CS255089B1 - Process for the production of peptides by continuous solid-state flow method - Google Patents

Abstract

The method of producing peptides by the solid-phase method in a column arrangement is discussed, the essence of which is that the polymer carrier, on which the gradual construction of the peptide chain takes place, is placed in a column, and it is washed with all solutions and solvents used in the synthesis in such a way that the washing direction is changed after each cycle. This arrangement prevents the frit from clogging with small parts of the resin, because the change in the washing direction prevents the settling of small resin particles, which could reduce the flow rate, and accelerates and simplifies the entire synthesis of peptides.

Description

The invention relates to a process for the production of peptides by the solid-phase continuous flow method.

Peptide synthesis by solid-phase method has been unusual in recent years and nowadays peptides are prepared almost exclusively by this method. In its now conventional configuration, all reactions are carried out on a polymeric support that is placed in a reaction vessel and all solutions and solvents are dispensed into the vessel, mixed with the polymeric resin, and after a selected time the solution or solvent is removed, e.g.

The modified procedure of placing the polymer support in the column and washing the column with the support with all of the selected solutions and solvents encountered the problem of gradual clogging of the bottom frit of the column with resinous resin particles, resulting in reduced column flow and increased proportional pressure on the column. has reached an acceptable flow rate. Such synthesizers required special designs that resembled high pressure liquid chromatography devices. Some authors have attempted to overcome this drawback by preparing special polymeric carriers which exhibited increased crumb resistance in terms of physical properties.

The above-mentioned disadvantages are eliminated by the method of production of peptides by the continuous flow method in the solid phase according to the invention, which consists in that the successive construction of the peptide chain is carried out on a polymer support placed in the column.

The process of the invention describes a process that does not require either special polymer supports or special equipment to exert high column pressures. Put enough polystyrene resins cross-linked with divinylbenzene (the most commonly used polymer carrier for solid phase peptide synthesis) in a normal glass column to fill the swollen resin volume with the entire column volume. All the reactions and washes involved in solid phase peptide synthesis are carried out in such a way that the wash direction of the polymer support column is changed after each cycle. In this arrangement, the frit does not gradually become clogged with tiny pieces of resin, as they are washed up or down alternately by the liquid stream.

In carrying out the synthesis according to the invention, the inventors have proven to be able to pressurize container vessels containing the solutions and solvents used in the synthesis to a slight overpressure (20 to 30 kPa) with, for example, nitrogen, and a connecting hose of inert material, e.g. Teflon, the polymer support column is washed with this overpressure. The flow is thus controlled by the overpressure of the gas. Both ends of the column are connected to a four-way valve, the third outlet of which is connected via a six-way valve to the storage vessels and the fourth outlet is disposed of. Switching the four-way valve after each cycle results in a change in the flow direction of the column.

The process according to the invention has been successfully verified in practice. The synthesis of peptides was simplified, raw materials were saved and the whole preparation of the desired peptides was accelerated.

The following examples illustrate, but do not limit, the method of the invention

Example 1

A 5g chloromethylated polystyrene resin cross-linked with 1% divinylbenzene and esterified Boc-Gly (Gly content 0.5 mmol / resin) was placed in a glass column (2 x 9 cm).

Peptide synthesis involves 8 cycles, consisting of the following steps: (1) TFA: DCM (1 :), 30 min, (2) DCM, to neutral, (3) TEA: DCM (1: 9), 3 min. (4) DCM, until neutral, (5) preformed HOBt esters (5 mmol) in 30 mL DCM / DMF (4: 1), (6) DCM, 5 min. If the test for free amino groups is positive, repeat steps 5 and 6. Each step involves washing the column with the polymer support with said solutions and solvents at a pressure of 20 kPa applied to the liquid storage vessel.

The following protected amino acids were condensed in each cycle: Boc-Thr (Bzl), Boc-Ser (Bzl), Boc-Thr (Bzl), Boc-Thr (Bzl), Boc-Ser (Bzl), Boc-Ser (Bzl) ), Boc-Gly

Boc-Phe. After synthesis, 7.1 g of peptidyl-resin were obtained. Peptidyl resin (1 g) was suspended in a mixture of TFMSA (1.5 mL), TFA (5 mL), thioanisole (2 mL) and stirred for 1 h at room temperature. temperature. The product was precipitated with 100 ml of absolute ether and extracted into 20% acetic acid.

After lyophilization, it was purified on a column (1 x 100 cm) containing polyacrylamide gel, mobile phase 1 M acetic acid. Fractions containing product units were pooled and lyophilized. The purified product had satisfactory amino acid analysis and acted as a single substance by thin-layer chromatography in 1-butanol: pyridine: acetic acid: water (30: 20: 6: 12) and paper electrophoresis in pH 2.5 buffer.

Yield: 157 mg of Phe-Pro-Gly-Ser-Ser-Thr-Thr-Ser-Thr-Gly.

Example 2

In a glass column (2 x 9 cm) was placed 5 g of benzhydrylamine resin (polystyrene) sulfurized with 1% divinylbenzene (amino group content of 0.6 mmol / g resin). The synthesis consisted of 3 cycles, which consisted of the same steps as in Example 1, except for the first cycle which proceeded to Step 3. The following amino acids were condensed in each cycle: Boc-Gly, Boc-Leu and Boc-Pro. 5.9 g of peptidyl resin were obtained after synthesis. The peptide was cleaved from the peptidyl resin (5.9 g) in 60 ml of liquid HF, extracted into 20% acetic acid and lyophilized. The crude product had satisfactory amino acid analysis and showed 5% impurity on analytical HPLC (based on integration of peak area at 210 nm).

Yield: 720 mg of Pro-Leu-Gly-NH4

Note:

The peptides obtained in the examples were synthesized according to the invention by changing the direction of flow of the mobile phase after each synthetic cycle (i.e., after the addition of another protected amino acid), thus avoiding clogging of the resin frit and the reaction components very carefully and while mixing perfectly. Peptide synthesis was carried out continuously.

Explanation of abbreviations used in the text:

Boc t-butyloxycarbonyl

TFA trifluoroacetic acid

DCM dichloromethane

TEA triethylamine

HOBt N-hydroxymenztriazole

DMF dimethylformamide

Bzl benzyl

TFMSA trifluoromethanesulfonic acid

HF hydrogen fluoride

HPLC high performance liquid chromatography

Gly glycine

Thr threonine

Ser serin

For proline

Phe phenylalanine

Leu leucine

Claims (1)

SUBJECT OF THE INVENTION A method for the production of peptides by a solid-phase continuous flow method, characterized in that the successive construction of the peptide chain is carried out on a polymer support placed in a column and the flow direction of the mobile phase always changes after the addition of another amino acid.