CZ279293A3 - Reaction vessel for multisequential synthesis of peptides and peptide libraries - Google Patents

Description

Reactor for multi-sequence synthesis of peptides and peptide libraries

Technical field

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a multispecific reactor and a peptide library on a solid support. J Γ * Ϊ '5 5 • 2 γλ ť! BACKGROUND OF THE INVENTION Synthesis of cn peptides

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Solid phase peptide synthesis according to R.B. Merrifield described, for example, in Stewart J.M. and Young J.D., Solid Phase Peptide Synthesis, Freeman, San Francisco, 1985, have introduced the possibilities of partial or complete automation in this field of peptide synthesis. Merrifield R.B., Stewart J.M. and Jarnberg N., Apparatus for the automated synthesis of peptides, US 3,531,258; Brunfeldt K. Roepstorff P. and Halstrom J., Reaction System, US 3,557,077; Kubodera T., Hara I. and Makabe et al., Apparatus synthesis of peptides or the like organic compounds, US 3,647,390; Won Kil Park and D. Regoli, Solid Phase Synthesis System, US 3,715,190; Bridgham J. et al. Automated polypeptide synthesis apparatus, US 4,668,476; Saneii H.H., Solid Phase Synthesizer, US 4,746,490 and hence a number of synthesizers realized or proposed. In principle, they differ in chemical synthesis methodology, most commonly in Fmoc or Boc strategies: Atherton E. and Sheppard R.C., 1989, Solid phase peptide synthesis. A practical approach. IRL Oxford University Press; Barany G. and Merrifield RB, 1980, Solid-phase peptide synthesis, The peptides, Gross and Meienhofer (Eds.) Pp. 1-284, Academic Press, or carrier, various types of polymers, cotton and the like, Alternatives Carriers and Methods in Solid Phase Synthesis; Lebl et al., Eichler, J. and Bienert, M .: Peptides 1990, Giralt and Andren (Eds.); pp. 153-155, or technical and design solutions.

The prior art synthesizer design solutions have focused on the simultaneous synthesis of the maximum amount of a given peptide or the synthesis of only one or a limited number of peptides of the highest possible quality and possibly the amount. More recently, these two approaches have been supplemented with the aim of obtaining all possible combinations of amino acid chains of a selected length and from pre-selected amino acids, as described in Lam K.S., Salmon S.E., Hersh E.M., Hruby V.J., Kazmierski W.M. and Knapp, R.J., A new type of synthetic peptide library for identifying ligandbinding activity, 1991, Nature vol. 354, pp. 82-84; Houghten R.A., Pinilla C., Blondelle S.E., Appel J.R. , Dooley C.T. and Cuervo J. H., Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery, 1991, Nature vol. 354 pp. 84-86. This so-called randomization yields a bank or library of peptides whose various activities are subject to further testing.

The previously known automatic version of the synthesizer consists of a cylinder mounted on a circular body, around whose circumference there are formed chambers with a solid support, each of which is provided on the underside with a frit with a drain channel and other necessary accessories. The cylinder is closed with the amino acids of solvents and reagents, the cleaning of the wells after synthesis and in particular the uniform distribution of the solid support into the wells in case of peptide library synthesis has not been satisfactorily resolved. The design of the reactor does not allow flexible changes in the volume of the support used.

head with solution inlets of this type of reactor however

SUMMARY OF THE INVENTION

The above-mentioned drawbacks are overcome by a cylinder-type multi-sequential peptide synthesis reactor and a peptide library from a top-down, rotating head with a central stirrer and with holes for solvent, amino acid and reagent solutions, and a center-conical reactor body underneath. According to the invention, radial recesses are formed in the central cone all over its surface from the center to the inlets, the number of which corresponds to the inlets formed in the bottom of the body along the circumference of the reactor between the tube walls and the center / cone. The inlets open into a frit-fed bottom end disposed therein in the bottom part. Drainage channels are formed under the frits. Amino acid solution and reagent inlet tubing are mounted in conduits located above the individual containers in a common plate. In this case, the common plate is mounted in the vertical direction above the head, to which it is mechanically connected and rotatable. The filling outlet is connected to the inlet leading to the conical part of the swivel head via a sump, a change-over valve and a pump.

The advantage of the reactor according to the invention is that it can be used for the synthesis of peptides by both major Fm processes. and Boc. The design remains the same for both procedures, changing only the number and type of solvents and reagents. In addition to this, the reactor allows the bound peptides to be cleaved from the support and collected in collector tubes. An essential advantage of this embodiment of the reactor is that it allows both peptide synthesis and peptide library synthesis using the flotation and sedimentation principles to uniformly distribute the solid support particles. Since conventional peptide synthesis along with randomization synthesis can be performed in this reactor, it is also possible to automate the preparation of peptides having only a randomized portion of their sequence.

The radial recesses formed on the surface of the central cone and the vessel inlets divide the reactor surface area into the same, sharply delimited sectors. The solid support, after mixing in solution, is evenly distributed to all vessels distributed around the reactor perimeter. The reactor is dismountable, which simplifies handling and especially cleaning of all its parts. The containers are replaceable and can be reused after cleaning. The height and volume of the containers can be varied as required. The board in which the holders for the supply of amino acid solutions and reagents are fixed is provided with a vertical movement. The position of the board in the upper position prevents contamination and adherence of the carrier at the mouth of the leads.

Overview of the drawings

1 shows one embodiment of a reactor according to the invention and FIG. 2 shows a variant of a central cone.

The exemplary embodiments of the invention have a central radial

The reactor for multis sequential synthesis of peptides and peptide libraries according to FIG. 1 is formed by a cylinder 1 mounted on a body 20 with a central cone 6. Between cone 6 and the wall of cylinder 1, inlets 20 are formed in the body 20 The central cone 6 has radial recesses 26 over its entire surface, which extend from the tip of the cone 6 to the individual inlets 2. Each recess 26 thus corresponds to one inlet 2. The exchangeable containers 4 are The reactor is sealed from above by a rotating head 5 provided with a drive 18. In the center of the head 5, a conical hollow part 10 with a central bore 10 is formed on its inner side. under which is located a rotary impeller 9, driven by an electric motor 22. d, through each replaceable container 4 outside the conical hollow part 10, openings are provided for guiding 11 of the inlet tubing 17. Outside of the conical hollow part 10, an opening is provided in the head 5 for draining the solution suspension and solid support 23 from the reactor. and the solution is further passed through the transfer valve 16 and the pump 14 through an inlet 13 through the orifice in the head 5 to the space of the conical hollow part 10 back to the reactor. The conduits 11 of the supply hoses 17 are rigidly connected to a common plate 12 equipped with a drive 19 for vertical movement and moving along the central cylinder 28 and the connecting rods 24. connecting the head 5 and the upper body 25. The body 25 is provided with a drive 18 for rotational movement of the head 5 and the common plate 12. The inlet hoses 17 are led from reservoirs of amino acid solutions (not shown) and reagents. Valves 21 each actuating one channel 7 are connected via a system of switch valves (not shown) to an inert gas reservoir, a vacuum source and a waste.

The number of containers 4 can be varied by replacing the body 20 and the lower part 8. Depending on the amount of filling required, the height and volume of the containers 4 can be varied. They can be cylindrical, square, conical or the like. Protrusions 27 may be formed on the inner surface of the containers 4 to disrupt and prevent the agglomerated carrier from entering the reactor space.

The reactor has three basic functions. First, it allows simultaneous multiple peptide synthesis by both the most important Fmoc and Boc methods. Secondly, it allows the synthesis of peptide libraries consisting of a combination of a given number of different amino acids of a given chain length again by both methods, and thirdly, it is suitable for the simultaneous cleavage of the peptides thus prepared, avoiding cross-contamination. For multiple synthesis of peptides and peptide libraries in both the Fmoc and Boc strategies, the reactor is equipped with an amino acid solution dosing device, which is then delivered via inlet tubes 17 to individual containers 4 according to the desired sequences. At the time of dispensing, the common plate 12 is placed in the lower position and the mouth of the feed tubing 17 passing through the conduits 11 is close to the inlets 2 in the reactor body 20, i.e. above the containers 4. After dispensing, the common plate 12 extends to the upper position so the orifice of the inlet tubing 17 is located in the upper portion of the reactor vessel and is free of contamination. Solvents and reagents are either sucked out of the reactor by vacuum channels 7, or by means of a pump 14 through a discharge outlet 23. During the reaction, deprotection, washing and other operations, the contents of the individual vessels 4 can be agitated by lowering the pressure of the inert gas supplied to the bottom of the reactor via the shut-off valves of block 21 under frits 3 and a switch valve block (not shown). The gas will only stir up the contents of the containers.

Various types of solid supports such as resin, cotton, cotton fabric and the like can be used in the synthesis of peptides in this reactor. In the synthesis of randomized peptides and peptide libraries, it is necessary to use such carriers that form a suspension in solution. The dosing and washing process is adapted to the type of carrier and the synthesis conditions.

Upon completion of the multiple peptide synthesis, the solid support and bound peptide vessels 4, as well as the reactor interior, are washed with a suitable solvent such as dimethylformamide and dried. The digestion reagent is then added and the resulting peptide solution is aspirated into tubes of a collector not shown.

During cleavage, possible mixing of the cleaved peptide solutions should be avoided. Therefore, each reactor vessel 4 must be closed from below by a frit 3 and one vessel 7 in the reactor body 20 corresponds to one channel 7.

When synthesizing peptide libraries, the required amount of solid support, such as a synthetic resin, is introduced into the reactor.

The first distribution of the carrier quantity into the individual containers is identical to the randomization step, i.e., the uniform distribution of the carrier to all containers.

The binding of the amino acids to the solid support takes place in the container 4 in a suitable solvent, where the beads are suspended and mixed with a low pressure inert gas stream which is fed under the individual frits 3 of the containers 4. The solvent quantity and inert gas pressure must be such that mixing of the contents within one container 4 and at the same time there must be no excessive rise and ripple of the level in the containers above the common interface and intermixing of the contents of adjacent containers.

After the addition of the solutions of the individual amino acids via the supply tubes 17, in this case the same amino acid is always added to the same container 4, the reactions of the first cycle are carried out. Washing, deprotection and activation are performed together with the same reagents for all amino acids in all vials 4 simultaneously. The dosing of reagents and solvents, mixing and aspiration is consistent with conventional multi-sequential synthesis described above. Upon completion of the first cycle, the solid carrier beads are again suspended in the liquid by means of an inert gas stream at a higher pressure and the entire cycle is repeated.

Randomization takes place by adding a suitable solvent to the reactor space so that the central cone is flooded with the solvent. Subsequent introduction of a higher pressure of inert gas into the passages 7 and using a stirrer 9 disperses the used solid support in the liquid in the reactor space and mixes. An even statistical distribution of the solid support in the reactor occurs after the flotation is interrupted by stopping the gas supply, stopping the stirring, and then evacuating the solution from the reactor. The beads sediment in the vessels and on the delimited areas of the central cone. By appropriately rinsing the central cone while continuously evacuating the reactor, the carrier adhering to the central cone 6 is flooded into the appropriate containers 4. By repeating the entire process several times, a peptide library containing peptides of the desired length is prepared.

Upon completion of the last cycle, a purge solvent is added to the reactor vessel with which the solid support is then sucked off by the charge outlet 23. The solid support beads are retained in the well 15 and the flushing solvent is returned via the transfer valve 16 and the pump 14 through the inlet 13 to the reaction vessel until all the solid support beads are retained.

Claims (2)

PATENT CLAIMS 1. A multi-sequential peptide and peptide library synthesis reactor, comprising a cylinder, a closed-top rotating head with a center stirrer and holes for the inlet of amino acid solutions, solvents and reagents, and product evacuation, and a bottom cone with a center cone reactor. a radial recess (26) is formed in the central cone (6) over its entire surface from the center to the inlets (2), the number of which corresponds to the inlets (2) formed in the body (20) around the reactor circumference between the cylinder walls (1) and the central cone (6), wherein the inlets (2) open into the sintered exchange containers (4) at the lower end provided with a frit (3) disposed in the lower part (8) in which channels (7) are formed under the frits (3), wherein inlet ducts (17) of amino acid and reagent solutions are fixed in the guide (11) of the common plate (12), while the common plate (12) is slidably mounted vertically over the head (5) with which it is mechanically rotatably connected, the filling outlet (23) being connected via a sump (15) to the changeover valve (16) and the pump (14) to the inlet (13) opening into the conical part (10) of the head (5). 2. A reactor for multis sequential synthesis of peptides and peptide libraries according to claim 1, characterized in that the replaceable vessels (4) are circumferentially provided with internal protrusions (27).