DEVICE FOR PARALLEL OLIGOMER SYNTHESIS, METHOD OF PARALLEL OLIGOMER SYNTHESIS AND USE THEREOF

Abstract

A device for parallel oligomer synthesis having a centrifuge with a plurality of reactor holders configured to retain reactors at an angle and a plurality of siphon based outflow holders are disclosed. A method of parallel solid-based peptide synthesis following the timing protocol of the device and a use of the device for parallel oligomer synthesis are also disclosed.

Claims

1. A device for parallel oligomer synthesis, the device comprising: a centrifuge, the centrifuge comprising a plurality of reactor holders, the reactor holders being configured to retain reactors at an angle, and a plurality of holders for siphon based outflows, a plurality of reactors, the reactors having upper ends and lower ends, the lower ends comprising nozzles, the reactors being positioned at an angle in the reactor holders, a plurality of siphon based outflows, the siphon based outflows having first ends and second ends, the first ends being connected to the nozzles of the lower ends of reactors, and the siphon based outflows being configured to work on a siphoning principle, a distribution rotor, the distribution rotor being provided with pre-activation compartments at its outer circumference, the pre-activation compartments being provided with grooves directed towards the outer edge of the distribution rotor, and configured to enable the transfer of contents from the pre-activation compartments of the distribution rotor to the reactors upon rotation of the distribution rotor, a lifting device, the lifting device being configured to enable the distribution rotor to move in vertical direction between an upper position and a lower position, a positioning device, the positioning device being configured to enable a rotation of the distribution rotor in the upper position independently from the centrifuge, a driving motor, the driving motor, the centrifuge and the distribution rotor having the same common axis, the driving motor being configured to enable a synchronized rotation of the centrifuge and the distribution motor in the lower position, an outflow channel, the outflow channel being positioned at the outer circumference of the centrifuge, the outflow channel arranged so that the siphon based outflows empty into said outflow channel continuous groove, the outflow channel further comprising a plurality of outflow openings, a system of outflow pipes, the outflow pipes being connected to outflow openings of the outflow channel and being positioned below the centrifuge, a dosing device, the dosing device comprising a distribution valve, the distribution valve having a plurality of ports, and a dosing pump, the dosing pump being configured to measure the exact quantity of liquid reagents the dosing pump being connected to the distribution valve, the distribution valve being connected to stock solutions of reactants and/or solvents and/or activating reagents and/or coupling reagents via plurality of ports of the distribution valve and via tubing, and a control device, the control device configured to retrieve a sequence of a peptide to be synthesized and to determine a sequence specific timing protocol, the control device being connected to said driving motor, said dosing device and said lifting and positioning device.

2. The device for parallel oligomer synthesis according to claim 1, wherein the centrifuge further comprises: a first disk made of inert material, the first disk being provided with the reactor holders on its inner circumference, and with a second set of openings for the siphon based outflows on its outer circumference, a second disk made of inert material, the second disk being seated on the same axle as the first disk and positioned below the first disk, and provided with fixation slots for fixing the reactors and with a first set of openings for the siphon based outflows, such that the plurality of reactors are situated in the reactor holders of the first disk and fixed in the fixation slots of the second disk, and a plurality of the siphon based outflows are connected to the plurality of reactors through the first set of openings in the second disk and fixed by the second set of openings in the first disk.

3. The device for parallel oligomer synthesis according to claim 1, wherein the siphon based outflow is the S-trap outflow comprising an S-shaped tube.

4. The device for parallel oligomer synthesis according to claim 1, wherein the siphon based outflow is the I-trap outflow comprising a channel connected to the nozzle of the reactor, a peek tubing comprising a tube with a first end and a second end, the first end being connected to the channel, the channel and the peek tubing being arranged so as to work on a siphoning principle, a cover having a first end and a second end, a channel holder and a channel system comprised in the channel holder, the first end of the cover being attached to the channel holder.

5. The device for parallel oligomer synthesis according to claim 4, wherein the channel system further comprises at least one stopcock and/or the channel holder is formed integrally with the fixation slots, forming a separate element, fixed to the lower disk.

6. The device for parallel oligomer synthesis according to claim 1 further comprising a selector device, and a plurality of storage containers for storing reagents, wherein the selector device is connected to the plurality of storage containers and to the dosing device via tubing.

7. The device according to claim 6, wherein the selector device comprises: coplanar layers of graphite pressed together by spring, plurality of intake ports connecting the selector device with the plurality of storage containers, and an outtake port connecting the selector device with the dosing device.

8. The device for parallel oligomer synthesis according to claim 6 further comprising a distribution device, the distribution device being connected via tubing to the selector device, dosing device and to the distribution rotor, the distribution device being preferably a solenoid valve connecting the dosing device with either the selector device or the distribution rotor.

9. The device for parallel oligomer synthesis according to claim 1, wherein the reactors are syringes, preferably made of plastic, and wherein the syringes comprise filters, the filters preferably made of sintered glass, plastic or metal mesh, or any porous material.

10. The device for parallel oligomer synthesis according to claim 1, wherein it further comprises a temperature control device, the temperature control device comprising an infrared radiator, microwave radiator, and temperature sensor with feedback control.

11. The device for parallel oligomer synthesis according to claim 1 further comprising a plurality of magnets, the plurality of magnets being attached to the inner side of the centrifugation drum, and a plurality of small magnets to be placed inside the reactors, in order to stir the contents of the reactors upon rotor movements.

12. The device for parallel oligomer synthesis according to claim 1, wherein the dosing device is a programmable syringe pump.

13. The device for parallel oligomer synthesis according to claim 1 further comprising: a centrifugation drum for placing the centrifuge, and a box, the box having a circular opening for placing the centrifugation drum and the system of outflow pipes.

14. The device for parallel oligomer synthesis according to claim 13 further comprising: a sealed cover positioned over the centrifugation drum, the sealed cover having a first part and a second part, the first part made of glass or plexiglass, and the second part made of the same inert material as the centrifuge.

15. A method of parallel solid-based peptide synthesis using the device according to claim 1, characterized in that it comprises the following steps: a) resin beads provided with functional groups suitable for immobilization of amino acids are placed in the reactor; eventually equipped with a magnet placed inside the reactor; b) the control device retrieves the sequence of a peptide to be synthesized and determines a sequence specific timing protocol; c) the dosing device dispenses measured quantity of first N-protected amino acid through the lifting and positioning device to the first one of the pre-activating compartments of the distribution rotor; d) the distribution rotor turns to enable the second pre-activation compartment a reception of contents of the dosing device through lifting and positioning device; e) steps c) and d) repeat at most until each pre-activation compartment contains N-protected amino acid solution according to the timing protocol; f) the dosing device dispenses measured quantity of a solution of carboxyl group activating compound through the lifting and positioning device to the first one of the pre-activating compartments of the distribution rotor; g) the distribution rotor turns to enable the second pre-activation compartment a reception of contents of the dosing device through lifting and positioning device; h) steps f) and g) repeat at most until each pre-activation compartment receives solution of carboxyl group activating compound according to the timing protocol; furthermore, color changing indicator may be added in this step for indication of the completion of the condensation step l); i) distribution rotor is lowered to position in which it makes contact with reactors in the centrifuge; j) the distribution rotor rotates in a synchronized rotation with the centrifuge, causing the contents of the pre-activation compartments to transfer by a centrifugal force via grooves into the reactors; k) distribution rotor detaches from reactors by lifting mechanism and reactors content is being stirred; l) condensation reaction of activated N-protected amino acids with functional groups suitable for immobilization of amino acids on the resin beads proceeds for predetermined time, or, alternatively, if color changing indicator was added, until the color change indicates complete reaction; m) the centrifuge with reactors rotates so that the liquid from the reactors is transferred by a centrifugal force via the siphon based outflows out of the reactors; n) the dosing device dispenses measured quantity of a solvent through the lifting and positioning device to each one of the reactors of the centrifuge, and the centrifuge with reactors rotates so that the liquid from the reactors is transferred by a centrifugal force via the siphon based outflows out of the reactors; o) the dosing device dispenses measured quantity of a deprotection agent solution through the lifting and positioning device to each one of the reactors of the centrifuge, where a deprotection of N-protected end of the amino acids takes place; p) the centrifuge with reactors rotates so that the liquid from the reactors is transferred by a centrifugal force via the siphon based outflows out of the reactors; q) repeating step m) in order to wash the resin beads from non-reacted reagents; furthermore, color changing indicator may be added in this step for indication of the completion of the condensation step l); r) repeating steps c) to p) for second and following N-protected amino acids according to the timing protocol, until the desired peptide sequence is completed; s) cleaving the final peptide from resin beads by a cleaving agent.

16. The method according to claim 15, wherein, in the step k) reactors content is being stirred by repetitive back and forth motion of the rotor or by a slow rotation under assembly of magnets when small magnets are placed in each reactor.

17. The method according to claim 15, wherein the step n) is repeated, preferably it is repeated at least twice, more preferably step n) is repeated 5 times.

18. A method for parallel oligomer synthesis comprising the step of providing the device according to claim 1.

19. A method for oligonucleotide or carbohydrate synthesis comprising the step of providing the device according to claim 1.

20. A method of peptide synthesis using the device according to claim 1, characterized in that it comprises the following steps: a) resin beads provided with functional groups suitable for immobilization of amino acids are placed in the reactor; eventually equipped with a magnet placed inside the reactor; b) the control device retrieves the sequence of a peptide to be synthesized and determines a sequence specific timing protocol; c) the dosing device dispenses measured quantity of first N-protected amino acid through the lifting and positioning device to the first one of the pre-activating compartments of the distribution rotor; d) the distribution rotor turns to enable the second pre-activation compartment a reception of contents of the dosing device through lifting and positioning device; e) steps c) and d) repeat at most until each pre-activation compartment contains N-protected amino acid solution according to the timing protocol; f) the dosing device dispenses measured quantity of a solution of carboxyl group activating compound through the lifting and positioning device to the first one of the pre-activating compartments of the distribution rotor; g) the distribution rotor turns to enable the second pre-activation compartment a reception of contents of the dosing device through lifting and positioning device; h) steps f) and g) repeat at most until each pre-activation compartment receives solution of carboxyl group activating compound according to the timing protocol; furthermore, color changing indicator may be added in this step for indication of the completion of the condensation step l); i) distribution rotor is lowered to position in which it makes contact with reactors in the centrifuge; j) the distribution rotor rotates in a synchronized rotation with the centrifuge, causing the contents of the pre-activation compartments to transfer by a centrifugal force via grooves into the reactors; k) distribution rotor detaches from reactors by lifting mechanism and reactors content is being stirred; l) condensation reaction of activated N-protected amino acids with functional groups suitable for immobilization of amino acids on the resin beads proceeds for predetermined time, or, alternatively, if color changing indicator was added, until the color change indicates complete reaction; m) the centrifuge with reactors rotates so that the liquid from the reactors is transferred by a centrifugal force via the siphon based outflows out of the reactors; n) the dosing device dispenses measured quantity of a solvent through the lifting and positioning device to each one of the reactors of the centrifuge, and the centrifuge with reactors rotates so that the liquid from the reactors is transferred by a centrifugal force via the siphon based outflows out of the reactors; o) the dosing device dispenses measured quantity of a deprotection agent solution through the lifting and positioning device to each one of the reactors of the centrifuge, where a deprotection of N-protected end of the amino acids takes place; p) the centrifuge with reactors rotates so that the liquid from the reactors is transferred by a centrifugal force via the siphon based outflows out of the reactors; q) repeating step m) in order to wash the resin beads from non-reacted reagents; furthermore, color changing indicator may be added in this step for indication of the completion of the condensation step l); r) repeating steps c) to p) for second and following N-protected amino acids according to the timing protocol, until the desired peptide sequence is completed; s) cleaving the final peptide from resin beads by a cleaving agent.

Description

BRIEF DESCRIPTION OF FIGURES

[0083] FIG. 1 shows a cross-sectional view of the device for parallel oligomer synthesis in the embodiment in which the device comprises all sub-systems.

[0084] FIG. 2 shows a perspective view of the device for parallel oligomer synthesis in the embodiment in which the device comprises all sub-systems.

[0085] FIG. 3 shows a cross-sectional detail view of the reactor in which the oligomer synthesis occurs together with the S-trap outflow system.

[0086] FIG. 4 shows a cross-sectional view of the centrifugation subsystemreactors fixed in the reactor holders and fixation slots.

[0087] FIG. 5 shows a perspective view of the centrifugation subsystemreactors fixed in the reactor holders and fixation slots.

[0088] FIGS. 6a and 6b show a top view and a perspective view of the distribution rotor.

[0089] FIG. 7 shows a cross-sectional view of the connection between the distribution rotor and the positioning mechanism and the lifting mechanism, which enable the movement of the distribution rotor.

[0090] FIG. 8 shows a perspective view connection between the distribution rotor and the positioning mechanism and the lifting mechanism, which enable the movement of the distribution rotor.

[0091] FIG. 9 shows a cross-sectional view of the centrifugation drum and the driving motor of the centrifuge.

[0092] FIG. 10 shows a perspective view of the centrifugation drum and the driving motor of the centrifuge.

[0093] FIG. 11 shows a cross-sectional view of the selector device.

[0094] FIG. 12 shows a perspective view of the selector device.

[0095] FIG. 13 shows a detailed view of the graphite layers within the selector device.

[0096] FIG. 14 shows a cross-sectional detail view of the reactor in which the oligomer synthesis occurs together with the I-trap outflow system.

[0097] FIG. 15a shows a cross-sectional detail view of the I-trap outflow system.

[0098] FIG. 15b shows a perspective view of the I-trap outflow system.

[0099] FIG. 16 shows a schematic view of the device for parallel oligomer synthesis in the embodiment in which the device comprises all sub-systems.

[0100] FIG. 17 shows a schematic view of the device for parallel oligomer synthesis in the embodiment in which the device comprises a solenoid valve in addition to all other sub-systems.

[0101] FIG. 18 shows an HPLC trace of a 21mer peptide

[0102] FIG. 19 shows a mass spectrum of a 21mer peptide

[0103] FIG. 20 shows an HPLC trace of a model YGGFL peptide

EXAMPLES

Example 1

[0104] In the following example an embodiment device for parallel oligomer synthesis will be described in the embodiment in which the device comprises all sub-systems in accordance with FIG. 1.

[0105] The synthesis itself takes place in reactors (FIG. 3), which are constituted by plastic syringes 101. Each of the reactors has an upper end 102 and a lower end 103. The upper end is an opened end and the lower end comprises a nozzle 104. Inside the reactor 101, a filter 105 is provided at the lower end 103 close to the nozzle 104 to provide a semipermeable membrane, which allows the resin beads, which are placed in the reactors 101 during the synthesis, to stay inside the reactor, while it allows the reagents to be washed out of the reactor 101. The filter 105 used in the example is a sintered plastic (polypropylene, Teflon), but may be also sintered glass, or metal.

[0106] The nozzles 104 of the reactors 101 are connected to S-trap outflows 201. Each of the S-trap outflows 201 has a tubular shape and comprises a first end 202 and a second end 203. The first end 202 of the S-trap outflow 201 is connected to the nozzle 104 of the lower end 103 of the reactor 101. The S-trap outflow 201 works on a siphoning principle, i.e. the second end 203 of the S-trap outflow 201 is positioned higher than the level of content in the reactor 101. The second end 203 of the S-trap outflow 201 also comprises a nozzle. The nozzle of the S-trap outflow 201 is positioned so that a liquid removed from the reactor 101 through the second end nozzle of the S-trap outflow 201 flows into a circular outflow channel 205.

[0107] As can be seen in FIGS. 4 and 5, the reactors 101 and the S-trap outflows 201 are positioned in a centrifugation subsystem comprising a centrifuge 301. The centrifuge in this example is a circular disk seated on a center axle 402. The centrifuge disk is in this example made of steel. The centrifuge is driven by a driving motor 401 connected to the axle 402 of the centrifuge 301 by a transmission 401b.

[0108] The centrifuge 301 shown in this example comprises two circular disksa lower disk 301a and an upper disk 301b, both made of steel and seated on a common axle 402 in the center of mass. The two disks 301a, 301b are positioned vertically at a distance, but physically connected by screws 308. The upper disk 301b comprises reactor holdersopenings 106, which are configured to retain the reactors 101 at an angle. The reactors 101 are positioned at an angle of 45 degrees in this example. The lower disk 301a comprises fixation slots 107, in which the nozzles 104 of the lower ends 103 of the reactors 101 are fixed. The reactors 101 are inserted from above, through the reactor holders 106 into the fixation slots 107.

[0109] The upper disk 301b is further provided with openings (not shown) for the second ends 203 of the S-trap outflows 201 to be retained in such a way that the nozzles of the second ends 203 of the S-trap outflows 201 are positioned in the circular outflow channel 205 and such that the content of the liquid in the reactor 101 reaches a level which is lower than the second end 203 of the S-trap outflow 201. In this way, the S-trap outflows 201 work on the siphoning principle, such that depending on the speed of rotation, the liquid may stay inside the reactor 101 or may be removed from it partially or completely. The liquid is removed from the reactor 101 through the nozzle of the S-trap outflow 201 and flows into the circular outflow channel 205, which is constituted by a groove positioned on the perimeter of the centrifuge 301 and aligned with the nozzles of S-trap outflows 201, such that the nozzles of S-trap outflows 201 are positioned in the circular outflow channel 205. The circular outflow channel 205 comprises outflow openings positioned on its bottom. The outflow openings of the outflow channel 205 are connected to outflow pipes, which provide an outflow system of the device 1 and drain away the liquid removed from the reactors 101 during the synthesis.

[0110] Above the centrifuge 301, a circular disk of the distribution rotor 302 made of plastic is positioned (FIGS. 6a, 6b). The distribution rotor 302 and the centrifuge 301 have the same common axis in the center of mass, however, the distribution rotor 302 is seated on its own axle 303 and it is connected to a positioning device 304, i.e. a driving motor to allow positioning of the distribution rotor 302 independently from the centrifuge 301. The distribution rotor 302 is further movable in vertical direction between a lower position and an upper position. The vertical movement is provided by a lifting device 305, which may be another motor or gas operated piston. In this example, the stepper motor is provided as the lifting device. The positioning and the lifting device can be seen in FIGS. 7 and 8. The distribution rotor 302 is driven by its own driving motor (positioning device 304) only when it is positioned in the upper position, when it is detached from the centrifuge 301. When the distribution rotor 302 is positioned in the lower position, the motor driving the distribution rotor (positioning device 304) is disengaged from the distribution rotor 302 and all actuation of the distribution rotor 302 is performed by the motor 401 driving the centrifuge 301 (FIGS. 9, 10) so that the synchronized rotation of the centrifuge 301 and the distribution rotor 302 is ensured.

[0111] The distribution rotor 302 comprises pre-activation compartments 306, which are positioned at the outer circumference of the distribution rotor 302, and which are provided with grooves 307 directed radially outwards, towards the outer edge of the distribution rotor 302. These grooves 307 enable the connection between the pre-activation compartments 306 of the distribution rotor 302 and centrifuge 301 when the distribution rotor 302 is in the lower position such that the contents of the pre-activation compartments 306 is transferred to the reactors 101 upon synchronized rotation of the distribution rotor 302 and the centrifuge 301 at medium rotation speed.

[0112] In this example, the pre-activation compartments 306 are filled in via a dosing device 501 (shown schematically in FIGS. 16, 17). The dosing device 501 comprises a distribution valve 502 and a dosing pump 504. The dosing pump 504, which is in this example a programmable syringe pump and is configured to measure the exact quantity of the liquid reagent to be dosed into the individual preactivation compartments, is connected to the distribution valve 502.

[0113] The distribution valve 502 comprises a plurality of ports, 12 ports in this example. The distribution valve 502 comprises at least one outtake port which is via tubing (not shown in figures) connected to the centrifugation subsystem (distribution rotor 302) and through which the individual pre-activation compartments 306 are filled in through the inlet port 709. The other ports of the distribution valve 502 are intake ports and provide for a connection with stock solutions of reactants and/or solvents and/or activating reagents and/or coupling reagents stored in storage containers placed in locations 710.

[0114] The dosing pump 504 is connected to the desired reactant via one of the ports of the distribution valve 502 to make the intake connection (while the other ports are closed) and a desired quantity may be sucked by the dosing pump 504 inside the dosing pump 504. Subsequently, the intake connection may be closed and an outtake connection between the dosing pump 504 and the distribution rotor 302 may be filled through the inlet port 709. In this way, the desired quantity of the reactant is transferred via tubing, and one of the pre-activation compartments 306 is filled in. This process is repeated with either the same reactant or other reactants until the desired number of pre-activation compartments 306 is filled in.

[0115] In this example, one of the intake ports of the distribution valve 502 is connected to a selection device 601 (FIGS. 11, 12), which is a valve configured to select the desired amino-acid. The selected amino acid is then transferred through the selector device 601, the distribution valve 502 and the dosing pump 504 into the individual pre-activation compartments 306 of the distribution rotor 302. The selection device 601 comprises a plurality of intake ports 602; 28 intake ports in this example. The intake ports are connected via tubing with the storage containers 701 containing various amino-acids. The selection device 601 further comprises an outtake port 603, which is connected via tubing with one of the intake ports of the distribution valve 502 of the dosing device 501. In order to retrieve the desired amino-acid from one of the storage containers 701, the connection between the dosing pump 504, one of the intake ports of the distribution valve 502, the outtake port 603 of the selector device 601 and one of the intake ports 602 of the selector device 601 is opened and the desired quantity of the desired amino-acid is sucked from the storage container 701 into the dosing pump 504. The desired quantity of the desired amino-acid is then transferred from the dosing pump 504 through the outtake port of the distribution valve 502 into one of the pre-activation compartments 306.

[0116] In this example, the selection device 601 comprises two coplanar, circular layers (604,605) of graphite pressed together by spring 606. The upper layer 604 comprises the plurality of intake ports 602, located close to the outer circumference of the upper layer. The lower graphite layer 605 comprises the outtake port 603 and an elongated groove 607. The outtake port 603 is located in the middle of the circular graphite layer 605. The elongated groove 607 has a first end 608 and a second end 609. The position of the first end 608 of the groove 607 is identical with the position of the outtake port 603. The groove 607 is elongated radially outwards from the position of the first end 608 of the groove 607 such that the position of the second end 609 of the elongated groove 607 is identical with position of one of the intake ports 602 in the upper graphite layer 604. As the intake ports 602 are positioned in a circular configuration along the outer circumference of the upper graphite layer 604, the elongated groove 607 allows to establish a connection between the outtake port 603 and any one of the intake ports 602 by rotating of one of the two coplanar graphite layers (604, 605) relatively to the other one. In order to ensure the non-crosscontamination of the desired reactant by the reactant from any other unselected port, this coplanar arrangement selector port is used in underpressure (suction) regimen, when the sealing of the two coplanar surfaces can be guaranteed. The detailed view of the coplanar structure of the selection device 601 is shown in FIG. 13.

[0117] The solutions of amino-acids are in this example stored in storage containers 701. The number of storage containers corresponds to the number of intake ports 602 of the selector device 601, i.e. 28 in this example. The storage containers 701 have unified cylindrical shape and are placed in a holder 702. The holder 702 has means for holding the storage containers 701.

[0118] As shown in FIGS. 1 and 2, the centrifuge 301 and the distribution rotor 302 are placed in a centrifugation drum 704 of a cylindrical shape with diameter slightly larger than the diameter of the centrifuge 301, such that the centrifuge 301 fits in the centrifugation drum 704. The centrifugation drum 704 is placed in a box 705, which has an upper part 706 with a circular opening 707 in this upper part 706, into which the centrifugation drum 704 is placed. In this example, the upper part 706 of the box 705 serves as a storage place for the dosing device 501. The holder 702 with the selector device 601 and the storage containers 701 is also attached to the upper part 706 of the box 705. The box 705 further retains the outflow pipes, which provide an outflow system of the device and drain away the liquid removed from the reactors 101 during the synthesis. These outflow pipes are combined into one outflow tube, which is positioned within the box 705.

[0119] The centrifugation subsystem is covered in the centrifugation drum 704 by a cover 708, which is rotatably attached to the upper part 706 of the box 705 and moves rotatably from an open position to a closed position and vice versa. The cover 708 is in this example comprises two concentric disksthe inner disk 708a is made of polypropylene and the outer disk 708b is made of glass to allow the operator to watch the operation of the device directly. The inner disk 708a of the cover is placed between the distribution rotor 302 and the positioning device 304 (and the lifting device 305), as both of them are placed above the distribution rotor 302 and outside of the centrifugation drum 704. There is at least one openinginlet port 709 in the inner disk 708a of the cover 708, through which the distribution rotor 302 of the centrifugation subsystem may be connected to the dosing device 501.

[0120] The system is controlled by a timing protocol of the program constructed for operation of the device 1, such that all sub-devices are connected to that control subsystem and operated by the timing protocol of the control subsystem.

Example 2

[0121] In another embodiment, with reference to FIGS. 14 and 15a, 15b, the device 1 for parallel oligomer synthesis comprises I-trap outflow instead of S-trap outflow. The I-trap outflow comprises a vertical channel 1001, connected to the nozzle 104 of the reactor 101, a peek tubing 1002 having a first open end 1012 and a second open end 1022, the first open end 1012 being connected to the vertical channel 1001, the peek tubing 1002 being vertically extended such that the second open end 1022 of the peek tubing 1002 is positioned higher than the level of contents of the reactor 101, and an inverted syringe as a cover 1003, having a first open end 1013 and a second end 1023 with the top, covering the peek tubing 1002 such that an interspace 1004 between the peek tubing 1002 and the cover 1003 is created.

[0122] The liquid from the reactor 101 cannot overcome the gravity based resistance of the peek tubing 1002 at the low speed of rotation (e.g. during liquid transfer from pre-activation compartments or during a synthesis shaking), or it is removed completely from the reactor 101 at high speed rotation by centrifugal forces. The liquid to be removed flows from the reactors 101 through the I-trap outflow and through a channel system 1005 that is positioned in a channel holder 1006. The channel holder 1006 and the fixation slots 107 are provided integrally. They form a separate element that is attached to the lower disc 301a. The cover 1003 of the peek tubing 1002 is connected to the channel holder 1006. The channel holder 1006 comprises a funnel 1007 arranged at one end of the channel system 1005 so that the channel system 1005 is through the funnel 1007 connected with the interspace 1004 created between the peek tubing 1002 and the cover 1003. The channel system 1005 further comprises at least one channel positioned horizontally 1010, i.e. in parallel with the planes of the lower 301a and the upper 301b, discs and at least one stopcock 1008, positioned close to the end of the channel system 1005 that is opposite to the end with the funnel 1007. The stopcock 1008 may be in closed position, when the liquid is retained in the system, or in open position, when the outflow of the liquid from the system is allowed. The liquid can be removed from the reactors 101 by the succession of fast centrifugation, in which the liquid is transferred from the reactors 101 through the vertical channel 1001 and the peek tubing 1002 into the interspace 1004 created between the peek tubing 1002 and the cover 1003, slow rotation, during which the liquid is flowing out of the interspace 1004, through the funnel 1007 into the channel system 1005 and out of the stopcock 1008, and finally short fast centrifugation, which removes the liquid from the channel system 1005 completely, preventing thus any potential crystallization of reagents in solutions.

[0123] This arrangement is advantageous because the operator may decide that he/she wants to collect and use the eluent collected from individual reactors 101for example for measuring absorbance or spectra of the solutions. Thus, this arrangement is particularly advantageous for the collection of solutions created by cleaving the finished synthetic product by cleaving reagents, for example by trifluoroacetic acid. After incubation of washed resin with cleaving reagent, the stopcock 1008 is turned into closed position and liquid is transferred by centrifugation through the peek tubing 1002 to the interspace 1004 created between the peek tubing 1002 and the inverted syringe as the cover 1003. Rotor may then be taken out of the machine, placed on the assembly of receiving flasks (e.g. Falcon tubes) arranged in a circular fashion and stopcocks 1008 are opened to let the liquid flow into the receiving flanks That solution may then be worked-up in a common procedure (precipitation, evaporation, lyophilization).

Example 3

[0124] In another embodiment, the device 1 for parallel oligomer synthesis comprises a distribution device (not shown) in addition to the components as described in Example 1 or Example 2. The distribution device connects the dosing pump 504 via the distribution valve 502 to either the selector device 601 or to the pre-activation compartments 306 of the distribution rotor 302. The distribution device is a solenoid valve with three ports switching between the connection of the dosing pump 504 with the outtake port of selector device 601 and the connection between the dosing pump 504 and the pre-activation compartments 306 of the distribution rotor 302 (through the distribution valve 502).

[0125] In this example, when the distribution device is enabled in the system, the distribution valve 502 comprises 12 ports for connection with the distribution device.

[0126] It is also possible to place a tubing with a predefined volume (which is larger than the volume being delivered to the reactor) between the dosing pump 504 and the inlet port 709 of the pre-activation compartments 306 to avoid the reagents entering the dosing pump 504 inner volume. In this arrangement, the distribution device is placed between selector device 601 and inlet port 709 of the centrifuge assembly. The distribution device is first opened in the direction to the dosing pump 504, the liquid is sucked from the distribution valve 502 into the tubing, the distribution device is opened in direction of delivery to one of the reactors 101 and the liquid is expelled into the reactor, without entering the syringe of the dosing pump 504 and contaminating the liquid in there. This schematic arrangement of this example can be seen in FIG. 17.

Example 4

[0127] In this example, the device 1 may further (in addition to the components described in the examples above) comprise a temperature control device, provided inside the centrifugation drum 704 (not shown in the figures). The temperature control device in this example comprises an infrared radiator and a microwave radiator, and a sensor with a feedback control. Such a temperature control device may provide temperatures up to 90 degrees Celsius and is controlled automatically by the control subsystem.

Example 5

[0128] In another example (not shown in the figures), the device 1 may further (in addition to the components described in the examples above) comprise a first plurality of magnets which are attached statically to the inner side of the centrifugation drum 704 above the reactors 101, such that they are distributed along the circumference of the centrifugation drum 704. Further, a second plurality of magnets is positioned inside each of the reactors 101. The size of the magnets placed inside the reactors 101 is smaller than the size of the magnets attached to the centrifugation drum 704. Because of the interaction of the magnets inside the reactors 101 with magnets attached to the centrifugation drum 704, the resin inside the reactors 101 is enabled to be mixed more thoroughly during the slow rotation of the centrifuge 301 in the synthetic process. The addition of magnets is especially advantageous for larger quantities of contents inside the reactor, when rotation and/or shaking of the centrifuge is not sufficient to mix the content to a desired extent.

Example 6

[0129] Synthesis of ValA10-insulin A-chain S-sulfonate (Mw 2687.8116 and 2689.9100) Sequence (21 residues): Gly-Ile-Val-Glu-Gln-Cys(SSO3H)-Cys-Thr-Ser-Ile-Cys(SSO3H)-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys(SSO3H)-Asn was synthesised using the device according to the present invention and the method according to the present invention. Preloaded Fmoc-Asn(Trt)-Wang-LL-resin (0.29 mmol/g), 100 mol scale, was used. The resin was purchased from IRIS Biotech GmbH. Resin was placed to two PP syringes of the 10 ml volume with frits (250 mol of the Fmoc groups).

[0130] The control device retrieved the sequence of Gly-Ile-Val-Glu-Gln-Cys(SSO3H)-Cys-Thr-Ser-Ile-Cys(SSO3H)-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys(SSO3H)-Asn and determined the sequence specific timing protocol. Two couplings per position were used (each condensation was performed twice for each step). The first coupling (45 min) was done with 1 ml of 0.5 M amino acid and 0.5 ml of 0.5M DIC in DMF. The second coupling (45 min) was done with 0.5 ml of 0.5 M amino acid and 0.5 ml of 0.5M DIC in DMF.

[0131] The dosing device dispensed 1 ml of first N-protected 0.5 M amino acid solution through the lifting and positioning device to the first one of the pre-activating compartments of the distribution rotor, the distribution rotor turned to enable the second pre-activation compartment a reception of contents of the dosing device through lifting and positioning device. The dosing device dispensed 0.5 ml of 0.5 M solution of diisopropylcarbodiimide (DIC) through the lifting and positioning device to the first one of the pre-activating compartments of the distribution rotor, the distribution rotor turned to enable the second pre-activation compartment a reception of contents of the dosing device through lifting and positioning device and the steps repeated to ensure the reception of 0.5 ml of 0.5 M solution of diisopropylcarbodiimide (DIC) to the second one of the pre-activating compartments of the distribution rotor according to the timing protocol.

[0132] The distribution rotor was lowered to position in which it makes contact with reactors in the centrifuge, and rotated with a speed of 100 rpm in a synchronized rotation with the centrifuge, causing the contents of the pre-activation compartments to transfer by a centrifugal force via grooves into the reactors. Then the distribution rotor detached from reactors by lifting mechanism and reactors content were stirred by repetitive back and forth motion for 45 minutes in order to complete the condensation reaction.

[0133] Then the centrifuge with reactors rotated with the speed of 1000 rpm so that the liquid from the reactors was transferred by a centrifugal force via the S-trap outflows out of the reactors. The dosing device dispensed 1 ml of DMF through the lifting and positioning device to each one of the reactors of the centrifuge, and the centrifuge with reactors rotated with the speed of 1000 rpm so that the liquid from the reactors was transferred by a centrifugal force via the S-trap outflows out of the reactors. This washing step was repeated 5 times.

[0134] In the next step, the dosing device dispensed 2 ml of 20% piperidine/2% DBU in DMF through the lifting and positioning device to each one of the reactors of the centrifuge, where a deprotection of N-protected end of the amino acids took place for 2 minutes before the centrifuge with reactors rotated so that the liquid from the reactors was transferred by a centrifugal force via the S-trap outflows out of the reactors. Then washing step occurred using 5 times 1 ml DMF as described above.

[0135] The above described procedure was repeated for the second and the following amino acids in the synthesized peptide sequence, until the desired peptide sequence was completed. The final peptide was cleaved from the resin using TFA/scavengers and precipitated in a cold diethyl ether. The precipitate, crude peptide in SH form, was immediately converted to S-sulfonate derivative using a protocol described in Kosinova et al. Biochemistry 2014, 53, 3392-3402. HPLC trace of the crude S-sulfonate product (178 mg) is shown in FIG. 18.

[0136] The identity of the product main in the main peak was confirmed by MS (negative mode, see FIG. 19). All main m/z signals in the spectrum belong to the product.

Example 7

Synthesis of YGGFL Mutants

[0137]

TABLE-US-00001 Sequences(24YGGFLmutants): YGGFL,GGYFL,GGYLF,GGFYL,GGFLY,GGLYF,GGLFY, GYGFL,GYGLF,GYFGL,GYFLG,GYLGF,GYLFG,GFGYL, GFGLY,GFYGL,GFYLG,GFLGY,GFLYG,GLGYF,GLGFY, GLYGF,GLYFG,YGGFL Rotorsize 25positions Numberofcouplings 1 perposition: Centrifugationtime 20sec Couplingtime 30min Resin Fmoc-inkamideresin s=0.5mmol/g,2420mg Fmoc-AA/oxyma/DMFvolume 0.4mL Fmoc-AA/oxymamolarity 0.5 Reagent(DIC)/DMF 1.0 molarity Reagentvolume 0.24mL

[0138] Sequences (24 YGGFL mutants): YGGFL, GGYFL, GGYLF, GGFYL, GGFLY, GGLYF, GGLFY, GYGFL, GYGLF, GYFGL, GYFLG, GYLGF, GYLFG, GFGYL, GFGLY, GFYGL, GFYLG, GFLGY, GFLYG, GLGYF, GLGFY, GLYGF, GLYFG, YGGFL were synthesised using the device according to the present invention and the method according to the present invention.

[0139] Fmoc-Rink amide resin s=0.5 mmol/g, 2420 mg was placed into the PP syringes with fits (2420 mg) and swollen in DMF. The resin was purchased from IRIS Biotech GmbH. The control device retrieved the sequence of first of the sequences above and determined the sequence specific timing protocol. Single coupling was applied using oxyma/DIC activation. The dosing device dispensed 0.4 ml of first N-protected 0.5 M amino acid solution through the lifting and positioning device to the first one of the pre-activating compartments of the distribution rotor, the distribution rotor turned to enable the second pre-activation compartment a reception of contents of the dosing device through lifting and positioning device. The dosing device dispensed 0.24 ml of 1.0 M solution of diisopropylcarbodiimide (DIC) through the lifting and positioning device to the first one of the pre-activating compartments of the distribution rotor, the distribution rotor turned to enable the second pre-activation compartment a reception of contents of the dosing device through lifting and positioning device and the steps repeated to ensure the reception of 0.24 ml of 1.0 M solution of diisopropylcarbodiimide (DIC) to the second one of the pre-activating compartments of the distribution rotor according to the timing protocol.

[0140] The distribution rotor was lowered to position in which it makes contact with reactors in the centrifuge, and rotated with a speed of 100 rpm in a synchronized rotation with the centrifuge, causing the contents of the pre-activation compartments to transfer by a centrifugal force via grooves into the reactors. Then the distribution rotor detached from reactors by lifting mechanism and reactors content were stirred by repetitive back and forth motion for 30 minutes in order to complete the condensation reaction.

[0141] Then the centrifuge with reactors rotated with the speed of 1000 rpm for 20 s so that the liquid from the reactors was transferred by a centrifugal force via the S-trap outflows out of the reactors.

[0142] The dosing device dispensed 1 ml of DMF through the lifting and positioning device to each one of the reactors of the centrifuge, and the centrifuge with reactors rotated with the speed of 1000 rpm so that the liquid from the reactors was transferred by a centrifugal force via the S-trap outflows out of the reactors. This washing step was repeated 2 times. In the next step, the dosing device dispensed 1 ml of 20% piperidine/2% DBU in DMF through the lifting and positioning device to each one of the reactors of the centrifuge, where a deprotection of N-protected end of the amino acids took place for 2 minutes before the centrifuge with reactors rotated so that the liquid from the reactors was transferred by a centrifugal force via the S-trap outflows out of the reactors. Then washing step occurred using 5 times 1 ml DMF as described above.

[0143] The above described procedure was repeated for the second and the following aminoacids in the synthesised peptide sequence, until the desired peptide sequence was completed. After completion of the synthesis, the resin was washed with methanol (2) and dried. The final peptide was cleaved from the resin by manual addition of 95%TFA/5% H.sub.2O (200 L per reactor) for 2 hrs. HPLC analysis was performed after dilution of the sample with 800 L of water.

[0144] An example of HPLC trace of the crude product is shown in FIG. 20. (H-YGGFL-NH.sub.2, column Symmetry C.sub.18 3.5 m, 4.675 mm, grad 5-65% MeCN/20 min).