DE1143516B - Process for the production of peptides - Google Patents

Description

GERMAN

PATENT OFFICE

F33780rVb / 12q

REGISTRATION DATE: APRIL 27, 1961

NOTICE THE REGISTRATION AND ISSUE OF EDITORIAL: 14. F E B R U A R 1963

It is known that peptides can be prepared by combining an amino acid protected on the carboxyl group with an amino acid protected on the nitrogen-containing group by acid amide bond forming agents, e.g. B. carbodiimides, or after conversion into their reactive derivatives by other conventional methods of peptide chemistry. For example, acyl groups, especially carbobenzoxy groups, and other substituents which have to be removed again after formation of the peptide bond are used as protective groups for the nitrogen-containing, ie mostly the NH _{2 group.} The N-protective group is split off in this way by hydrogenation or by the action of strong acids, such as hydrohalic acids, in glacial acetic acid. You have to accept that a hydrogenative cleavage in some peptides, z. B. S-containing, causes difficulties or that the action of concentrated acids partially leads to cleavage of the peptide group formed.

There is therefore an urgent need for such protective groups after the formation of Peptide bond are easily cleaved from the nitrogen-containing group without causing this cleavage necessary measures further changes occur in the molecule formed.

Process for the production of peptides

Applicant:

Paint factories Bayer Aktiengesellschaft,
Leverkusen

Dr. Elisabeth Dane, Gauting,

Fritz Drees and Dipl.-Chem. Peter Konrad, Munich, have been named as the inventor

It has now been found that peptides can be produced by protecting amino acids or their functional derivatives on the NH _a group by condensation with / 3-dicarbonyl compounds,

ao the optionally modified carboxyl group of the resulting derivatives in known per se Way converted into the carbonamide group and then the protective group by mild treatment split off with acids.

The method according to the invention can be illustrated by the following formulas, which exemplify the Show production of glycyl-glycine, explain:

C = O
OC ₂ H ₅

+ H ₂ N-CH ₂ -COOK

N-CH ₂ -COOK

C = O
OC ₂ H ₅

H ₂ C-COOK

309 508/270

H ₂ C - CONH - CH ₂ - COOC ₂ H ₆

• Κ

+ ClCH ₂ CN
CH ₂ -COOCH ₂ CN

H ₂ NCH ₂ COOC ₈ Hb

H ₂ N - CH ₂ - CO - NH - CHj - COOH

The in the inventive implementation of the amino acids or their functional derivatives, for example their esters or their salts, reaction products obtained with α-dicarbonyl compounds are surprisingly so stable that they can be used for peptide syntheses. she thus differ from those from amino acids in alkaline solution with benzaldehyde and substituted ones Benzaldehydes obtained Schiff's bases. These already disintegrate in moist air (Bergmann and Employee; Ber., 58, p. 1034 [1925]; Hoppe Seyler's magazine for »Physiol. Chemie ", 152, p. 282 [1926]). Also has z. B. Th. Wieland tries to use benzylidene glycine potassium in a peptide synthesis. However, he stopped this attempt because it was unsatisfactory (Th. Wieland et al., A. 576, P. 104, [1952]). It has also been described (Angew. Chemie, 69, p. 363 [1957]) that products from a a-dicarbonyl compound, namely pyruvic acid, and α-amino acids are unsuitable for peptide synthesis.

From the IR spectrum it can be seen that the derivatives prepared with / J-dicarbonyl compounds of amino acids are partly in the enamine form and thus through hydrogen bonds are stabilized.

As / 3-dicarbonyl compounds, 1,3-diketones, jS-ketoesters and / J-keto acid amides used will. Examples of such compounds are: benzoylacetone, acetylacetone, ethyl acetoacetate, and acetoacetic anilides and ethyl cyclopentanone (2) carboxylate and salicylaldehyde, which is enolized as / J-dicarbonyl compound can understand.

The naturally occurring α-amino acids, their optically active ones, can be used as amino acids Forms or racemates, their salts, esters or amides and peptides can be used as well as such Amino acids that are only known from syntheses, such as ε-aminocaproic acid.

The amino acids are expediently dissolved in the solution of an alkali metal or alkaline earth metal hydroxide a suitable solvent, for example in lower alcohol. You can do it through others Conventional measures convert them into their salts, which are used in solution or suspension for the reaction will. It is also expedient to use the esters or amides of the amino acids in a solvent or diluent for reaction with the / J-dicarbonyl compound. The amino acid or its Derivatives are then made with the / J-dicarbonyl compound added to the solvent or diluent. It is advisable to heat for a short time, whereby in most cases, the reaction product of α-dicarbonyl compound and amino acid or derivative precipitates in crystalline form. You can, if necessary, separate from the solution and after a Use purification by recrystallization for further reaction; but you can also do it at Use choice of a suitable solvent directly for further implementation.

If you start from the free amino acids or their salts, you can now use the formed Produce reactive derivatives such as active esters. On the other hand it is too possible to react such active esters with the /? - dicarbonyl compound. In both cases one arrives to the amino acid derivatives activated on the carboxyl group and protected on the amino group, the amino acids or peptides now protected by the carboxyl group to the desired peptides are implemented.

For example, you can glycine in methanolic potassium hydroxide solution with benzoylacetone to the potassium salt of a React condensation product, which is mixed with chloroacetonitrile (cf. R. Schwyzer, HeIv., 38, p. 69 [1955]) in ethyl acetate into the benzoylacetone derivative of glycine cyanomethyl ester convicted. This cyanomethyl ester is then mixed with glycine ethyl ester in ethyl acetate Benzoylacetone derivative of glycyl glycine ethyl ester reacted from which by the action of alcoholic Hydrochloric acid benzoylacetone is split off and its ester group by mild alkaline hydrolysis is saponified so that glycylglycine is obtained as the end product.

The compound occurring as an intermediate in the peptide synthesis can, on the other hand, also by Implementation of benzoylacetone with glycyl glycine ethyl ester can be obtained, whereby their constitution is proven.

example 1

0.93 g (12.4 mmol) of glycine were hot in 5 ecm (12.4 mVal) 2.48N methanolic potassium hydroxide solution dissolved and the solution with the warm solution of 2.00 g (12.4 mmol) of benzoylacetone in 4 ecm of ethanol

(99%) added. A yellowish, crystalline precipitate separated out immediately from the light brown-red solution, which no longer went noticeably into solution when heated. A thick, yellowish, fine-needle crystal slurry had deposited within 48 hours. 2.70 g of K salt of the condensation product of glycine and benzoylacetone, ie 85% of theory, are obtained. After repeated recrystallization from ethanol (99%), the colorless, cotton wool needles melted at 250 to 255 ^° C. The salt is easily soluble in water. If it is left to stand for a long time in a sulfuric acid solution, it is split into the components benzoylacetone and glycine. It is easily soluble in methanol, slightly soluble in benzene, chloroform, acetone, ethyl acetate and ether.

IO The dried salt is hygroscopic. It was burned _{with V 2} O _{5 for analysis.}

C ₁₂ H ₁₂ NO ₃ K (257.4)

Calculated ... C 55.99, H 4.70, N 5.44;
Found ... C 56.20, H 5.07, N 5.31.

Corresponding condensation products are obtained from those listed in Table I in the same way Components. If amino acid esters are used, these are set according to the below for Glycyl-glycine-ethyl ester described method with the / f-dicarbonyl compound around, when using Amino acid ester hydrochlorides, 1 equivalent of methanolic potassium hydroxide solution is added to the batch.

Table I.

Acetoacetic ester F. yield the same ⁰ C % Glycine the same 175 94 DL-alanine the same 177 to 179 60 DL serine the same 136 85 DL-methionine Acetoacetic anilide 150 to 155 74 Glycyl-glycine-ethyl ester 113 75 Glycine Acetoacetic acid-2-chloroanilide 140 to 145 92 (Decomposition) Glycine Acetoacetic acid-2-anisidide 204 90 Acetoacetic acid-2-aminopyridide (Decomposition) Glycine 209 80 Glycine Benzoylacetone 224 93 the same (Decomposition) Glycine methyl ester Acetylacetone 89 75 DL serine the same 205 to 210 Glycine 235 to 240 DL-alanine the same 181 to 185 73 the same (Decomposition) Glycine methyl ester Acetaldehyde dimethyl acetal 62.5 78 Glycine ethyl ester Acetylacetone 66 80 Glycine Salicylaldehyde 221 to 222 80 Glycil-glycine-ethyl ester 121 to 122 88 Glycine 220 87 p-methoxy-benzoylacetone (Decomposition) yellow or red Glycine the same 275 to 280 80 the same (Decomposition) Glycine methyl ester the same 98 to 100 78 Glycyl-glycine-ethyl ester 148 to 149 97 DL-alanine Salicylaldehyde 265 to 272 84 (Decomposition) O-benzyl-DL-serine 183 97 (Decomposition)

The above-mentioned potassium salt obtained from glycine and benzoylacetone is in the following manner with Chloracetonitrile converted to the corresponding cyanomethyl ester:

0.515 g (2 mmol) of the salt were refluxed for ₂ hours with 3 ecm chloroacetonitrile and 4 ecm ethyl acetate. The suspended salt dissolved and a finely crystalline, colorless precipitate separated out. 0.14 g (potassium chloride).

The yellow mother liquor was brought to dryness in vacuo. The remaining yellow crystals were taken up hot in ethyl acetate. On cooling, well-educated, almost colorless needles 14O ⁰ C left of mp. From (0.155 g). The yellow mother liquor of the above product was washed with water and dried with sodium sulfate. After the ethyl acetate had been stripped off, yellow crystals remained, from which after the recrystallization

Sieren from ethanol-water further 0.110 g of the product of mp. 139 to 141 were obtained ^0C. The total yield is 0.265 g (52% of theory).

The cyanomethyl ester thus obtained is used to form a peptide with glycine ethyl ester in the following Wise implemented:

0.10 g (0.387 mmol) of the above Cyanomethyl ester was dissolved in 5 ecm of ethyl acetate in the warm and, after cooling, with 0.160 g (1.55 mmol) of glycine ethyl ester were added. After about 30 minutes, the colorless solution began to soften colorless, crystalline, voluminous precipitate of the glycyl-glycine-ethyl ester-benzoylacetone derivative to be deposited. After about 2 hours it was filtered off with suction and washed with petroleum ether. 0.035 g. Colorless needles from m.p. 132 to 133 ° C. Due to the mixed melting point, these needles are identical to the product described below (see below). Separated from the mother liquor after the addition of 20 ecm petroleum ether from a further 0.070 g of the product, so that the total yield 0.105 g (86.5% of Theory) fraud.

This connection can also be obtained as follows:

0.81 g (0.005 mol) of benzoylacetone were dissolved in 5 ecm of ethanol (99%) and 0.80 g (0.005 mol) of glycylglycine ethyl ester, dissolved warm in 3 ecm of ethanol (99%), was added. The yellow solution was briefly heated to boiling. On cooling to 0 ^° C., a yellowish precipitate separated out (0.3 g). A considerable amount of the needle-like product could still be obtained from the mother liquors.

The substance was recrystallized several times from very little ethanol (96%). The colorless needles then melted at 134.5 ° C. The product is easily soluble in methanol, ethanol, benzene, acetone, something also in water. It is sparingly soluble in ether and petroleum ether.

C ₁₆ H ₂₀ N ₈ O ₄
Calculated
found

(304.3)

... C 63.15, H 6.62, N 9.21;

... C 63.04, H 6.72, N 9.45.

By treating this product with dilute alcoholic hydrochloric acid and then alkaline Saponification is produced in the usual way glycylglycine.

Example 2

892.5 mg glycine (11.9 mmol) were in 5 ecm methanolic potassium hydroxide solution (11.9 meq) on the water bath brought into solution. After cooling, the solution remained clear. 1.86 g of ethyl cyclopentanone carboxylate were then obtained (11.9 mmol) was added while swirling the flask. It parted instantly a thick, colorless precipitate forms. When heated on the water bath, the substance almost went completely in solution. The cooled contents of the flask solidified after rubbing with a glass rod back to a solid mass. The sucked off crude product was with methanol and then with Ether rubbed through. Yield 2.58 g (86% of theory).

The compound is the potassium salt of the glycine cyclopentanone (2) carboxylic acid (1) ethyl ester derivative ; Potassium can be detected with perchloric acid. For cleaning, the salt was repeatedly removed from absolute Recrystallized ethanol. It decomposes when heated, giving it a dark color.

The potassium salt is easily split into its components by dilute acids: With 2,4-dinitrophenylhydrazine solution According to Shriner, the salt gives 2,4-dinitrophenylhydrazone after a few minutes of the ketoester.

Corresponding ones are obtained in the same way and taking into account the remark before Table I Condensation products from the following components:

Table II

Cyclopentanone- (2) -carboxylic acid- (l) -ethyl- the same F. yield ester the same ° C % Glycine methyl ester hydrochloride the same 52 80 Glycine ethyl ester the same 76 96 Glycine ethyl ester hydrochloride 75 to 76 78 DL-alanine the same 150 to 151 (Decomposition) Isoleucine the same 200 to 205 (Decomposition) DL serine the same 160 to 165 93 the same (Decomposition) L-glutamic acid 288 to 289 86 (Decomposition) Glycyl-glycine-ethyl ester 149 to 150 63 O-benzyl-DL-serine 162 to 165 89 (Decomposition)

The above-mentioned potassium salt of glycine-cyclo 1.25 g of the potassium salt (5 mmol) were in 10 ecm

Pentanone (2) carboxylic acid (l) ethyl ester derivative is suspended in 65 ethyl acetate with 6 ecm of chloroacetonitrile with chloroacetonitrile to form the glycine cyanomethyl ester derivative of the cyclopentanone carboxylic acid ester in the follow

implemented the way:

(about 0.08 mol) are added and the mixture is refluxed on a water bath for 1 hour. After this Time a fine powdery precipitate had

9 10

divorced. He was sucked off and gave both with other methods for the preparation of the peptide

Perchloric acid as well as a bond with silver nitrate solution, ζ. B. the carbodiimide method, can positive reaction. The yield of potassium chloride is also due to the through / 3-dicarbonyl compounds was 345 mg (4.6 mmol). use protected amino acids and their salts,

In the ethyl acetate filtrate, the solvent at 5, as Examples 3 and 4 show. 40 ° C stripped off in vacuo, the oily-crystalline

The residue was taken up again in 8 ecm of ethyl acetate. Example 3

It was shaken out twice with 5 ecm of water each time

and then dried over sodium sulfate. 0.200 g (0.776 mmol) of the benzoylacetone

After the solvent was removed a second time, io protected potassium glycine were added with 0.108 g 1.16 g (92% of theory) remained light (0.776 mmol) glycine ethyl ester hydrochloride in 10 ecm yellowish-colored needles with a crude melting point of chloroform were stirred for 1 hour at room temperature.

85 to 89 ° C. After two recrystallization, the mixture was then cooled to 0 ° C. and 0.170 g

sizing from aqueous ethanol (1: 1) melted the (0.928 mmol) dicyclohexylcarbodiimide added;

Substance at 89 to 90 ° C. Stirring was continued. After an hour they were

The compound is easily soluble in ether, benzene, precipitated potassium chloride and the urea

Acetone, methanol, chloroform and tetrahydrofuran; sucked. The chloroform mother liquor also evaporated

it is difficult to dissolve in cold ethanol and in petroleum room temperature. Yellowish crystals remained

ether and is insoluble in water. return. This was made up in about 12 ecm of ethyl acetate

With iron (III) chloride solution, a blue 20 is obtained and the solution is undissolved from the small amount

Color reaction. Part filtered off. The remaining yellowish,

CHNO (252 26) product still contaminated by urea (0.340 g)

calculated C 57 12 H 6 39 N 11 11 was recrystallized twice from ethanol (96% strength).

found '.'. '. C 51.26, H 6.48, N ll, 0L E> he colorless platelets melted at 131 to 133 ° C.

35 The mixed melting point with the benzoyl

This cyanomethyl ester is glycol-glycine-ethyl ester protected with glycine ethyl ester (cf. Beizum Glycylglycine ethyl ester derivative of Cyclopenta game 1) gave no depression. The work-up too non- (2) -carboxylic acid- (l) -ethyl ester in the following glycyl-glycine happens as indicated above. Wise implemented:

140 mg of the cyanomethyl ester (0.55 mmol) were 30 Example 4

dissolved in 1 ecm of ethyl acetate and the solution in the

Cold with 113 mg glycine ethyl ester (1.1 mmol) 1.256 g (0.005 mol) of the prepared according to Example 2,

offset. After one hour the contents of the flask were blocked by cyclopentanone (2) carboxylic acid (1) ethyl ester solidified into a colorless, solid mass. The protected potassium glycine were in 2 ecm ice water sucked and dissolved needles 35 washed with absolute ether. 3 ecm were slowly weighed while cooling air dry 150 mg (91% of theory). They added dilute hydrochloric acid (about 0.006 mol), taking melted after a single recrystallization from immediately a thick, colorless precipitate precipitated out absolute ethanol at 147 to 148 ° C. after a short time a little agglomerated. It was

_ The compound is sparingly soluble in water and quickly sucked off and washed with plenty of ice water. Ether, soluble in hot ethanol and in hot 40. After no more chloride ions in the washing water Benzene; It dissolves easily in tetrahydrofuran and was detected again with cold Chloroform. Ethanol (absolute) and dry ether through

140 mg (0.47 mmol) of the above, wash. There were 0.735 g (68% of theory) of one obtained by cyclopentanone (2) carboxylic acid (l) ethyl ester colorless, finely crystalline powder. The sheltered one Glycyl glycine ethyl esters were in 1 ecm 45 settlement point was about 146 to 148 ° C. The acid Ethanol (96%) when heated on a water bath is somewhat soluble in tetrahydrofuran and ether, hardly at all brought into solution and to this solution 0.5 ecm soluble in water, alcohol, benzene and ethyl acetate, alcoholic hydrochloric acid (0.65 mVal) given. That when trying to recrystallize it or in a vacuum Solvent was immediately dried in vacuo until it decomposes.

incipient crystallization withdrawn. To complete 50 1.07 g of the freshly prepared acid (5 mmol) were Constant deposition was converted into an ice-cooled solution of 20 ecm chloroform with 10 ecm of ether offset. With the obtained from the mother liquor and 0.6 ecm glycine ethyl ester (0.6 mmol) added. Proportions, the yield was 0.082 g (89% of after stirring for 10 minutes were added to Theory). The colorless needles melted at 180 ° until the solution was 1.1 g (5.3 mmol) of dicyclohexylcarbodiimide 185 ° C; given after single recrystallization from etha- 55. With ice cooling, it was still P / 2 hours nol ether, the melting point was 183 to 184 ° C. Stirring was continued. It had 0.41 g of dicyclohexyl die Compound proved to be deposited by melting point and urea. The chloroform was im Mixed melting point as identical to glycyl-glycine vacuum removed and the residue in again ethyl ester hydrochloride. This can be added according to the known 6 ecm chloroform. It was divorced Methods can be converted into glycyl glycine. 60 more 0.04 g of urea from. After the solution

Analogously, as described in Examples 1 and 2, evaporated medium, the residue was with higher peptides can also be built up. Washed through with dry ether. 0.71 g is obtained Implementation of the colorless needles protected by benzoylacetone (47.5% of theory). The crude glycine cyanomethyl ester (Melting point 139 to 141 ° C, see product melted at 130 to 140 ° C. After one time Example 1) with glycyl glycine ethyl ester was recrystallization from ethanol (96%) in good 5g Yield of the protected by benzoylacetone, melting point at 149 to 150 ° C. Talking glycyl-glycyl-glycine ester (melting point A mixed melting point with the

173 ° C). pentanone (2) carboxylic acid (l) ethyl ester protected

Glycyl glycine ethyl ester (Table II) gave no depression. The processing to glyeyl-glycine happened, as you can read above.

Example 5

1.3 g of cyclopentanone (2) carboxylic acid (l) ethyl ester-DL-alanine-potassium (5 mmol) were in 10 ecm ethyl acetate and 7 ecm chloroacetonitrile for 1 hour on the Steam bath heated. The potassium salt initially went almost completely into solution. After 15 minutes a fine, colorless precipitate began to separate out. To complete the implementation the reaction mixture was stirred with the magnetic stirrer at a temperature of 80 ° C. for a further 2 hours. After suctioning off, 350 mg of potassium chloride (95% of theory) are obtained.

The filtrate was concentrated in vacuo, the residue was taken up in ethyl acetate, once with dilute Sodium bicarbonate solution, extracted twice with water and dried over sodium sulfate. After the solvent had been removed again, 1.26 g (95% of theory) remained slightly yellowish in color Needles from the raw melting point 86 to 87 ° C return. After recrystallization from aqueous ethanol (1: 1) the cyclopentanone (2) carboxylic acid (l) ethyl ester-DL-alanine cyanomethyl ester melted unchanged at 86 to 87 ° C.

The compound is easily soluble in ether, benzene, ethanol, chloroform, ethyl acetate and in THF; she Difficult to dissolve in water and in petroleum ether.

2.795 mg substance, 1.690 mg H ₂ O, 6.023 mg CO ₂ ; 2.967 mg substance, 0.285 ecm N ₂ (24 ° C / 721 torr).

sets and then the solvent is stripped off in vacuo. The remaining yellow oil could cannot crystallize even after a long period of time. For further implementation it was no longer specially treated. It consists of cyclopentanone - (2) - carboxylic acid - (1) - ethyl ester - dl - alanylglycine potassium.

The potassium salt obtained in the alkaline saponification of the protected dipeptide ester C ₁₅ H ₂₄ N ₂ O _{5 (not}

ίο crystalline) was taken up in 6 cc Essigester, mixed with 9 cc chloroacetonitrile and stirred for 4 hours at a temperature of 65 ⁰ C with magnetic stirring in an oil bath. The mixture immediately turned an intense yellow. A small amount of precipitate was filtered off with suction and the filtrate was concentrated in vacuo. The brownish oil was taken up in ethyl acetate, washed twice with water and, after drying over sodium sulfate, concentrated again in vacuo. Preserves when cooling in an ice bath and rubbing

ao one 1.24 g of brownish crystals (59% of theory, based on protected dipeptide ester C ₁₅ H ₂₄ N ₂ O ₅ ).

After recrystallizing twice from ethanol- Water melted the colorless needles at 122 to 24 ° C.

The cyclopentanone (2) carboxylic acid (l) ethyl ester-DL-alanyl-glycine-cyanomethyl ester is readily soluble in ethyl acetate, benzene, acetone, chloroform, it is insoluble in ether, petroleum ether and water.

2.857 mg substance, 5.859 mg CO ₂ , 1.717 mg H ₂ O; 2.561 mg of substance, 0.318 ccmN ₂ (25 ° C / 713 torr).

C ₁₅ H ₂₁ N ₃ O ₅ (323.34)
Calculated ... C 55.72, H 6.54, N 13.00; Found ... C 55.96, H 6.72, N 13.34.

C ₁₃ H ₁₈ N ₂ O ₄ (266.29)

Calculated ... C 58.62, H 6.81, N 10.52; found ... C 58.81, H 6.77, N 10.55.

533 mg of the cyanomethyl ester C ₁₃ H ₁₈ N ₂ O ₄ (2 mmol) were dissolved in 3 ecm of ethyl acetate, and 210 mg of freshly distilled glycine ethyl ester were added. After standing for 3 hours at room temperature, the solvent was stripped off. A colorless crystal mass remained, which weighed 550 mg and melted ^{at 98 to 110 ° C.} Yield 88% of theory.

The protected dipeptide ester was recrystallized several times from methanol-water. The needles dried over phosphorus pentoxide had a melting point of 109 to 110 ^° C.

The cyclopentanone (2) carboxylic acid (l) ethyl ester-DL-analyl-glycine-ethyl ester is easily soluble in ethanol, methanol, benzene, acetone, chloroform, THF and sparingly soluble in ether, petroleum ether and water.

3.320 mg substance, 7.030 mg CO ₂ , 2.301 mg H ₂ O; 3.514 mg substance, 0.288 ecm N ₂ (23 ° C / 720 torr).

C ₂₅ H ₂₄ N ₂ O ₆ (312.36)
Calculated ... C 57.67, H 7.74, N 8.97; found ... C 57.78, H 7.75, N 8.97.

2.06 g of the protected dipeptide ester C ₁₅ H ₂₄ N ₂ O ₅ (6.6 mmol) were mixed with 5 ecm methanolic potassium hydroxide solution (8.3 meq), 3 ecm methanol and 0.5 ecm water and stirred with a magnetic stirrer. After 10 minutes, the starting product had completely dissolved. After a further 15 minutes, the reaction mixture was treated with 1.3 ecm η-hydrochloric acid

670 mg of the protected cyanomethyl ester C ₁₅ H ₂₁ N ₃ O ₅ (2.08 mmol) were dissolved in 2 ecm ethyl acetate and 0.6 ecm freshly distilled glycine ethyl ester was added. The mixture stood at room temperature for 2 hours. After cooling in an ice bath and scratching with a glass rod, the contents of the flask solidified to a solid, colorless mass which, after suctioning off and washing with a little cold ethyl acetate, weighed 490 mg (65% of theory) and melted ^{at 122 to 27 ° C.} After recrystallization from ethanol-water of the melting point of the cyclopentanone (2) -carboxylic acid (l) ethyl ester-DL-alanyl-glycyl-glycine ethyl ester was 123-26 ⁰ C.

420 mg of the above protected tripeptide ester C ₁₇ H ₂₇ N ₃ O ₆ (1.13 mmol) were mixed with 2 ecm of ethanol (96%), 1 ecm of ethanolic hydrochloric acid (1.3 m-VaI) and two drops of water. When heated gently on the steam bath, the ester went completely into solution. The solvent was then slowly drawn off in vacuo. The split off cyclopentanone (2) carboxylic acid (1) ethyl ester was removed by triturating the residue several times with ether. In the end, a solid, slightly yellowish-colored product is obtained which quickly absorbs moisture in the air.

It was dissolved in a little methanol and 1 ecm of methanolic potassium hydroxide solution was added (1.6 meq). To 30 minutes was concentrated, the colorless, powdery residue was taken up in a little water and with

- Ethanol precipitated again. After twice falling over the product showed a decomposition point of 21O ⁰ C.

The DL-alanyl-glycyl-glycine was found to be chromatographic pure.

2.365 mg substance, 3.400 mg CO ₂ , 1.383 mg H ₂ O; 2.712 mg substance, 0.501 ecm N ₂ (25 ° C / 117 Torr).

C ₇ H ₁₃ N ₃ O ₄ (203.19)
Calculated ... C 41.37, H 6.45, N 20.68;
Found ... C 39.23, H 6.54, N 19.96.

Example 6

1.48 g of L-alanine (16.6 mmol) were dissolved in 10 ecm of methanolic potassium hydroxide solution (16.6 meq) on the steam bath. 2.6 g of cyclopentanone (2) carboxylic acid (1) ethyl ester were added to the low-boiling solution. It initially fell out of a colorless precipitate which was again only after 20 minutes of stirring at a temperature of 7O ⁰ C in solution. The reaction mixture had turned an intense yellow color. After a further 5 minutes, the mixture was cooled in an ice bath. The potassium salt C ₁₁ Hi ₆ NO ₄ K is difficult to precipitate.

_ After one recrystallization from absolute ethanol, 2.65 g of fine, colorless needles are obtained (60% of theory).

The potassium salt is easily soluble in water, it dissolves differs in cold ethanol, methanol and is insoluble in ether and petroleum ether.

2.65 g of the above potassium salt (10 mmol) were suspended in 10 ecm of ethyl acetate, 9 ecm of chloroacetonitrile were added and the mixture was stirred at 80 ° C. with a magnetic stirrer for _{2 hours.} The finely crystalline precipitate was filtered off with suction and the filtrate was concentrated in vacuo. The oily residue was taken up in ethyl acetate, washed once with dilute sodium hydrogen carbonate solution and twice with water and the organic phase was dried over sodium sulfate. After removing the ethyl acetate in vacuo, the residue quickly solidified to a crystalline mass. 2.41 g of crude product (81% of theory). _

After recrystallizing twice from ethanol- Water melted the cyclopentanone (2) carboxylic acid (l) ethyl ester-L-alanine cyanomethyl ester at 61 to 62 ° C. It is soluble in ethyl acetate, alcohol, benzene, chloroform and insoluble in water and petroleum ether.

1.33 g of the above cyanomethyl ester (5 mmol) were dissolved in 3 ecm of ethyl acetate with gentle warming solved. 1 ecm of freshly distilled glycine ethyl ester was added to this. After p / 2 hours it was off the reaction mixture deposited a colorless, crystalline substance. After the suction one obtains 1.35 g of cyclopentanone (2) carboxylic acid (l) ethyl ester-L-alanyl-glycine ethyl ester (86% of theory) from the crude melting point 132 to 35 ° C.

The needles recrystallized from ethanol-water melted at 138 to 40 ^° C.

The compound is easily soluble in ethanol, methanol, benzene, acetone, chloroform, THF; she is sparingly soluble in ether, petroleum ether and water.

1 g of the above protected dipeptide ester (3.2 mmol) was dissolved in 4 ecm of ethanolic hydrochloric acid (5.2 mVal) dissolved with gentle warming. Then the solvent was immediately in vacuo deducted. The oily residue was taken up in a little water and used to remove the cyclopentanone (2) carboxylic acid (1) ethyl ester shaken out several times in ether. 9 ml of sodium hydroxide solution were added to the aqueous solution of the dipeptide ester chlorohydrate given. After 30 minutes, it was neutralized with hydrochloric acid and the solution in the desiccator Brought dry. The residue taken up in a little water can be reprecipitated with ethanol. After falling three times, the product was free from sodium chloride.

The L-alanyl-glycine was found to be chromatographic pure.

The optical rotation gave a value of % - + 49.8 (c = 2.59).

3.681 mg substance, 5.583 mg CO ₂ , 2.312 mg H ₂ O; 3.344 mg substance, 0.601 ecm N ₂ (27 ° C / 725 torr), 2.931 mg substance, 0.520 ecm N ₂ (23 ° C / 722 torr).

C ₅ H ₁₀ N ₂ O ₃ (146.1)

Calculated ... C 41.10, H 6.90, N 19.18; found ... C 41.39, H 7.03, N 19.51,

N 19.44.

Claims (1)

Claim: Process for the preparation of peptides, characterized in that amino acids or their functional derivatives are protected on the amino group by condensation with β-dicarbonyl compounds, the optionally modified carboxyl group of the enamine derivatives formed is converted into the carbonamide group in a manner known per se and then the protective group mild treatment with acids splits off. Considered publications: Angewandte Chemie, 69 (1957), p. 363. © 309 509/270 2.63