CS260641B1 - Human modified c-peptide and method of its preparation - Google Patents

Description

The invention relates to a modified human C-peptide for radioimmunoassay and for the preparation of antibodies and a method for its production.

It is known that human C-peptide (AA33-63 human proinsulin) is cleaved from the proinsulin molecule by specific proteases to produce the active hormone insulin. Both insulin and C-peptide are then secreted by pancreatic β-cells in an equimolar ratio to portal blood (Středa M .: Diabetologie, Avicenum, Praha, 190 pp. (1985); Kemmler W. et al .: J. Biol. Chem, 248 , 4,544 (1973)).

Radioimmunoanalytical determination of C-peptide level in blood serum, resp. The urine of patients provides information on β-cell function, and is a reliable modern method of diagnosing diabetes mellitus (Melani F. et al., Proc. Natl. Acad. Sci. USA, 67, 148 (1970)).

This radiomunoanalytical (RIA) C-peptide assay has a number of advantages over other diagnostic methods of diabetes, such as glucose tolerance test or insulin level determination:

1. measuring the C-peptide level directly determines the production of insulin proper;

2. unlike insulin, there is no variable uptake of C-peptide in the liver;

3. Patients receiving exogenous insulin may also be monitored.

A disadvantage of the human modified C-peptides synthesized so far is that it is not possible to quantitatively follow direct methods of ligand binding to a macromolecular carrier.

The subject of the invention is a human modified C-peptide of the formula:

H — X — Glu ³³ —Ala — Glu — Asp — Leu — Gln — —Val — Gly ⁴⁰ —-Gin — Val — Glu — Leu — Gly ⁴⁵ - —Gly — Gly — For — Gly — Ala — Gly — Ser— —Leu — Gin — For — Leu — Ala — Leu — Glu— —Gly — Ser — Leu — Gin ⁶³ —Y — OH, where

X is tyrosine and

Y is, for example, norvaline or norleucine.

For example, the substituent X may be Tyr or any amino acid allowing the introduction of a radioactive atom and Y a natural amino acid, ie an amino acid not found in the human body. This amino acid, e.g., Nva, Nie and the like, can be used to monitor binding to a macromolecular carrier.

The present invention further provides a method of making a human, modified C-peptide, comprising synthesizing the peptide on a benzhydrylamine or chloromethylated polymeric support in a known manner at room temperature.

An overview of the individual synthesis steps is shown schematically in Tables 1 and 2.

The modified C-peptide was prepared by two methods of solid phase (Stewart JM; Young JD: Solid phase peptide synthesis, Pierce Chemical Company Rockford, 176 p. (1984)), starting with 9-fluorenylmethyloxycarbonyl- ( Fmoc), respectively. t-butyloxycarbonyl- (Boc) amino acid derivatives. The resulting product was cleaved from the resin and purified by conventional purification methods - e.g., electrophoretic, gel and ion exchange chromatography, high performance reverse phase liquid chromatography (HPLC). Purity was verified by electrophoresis, HPLC, and amino acid analysis. Immunoreactivity was verified by radioimmunoassay.

The modified C-peptide is immunoreactive and has been used to prepare polyclonal rabbit antisera. It can be labeled with, for example, ¹²⁵ I radioactive ^reagents and can thus be used to prepare a complete RIA kit for the C-peptide assay.

The following is an example of a method of making a modified human C-peptide of the invention.

He did

Preparation of the modified C-peptide of the invention using Fmoc-amino acid derivatives

1.0 g of benzhydrylamine resin was used and bound to an acid-labile hanger (3- / 4-hydroxymethylphenoxy / propionate) with the first C-terminal amino acid Nva, substitution being 0.4 mmol / g resin (Albericio F .; Barany G.). Int. J. Peptide Protein Res, 26, 92 (1985).

The α-amino groups of the amino acids were protected by the Fmoc- group, the Glu and Asp side chains were protected by the OBuMt-butyl ester group and the hydroxyl group by Ser was protected by BuHt-butyl ether-). The carboxyl groups were activated as N-hydroxybenztriazole esters (HOBt) (symmetric anhydrides were used for Gln). The synthesis was carried out according to the procedure, see Tab. 1. The Fmoc group was cleaved with a 1: 1 solution of piperidine in dimethylformamide (DMF). Dicyclohexylcarbodiimide was used as condensing agent. Completeness of condensation was monitored by ninhydrin test (for Pro chloranil test).

The finished peptide (and the post-chain amino acid protecting groups) was cleaved from the resin with a 45% solution of trifluoroacetic acid (TFA) in dichloromethane (2 ti), and precipitated with ether, and the resin was further extracted with 30% acid. acetic acid, water and lyophilized. The peptide was purified on Biogel P4, DEAE Sephadex and high performance reverse phase liquid chromatography (HPLC).

The yield after gel filtration was 0.904 g of peptide, i.e. 70%. The peptide purified by ion-2B0B4X chromatography and preparative HPLC is electrophoretic (with relative mobility to glycine in a pH-2.4 buffer, E = 0.25) and HPLC uniform. The result of the amino acid analysis corresponds to the amino acid composition:

Theoretical representation Analysis

Asp 1 0.83 Ser 2 1,8 Gin + Glu 8 7.6 For 2 2.2 Gly 7 6.5 Ala 3 3.0 Wall 2 1.7 Nva 1 0.97 Leu 6 5.97 Tyr 1 0.7

The peptide was bound to bovine serum albumin and the conjugate was used to prepare rabbit and guinea pig antiserum.

Example 2

Preparation of the modified C-peptide of the invention using Boc-protected amino acids

0.5 g of the chloromethylated resin to which N @ 1 was substituted in the substitution of 0.32 mmol / g resin were used, the .alpha.-amino groups of the amino acids were protected by the Boc group, the carboxyl groups of the Glu side chains, Asp by OBzl- (benzyl ester). The hydroxy group was Bzl (benzyl ether) and the amino acids were activated as esters of N-hydroxybenztriazole or as symmetrical anhydrides The synthesis was carried out according to the procedure in Table 2. The Boc group was cleaved with 45% trifluoroacetic acid in dichloromethane. Condensation was monitored by ninhydrin or chloranil and acetylation tests.

The finished peptide (and side chain protecting groups) was digested with liquid hydrogen fluoride for 1 h in the presence of 3% thioanisole, which was then extracted with ethyl acetate, the peptide was extracted with 30% acetic acid, water and lyophilized. A mixture of peptides (0.493 g, in which the entire C-ipeptide was detected) was obtained. The immunoreactivity of the product was 60% of the activity of the commercial C-peptide (by BIODATA). rabbit antiserum.

The human modified C-peptide of the invention is suitable for radioimmunoassay and for the preparation of antibodies, e.g., rabbit and guinea pig antisera.

Table 2

Table 1

step Fmoc-preparation Reagents (~ 15 ml) min. 1 CH 2 Cl 2 (2X) 3 2 DMF (3X) 3 3 DMF: Piperidine (2X) 20 May 4 (1: 1) DMF (3X) 3 5 dioxane: H2O (2: 1) (2X) 10 6 DMF (4X) 5 7 CH2Cl2 (or DMF) (3X) 5 8 HOBt ester Fmoc-AK ch 15 Dec symmetric anhydride and more 9 (3 eq) in CH 2 Cl 2 (or CH 2 Cl 2: DMF) ninhydrin assay 5 10 repeat condensation 11 if necessary (step 7—9) DMF (6X) 8 12 2-Propanol (5X) 8 13 CH2Cl2 (5X) 8

Boc preparation

step Reagents (~ 15 ml) min. 1 CH 2 Cl 2 (4X) 7 2 45% TFA in CH 2 Cl 2 (IX) 2 3 45% TFA in CH 2 Cl 2 (IX) 20 May 4 CH 2 Cl 2 (3X) 5 5 8% TEA in CH 2 Cl 2 (2X) 3 6 CH 2 Cl 2 (4X) 7 7 HOBt ester Boc-AK or symmetry. anhydride (3 eq) in CH 2 Cl 2 40 and more 8 ninhydrin test 5 9 CH 2 Cl 2 (3X) 5 10 2-Propanol (3X) 5 11 12 CH2Cl2 (4X) repeat the condensation if necessary (steps 7-11) 7

Note:

TEA = triethylamine AK = amino acid

Claims (2)

SUBJECT 1. A human modified C-peptide of the formula: H — X — Glu ³³ —Ala — Glu — Asp — Leu — Gin — —Val — Gly ⁴⁰ —Gin — Val — Glu — Leu — Gly ⁴⁵ - —Gly — Gly — For — Gly — Ala — Gly — Ser— - Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-vLeu-Gin ^63- Y-OH where OF THE INVENTION X is tyrosine and Y is norvaline or norleucine. 2. A process for preparing a human modified C-peptide according to claim 1, wherein the peptide is synthesized on a benzhydrylamine or chloromethylated polymeric support at room temperature. Severography, n. Race 7, Most Price 2,40 Kčs