CS262081B1 - Equipment for automatic production of a number of peptides by solid phase synthesis - Google Patents

Abstract

A device for the automatic production of a series of peptides by solid-phase synthesis, consisting of storage bottles in which there is an overpressure of 20 to 50 kPa of inert gas, connected via a distribution valve to series-connected reactors, which are connected via a four-way valve to the waste, and the reservoir is connected via a four-way valve and a peristaltic pump to the distribution valve and reactors. The device has been successfully used for the synthesis of a series of peptides simultaneously.

Description

(54) Equipment for the automatic production of a range of peptides by solid-phase synthesis

A device for the automatic production of a series of peptides by solid-phase synthesis, consisting of storage bottles in which there is an overpressure of 20 to 50 kPa of inert gas, connected via a distribution valve to series-connected reactors, which are connected via a four-way valve to the waste, and the reservoir is connected via a four-way valve and a peristaltic pump to the distribution valve and reactors.

The device has been successfully used to synthesize a number of peptides simultaneously.

Fig. <

The invention relates to a device for the automatic production of a number of peptides by solid-phase synthesis.

Currently, in basic research of new peptide preparations, there is a requirement to obtain large sets of relatively small amounts of peptides for preliminary testing. Both the classic solid-phase peptide synthesis in a stirred reactor and the continuous flow device, which requires equipment similar to high-performance liquid chromatography, do not comply with this trend, because they synthesize larger amounts of only one peptide at a time. The attachment of protected amino acids to the polymer carrier is carried out by washing cycles, which are usually controlled by an automatic device. In order to be able to control the peptide synthesizer automatically, it is necessary to use compactible cycles, so that all synthesis steps are identical with the exception of the attachment of the amino acid. In this way, individual peptides are synthesized by constantly repeating identical cycles. However, current devices do not allow the preparation of several different peptides side by side, so that each and every smallest amount of the desired peptide must be prepared separately. This is especially time-consuming, while the consumption of energy and raw materials (especially solvents) is considerable.

The above disadvantages are eliminated by a device for the automatic production of a number of peptides by solid-phase synthesis, consisting of pressurized storage bottles, a distribution and multi-way valve, flow-through reactors and a peristaltic pump according to the invention, the essence of which lies in the fact that the storage bottles, in which there is an overpressure of 20 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 the distribution valve and reactors.

The device according to the invention enables the simultaneous production of a number of different peptides and thus significantly shortens the time for their preparation. This has the main advantage that, for example, a smaller amount of a number of peptides is immediately available, needed for clinical trials, further synthesis or research work. The device according to the invention allows for more efficient use of all raw materials, energy and operating personnel, because, especially when the device is miniaturized and connected to a computer, the entire production is controlled by a minimum number of people and from one place. The device according to the invention significantly speeds up and simplifies the synthesis of peptides.

The attached drawings show the device according to the invention. Fig. 1 shows a cross-section of one flow reactor, Fig. 2 shows a schematic drawing of a series of reactors connected in series, as verified in practice by the authors of the invention 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 are prepared, such as DMF, DCM, DIEA in DCM, TFA in DCM, etc. From the storage bottles 1, the desired solutions are filled into the reactors 4, which are connected in series and the number of which is not limited, through Teflon tubes 7 via a distribution valve 2. In a specific case, the device can also be used for only one reactor 4, while the size of the reactors 4 is governed by the need for the amount of peptides to be prepared. Teflon tubes 7 lead from the reactors 4 through a four-way valve 3 to the waste 8, where the no longer needed solvents are collected. The reservoir 6, in which the protected amino acid is activated, is connected to the reactors 4 through Teflon tubes 7, via a four-way valve 3 and a peristaltic pump 5 and a distribution valve 2.

The device according to the invention operates in such a way that the respective solution is dosed from the storage bottles 1, which are under the pressure of an inert gas, the value of which is balanced by safety pressure valves, through the distribution valve 2 into the reactors 4. In the reactor 4 there is a polymer carrier, modified with benzhydrylamine groups or chloromethylated groups esterified with a carboxy-terminal amino acid, to which active protected amino acids are gradually condensed, which are prepared in advance and collected in the reservoir 6. These protected amino acids are led from the reservoir 6 through the four-way valve 3 and the peristaltic pump 5 and the distribution valve 2 into the reactors 4. After dosing the protected amino acid, the four-way valve is switched so that the ACA recirculates in the circuit formed by the peristaltic pump. tic pump 5, distribution valve 2, reactor 4 and four-way valve 3. After the reaction, which is monitored by the ninhydrin test, is completed, the four-way valve is switched to its original position and the ACA is washed out of the reactors 4 and the relevant contaminated part of the equipment with solvent directly into the waste 8. The reactors are determined by the type of protected amino acid that needs 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, equipped with two meshes inside, between which there is a polymer carrier, and on the outside it has 2 conical 'ends' that allow the reactors to be connected in series. However, reactor 4 itself can be made of glass or other suitable material, while it is always necessary to meet the requirement of precise manufacturing of the ends that fit together. The size of the reactors is determined by the amount of peptides being prepared.

In the device according to the invention, peptides are synthesized on a solid phase using flow reactors connected in series, in which a small amount of polymer carrier is placed, and thus it is possible to prepare a larger number of peptides in an amount sufficient for indicative biological tests. During the actual verification of the device according to the invention, polypropylene flow reactors with double meshes preventing carrier mixing, which are commonly used for pre-chromatography in high-performance liquid chromatography (hereinafter referred to as HPLC), proved to be effective. In the device according to the invention, a polymer carrier is placed in the reactor. The reactors are equipped with ends similar to the tip of a disposable syringe, which can be inserted into each other. Liquid can be forced through such a series of reactors in both directions by simply applying a slight excess pressure of the carrier gas (air, nitrogen). The synthesis can be interrupted at any time by removing the excess pressure.

Individual reactors can be removed from the series or, conversely, a new reactor can be added. 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 including one additional amino acid. In principle, however, the device can be used in the synthesis of a large set of peptides (more than one hundred). The reactors will then be connected to include the desired amino acid regardless of the number of amino acids added.

The following examples of embodiments of the device according to the invention are merely illustrative, but not limiting.

Example 1

150 mg of benzhydrylamine polymer support (0.8 mmol/g) is placed in polypropylene reactors (20 x 4 mm, free volume 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 samples were taken was the last in the series in the direction of reagent flow. In the case of a positive test for a free amino group after the condensation, steps 6 and 7 were repeated and a catalytic amount of dimethylamidopyridine (100 mg) was added to the solution of the active component. The following protected amino acids were added in individual cycles:

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 removed one cycle at a time (inclusion of one amino acid). The exceptions were the first and last reactors in the direction of reagent flow, which were left in series throughout the synthesis and only one reactor was used to exclude tyrosine from the sequence. The weight gain was approximately 100 milligrams per reactor. The individual peptidyl resins were cleaved with liquid hydrogen fluoride with the addition of 5% p-cresol, 1 hour at 0 °C. After evaporation of the hydrogen fluoride, 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 on an HPLC chromatograph and the amino acid composition was determined. The results are shown in Tables 1 and 2.

TABLE 1

Amino acid analyses of individual novel peptides

Peptide Ak Amino Acid Analysis

No. omit. Val Tyr Arg Asp Shit 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 Shit 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 Val — 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 and physical data of individual new peptides Peptide No. If omitted. Rt Yield (mg) (min.) Content* (%) Rt (min.) Content** (%)

1 Leo 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 Shit 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 Val 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.) and peptide content (%) obtained high-performance liquid chro-

matography under the following conditions:

(*) isocratic 40% methanol in water containing 0.1% TFA, flow rate 25 ml/hour, 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 content of phase A 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 no. 10 and 11 were synthesized in the first and last reactor.

Explanation 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)).