CS209740B1 - Synthetic substrates for proteolytic enzymes - Google Patents

Abstract

The present invention relates to synthetic substrates for proteolytic enzymes of the general formula wherein A and B, the same or different, denotes aromatic amino acid residues, especially phenylalanine, tyrosine or tryptophan, X and Y, the same or different, represent residues aliphatic amino acids, especially glycine, alanine or leucine and R is methyl or ethyl.

Description

The invention relates to synthetic substrates for proteolytic enzymes of the general formula:

NH

CO-His-A-BXY-OR wherein A and B, the same or different, denote aromatic amino acid residues, especially phenylalanine, tyrosine or tryptophan, X and Y, the same or different, denote aliphatic amino acid residues, especially glycine, alanine or leucine and R are methyl or ethyl.

The invention relates to synthetic substrates for proteolytic enzymes of the general formula IJ-CO-His-ABXY-OR O ^NM wherein A and B, the same or different, denote aromatic amino acid residues, in particular phenylalanine, tyrosine or tryptophan; X and Y, identical or different, denote aliphatic amino acid residues, in particular glycine, alanine or leucine, and R denotes methyl or ethyl.

Synthetic substrates of the above formula are cleaved by proteolytic enzymes such as pepsin, gastricsin, cathepsin D, etc., in the acidic pH range of 1 to 6 are useful, for example, in the clinical diagnosis of gastric juice, enzyme production, enzymatic research, etc.

The specificity of the enzymatically cleaved enzymatic substrates in the acidic pH range is characterized mainly by the peptide bond of the two aromatic amino acids, most commonly a phenylalanine-tyrosine or phenylalanine-tryptophan combination. The first types of said substrates were N-acyl dipeptides of said aromatic amino acids with different aliphatic acyl residues. Fruton et al, Adv. Enzymol. 44, 1-36 (1976), synthesized tripeptides and later higher peptides with the N-terminal benzyloxycarbonyl residue of histidine. This additional part created a new type of synthetic substrates, the so-called cationic substrates. due to the presence of an ionized imidazole residue of histidine in the acidic pH range.

Later, it has been found that the active sites of the enzyme (generally) include 5 to 7 amino acid residues; the corresponding length of the synthetic substrates was thus determined.

It has been found that when the hydrophobic residue, e.g., benzyloxycarbonyl, is replaced in the N-terminal part of the substrate by a hydrophilic pyroglutamic acid residue in combination with histidine, i.e. the pyroglutamyl-histidine sequence in 4-6 amino acid residues, not only high solubility is achieved 3 moles, but mainly the kinetic constants of the synthetic substrates are particularly preferred. This treatment in the N-terminal part is of additional importance as it protects the substrate from enzymatic degradation. The kinetic constants of the new synthetic substrates have values / e.g. Michaelis constants u / K _m 1.0 to 1.3 mm, in the catalytic constants k _cat 40 SecB

The present invention provides synthetic substrates for proteolytic enzymes formula _of Anh ^ ^co_Hís ~ ^{and 'b} ~ ^{x' y 'or} wherein the symbols A and B, identical or different, denote radicals of aromatic amino acids, especially feny1alaninu, tyrosine, or tryptophan, X and Y , the same or different, denotes aliphatic amino acid residues, in particular glycine, alanine or leucine, and R denotes methyl or ethyl.

The advantage of the synthetic substrates according to the invention is that they can be determined by the ninhydrin method (but also by other color reactions sensitive to - (- amino)). Compared to known cationic substrates which also utilize color ninhydrin reactions, e.g. the Fruton substrates, the substrates of the invention have 2 orders of magnitude higher catalytic constant to _cat . The sensitivity of the assay method is also made possible by a 2 to 3 fold higher extinction coefficient of the C-terminal cleavage product, eg, phenylalanyl-alanyl-leucine methyl ester, compared to a chromogenic substrate using changes in absorbance of 4-nitrophenyl-alanine.

Preferred kinetic constants of the synthetic substrates of the invention allow, for example, the determination of pepsin or other similar enzymes at very low concentrations,

1x10 ”9 mol / liter.

The starting materials used to synthesize the substrates are either known and commercially available compounds or can be prepared analogously to the literature. Synthesis of individual intermediates and final products is feasible by methods generally known from amino acid and peptide chemistry. The end products are substances crystallizing from common organic solvents, well characterized by melting point and amino acid analysis.

Further details of the preparation of the synthetic substrates according to the invention can be seen from the following examples:

Examples

EXAMPLE 1 a) Benzyloxycarbonylphenylalanyl-phenylanyl-glycyl glycine methyl ester.

To a solution of 4.46 g of benzyloxycarbonylphenylalanyl-phenylalanine and 1.4 g of N-hydroxybenztriazole in 20 ml of dimethylformamide was added a solution of 1.82 g of methyl glycyl glycine ester hydrochloride and 1.4 ml of N-ethylpiperidine in 20 ml of dimethylformamide. To the mixtures cooled to 0 ° C was added 2.28 g of N, N'-dicyclohexylcarbodiimide and the mixture was stirred at 0 ° C for 2 hours. After standing for 20 hours at room temperature, the precipitated N, N * -dicyclohexylurea was filtered off, the filtrate was evaporated in vacuo, the oily residue was dissolved in a mixture of 150 ml of ethyl acetate and 10 ml of water, the organic phase was extracted with 1 M acid. hydrochloric acid, water 5% sodium bicarbonate solution, dried over anhydrous sodium sulfate and evaporated in vacuo. The residue was crystallized from 2-propanol and petroleum ether. 4.39 g (76% of theory) of the product having a melting point of 144 DEG -146 DEG C. were obtained.

b) Phenylalanyl-phenylalanyl-glycyl-glycine methyl ester hydrobromide.

To a solution of 2.8 g of the preceding product in 2 ml of glacial acetic acid was added 5 ml of a 35% solution of hydrogen bromide in glacial acetic acid and after 1 hour at room temperature 100 ml of ether was added to the reaction mixture. The precipitated hydrobromide was filtered off, washed with ether and dried in a desiccator over sodium hydroxide and phosphorus pentoxide. 2.5 g (95% of theory) of hydrobromide, m.p. 196-198 ° C, were obtained.

c) Pyroglutamyl-histidyl-phenylalanyl-phenylalanyl-glycyl-glycine methyl ester.

To a solution of 1.54 g of pyrogututyl-histidine hydrazide in a mixture of 30 ml of dimethylformamide and 2 ml of conc. hydrochloric acid, cooled to -20 ° C, was added a solution of 0.385 g of sodium nitrite in 1.54 ml of water. After stirring at -20 ° C for 10 minutes, the pH of the reaction solution was adjusted to 6.9 with N-ethylpiperidine, and a solution of phenylalanyl-phenylalanyl-glycyl-glycine methyl ester hydrobromide and 0.7 mL of N-ethylpiperidine in 30 mL of dimethylformamide was added. The reaction mixture was then allowed to stand for 20 hours at 0 ° C under vacuum. evaporated. The residue was dissolved in 12 mL of methanol and the product was purified by column chromatography on silica gel, equilibrated with methylene chloride and methanol (4: 1), at a flow rate of 20 ral / hr. · Homogeneous fractions (positive for Pauly reagent) were combined, vacuum evaporated and the residue was crystallized from 2-propanol and ethyl acetate. 1.4 g (36% of theory) of the product having a melting point of 143 DEG -146 DEG C. were obtained.

Amino acid analysis: glutamic acid (1.07), histidine (1.00), phenylalanine (1.89), glycine (2.02).

Example 2

Chromatography of the products was performed on a thin layer of silica gel in butanol-acetic acid-water (4: 1: 1) (Sj) and butanol-acetic acid-pyridine-water systems (15: 3: 10: 6) (S ₂ ).

a / Benzyloxycarbonylphenylalanyl-phenylalanyl-alanyl-alanine methyl ester

An analytical sample, m.p. 187-192 ° C, was prepared analogously to Example 1a (m.p. 187-192 ° C), and melted at 117-218 ° C after crystallization from 2-propanol.

(b) Phenylalanyl-phenylalanyl-alanyl-alanine methyl ester hydrobromide

It was prepared analogously to the hydrobromide in Example 1b). Rf 0.64 / Si; 0.74 (S2).

C/. Pyroglutamyl-histidyl-phenylalanyl-phenylalanyl-alanyl-alanine methyl ester

It was prepared in analogy to the corresponding hexapeptide given in Example 1c) in a yield of 33%, mp 209-211 ° C.

Amino acid analysis: glutamic acid (1.02), histidine (0.96), phenylalanine (2.04), alanine (1.98).

Example 3 a) Benzyloxycarbonylphenylalanyl-phenylalanyl-alanyl-1-eucine methyl ester

It was prepared analogously to Example 1a) in a yield of 60% of theory with m.p.

Mp 199-202 ° C. An analytical sample melted at 201-203 ° C after crystallization from ethyl acetate.

(b) Phenylalanyl-phenylalanyl-alanyl-leucine methyl ester hydrobromide

It was prepared analogously to the hydrobromide in Example 1b). Rf 0.63 (Si); 0.84 (S2).

c / Pyroglutamyl-histidyl-phenylalanyl-phenylalanyl-alanyl-leucine methyl ester

It was prepared analogously to the hexapeptide shown in Example 1c) in a yield of 26 from theory, m.p. 240-244 ° C. An analytical sample melted at 244-246 ° C after crystallization from methanol and 2-propanol.

Amino acid analysis: glutamic acid (1.03), histidine (1.03), phenylalanine (1.99), alanine (0.96), leucine (0.96).

Claims (1)

SUBJECT Synthetic substrates for proteolytic enzymes of general formula, _NH > - CO-Hi-A-BXY-OR OF THE INVENTION wherein the symbols A and B, the same or different, denote aromatic amino acid residues, especially phenylalanine, tyrosine or tryptophan, X and Y, the same or different, denote aliphatic amino acid residues, especially glycine, alanine or leucine, and R denotes methyl or ethyl.