DE1543593B - Process for the preparation of histidine-containing peptides - Google Patents

Description

X, C-CH

X = F, Cl, Br

NH-Ac

or

a combination

of halogens.

The synthesis of histidine-containing peptides presents particular difficulties for several reasons: The nix-acyl derivatives of histidine and those containing histidine N-acyl peptides have the character of zwitterions with the disadvantages associated with them, such as poor solubility in aprotic solvents on the one hand and solubility in aqueous acids on the other.

It is already known that the imino group of histidine can be reversibly blocked. Acyl radicals such as the benzyloxycarbonyl radical or the tosyl radical were used for this purpose. The disadvantage of these methods, however, is that these radicals are split off under alkaline conditions when ester saponification becomes necessary and, moreover, can transfer the acyl radical as activated acid amides. The N ^lm -benzyl or N ^lm -trityl derivatives also used do not have any effect on reducing the basicity. As a result, the problematic solubility in acids is retained.

It has now been found that histidine-containing peptides can be produced by working on the imino group of histidine by 2,2,2-trihalo-l-acylaminoethyl protective groups substituted histidines or histidine-containing peptides with a free amino group and Amino acids or peptides with a free carboxyl group condensed with one another or vice versa Histidines or histidine-containing peptides with a free carboxyl group and amino acids or Peptides with a free amino group condensed with each other and optionally all or some of the splits off existing peptide protecting groups.

An imino protective group for the histidine is supposed to

a) reduce the basicity of the imidazole ring,

b) be stable under the reaction steps customary in peptide syntheses and

c) be easy to introduce and cleavable again after the peptide synthesis has ended, without peptide bonds to be attacked.

ίο These protective groups can be used as acyl components only those are used which are derived from the NH group to which they are attached, without simultaneous hydrolysis can be cleaved from peptide bonds.

Such radicals are the acyl radicals previously used in peptide chemistry, such as the benzyloxycarbonyl radical.

The introduction of these residues, for example, in Na-acyl-histidines, e.g. B. tert-Butyloxycarbonyl-L-histidine methyl ester or histidine-containing N-acyl peptides or their esters, such as N-benzyloxycarbonyl-L-valyl-L-histidine or its methyl ester, is achieved in high yield by reaction with N-acyl-1-trihaloacetaldimines (II), as such or in the reaction medium from 2,2,2-trihalo-1-ethane-sulfonyl-N-acyl-ethylamines or 1,2,2,2-tetrahalo-N-acylethylamines or other 2,2,2-trihalo-N-acyl-ethylamines, the in the 1-position carry a residue that can be eliminated as an anion (III), can be generated by the action of a base such as triethylamine. 1,2,2,2-tetrahalo-N-benzyloxycarbonyl-ethylamine can analogously to the manner described in J. Ind. Chem. Soc, Vol. 13, p. 117 (1936) getting produced.

X ₃ C-CH = N-Ac X ₃ C-CH-Y

NH - Ac

(H) (III)

In II and III, X has the above meaning and Y denotes chlorine, bromine, sulfonyl-alkyl, ureido-alkylsulfonyl SO ₂ CH ₂ CH ₂ - NH - CO - NH ₂ or the azide radical.

The N-acyl-N ^im -2,2,2-trihalogen-1-acylaminoethyl-histidines obtained in this way or the corresponding peptide derivatives have only low basicity and show a much greater solubility in organic solvents than those not substituted in the imidazole ring Links. So is z. B. N-benzyloxycarbonyl-L-valyl-L- (N ^lm -2,2,2-trifluorol-benzyloxycarbonylamino-ethyl) histidine is sparingly soluble in water at pH 4, while the N ^lm -unsubstituted compound is soluble at pH 4 is. The same N ^im -substituted compound cannot be shaken out even from a solution in chloroform with 0.5 mol of citric acid. In contrast, it is soluble in dilute aqueous hydrochloric acid.

Of the tri-halo-acylaminoethyl groups are the trifluoro compounds are particularly preferred when alkaline hydrolysis occurs in the course of peptide syntheses is made.

The condensation methods customary in peptide chemistry are used for the production of the peptides according to the invention such as the carbodiimide or azide method and, for example, the mixed method Anhydrides or the activated ester in question.

The cleavage of the new protective groups succeeds under those conditions which are necessary for the cleavage characteristic of the acyl residues present in them

3 4

are. Compounds of 20 ml of absolute dimethylformamide in solution are primarily formed here. Then

Structure is added 1.0 g (10 mmol) of triethylamine in 10 ml of abso-

NJ ₇ -CH ₂ - CH - CO— lutem dimethylformamide added. After 2 hours

Ii j; I let stand at 20 ° C, the precipitated triethyl

\ / NH ⁵ ammonium chloride filtered off, the solution in the oil pump

I evaporated in vacuo, the residue taken up in ethyl acetate, the ethyl acetate solution with 0.5MoI lemon

X ₃ C CH NH ₂ acid and water, dried with sodium sulfate and evaporated. The residue will be off

which are recrystallized by adding water or alcohols in benzene / petroleum ether. Yield 88%,

Trihalogenacetaldehyd hydrate or in trihalo mp. 9O ⁰ C, sinter from 75 ° C. [oc] | f ₆ : -8 ° C (c = 1 in

acetaldehyde hemiacetals, ammonia and the histidine methanol),

containing peptide with free imidazole ring broken down. The same connection can also be established as follows

So z. B. der2,2,2-trifluoro-l-benzyloxycarbonyl- are:

ethyl residue by catalytic hydrogenation in methanol 15 0.633 g of the methyl ester prepared according to Example 1

with the addition of a little glacial acetic acid or by heating, 15 ml of dioxane, 5 ml of water and 2.5 ml of in

with trifluoroacetic acid (45 minutes reflux) and dige-NaOH at 20 ° C for 2 hours. Then will

can be removed with water or methanol. evaporated in vacuo (bath up to 30 ° C) and in water

The compounds prepared according to the method dissolved. With 0.5 mol of citric acid, the pH is adjusted to 4

can be used both as a drug and as an intermediate pro-20 acidic, whereby the N-benzyloxycarbonyl-l-valyl-L-

products can be used for further peptide syntheses. (N ^lm - 2,2,2 -trifluoro-1 - N - benzyl oxycarbonylamino-

ethyl) histidine precipitates. Yield 92%, mp. 90 C ⁰

Example 1 after sintering from 75 ° C.

N-Benzyloxycarbonyl-L-valyl-L- (N ^lm -2,2,2-trifluor- ₂₅ Example 4

l-N-benzyloxycarbonylamino-ethyty-histidine-methyl-

ester N-benzyloxycarbonyl- L- (N ^im -2,2,2-trichloro-lN-ben-

■ _Λ , ο -ι ι τ -a -IUi χι t. 1 u 1 zyloxycarbonylamino-ethyO-histidine-methyl ester

0.6 g 2,2,2-fluorine-1-chloro-N-benzyloxycarbonyl-

ethylamine are abs in 15 ml. Tetrahydrofuran ge This compound is made from N-benzyloxycarbonyl-

solves. To this, 0.21 g of triethylamine are added and 30 L-histidine methyl ester and 1,2,2,2-tetrachloro-N-benzyl-

from the precipitated triethylammonium chloride to oxy-carbonyl-ethylamine in tetrahydrofuran with addition

5 minutes in the solution of 0.8 g of N-benzyloxycar- set of triethylamine prepared analogously to Example 2,

bonyl-L-valyl-L-histidine methyl ester in 15 ml of abs. Yield 85 ° / ₀ , m.p. 65 to 70 ° C.
From tetrahydrofuran. After standing for 2 hours

Room temperature (2O ⁰ C) the filtrate in a vacuum 35 B is ρ ie 1 5

evaporated the residue dissolved in chloroform, the _N -Benz _y loxycarbonyl-L-valyl-L- (Ni - 2,2,2-trichloro-

Chloroform solution with 0.5 mol of citric acid and lN-benzyloxycarbonylamino-ethy ^ -histidine
Washed water, dried with sodium sulfate and

evaporated. The residue is made from benzene / petroleum- The compound is made from N-Ben-

ether recrystallized. Yield 83 ° / ₀ , m.p. 62 to 40 zyloxycarbonyl-L-valyl-L-histidine hydrochloride with

64 ⁰ C. [offs: -10.0 (c = 1 in methanol). 1,2,2,2-tetrachloro-N-benzyloxycarbonyl-ethylamine obtained. Yield 90%, mp. 55 to 6O ⁰ C.
Example 2

N-Benzyloxycarbonyl-L-valyl-L- (N ^lra -2,2,2-trifluoro- e 1 s ρ 1

lN-benzyloxycarbonylamino-ethyrj-histidine-methyl- N-benzyloxycarbonyl-L-valyl-L- (N ^lm -2,2,2-trifluoro-

ester * l-N-benzyloxycarbonylamino-ethyO-histidyl-L-leu-

... _r / ~>, in χι τ. 1 . 1 . . cin-benzyl ester

0.805 g (2 mmol) N-benzyloxycarbonyl-L-valyl-L-

histidine methyl ester and 0.54 g of 2,2,2-trifluoro-1-chloro-0.62 g (1 mmol) of N-benzyloxycarbonyl-L-va-lyl-L N-benzyloxycarbonyl-ethylamine in 15 ml of abso- 50 (N ^im - 2,2,2 - trifluoro -1 - N - benzyloxycarbonylaminolutem tetrahydrofuran. Add 0.202 g of ethyl) histidine and 0.393 g (1 mmol) of L-leucine-benzyl-triethylamine, dissolved in 5 ml of absolute tetrahydrofuran - Ester-p-toluenesulfonate are added in 15 ml of absolute tefuran and trahydrofuran is suspended for 2 hours at room temperature. Then 0.1 g of tri- (2O ⁰ C) are added. It is then evaporated in vacuo, ethylamine (ImMoI) is dissolved in 5 ml of absolute tetrahydrate residue in chloroform and furan is added with water, a clear solution being formed. Cool the triethylammonium chloride formed was shaken at -20 ⁰ C., and 0.246 g (1.2 mmol) Dicycloanschließend with 0.5 moles of citric acid and water hexylcarbodiimid and 0.23 g (2 mmol) of N-Hydroxygewaschen, dried with sodium sulfate, evaporated succinimide. This method corresponds to the process and the residue from benzene / petroleum ether is recrystallized in patent application F 48 611 IVb / 12 q siert. Yield 85%, melting point 62 to 64 ° C. 60 (Fw 5020 A). After stirring for ^{2 hours at -20.degree}

is stored overnight at + 4 ° C and removed from

Example 3 filtered off dicyclohexylurea which has precipitated. The Lö-

N-Benz _y loxycarbonyl-L-valyl-L- (Ni ™ -2,2,2-trifluoro ^sun S ^is evaporated ⁱⁿ vacuo, the residue in

l-N-benzyloxycarbonylamino-ethyl) -histidine added to ethyl acetate and the ethyl acetate solution with

, · "■ 65 0.5 mol citric acid, saturated sodium hydrogen

• Washed 2.12 g (5 mmol) of N-benzyloxycarbonyl-L-valyl-L-hicarbonate solution and water and

stidine hydrochloride and 1.44 g (5 mmol) 2,2,2-Trifluoi- evaporates. The residue is made from benzene petroleum ether

1-chloro-N-benzyloxycarbonyl-ethylamine are recrystallized in. Yield 80 to 85%, m.p. 70 ⁰ C.

I 543 593

■ The tripeptide derivative is used to remove all protective groups subjected to catalytic hydrogenation in methanol using Pd-charcoal as a catalyst. The free tripeptide is identical to an authentic comparison sample.

Example 7

N-tert-butyloxycarbonyl- L- (N ^lm -2,2,2-trifluoro-

l-N-benzyloxycarbonylamino-ethyO-histidine-methyl-

ester

0.8 g (3 mmol) of N-tert-butyloxycarbonyl-L-histidine methyl ester and 0.8 g (3 mmol) of 2,2,2-trifluoro-1-chloro-N-benzyloxycarbonylethylamine are dissolved in 15 ml of absolute tetrahydrofuran dissolved, whereupon 0.303 g of triethylamine in 5 ml of absolute tetrahydrofuran is added. ^{After standing for 2} hours at 20 ° C., it is evaporated in vacuo, taken up in chloroform, washed with water, 0.5 M citric acid solution and water, dried with sodium sulfate and evaporated. The residue is recrystallized from benzene / petroleum ether. Yield 90 ° / _0, mp. 35 to 4O ⁰ C.

Example 8

Na-tert-butyloxycarbonyl-N ^lm - (2,2,2-trifluoro-l-tert-butyloxycarbonylamino-ethyl) -L-histidine-methyl-

ester

a) 0.363 g of 2,2,2-trifluoro-1-ethanesulfonyl-N-tert-butyloxycarbonyl-ethylamine and 0.336 g of N-tert-butyloxycarbonyl-L-histidine methyl ester are dissolved in 5 ml of abs. Dissolved tetrahydrofuran and mixed with 0.14 ml of triethylamine. After standing overnight at room temperature, it is evaporated in vacuo, the residue is taken up in ethyl acetate and the ethyl acetate solution is washed successively with dilute citric acid solution, with sodium hydrogen carbonate solution and water, dried with sodium sulfate and evaporated. Yield 0.50 g (86%), 50 to 6O ⁰ C. mp.

C ₁₉ H ₂₉ F ₃ N ₄ O ₆ (466.5)
Calculated: C 49.04 H 6.27 N 12.02
Found: C 49.02 H 6.32 N 11.94

N «-tert-butyloxycarbonyl-N ^lm - (2,2,2-trifluoro-1-tert-butyloxycarbonylamino-ethyl) -L-histidine

b) 0.933 g of N-tert-butyloxycarbonyl-N ^im - (2,2,2-trifluoro-1-tert-butyloxycarbonylamino - ethyl) - L-histidine methyl ester are dissolved in 12 ml of dioxane and 6 ml of 0.5 η NaOH 1 Stirred for hour. After the dioxane has been evaporated off in vacuo, the pH is brought to 4 with 0.5 mol of citric acid solution and the precipitate is filtered off. It is dissolved in ethyl acetate and, after washing the solution with water and drying with sodium sulfate, it is evaporated. 1 vol: from ether / petroleum ether 81 recrystallized, the yield is 0.68 g (75 ° / _0), mp to 115 ⁰ C, Rf 0.39 (thin layer chromatography with benzene-Essigester-glacial acetic acid-water, 10:10. 2 .).

C ₁₈ H ₂₇ F ₃ N ₁ O ₆ (452.5)

Calculated: C 47.83 H 6.02 N 12.40
Found: C 47.79 H 6.05 N 12.44

Preparation of the starting material 2,2,2-trifluoro-1-acetoxy-N-tert.-butyloxycarbonyl-ethylamine cc) 2,2,2-trifluoro-1-acetoxy-N-tert.-butyloxycarbonyl

ethylamine

1.3 g of 2,2,2-trifluoro-1-hydroxy-N-tert-butyloxycarbonylethylamine are dissolved in 5 ml of abs. Dissolved pyridine and treated with 0.66 g of acetic anhydride. After 30 minutes at room temperature is "15 minutes heated to 5O ⁰ C and allowed to stand overnight. Then ίο is evaporated in vacuo and the residue taken up in ethyl acetate. The ethyl acetate solution is washed with aqueous citric acid, sodium hydrogen carbonate solution and water and dried with sodium sulfate. After evaporation in vacuo, the residue crystallizes on trituration. Yield 1.4 g (93%), m.p. 52 to 54 ° C.

C ₉ H ₁₄ F ₃ NO ₄ (257.2)

Calculated: C 42.02 H 5.49 N 5.45
Found: C 42.25 H 5.74 N 5.29

ß) 2,2,2-trifluoro-1-ethylmercapto-N-tert-butyloxycarbonylethylamine

To 4 g of 2,2,2-trifluoro-1-acetoxy-N-tert-butyloxycarbonyl-ethylamine in 50 ml of abs. Tetrahydrofuran is given 2.8 g of ethyl mercaptan and 10 ml of triethylamine. After standing overnight at room temperature, the mixture is evaporated in vacuo and the residue is taken up in ethyl acetate. The ethyl acetate solution is successively with dilute hydrochloric acid, sat. Washed sodium hydrogen carbonate solution and water, dried with sodium sulfate and evaporated. From petroleum ether to crystallize under refrigeration 3 g (70%), mp. 47 ⁰ C. The IR spectrum and the proton nuclear magnetic resonance spectrum consistent with the structure of the compound same.

C ₉ H ₁₆ F ₃ NO ₂ S (259.3)
Calculated: C 41.68 H 6.22 N 5.41
Found: C 41.55 H 6.10 N 5.25

«) 2,2,2-Trinuor-1-ethanesulfonyl-N-tert-butyloxycarbonyl-ethylamine

3 g of 2,2,2-trifluor-1-ethylmercapto-N-tert-butyloxycarbonylethylamine are dissolved in 35 ml of glacial acetic acid and 7 ml of acetic anhydride and with ice cooling with 3 g 34 percent hydrogen peroxide added. One lets gradually come to room temperature and work up after standing overnight. The solution is evaporated in vacuo at room temperature, the residue taken up in ethyl acetate, from Insolubles are filtered off and the solution is evaporated again. The residue crystallizes at Rub with petroleum ether. Yield 1.2 g (35%), melting point 134 ° C. The IR and proton resonance spectrum are consistent with the structure of the compound.

C ₉ H ₁₈ F ₃ NO ₄ S (291.3)

Calculated: C 37.10 H 5.54 N 4.82
Found: C 37.09 H 5.73 N 4.73

The 2,2,2-trifluoro-hydroxy-N-tert-butyloxycarbonylethylamine used in cc) is prepared from tert-butyloxycarbonylamide and gaseous fluoral analogously to Chem. Ber. 99, (1966), p. 1944, where care must be taken to ensure that it is acid-free. Yield 77%, mp. 117-118 ⁰ C (Essigester / petroleum ether).

Claims (2)

Patent claims: 1. Process for the preparation of histidine-containing Peptides, characterized in that one on the imino group of the histidine by 2,2,2-trihalo-1-acylamino-ethyl protective groups substituted histines or histidine-containing peptides with a free amino group and amino acids or peptides with a free carboxyl group condensed with one another or vice versa corresponding Histidines or histidine-containing peptides with a free carboxyl group and amino acids or Peptides with a free amino group condensed with each other and optionally all or some splits off the existing peptide protective groups. 2. The method according to claim 1, characterized in that the trihalogen compounds Trifluoro and the trichloro compounds can be used. The 2,2,2-trihalo-1-acylaminoethyl groups used in the process according to the invention meet these requirements (I)