DK173633B1 - Method and cannula for pipetting robot for fully automatic simultaneous synthesis of multiple polypeptides - Google Patents

Abstract

Process and apparatus for the completely automatic simultaneous synthesis of several polypeptides, where up to 96 different polypeptides are synthesised by the solid-phase synthetic method in a robot pipetter.

Description

in DK 173633 B1

The invention relates to a method for fully automatic simultaneous synthesis of multiple polypeptides according to the solid synthesis method as well as a pipetting robot needle.

5 For rapid assessment of structure-effect relationships of biologically active peptides in receptor binding studies and rapid epitope study for the immunology of peptides and proteins, relatively small amounts (less than 20 mg of each) of many 10 different peptides are used. The preparation of these peptides is conveniently done according to the solid phase peptide synthesis.

This synthesis is based on that of R.B. Merrifield developed method (G. Barany, R.B. Merrifield in The Peptides, Analysis, Synthesis, Biology, vol. 2, 3-284 15 (1980), published by Gross, Meienhofer Academic Press,

New York), which builds up the peptide chain in stages. The synthesis steps can be summarized as follows: a) Binding of the first amino acid in peptide chain 20 to a polymeric carrier via an anchor group, b) stepwise condensation of the other amino acids of the peptide chain, c) intermediate steps between the individual condensations consisting of washing, cleavage of protecting groups and neutralization, 30d) optionally acylation of terminated amino groups, and e) cleavage of the peptide from the carrier.

2 DK 173633 B1

For this peptide synthesis, a synthesis time of up to 18 hours, usually up to 4 hours per day, is to be expected. amino acid. (The individual condensations usually require 1 to 2 hours of reaction time; between the 5 condensations usually require about 10 intermediate steps, to which should be estimated about 2 to 15 minutes per step). Thus, the production of peptides with a greater number of amino acids is very time consuming, labor intensive and expensive.

10 For the solid phase synthesis of analogous peptides, R.A. Houghten (Proc. Natl. Acad. Sci., USA, Vol. 82, pages 5131-5135, August 1985, Immunology) described a method. According to this, polymeric carrier material for the synthesis is filled into small porous polypropylene bags in portions of 50-100 mg, the bags are fused together, the common intermediate steps of the syntheses (washing, neutralization and cleavage of protecting groups) are carried out simultaneously on all bags in a single reaction vessel, and the individual condensations are carried out separately. The method can be performed manually or partially automatically using a peptide synthesizer.

The disadvantage of the method described is that the handling of the bags is a bit complicated, that the bags cannot be reused, that the bags have to be sorted for condensation of the various peptides and that no control samples can be taken during the synthesis.

The object of the invention is to provide a method and a cannula which enables automatic simultaneous synthesis of multiple polypeptides and avoids the above-mentioned disadvantages.

The task is solved by varying the aforementioned solid phase synthesis method so that it can be carried out by means of a correspondingly adapted pipette-robot robot. Pipetting robots have so far been used for serial analysis. For example, the pipetting robot, RSP 5052 from the company TECAN, can be used.

A pipetting robot has the following outer components: At least one arm with metering pipette, one holder with storage containers, and one microtiter plate that can hold up to 96 wells. The robot arm brings the reagents from the containers into the individual wells of the microtiter plate and, when necessary, sucks liquids out of the 10 wells. The metering pipette cannula may also be made so that it is separated into two parts by a dividing wall running from above and below. (With this divided cannula it is possible to perform two different dosages or one dosing and one suction with a 15 arm). The workflow of the device is controlled by a computer program.

The solid phase peptide synthesis of the invention is carried out in such a pipetting robot as follows: In the wells of the microtiter plate, support materials (preferably granulated support material) are placed. The carrier material may be attached to the starting portion of the individual desired peptides. The fluids needed for the reactions and washing steps are poured into the storage containers.

If the peptide is to be separated from the carrier after synthesis is completed and / or if free amino groups are to be acylated, the reagents required for these reactions must also be prepared in the storage containers. From the required reaction times, it is seen that it is appropriate to use a microtiter plate containing no more than 30 wells. Thus, in a program run, a maximum of 96 different polypeptides can be synthesized. According to the program adapted to the synthesis of these peptides, the robot brings the reagents and washing liquids to the individual wells and suction the liquid over the carrier material after appropriate residence times.

The method and the necessary adaptation of the apparatus to the method are explained in more detail on the basis of the two-armed pipetting robot RSP 5052 from the company Tecan. However, the application of the method is not limited to this apparatus. Other types of pipetting robots, especially also single or multi-armed robots, can be adapted to the method of the present invention.

A 96-well microtiter plate is selected. For example, a well contains. 10 mg of resin that can be applied with an amino acid and slightly more than 300 μΐ fluid. The amount of resin corresponds to approx. to 5 µmol of amino acid or is suitable for the preparation of ca. 5 µmol peptide. Usual carrier materials on a polystyrene or polyacrylamide basis are useful. It is convenient to construct peptides containing a maximum of 20 amino acids.

The reagent solutions and washing liquids 20 used for this purpose are prepared in the storage containers provided for this purpose. The apparatus 1 arm 1 is equipped with a metering pipette, arm 2 with a suction cannula with flushing device. This flushing device is preferably connected to a separate standing storage container for the solvent used. The synthesis is done according to the program programmed in the connected PC.

The dosage of all reagent solutions taken from open storage containers is done with arm 1.

Before changing the dosing pipette from one reagent solution 30 to another, the dosing pipette is rinsed with solvent in a special rinsing position. Extraction of reagents and washing fluids takes place with arm 2 through a filter-equipped cannula. To prevent resin loss and contamination of the neighboring wells, the outside of the cannula is rinsed with solvent by a feed located on the outside of the cannula after each suction procedure. The next washing procedure is started at the same time as this solvent. Thereafter, the cannula is rinsed in a cannula flush position.

For separating the peptide from the resin, the wells are fed e.g. trifluoroacetic acid through arm 1.

After cleavage, the solution is suctioned with the suction cannula and transferred to another microtiter plate from which the processing takes place.

As mentioned above, the suction cannula can be fitted with a filter. This filter is located at the lower end of the cannula and prevents suction and entrainment of resin.

The gate may conveniently consist of a stainless steel mesh which e.g. fastened with a soldered ring. Another option is to fit a metal interplate or ceramic interplate into the opening of the cannula. If a cannula is used without a filter, the entrainment and aspiration of resin 20 can be largely avoided by slow aspiration. This, however, significantly prolongs the time of aspiration.

Rinse the outside of the suction cannula with solvent after each suction procedure. Conveniently, this is effected by feeding the solvent into a conduit (hose or tube) running along the cannula and positioned so that the aperture is barely above the lower end of the cannula. It is important that the solvent rinse the entire circumference of the lower end of the cannula.

The device arm 2 may be provided with a comb instead of the suction cannula. Such a comb comprises several suction cannulas (usually 4-12 needles) of the embodiment described above. By means of such comb DK 173633 B1 6, several wells can be operated simultaneously, leading to a significant time saving.

Usually, DMF or N-methylpyrrolidone is used as the solvent. The wells and the optional flushing device, if any, must also be made of solvent-resistant material such as polypropylene or teflon. In the commercially available appliances, the dosing pipettes and suction cannulas are made of stainless steel. This material 10 is suitable for the process of the invention.

In this synthesis, if a greater amount of resin is used per peptide (e.g., 50 mg of resin), larger well microtiter plates should be used. These microtiter plates are standardized and commercially available. They then contain a correspondingly lower number of wells.

Instead of a two-arm pipetting robot, a one-arm apparatus can also be used. In this case, the suction cannula must have a division into free volumes. With one part suction of washing and reagent solutions takes place, the other part fulfills the function of arm 1 from the two-arm apparatus, ie. performs dosing of the reagent solutions. The protection against resin aspiration is as described by the two-arm apparatus. Also, this cannula is flushed as described above.

The process is carried out, for example, with the following substances (quantity declaration per well): Starting material is 10 mg with Fmoc amino acids resin attached (grain size 200-400 mesh); Fmoc-protected amino acids are used in up to ten times the excess for each coupling step, ie. 200 µl of a DMF solution of 50 µmol of Fmoc amino acid and 50 µmol of 1-hydroxybenzotriazole and 100 µl of a DMF solution of 75 µmol of Ν, Ν-dicyclohexylcarbodiimide are added; the switching time is approx. one hour. Cleavage of the Fmoc protecting group is done by using 300 μΐ of a 40% solution of pipe-ridin in DMF. The cleavage time is approx. 20 minutes.

5 The washing steps are performed with 300 µl DMF.

The final peptide cleavage from the carrier can be effected in the wells by manual or automatic addition of 300 µl trifluoroacetic acid (20 minutes reaction time). The acylation of free groups (NH 2, OH) 10 can be done analogously by the addition of suitable acid anhydrides, e.g. acetic anhydride and pyridine. After completion of the reaction, the solution is sucked off and led to reprocessing.

As seen in the above explanation, all steps of peptide synthesis are performed in open containers. Nevertheless, in carrying out the synthesis of the invention, the peptides are obtained in very high purity.

FIG. 1 shows an example of a needle for a pipetting robot with an arm: (1) needle; (2) and (3) the needle and suction side of the needle; (4) hose for rinsing solvent for rinsing; (5) filter.

The following example illustrates the process of the process, describing the build-up of a peptide in a well. The purity of the product is noticed.

Example

44 different undecapeptides 30 were synthesized by the Tecan pipetting robot RSP 5052. In any case, starting material was a tetrapeptide Fmoc-Arg (Mtr) -Gin-Arg- (Mtr) -Tyr (tBu) -linker-linked tetrapeptide linker. polystyrene (attached about 0.5 DK 8 mmol / g resin). 44 times, 10 mg of this resin was introduced into the wells of a microtiter plate (5 µmol peptide) and the synthesis started. By way of example, the synthesis of: 5

Ac-His-Tyr-Ile-Leu-Asn-Ile-Thr-Arg-Gln-Arg-Tyr-NH 2

The synthesis of the other 4 peptides proceeded analogously.

10 Synthesis cycle: 300 µl piperidine (40%) in DMF (5 minutes) 300 µl piperidine (40%) in DMF (20 minutes) 10 times washing each time with 300 µl DMF (2 minutes) 200 µl of a DMF solution which is 50 ymolar with respect to Fmoc-Thr (tBu) -OH and 50 ymolar with respect to HOBt 20 and 100 µl of a DMF solution 75 ymolar with respect to DCC (1 hour) 25 times wash with 300 µl DMF (2 minutes).

In identical synthesis cycles, similar solutions of:

Fmoc-Ile-OH

5

Fmoc-Leu-OH

Fmoc-Asn-OH

Fmoc-Ile-OH

Fmoc-Tyr (tBu) -OH

Fmoc-His (Trt) -OH.

15 with the same concentrations. The final acetylation was carried out as usual with 300 µl of a DMF solution 40 ymolar in acetic anhydride and pyridine after Fmoc digestion.

The resin cleavage occurred with 300 µl trifluoroacetic acid / 1% anisole for 30 minutes, washing 5 times with 300 µl trifluoroacetic acid. The combined trifluoroacetic acid solutions were stored under moisture exclusion for 1 hour at 50 ° C and the trifluoroacetic acid was distilled off under vacuum. The residue was treated with ether in an ultrasonic bath, the ether was decanted, the residue dissolved in water and lyophilized. HPLC chromatogram (RP18 column) (Fig. 2).

10 DK 173633 B1 Running agent A Water / acetonitrile / trifluoroacetic acid 95/5 / 0.2 Running agent B Water / acetonitrile / trifluoroacetic acid 5 20/80 / 0.2 FAB mass spectrogram: M + H: 1518.

Claims (6)

A method for fully automatic simultaneous synthesis of multiple polypeptides according to the solid phase synthesis method, characterized in that a pipetting robot is used, whereby polymeric carrier material which may be attached to the starting portions of the desired peptides is placed in wells on microtiter plates, after which the peptides are built into wells. according to the known solid phase synthesis method, and any free amino groups and / or hydroxy groups of the peptides are acylated, and / or the peptides are then separated from the carrier, the reagents or washing liquids required for the individual stages being supplied to the wells of the corresponding storage containers via one or more robotic arms with cannulas, and the reagents or washing liquids present in the wells present in the wells are aspirated with a robotic arm with cannula after the sufficient residence time, the individual steps of the process being controlled by that of the computer connected for the robot, programmed p rogram. 2. A method according to claim 1, characterized in that the cannula is rinsed on the outside each time between aspirating the liquid and adding reagents. 3. A process according to claim 1 or 2, characterized in that the reagents are used for up to 20 times the excess of the stoichiometric amounts required. 4. Pipette robot needle, characterized in that its upper end is connected to the robot arm's metering device and the lower end of which is equipped with a filter and / or a flushing device, whose DK 173633 B1 upper end is connected to the robot arm .., and the lower end of which reaches down to barely above the lower end of the cannula, and whose opening is made such that the lower end of the cannula can be flushed. A cannula according to claim 4, characterized in that the cavity is divided into two parts by a dividing wall running from above and below. A cannula comb, characterized in that it consists of several needles according to claim 4 or 5.