DE1917939A1 - Process for the synthesis of cystine peptides - Google Patents

Description

FARBWERKE HOECHST AG., Formerly Meister Lucius & Brüning File number: - Fw 6049

Date: March 21, 1969
Dr.Tg/fe

Process for the synthesis of cystine peptides

The trityl group has already been used several times for the intermediate protection of the mercapto function of cysteine in the construction of higher peptides. To convert the S-protected cysteine peptides into the cystine peptides, the trityl group was split off with strong acids or with mercury or silver salts and subsequently oxidized, for example with atmospheric oxygen, iodine or diiodoethane. Furthermore, an S-trityl bond can also be cleaved with iodine in ethanol or methanol (J. Chenu Soc. 74 , (1952), page 1862; Helvetica Chimica Acta _51 (1963), pages 2061-2064). However, this method can only be used with relatively small peptides protected on the amino or carboxyl group, since higher peptides and above all unprotected on the amino and carboxyl groups are no longer soluble in methanol. In addition, the iodoxydation in methanol proceeds very slowly (1 hour at room temperature) in spite of an excess of iodine, and peptide methyl esters are formed as by-products. The use of water, on the other hand, was believed to have to be avoided, since it was assumed that the S-trityl group was resistant to iodoxydation in aqueous solvents, which is known from J. Amer. Chem. Soc. j37 (1965), pages 4923 and 4927, where Z-Cys- (Trit) -Cys (DPM) -NH-NH ₂ and H-Ala-Gly ~ VaI-Cys (Trit) -Ser-OCHo in hydrous dioxane linked to the corresponding heptapeptide by means of iodine. The addition of water to organic solvents has also been avoided because methionine and tyrosine are attacked by iodine in aqueous solvents (HeIv. Chim. Acta 51 (1968) pages 2061-2064).

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Surprisingly, it has now been found that at room temperature in aqueous acetic acid, the splitting off of the S-trityl group and the simultaneous oxidation to cystine peptides proceed much more rapidly than e.g. in methanol. Methionine and tyrosine are also not oxidized under these conditions.

The invention thus relates to a method for production Cystine-containing peptides, characterized in that a peptide with at least one S-TrityIcySteinruppe is obtained by oxidation with 1-2, preferably 1 molar equivalent of iodine in aqueous preferably 40-95% acetic acid removes the trityl group with the formation of Cleaves cystine peptides.

The yield depends on the water content of the acetic acid and the amount of iodine added (see Table 1).

Tabel

Elimination of S-trityl groups and simultaneous oxidation of H-Leu-Cys (Trit) -Gly-OH with iodine to form the corresponding cystine peptide in acetic acid (reaction time: 15 minutes at room temperature).

acetic acid Equivalent iodine j ! m prey 50 percent 1 92% 50 percent 1.5 87.7% 80 percent 1 89.2 % 90 percent 1 83.6% 95 percent 1 80.3% 95 percent 1.5 29.5 % 100 percent 1 18.7% 100 percent 2 30.0%

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As can be seen from Table 1, the yield falls with increasing acetic acid concentration. An excess of iodine in aqueous Acetic acid also surprisingly reduces the yield. Methionine, on the other hand, becomes very slow in aqueous acetic acid attacked. Two drops of a 0.In J / KJ solution are 100mg Methionine in 10 ml 40 percent Acetic acid does not completely decolorize even after one hour, while in aqueous solution it does after 5 seconds the brown iodine color of the added I / KI solution disappears completely.

It is advantageous to oxidize with an aqueous 0. In Jp / KJ solution I. or an oil / acetic acid solution. The S-trityl-cysteine derivatives are dissolved or suspended in aqueous acetic acid and mixed with the iodine solution. The mixture is stirred for 15 minutes at room temperature and works up appropriately. Oxidation-stable solvents such as dioxane, methanol or ethanol can if necessary as Solubilizers are added.

As further building blocks of the peptides containing S-trityl-cysteine, all amino acids found in naturally occurring peptides can be used in their L or D form Semi-synthetically accessible amino acids such as ^ -Methyl- ä alanine ^ -Methyl-3,4-dioxy-L-phenylalanine or ß-chloro-L-alanine is possible. Further functional groups can be protected according to the usual rules in peptide chemistry (cf. E. Schröder and K. Lübke, The Peptides, New York and London 1965, Volume I, especially pages 3-75), if this appears necessary *

The products of the process can be used as therapeutic agents or as intermediates for the production of other therapeutically valuable peptides, such as oxytocin, vasopressin, insulin or Thyreocalcitonin can be used.

The abbreviations of the amino acids used in the foregoing and in the following correspond to the international nomenclature, also

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the following abbreviations are used:

Boc = tert-butyloxycarbonyl

Tritol = trityl (= triphenyl)

ONB = 4-nitro-benzyl ester

DCC = dicyclohexylcarbodiimide

DCHA = dicyclohexylamine

TLC = thin layer chromatography

The invention is illustrated in more detail by the following examples.

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Experimental part

I. Leu-Cys-GIy

Leu-Cy s-GIy
1. Boc-Cys (Trit) -Gly-ONB

25.4 g of H-Gly-ONB-HBr and 12.25 ml of triethylamine are added to a solution, cooled to -10, of 40.2 g of Boc-Cys (Trit) -OH, stir for 10 minutes at -10 and then add a solution of 19.3 g of DCC in methylene chloride. The mixture is stirred for 4 hours at 0 and left stand overnight at room temperature. On the other day, the precipitate is filtered off with suction and the filtrate is concentrated in vacuo. The residue is taken up in ethyl acetate and extracted with 2N citric acid, sodium bicarbonate solution and water, dried with sodium sulfate and concentrated the ethyl acetate solution. The remaining oil is dissolved in acetone and chromatographed on aluminum oxide (Woelm, neutral). Yield 49.5 g oil (87%); DC: Uniform.

2. H-Cys (Trit) -Gly-ONB-tosylate

4.65 g of Boc-Cys (Trit) -GIy-ONB are dissolved in anhydrous trifluoroacetic acid solved. The mixture is left to stand at room temperature for 1 hour and then concentrated in vacuo. With ether becomes 2 times distilled and the residue dissolved in ethyl acetate. 1.7 g of p-toluenesulfonic acid monohydrate are added and it is precipitated Ether out the tosylate.

Yield 3.7 g (71.5%), m.p. 137-140 °, [cO] ^ + 37.95 ° (c = 2, methanol).

3. BoC-LeU-CyS (TrIt) -GIy-ONB

To a solution, cooled to 0 °, of 14.8 g of Boc-Leu-OH in 100 ml of methylene chloride are added to a likewise cooled solution

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14.8 g of Boe-Leu-OH ± n 100 ml of methylene chloride are added to a likewise cooled solution of 7 g of DCC in a little methylene chloride. The mixture is left to stand at 0 for 2 hours, the precipitate is filtered off with suction, the filtrate is cooled and 25 g of H-Cys (trit) -Gly-ONB tosylate and 4.8 ml of triethylamine are added. You leave the approach overnight. Stand in the refrigerator, concentrate the next day in a vacuum and distribute the residue between ethyl acetate and water. The ethyl acetate phase is shaken out as in I 1, dried and concentrated. The residue is triturated with ether. Yield 25.6 g (97%), m.p. 178-181 °, \ _s (j] _J) -13.1 ° (c = 2, methanol)

4. H-Leu-Cys (TrIt) -GIy-OH-IH ₂ O

16.6 ml of 2N sodium hydroxide solution are added dropwise to a suspension of 25.6 g of Boc-Leu-Cys (Trit) -GIy-ONB in 120 ml of dioxane / water (8: 2) over a period of about 4 hours (thymolphthalein as an indicator). When the addition is complete, it is neutralized with 2N citric acid and concentrated in vacuo. The residue is partitioned between ethyl acetate and 2N citric acid. The ethyl acetate phase is washed with water, dried with sodium sulfate and concentrated. The residue is dissolved in trifluoroacetic acid. After standing for 1 hour, the trifluoroacetic acid is well distilled off in vacuo and re-distilled twice with ether. The residue is triturated with sodium acetate solution, a crystalline substance precipitating, which is suctioned off and from 70 percent. Alcohol is recrystallized. Yield 13.8 g (75%), melting point 161-165 °, {o6] _D -2.2 (c = 1, glacial acetic acid), TLC: uniform

5. Leu-Cys-GIy

-Cvs-GIy

, 0.
-Cys-GIy

5 CHoCOOH
Leu- '"" • ~ "■ *"

a) To a solution of 551.7 mg H-Leu-Cys (Trit) -Gly-OH-1 HO (1 mmol) in 5 ml of glacial acetic acid are added to 15 ml of water and 10 ml

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a 0.1 η J _o / glacial acetic acid solution. The reaction mixture is stirred for 15 minutes at room temperature and then concentrated in vacuo. The remaining residue was distributed between water and ether. The aqueous phase is stirred with Amberlite IR 45 (loaded with acetate ions) until the. Solution no more iodide can be detected. The exchanger is filtered off with suction and the filtrate is concentrated in vacuo, the residue is triturated with ether and filtered off with suction. Yield 281 mg (92%), m.p. 212-215 (decomp.) TLC: uniform

b) To a solution of -551.7 mg of H ~ Leu-Cys (Trit) -Gly-OH.1H ₃ O (1 mmol) in 5 ml of glacial acetic acid are added 5 ml of water and 10 ml of a 0.1 η aqueous J “/ KJ -Solution. The reaction mixture is concentrated in vacuo and the residue is partitioned between water and ether. The aqueous phase is concentrated in vacuo. The residue is freed from inorganic constituents in 5 percent acetic acid by filtration through Sephadex G IO (column 90 χ 1.5 cm). Yield 277 mg (91%) TLC: Uniform and identical to the material obtained from Experiment 5a.

II. VaI-Cys-AIa

VaI-Cys-AIa

1. BoC-VaI-CyS (TrXt) -OH-DCHA

To a suspension of 7.25 g of S-trityl-cysteine (20 mmol) in ml of dioxane are added to 10 ml of 2N NaOH, the mixture is stirred until everything is dissolved and then 3.15 g of Boc-VaI-OSu (10 mmol) are added, and the mixture is stirred Day at room temperature and the next day narrow the neutralized Reaction mixture. The residue is partitioned between ethyl acetate and 2N citric acid. Failure, used in excess S-trityl-cysteine is suctioned off and the ethyl acetate phase shaken out with water, dried with sodium sulfate

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and narrowed. There remains 3 g of oil.

The oil is dissolved in ether. Insolubles are filtered off. Now DCHA is added until it has a basic reaction, insoluble material is filtered again and the DCHA salt is then precipitated with petroleum ether.
Yield 5.7 g (76.6%), amorphous, TLC: uniform

2. BoC-VaI-CyS (TrIt) -AIa-ONB

An ethyl acetate solution of 5.7 g of Boc-Val-Cys (Trit) -OH DCHA (7.66 mmol) was extracted with 2N citric acid and water, dried with sodium sulfate and concentrated. The residue is dissolved in methylene chloride and 3.74 g H-Ala-ONB tosylate (9.4 mmol) was added. Add while stirring now at 0 ° 1.24 ml of triethylamine (9.1 mmol), 2.1 g of N-hydroxysuccinimide (13.2 mmol) and a cold solution of 2.0 g DCC in methylene chloride. The mixture is left to stand at 0 ° for 2 hours and at room temperature overnight and is worked up as for I 1. Yield 5.15 g (87.5%), melting point 175-177 ° For purification, the substance was dissolved in ethyl acetate and Chromatographed over neutral aluminum oxide (Woelm, Aktivst. I).

Yield 4.0 g (68%), melting point 185-186 °, [σό] _β -19.6 ° (c = 2, dimethylformamide).

3. H-Val-Cys (Trit) -rAIa-OII, 2.5 CH ₃ COOH

6 + 6 g of Boc-Val-Cys (Trit) -Ala-ONB are saponified in sodium hydroxide solution as in I 4 ml and then treated with trifluoroacetic acid. Work-up as for 14. Avis yield 4.6 g, recrystallized from glacial acetic acid / ether, mp.

146-7 °.

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4. Val-Cys-Ala

j, 1 CH., COOH, 1 H ₀ O

VaI-CVs-Ala

To a suspension of 683.8 mg H-Val-Cys (Trit) -Ala-OH, CHoCOOH in 30 ml of a mixture of acetic acid / water 1: 2, 10 ml of an O, In Jg / glacial acetic acid solution are added, and the mixture is stirred for 15 minutes at room temperature and works up as in I 5a. Yield 285 mg (86.5%), m.p. 227-230 ° (dec.) DC: Uniform

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Claims (1)

- 10 - Fw 6049 Claim The invention thus relates to a process for the production of cystine-containing peptides, which is characterized in that one from a peptide with at least one S-Tritylcystexngruppe by oxidation with 1-2, preferably 1 molar equivalent of iodine in aqueous preferably 40-95% acetic acid to form the trityl group split off from cystine peptides » 009842/1887