Example 2

a) N-carbobenzoxy-serine

The connection was made according to the instructions of St. G u 11 m a η n, R. A. B ο i s s ο η a s, HeIv. chim. Acta, 41, 1852 (1958).

b) N-carbobenzoxy-serine-p-nitrobenzyl ester

The compound is obtained as described under 1 b), from 47.8 g of N-carbobenzoxyserine, 42 ml of triethylamine, 64.8 g of p-nitrobenzyl bromide and 300 ml Ethyl acetate. From ethyl acetate / petroleum ether it forms colorless crystals with a melting point of 115.5 to 116.5 ° C. Yield 73.3 g (98% of theory).

The compound was hydrogenated to determine the optical purity. In this case, pure optically originated L-serine: [a] ² ₀ °: + 14.63 + 0.5 °; [«] / ° _s : +17.69 + 0.5 ° (c = 2; ln-hydrochloric acid). Starting material: [a]? : ± 14.64 ± 0.5 °; [*) »: + 17.69 ± 0.5 ° (c = 2; ln-hydrochloric acid).

c) N-carbobenzoxy-serine-tert-butyl ether-p-nitrobenzyl ester

The compound is obtained, as described under 1 c), from 40 g of N-carbobenzoxy-serine-p-nitrobenzyl ester, 360 ml of isobutene, 400 ml of methylene chloride and 5 ml of conc. H ₂ SO ₄ . It forms Essigester / petroleum ether colorless needles of melting point 68 to 70.5 ⁰ C. Yield 41 g (89.5% of theory).

d) Serine tert-butyl ether · ¹ U H ₂ O

The compound is formed, as described under 1 d), by hydrogenating 20 g of N-carbobenzoxyp-nitrobenzyl-serine tert-butyl ether. It was reprecipitated from methanol / acetone and dried at 50 ° C. in vacuo over P ₂ O ₅ . Melting point 25O ⁰ C (Z). Yield 6.9 g (90% of theory).

e) N-carbobenzoxy-serine-tert-butyl ether-dicyclohexylamine salt

The connection is established as described under 1 e),

from 19 g of N-carbobenzoxy-p-nitrobenzyl-serine-tert.-

butyl ether and 22 ml of 2N NaOH. Obtained from ethanol colorless needles with a melting point of 149 to 150 ° C. Yield 17.9 g (85% of theory).

Example 3
a) N-carbobenzoxy-tyrosine ethyl ester

48.7 ml of carbobenzoxychloride are added to a solution of 131 g of tyrosine ethyl ester in 640 ml of CHCl _{3 with cooling and stirring.} Then 202 g of Na ₂ CO ₃ · 10 H ₂ O (0.706 mol) in 385 ml of water are added and finally another 48.7 ml of carbobenzoxychloride are added. The precipitated product is filtered off with suction, washed neutral with water, dried and recrystallized from ethyl acetate / petroleum ether. A second fraction is obtained from the chloroform solution. The yield is 205.7 g (95.7% of theory) with a melting point of 88 to 91 ° C.

b) N-carbobenzoxy-tyrosine

80 g of N-carbobenzoxy-tyrosine-ethyl ester in dioxane-water are saponified with 233 ml of 2N NaOH for 2 hours (indicator phenolphthalein). With In-HCl is adjusted to pH 8, evaporated on a rotary evaporator at 3O ⁰ C, the dioxane, diluted with water and acidified at ⁰ +5 C with 5N H ₂ SO ₄ with stirring. The colorless oil which precipitates completely crystallizes on cooling and seeding. The crystals are filtered off with suction, dissolved in KHCO ₃ solution and reprecipitated with 5N H ₂ SO ₄ . After drying over P ₂ O ₅ , the product melts at 92 to 95 ° C (lit .: 101 ° C). The yield is 67 g (91.2% of theory).

c) N-carbobenzoxy-tyrosine-p-nitrobenzyl ester

It is produced as described under 1 b) by reacting 62 g of N-carbobenzoxy-tyrosine with 64.8 g of p-nitrobenzyl bromide in 42 ml of triethylamine and 300 ml of ethyl acetate. After 8 hours of reaction, the solution is diluted with methylene chloride. After working up, it is recrystallized from ethyl acetate / benzene / petroleum ether. Needles with a melting point of 117 ° to 119 ° C. are obtained in a yield of 84 g (95 ° / ₀ of theory).

The product was hydrogenated to determine the optical purity. This resulted in optically pure L-tyrosine [α] ² _ο ^ο : -10.70 ± 0.5 °; [a] / &: -12.25 ± 0.5 ° (c = 2; in hydrochloric acid). Starting material: [<x] f : -10.3 ± 0.5 ° (in In-HCl).

d) N-carbobenzoxy-tyrosine tert-butyl ether p-nitrobenzyl ester

The product is obtained as described under 1 c) by reacting 20 g of N-carbobenzoxy-tyrosine-p-nitrobenzyl ester with 200 ml of isobutene in 120 ml of methylene chloride and 1.5 ml of conc. H ₂ SO ₄ . From ethyl acetate / benzene / petroleum ether, colorless needles with a melting point of 73.5 to 74.5 ° C. are obtained in a yield of 17.9 g (80 70 of theory).

e) Tyrosm-tejt.butylether

^x / ₄ H ₂ O

The 'compound is formed, as described under 1 d), by hydrogenating 20 g of N-carbobenzoxy-tyrosintertbutyl ether-p-nitrobenzyl ester. It leaves in a yield of 8.3 g (87 ° / ₀ of theory) is obtained from water / methanol of melting point 248 to 249.5 ° C (Z).

f) N-carbobenzoxy-tyrosine-tert-butyl ether-dicyclohexylamine salt

The compound is, as described under 1 e), by saponifying 22.5 g of N-carbobenzoxy-tyrosintert.butylether-p-nitrobenzyl ester with 9 ml of ίη-HaOH. Colorless needles with a melting point of 160 to 161.5 ° C. are obtained from ethanol in a yield of 22.8 g (92 ° / ₀ of theory).

Claims (1)

Claim: Process for the preparation of hydroxyamino acid tert-butyl ethers and their N-a-carbobenzoxy compounds, characterized in that that N-oc-carbobenzoxyhydroxyamino acids are esterified with p-nitrobenzyl bromide, the Reacts product with isobutene in the presence of catalysts and through catalytic reduction the carbobenzoxy radical and the p-nitrobenzyl radical or hydrolytically only the p-nitrobenzyl radical splits off.