CZ296065B6 - Shortened derivatives of human insulin - Google Patents

Abstract

In the present invention, there are disclosed human insulin shortened derivatives of the general formula I X - Gly - Y (I), wherein X represents insulin desoctapeptide, Y denotes Phe-Phe-PheNHi2 or Phe-Phe-N(CHi3)PheNHi2 or Phe-N(CHi3)-Phe-PheNHi2. Disclosed is also a process for preparing such derivatives, wherein the insulin desoctapeptide is condensed with tetrapeptides of the general formula Gly-Phe-Phe-Phe-NHi2 or Gly-Phe-Phe-N(CHi3)PheNHi2 or Gly-Phe-N(CHi3)-Phe-PheNHi2 in the presence of trypsin tosylphenyl alaminochloroketone in 50 - 65 percent dimethylformamide at a temperature ranging from 4 to 25 degC for a period of 3 to 24 hours, whereupon the obtained products are isolated, especially using high-performance liquid chromatography to reverse phase.

Description

The invention relates to novel semisynthetically prepared truncated human insulin derivatives.

BACKGROUND OF THE INVENTION

The shortening of the carboxyterminal B-chain sequence of human insulin is possible, but has limits in relation to the biological effect. The critical length of the B chain of insulin is 25 amino acids, provided that the terminal carboxyl of phenylalanine 25 is amidated

23 24 25 26 27 28 29 30

----- Arg-Gly-Phe-Phe-Tyl-Thr-Pro-Lys-Thr

Another solution is to preserve the sequence B ²² -B ²⁶ , in the case of human insulin analogues prepared semisynthetically from desoctapeptide insulin and tetrapeptides, the impact of subsequent replacements made in a given region of the molecule is more visible than despentapeptide human insulin analogues (CZ Utility Model 6381). In the CZ Utility Model no. 6381, attention was focused on destetrapeptid insulins, in which the carboxy terminal portion of the B-chains B ²³ -B ²⁶ was modified peptide bond, i.e. has been substituted for hydrogen - NH - methyl group. The aim was to provide greater metabolic stability against endopeptidase cleavage. Introducing the N-methylated amino acid (N (CH ₃ ) phenylalanine) at each position will undoubtedly increase the resistance to chymotrypsin binding, but will be reflected in the spatial arrangement of that part of the chain that is considered critical to the insulin analog interaction with the receptor. By introducing an N-methylated amino acid, the peptide bond loses the ability to form a hydrogen bridge. The change in peptide bond conformation is associated with a change in the local flexibility of the peptide chain and increases the hydrophobicity of the peptide.

Three analogues of sequence B ²³ -B ²⁶ were prepared by the solid phase peptide synthesis procedure and were coupled to the carboxyl of arginine B ²² by enzyme-catalyzed semisynthesis. The products were isolated and characterized.

SUMMARY OF THE INVENTION

The present invention provides truncated human insulin derivatives of formula (I)

X-Gly-Y (I) where X = desoctapeptidine insulin,

Y = Phe-Phe-PheNH ₂ (Compound I) or

Phe-Phe-N (CH ₃ ) PheNH ₃ (Π) or Phe-N (CH ₃ ) -Phe-PheNH ₂ (ΠΙ).

Tetrapeptides that are prepared by solid phase synthesis are condensed with desoktapeptide insulin in the presence of tosylphenylalanine chloroketone trypsin in 50-65% DMF at 4-25 ° C for

3-24 hours. The resulting products are isolated, in particular by reverse-phase high performance liquid chromatography.

-1 CZ 296065 B6

Examples

Explanation of abbreviations

Boc - t-Butoxycarbonyl,

DIEA - Diisopropylethylamine,

DCM - Dichloromethane,

HOBt ester - Hydroxybenztriazole ester,

DCC - Dicyclohexylcarbodiimide,

DMF - Dimethylformamide

DMAP - Dimethylaminopyridine,

TFA - Trifluoroacetic acid

RP-HPLC - High performance reverse phase liquid chromatography,

TPCK - Tosylphenylalanine chloroketone

Example 1

Tetrapeptides were synthesized on solid phase according to Merrifeld. A 4-methylbenzhydrylamine (MBHA) resin (0.57 mmol / g substitution) was used for the synthesis. The t-butyloxycarbonyl (Boc) group was used as a semi-permanent N-terminal protection. The synthesis was carried out on a 0.3 mmol scale. The following synthesis protocol was used. The resin was neutralized by repeated washing with 10% DIPEA in DCM (2 x 5 min) then washed with DCM (3 x 1 min). A triple molar excess of the protected amino acid HOBt ester was then added to the resin reactor. The reaction was allowed to proceed for 1 hour. (The ester was prepared from Boc-amino acid and HOBt by DCC in DCM-DMF (9: 1), reaction time was 30 minutes at 0 ° C. Precipitated dicklohexylurea was filtered from the ester formed, washed with DCM and the filtrate concentrated at room temperature. and then added DMF to a final concentration of approximately 0.1 M. Exceptions were made with Boc-N (CH ₃ ) Phe condensation, with either ByBrop / DMAP or HBTU / DIEA condensation. condensation of unmethylated amino acids If the test was negative, the next reaction step was followed, if positive, the condensation reaction was repeated After completion of the peptide chain construction, the N-terminal Boc group was cleaved with 50% TFA in DCM for 25 min. washed with DCM, 4 x 1 min, the DCM volume was 50 ml each, the resin was dried to constant weight. This, together with the cleavage of the side chain protecting groups, was carried out with liquid hydrogen fluoride mixed with anisole (9: 1) at 0 ° C. The hydrogen fluoride was blown out with nitrogen at 0 ° C and the anisole was extracted with cold diethyl ether (4 x 50 mL). The released peptides were extracted with 20% acetic acid (5 x 50 mL), the resin filtered off and the peptide solution lyophilized. Peptide purity was checked by amino acid composition determination, RP-HPLC and mass spectrometry.

Example 2

Preparation of [Gly ²³ -Phe ²⁴ -Phe ²⁵ -Phe ²⁶ -NH 2] destetrapeptide B ²⁷ -B ³⁰ insulin. The following protocol was used to prepare this truncated analog. 23 mg of tetrapeptide was mixed with 195 μΐ DMF and 55 μΐ 0.05 M and CaCL. 22 mg of insulin desoctapeptide (DOI) was added and the reaction mixture was gently shaken. 3 mg TPCK-trypsin was dissolved in 50 μΐ 0.05 M CaCl 2 and added to the reaction mixture. The pH of the reaction mixture was adjusted to 7.0 with N-methylmorpholine. The reaction was allowed to stir for 3 to 24 hours, and the conversion was monitored. The reaction was stopped by addition of 1 ml acetone. The precipitate was centrifuged and the supernatant collected, and the precipitate was washed again with 1 ml acetone. The sediment was dissolved in 1.2 mL of 10% acetic acid and the resulting solution

-26096065 B6 was collected and lyophilized. The resulting product was characterized by RP-HPLC, mass spectrometry and capillary electrophoresis.

Example 3

Preparation (Gly ²³ -Phe ²⁴ -Phe ²⁵ -N (CH 3) Phe ²⁶ -NH 2] destetrapeptide B ²⁷ -B ³⁰ insulin. 10 mg of peptide was dissolved in 98 μΐ DMF and 27 μΐ 0.05 M CaCl ₂ was added. added 9.1 mg DOI, mixed gently and added 1.5 mg TPCK trypsin dissolved in 25 μΐ 0.05 M CaCl ₂ followed by the procedure of Example 2. The reaction time was at least 3 hours, the temperature was between 5-29 Deň: 32 ° C.

Example 4

Preparation of [Gly ²³ -Phe ²⁴ -N (CH 3) Phe ²⁵ -Phe ²⁶ -NH 2) destetrapeptide B ²⁷ -B ³⁰ insulin. 11 mg of the respective tetrapeptide was dissolved in 110 μΐ DMF and 30 μΐ 0.05 M CaCl _{2 was added} . The reaction time was at least 3 hours at 5-25 ° C.

Example 5

Capillary zone electrophoresis (CZE). Purity analysis of prepared peptides was performed by CZE method with high voltage source 0-15 kV. The separation space was a quartz capillary with an outer polyimide coating with an inner diameter of 0.056 mm, an outer diameter of 0.200 mm, a total length of 315 mm and an effective length (from start to detector) of 200 mm. The peptides were analyzed in 0.5 M acetic acid or in a solution of 0.04 M Tris and 0.04 M Tricine pH 8.1. The sample was applied manually and the measurement was performed at room temperature. The separation was carried out in the constant voltage mode of 10.0 kV and the current reached 9.5-10.8 μΑ. Detection was performed spectrophotometrically at 206 nm.

Example 6

Mass spectra of individual peptides. The measurement was performed by the FAB method, which consists in ionizing the test samples using accelerated xenon atoms. The sample was placed on a glycerin matrix. Spectra sensing was performed by multichannel analysis at low resolution. A three-sector mass spectrometer YAB-EQ with BEQQ geometry manufactured by VG Analytical Manchester was used. The ion source operated at an accelerating voltage of 8 kV.

Example 7

Amino acid composition of peptides. The peptides were hydrolyzed in 6 M HCl at 110 ° C for 20 hours and the individual amino acids separated on a D-500 (Durrum corp.) Proline and cystine representation was not evaluated.

Industrial applicability

The truncated human insulin derivatives according to the invention can be used for basic biochemical and pharmacological research and as substances having a hormonal action.

-3GB 296065 B6

Claims (2)

A truncated human insulin derivative of the formula I X - Gly - Y (I) where X = desoctapeptidine insulin, Y = Phe-Phe-Phe NH ₂ or Phe-N _{(CH3) 2} or PheNH Phe-N (CH ₃₎ -Phe-PheNH ₂ 2. A method for producing truncated human insulin derivative according to claim 1, characterized in that the insulin desoktapeptid condensed with a tetrapeptide of formula Gly-Phe-Phe-PheNH ₂ or Gly-Phe-Phe-N _{(CH3) 2} or Gly PheNH -Phe-N (CH ₃ ) -Phe-PheNH ₂ in the presence of tosylphenylalamine chloroketone trypsin in 50-65% dimethylformamide at 4-25 ° C for 3 to 24 hours, whereupon the resulting products are isolated, in particular by high performance reverse phase liquid chromatography . End of document