DE4403967C2 - Method and device for producing peptides, peptoids and oligonucleotides and their use - Google Patents

Description

The present invention relates to a method for producing peptides, peptoids and oligonucleotides and their use through fully automatic, simultaneous and multiple solid phase synthesis and a device for performing the method and its use according to the claims. Areas of application of the invention are chemical and pharmaceutical industry and laboratory equipment manufacturing.

The state of the art is subsequently to be explained using the simultaneous multiple solid phase peptide synthesis.

For fully automated solid phase peptide synthesis, 30 years the method developed by Merrifield (e.g. R. B. Merrifield, J. Am. Chem. Soc., 85 (1963) 2149; G. Barany, R. B. Merrifield in The Peptides, Analysis, Synthetics, Biology, Vol. 2, 1-284 (1980), ed. Gross, Meierhofer Academy Press, New York) and further developed versions (e.g. DE 36 31 662, DE 38 28 576, DE 40 05 518, DE 40 06 349) applied. Thereby becomes Mixing of the reagents and to accelerate the Exchange reaction between solid phase / carrier z. B. resin and Reagent mixture usually shaken the reaction vessel or pumped the reagent mixture. For all diamonds As a rule, research processes must be insufficient mechanical stability of the resins are taken into account. "Therefore magnetic stirrers were hardly used for this. Also the already known floating stirrers, which are either free-floating (US 44 98 785), be guided from above (US 47 59 635) or by a guide rod with the Lids connected (US 44 65 377) are unlikely to be advantageous or for the Task to be unsuitable " Transition to simultaneous, multiple solid phase synthesis occur this connection problems that were previously insufficient have been resolved.

The Zinsser devices previously available on the market Analytics or Abimed for simultaneous, multiple peptide synthesis There are various ways to deal with these problems overcome (DE 40 06 349 A1, DE 38 28 578 A1). On the one hand, after the resin has settled, from the supernatant of the reagent mixture over the resin aspirated certain volume through a cannula and immediately afterwards splashed back. This requires an additional volume Formation of the surplus and associated with it unnecessarily large excess of reagents. Adequate mixing is only achieved with very small amounts of resin. Already with 20 mg  Resin can cause problems when mixing the resin Reagents are coming, and with even larger amounts of resin, it drops Efficiency of this mixing method decreases very quickly. On the other ways try different solvents Mix density so that the resin is free in the mixture swims. The disadvantage here is the reduction in active Reagent concentrations by the second solvent. The necessary volume of the second solvent for the free swimming of the resin is also dependent of the resin and also of the peptide sequence changes accordingly even during synthesis. Hence the free swimming is the Hardly to guarantee resin in all cases. The The efficiency of the mixing is also low and decreases also with increasing amount of resin.

The invention is based, a method and a task Device for simultaneous, multiple solid phase synthesis to provide, which avoids the disadvantages mentioned above become. These disadvantages also apply to the Solid phase syntheses of other bioorganic polymers.

The object is achieved by the in claims 1 to 8 outlined measures resolved. Mixing the reagents with the carrier is carried out according to the invention by a suspended Magnetic stirrer in the individual reaction vessels, what in particular with insufficient mechanical stability of the resins from decisive advantage is. This creates a possible abrasion the resins that clog fries or pipettes can, avoided.

The method according to the invention is implemented by a device according to claims 5 to 7. For this purpose, in a circular coil system under each Reaction vessel or a rotating field around each reaction vessel generated. The current flowing through the windings builds in rotating magnetic field, which is on one in this system  located magnetic stirrer generates a torque, the Control the speed of the stirrer. Usually it will cylindrical reaction vessel inside the coil system brought, then the magnetic stirrer is rotated into the Magnetic field if it is below the level of the Magnetic field, and can now be more freely in the suspension move. After switching off the magnetic field, stirrers and Resin to the bottom and the supernatant solutions can either be from Pipetted off at the top or more advantageously by a frit in the bottom aspirated through the reaction vessels or through a Shielding gas, e.g. B. nitrogen, pressed through the frit become.

In the simple variant, the device for carrying out the process also as efficient and gentle washing and Filtration unit, preferably for biopolymers, carrier-bound Biopolymers, cell suspensions and other sensitive biological materials are used.

An effective resin / reagent mixing is achieved by the invention thereby the reliability of the individual syntheses and individual synthesis steps elevated. This leads to a purer raw product with a reduced use of Reagents. For example, in the synthesis of the 18-mer peptide monochloroacetyl GKKRPKPGGGWNTGGFRY-OH, compared to an analog synthesis without Magnetic stirrer, an increase in yield from 50% to 80% (target sequence of the raw product) reached.

The invention is intended to be described below using an exemplary embodiment are explained in more detail, but not by the invention is restricted.

embodiment

A steel plate with the dimensions width 195 × length 360 × Height 35 mm has 5 × 10 = 50 through holes. The holes have a diameter of 18 mm and are at a distance of 30 mm from each other. The spools to generate the rotating field are circular around each borehole  arranged below the steel plate.

The holding device for the reaction vessels with a Outside diameter of 16 mm is above the Steel plate. The reaction vessels are arranged so that their floor at the top of the steel plate, inside the planar unit or about 5 mm below the planar unit.

Claims (8)

1. A process for the preparation of peptides, peptoids and oligonucleotides by fully automatic, simultaneous and multiple solid phase synthesis, characterized in that
the individual carrier-reagent mixtures or carrier-washing mixtures in all reaction vessels are stirred by a magnetic stirrer suspended,
distributes the reagents or washing liquids using a pipetting robot and
if necessary, the reaction vessels are tempered and / or vibrated. 2. The method according to claims 1, characterized in that one the reagents or washes from all of them simultaneously Reaction vessels through a filter frit located in the bottom removed, preferably by applying a vacuum or by an excess pressure generated with a protective gas. 3. The method according to claims 1 and 2, characterized in that the reaction vessels at 0 to 120 ° C, preferably at 30 to 50 ° C tempered. 4. The method according to claims 1 to 3, characterized in that one the vibration of the reaction vessels by ultrasound causes. 5. Device for performing the method according to claims 1 to 4, characterized by in a planar unit summarized, for the generation of rotating magnetic fields circularly arranged coil systems, which are above or below that fixed in a holding device Reaction vessels is located through vertical holes in the  planar unit in the respective center of the coil systems and by positioning the bottom of the cylindrical Reaction vessels above, inside or below the planar Unit. 6. The device according to claim 5, characterized by in the planar unit arranged temperature control devices. 7. Device according to claims 5 and 6, characterized by arranged heating coils, surface heating, heating foils or Thermostat. 8. Use of the device according to claims 5 and 6 as a washing and filter unit for biopolymers, carrier-bound biopolymers and cell suspensions.