CS262081B1 - Device for automatic preparing peptides by synthesis on solid phase - Google Patents

Abstract

Equipment for automatic production of series peptide synthesis by solid phase synthesis consists of storage bottles in which it is overpressure of 20 to 50 kPa of inert gas through the distribution valve in series connected reactors that are connected via a four-way valve with waste, and a container it is connected via a four-way valve and peristaltic pump with distribution valve and reactors. The device has been successfully used for synthesis of a number of peptides simultaneously.

Description

(54) Equipment for the automated production of a variety of peptides by solid phase synthesis

Installations for the automated production of a series of peptides by solid phase synthesis, consisting of storage cylinders in which an overpressure of 20 to 50 kPa of inert gas is connected through a distribution valve to series connected reactors connected via a four-way waste valve and the container is interconnected via a four-way valve and peristaltic pump with a distribution valve and reactors.

The device was successfully used for synthesis of many peptides simultaneously.

Giant. <

The present invention relates to an apparatus for automatically producing a series of peptides by solid phase synthesis.

Currently, in the basic research of new peptide-based preparations, it is desired to obtain large amounts of relatively small amounts of peptides for preliminary testing. Both the classical performance of solid phase peptide synthesis in a stirred reactor and a continuous flow device that requires an apparatus similar to a high performance liquid chromatography device are in keeping with this trend because they synthesize larger quantities of only one peptide at a time. Attachment of the protected amino acids to the polymeric carrier is accomplished by washing cycles, which are generally controlled by the automation. In order to be able to automatically control the peptide synthesizer, it is necessary to use compatible cycles so that all synthesis steps are identical except for the amino acid coupling. In this way, individual peptides are synthesized by repeating identical cycles. However, current devices do not allow the preparation of several different peptides side-by-side, so that even the smallest amount of peptides required must be prepared separately. This is mainly time consuming, while the energy consumption of raw materials (especially solvents) is considerable.

These disadvantages are overcome by a solid-phase synthesis process for the production of a variety of peptides consisting of pressurized storage cylinders, a distribution and multi-way valve, flow reactors, and a peristaltic pump according to the invention. up to 50 kPa of inert gas are connected via a distribution valve to series connected reactors, which are connected via a multi-way valve to the waste, and the reservoir is connected via a multi-way valve and a peristaltic pump to a distribution valve and reactors.

The device according to the invention makes it possible to produce a number of different peptides at the same time, thus significantly reducing the time required for their preparation. This has the main advantage that, for example, fewer peptides are needed, for example, for clinical trials, further synthesis or research work. The device according to the invention makes more efficient use of all raw materials, energy and labor, because especially in miniaturization of the device and its connection to the computer, the whole production is controlled by a minimal number of people and from one place. The device according to the invention substantially speeds up and simplifies the synthesis of peptides.

The device according to the invention is shown in the accompanying drawings. Fig. 1 is a cross-sectional view of one flow reactor; Fig. 2 schematically illustrates a series connection of a series of reactors as verified in practice by the inventors in the preparation of a number of new peptides.

The device according to the invention consists of storage bottles 1 in which various solvents and reagent solutions such as DMF, DCM, DIEA in DCM, TFA in DCM and the like are prepared. From the storage bottles 1 the desired solutions are filled with Teflon tubing 7 through the distribution valve 2. to reactors 4 which are connected in series and whose number is not limited. In the specific case, the apparatus can be used for only one reactor 4, the size of the reactors 4 being determined by the amount of peptides to be prepared. From the reactors 4, the Teflon tubes 7 pass through the four-way valve 3 to the waste 8, where the unnecessary solvents are collected. The container 6, in which the protected amino acid is activated, is connected by Teflon tubing 7, via a four-way valve 3 and a peristaltic pump 5 and a distribution valve 2 to the reactors 4.

The device according to the invention operates by dispensing the respective solution via the distribution valve 2 into the reactors 4 from the storage cylinders 1 which are under the pressure of an inert gas whose value is equalized by pressure relief valves. In the reactor 4 there is a polymeric carrier treated with benzhydrylamine groups or chloromethylated carboxyterminal amino acid esterified groups to which active protected amino acids are condensed sequentially, which are pre-prepared and collected in reservoir 6. These protected amino acids are passed from reservoir 6 via a four-way valve 3 and a peristaltic pump 5 and a distribution valve 2 to the reactors 4. dosing of the protected amino acid is switched by the four-way valve so that the ACA recirculates in the peristal-forming circuit. After completion of the reaction, which is monitored by the ninhydrin test, the four-way valve is brought back to its original position and the ACA is washed out of the reactors 4 and the respective contaminated part of the device with solvent directly to the waste 8. The reactors are determined by the type of protected amino acid to be condensed and therefore the number of reactors 4 connected in series is operatively changed before each reaction cycle.

Reactor 4 is a mass-produced polypropylene chromatography column, provided with two meshes inside, with a polymeric support between them and externally having 2 conical terminals that allow serial connection of the reactors. However, the actual reactor 4 can be made of glass or other suitable material, while always meeting the requirement of precisely forming the interlocking terminals. The size of the reactors is determined by the amount of peptides being prepared.

In the device according to the invention, solid phase peptides are synthesized using series-connected flow reactors in which a small amount of polymeric carrier is placed, and thus it is possible to prepare a plurality of peptides in an amount sufficient for indicative bioassays. In the actual verification of the device according to the invention, polypropylene flow reactors with double-meshed, anti-carrier mixers have been found to be commonly used for pre-chromatography in high performance liquid chromatography (HPLC). In the apparatus of the invention, a polymeric carrier is placed in the reactor. The reactors are equipped with tips similar to the tip of a disposable syringe that can be inserted. With this series of reactors, the liquid can be forced in both directions by a slight overpressure of the carrier gas (air, nitrogen). The synthesis can be interrupted at any time by canceling the overpressure.

The individual reactors can be removed from the series or, on the contrary, a new reactor can be included. This advantage is most often used in the synthesis of similar peptides that differ in one amino acid, possibly by omitting an amino acid or by adding one extra amino acid. In principle, however, the device can be used to synthesize a large set of peptides (more than cost. The reactors will then be coupled to rank the desired amino acid independently of the number of amino acids attached.

The following examples illustrate, but are not limited to, the apparatus of the present invention.

Example 1

150 mg of benzhydrylamine polymer support (0.8 mmol / g) was charged into polypropylene reactors (20 X 4 mm, free content 0.7 ml). The reactors are labeled and connected in series. The synthesis cycles consist of the following washing steps:

1. 50% TFA in DCM

2. DCM

3. 10% DIEA in chloroform,

4. DCM

5. DMF,

6. a solution of 5 mmol of HOBt amino acid ester in DMF,

7. DCM.

The first synthetic cycle begins with step 2. The reactor from which the samples were taken was the last in the reagent flow direction. In the case of a positive test for the free amino group after condensation, steps 6 and 7 were repeated and a catalytic amount of dimethylamidopyridine (100 mg) was added to the active component solution. The following protected amino acids were added in each cycle:

BocLeu, Boc-Pro, Boc-Asn

Boc-Arg (Tos), Boc-Ser (Bzl),

Boc-Asp (OBzl), Boc-Arg (Tos)

Boc-Tyr (Z), Boc-Tyr (Z), and Boc-Val.

One reactor was sequentially phased out for one cycle (inclusion of one amino acid). The only exception is the first and last reactor downstream of the reagent, which was left in series for the synthesis and only one reactor was used to exclude tyrosine from the sequence. The weight gain was about 100 milligrams per reactor. Individual peptidyl resins were digested with liquid hydrogen fluoride with the addition of 5% p-cresol, 1 horine at 0 ° C. After the hydrogen fluoride was evaporated, the p-cresol was extracted with cold ethyl acetate and the peptide was dissolved in 20% acetic acid. After lyophilization, the crude products (20.2 to 47.2 mg) were analyzed by HPLC chromatography and the amino acid composition was determined and the results are shown in Tables 1 and 2.

TABLE 1

Amino acid analysis of individual new peptides

Peptide If Amino Acid Analysis

C. omits. Wall Tyr Arg Asp Ser For Leu 1 Leu 0.91 1.83 2.13 2.08 2.15 1.04 1.01 2 For 0.94 1.74 2.07 1.00 - 1.10 3 Asn 0.91 1.71 2.09 1.08 1.04 1.08 1.10 4 Arg 0.94 1.88 0.95 2.08 0.97 1.04 1.12 5 Ser 0.97 1.84 2.06 2.05 - 1.01 1.06 6 Asp 0.90 1.78 2.10 1.15 1,02 1.09 1.12 7, Arg 0.93 1.56 1.12 2.12 0.98 1.10 1.16 8 Tyr 0.88 0.84 2.06 2.08 0.97 1.06 1.10 9 Wall - 1.67 2.10 2.04 0.94 1.09 1.15 10 ! - 0.87 1.78 2.04 2.08 2.10 0.97 1.13 1.14 11 / - 0.90 1.79 2.06 0.96 1.09 1.09

TABLE 2 Analytical a physical data of individual new peptides Peptide C. If omits. Yield Rt (mg) (min) Content* (%) Rt (min) Content** (%)

1 Leu 35.2 4.10 84.6 6.34 92.6 2 For 20.2 6.37 88.4 12.54 86.2 3 Asn 24.1 8.87 94.9 20.29 93.8 4 Arg 37.3 12.72 85.9 25.58 89.9 5 Ser 47.2 11.95 93.7 26.49 94.8 6 Asp 42.1 11.18 90.0 26.95 91.0 7 Arg 39.4 14.26 89.1 28.65 85.0 8 Tyr 21.4 8.34 93.6 19.61 86.3 9 Wall 44.8 8.88 85.9 21.72 94.5 10 *** - 35.0 12.63 88.4 27.09 89.3 H »» · - 27.0 12.71 87.9 27.10 87.8 Retention times (min) a peptide content (%) gained high-efficiency liquid chro-

with the following conditions:

(*) Isocratically 40% methanol in water containing 0.1% TFA, flow rate 25 ml / h, 150 x 3 mm column packed with silica gel with hydrophobic carbon residues, UV detection at 220 nm, (**) gradient phase A methanol, phase B water containing 0.1% TFA, gradient profile:

time (min) 0 15 35 36 phase A content in B (%) 23 38 38 23 flow rate 0.1 ml / min, column 150 x 2.1 mm, UV detection 210 nm and 280 nm, (***) peptides Nos. 10 and 11 were synthesized in the first and last reactors.

Explanations of abbreviations used in the text: Arg - arginine

Leu - leucine

ACA - activated protected amino acid Pro - proline

DMF - dimethylformamide Ser - serine

DCM - dichloromethane Tyr - tyrosine

TFA - trifluoroacetic acid Val - valine

DIEA - dlisopropylethylamine Boc - t-butyloxycarbonyl

HOBt - hydroxybenztriazole OBzl - benzyloxy

Asn - asparagine Z - benzyloxycarbonyl

Asp - aspartic acid

Claims (1)

SUBJECT Apparatus for the automated production of a series of peptides by solid phase synthesis, consisting of pressurized storage cylinders, distribution and multi-way valves, flow reactors and a peristaltic pump, characterized in that the storage cylinders (1) have an overpressure of 20 to 50 kPa , OF THE INVENTION are connected via a distribution valve (2) to series connected reactors (4) which are connected via a four-way valve (3) to the waste (8) and the reservoir (6) is connected via a four-way valve (3) and peristaltic pump (5) with a distribution valve (2j and reactors (4)).