JP2005206525A - Automatic continuous peptide synthesizer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an automatic continuous peptide synthesizer based on liquid peptide synthesis method, capable of maintaining the reaction efficiencies of respective reagents at high levels through suppressing amino acid reagent loss to a minimum. <P>SOLUTION: The synthesizer is such that an inner case of raised airtightness is installed in the body case and treatment operations are carried out in the inner case. This synthesizer comprises a plurality of amino acid containers 6, a dispensing injection nozzle 18, reagent bottles 6A, a plurality of reaction vessels 31, a level sensor 34, an interface sensor 35, nozzles 33, 46 and 47, a retrieving vessel 44, dispensing injection nozzles 49, 50, 51 and 18, a lower layer(1) solvent bottle 63, a lower layer(2) reagent bottle 64, a lower layer(3) reagent bottle 65, a flow path 81, a nozzle 33, a flow path 85, a pump and a three-way solenoid valve. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a peptide continuous automatic synthesizer, and more particularly to a peptide liquid phase synthesizer using a solvent set in which a compatible state and a separated state reversibly change with temperature.

  The present inventors previously devised a liquid-phase peptide synthesis method using a solvent system that can control the temperature reversibly between a compatible state and a phase-separated state (Japanese Patent Laid-Open No. 2003). -62448, JP2003-183298).

  The present invention is an apparatus for continuously carrying out the above liquid phase peptide synthesis method, and in particular, in order to maintain the reaction efficiency of each reagent at a high level, the loss of the liquid carrier and the amino acid reagent can be minimized. We intend to provide a continuous automatic synthesizer with an emphasis on what we can do.

  Thus, the gist of the present invention is that a plurality of amino acid containers held by an amino acid table that moves horizontally at a predetermined timing, each amino acid container and a reagent bottle are sequentially moved at a predetermined timing, and A dispensing nozzle for an amino acid container that moves up and down at a predetermined timing, and a plurality of reaction containers that are held on a support table and that are rapidly heated and cooled within a predetermined range by an appropriate temperature adjustment mechanism A liquid level sensor and an interface sensor for detecting a liquid level and an interface of the liquid in the reaction container, a plurality of recovery containers held on a support table, and a lower end of the reaction container disposed on the reaction container. A nozzle that faces the bottom, a nozzle that is disposed in the collection container, a nozzle whose bottom end faces the bottom of the collection container, and a nozzle that faces a position at a predetermined height from the bottom, and at a predetermined timing Two dispensing nozzles for the reaction container that are sequentially moved onto the reaction container, a dispensing nozzle for the collection container that is sequentially moved onto each collection container at a predetermined timing, and the reaction One of the two dispensing nozzles for the container, a dispensing nozzle for the amino acid container, a flow path connecting the plurality of lower layer reagent bottles, and the two dispensing nozzles for the reaction container The other, a dispensing container dispensing nozzle, a flow path connecting a plurality of upper layer reagent bottles, a nozzle disposed in the reaction container, and two nozzles disposed in the collection container There is provided a continuous peptide automatic synthesizer characterized in that a pump and a three-way solenoid valve are arranged at appropriate locations in each of the flow channels, and the liquid is sent at an appropriate timing.

  According to the present invention, the liquid phase peptide synthesis method previously devised by the present inventors can be carried out efficiently, and in addition, the reaction of each reagent can be carried out with minimal loss of the liquid carrier and amino acid reagent. Efficiency can be maintained at a high level.

Examples of the present invention will be described below.
1 is a schematic configuration diagram of the entire apparatus, FIG. 2 is a front view of the upper side of the apparatus, FIG. 3 is a front view of the lower side of the apparatus, and FIG. 4 is a right side view in which the lower side of the apparatus is partially omitted. FIG. 5 is a plan view of the apparatus, and FIG. 6 is a perspective view of a reaction container, a recovery container, and a dispensing nozzle.

In the figure, 1 is a main body case. In addition, a storage chamber 2 for reagent bottles and waste liquid bottles and a storage chamber 3 for cooling water circulators are formed on the lower side of the main body case 1. In addition, an inner case 4 that houses an amino acid container, a reaction container, a recovery container, a dispensing nozzle, and a liquid feed line connecting them is disposed on the upper side of the main body case 1. In the inner case 4, since the reaction of the peptide goes wrong due to moisture, the confidentiality is enhanced and the pressure is increased to fill the inert gas such as N ₂ .

  Reference numeral 5 denotes an amino acid table which holds a plurality of amino acid containers 6, 6, 6. In the present embodiment, the amino acid table 5 is configured to hold the amino acid containers 6, 6, 6... In 4 rows and 5 columns. The amino acid table 5 includes a drive motor 7, a drive pulley 8, a driven pulley 9, a belt 10 wound around the drive pulley 8 and the driven pulley 9, and a coupling member that connects the belt 10 and the amino acid table 5. 11 is moved in the horizontal direction at a predetermined timing. Reference numerals 12 and 13 are guide members of the amino acid table 5.

  14, 14, 14... Are stirring stirrers placed in the respective amino acid containers 6, 6, 6..., And are rotated by a rotation drive mechanism 15 disposed below the amino acid table 5. The rotation drive mechanism 15 may be a known one, and a magnet provided below the bottom of each amino acid container 6, 6, 6... That rotates using the drive motor 16 and the drive motor 16 as a drive source. It is composed of attached circular rotary drive plates 17, 17, 17.

  Reference numeral 18 denotes a dispensing nozzle for an amino acid container, which is held by a dispensing nozzle holder, which will be described later, and sequentially moves onto each of the amino acid containers 6, 6, 6... And the reagent bottle 6A at a predetermined timing. It is made to move up and down at the timing of. The dispensing nozzle 18 is provided with a container sensor 18A for detecting the presence or absence of a container.

  Reference numeral 19 denotes a movement operation mechanism for the dispensing nozzle 18, which comprises a horizontal movement operation unit and a vertical movement operation unit. Reference numeral 20 denotes a horizontal movement operation unit, which includes a gantry 21 erected in the inner case 4, a support unit 22 mounted horizontally on the gantry 21, and a vertical movement operation provided on the front surface of the support unit 22. A guide member 23 that horizontally guides the support portion of the support portion, a drive motor (not shown) fixed to the support portion 22, a drive pulley 24, a driven pulley 25, and a loop between the drive pulley 24 and the driven pulley 25. And a moving operation mechanism constituted by the belt 26 and a coupling member 27 that couples the belt 26 and the support portion in the vertical movement operating portion.

  Reference numeral 28 denotes a vertical movement operation part, which is supported by the guide member 23 so as to be movable in the horizontal direction along the front surface of the support part 22 and is driven by a movement operation mechanism provided in the support part 22. And a dispensing nozzle holder 30 that can be moved up and down along the front surface of the support portion 29 by a lifting operation mechanism (not shown).

  31, 31, 31... Are reaction vessels set in parallel on a support table 32 provided in the inner case 4. In the present embodiment, the number of reaction vessels 31, 31, 31... Is four, but may be more or less. Further, the reaction vessel 31, 31, 31... Is provided with a nozzle 33 whose lower end faces the bottom of the reaction vessel via a nozzle holder described later.

  Reference numeral 34 denotes a liquid level sensor, and 35 denotes an interface sensor, which are arranged corresponding to each of the reaction vessels 31, 31, 31. The reaction vessels 31, 31, 31... Are subjected to rapid temperature control in the range of 5 to 80.degree. Further, the temperature adjusting mechanism 36 is cooled by circulating cooling water (not shown) by a cooling water circulator 38 in a jacket 37 including the reaction vessels 31, 31, 31. It is designed to be heated. Incidentally, 38a is a cooling water inflow pipe, and 38b is a cooling water outflow pipe.

  40, 40, 40... Are stirring stirrers placed in the respective reaction vessels 31, 31, 31..., And are rotated by a rotation drive mechanism 41 disposed below the support plate 32. The rotary drive mechanism 41 may be a known one, and a magnet provided below the bottom of each reaction vessel 31, 31, 31... That rotates using a drive motor 42 and the drive motor 42 as a drive source. It is composed of attached circular rotating plates 43, 43, 43.

  44, 44, 44 ... are collection containers set in parallel on a support table 32 provided in the inner case 4. Reference numeral 32a denotes a rack for holding the collection containers 44, 44, 44. Further, in this embodiment, the number of the collection containers 44, 44, 44... Is four corresponding to the number of the reaction containers 31, 31, 31. In addition, the recovery containers 44, 44, 44... Have a nozzle 46 whose lower end faces the bottom of the recovery container, and a nozzle 47 that faces a predetermined height from the bottom via the nozzle holder 45. Is arranged. The nozzle holder 45 spans the reaction container 31 and the recovery container 44, and is provided with circular holes 45a and 45a at the center of the portion covering the reaction container 31 and the recovery container 44. is there.

  48, 48, 48... Are stirring stirrers placed in the respective collection containers 44, 44, 44..., And are rotated by a rotation drive mechanism (not shown) disposed below the support plate 32. Is. Since the rotation drive mechanism is the same as the rotation drive mechanism 41, description thereof is omitted.

  49 and 50 are reaction vessel dispensing nozzles held by a later-described dispensing nozzle holder, which are sequentially moved onto the reaction vessels 31, 31, 31... At a predetermined timing. .

  Reference numeral 51 denotes a collection container dispensing nozzle held by a later-described dispensing nozzle holder, which sequentially moves onto the collection containers 44, 44, 44... At a predetermined timing.

  52 is a mechanism for moving the dispensing nozzles 49, 50, 51, and above the reaction vessels 31, 31, 31... And the collection vessels 44, 44, 44. A support plate 53 disposed horizontally; a guide member 54 provided on the upper surface of the support plate 53; a support substrate 55 supported by the guide member 54 so as to be movable in the horizontal direction along the upper surface of the support plate 53; Dispensing nozzle holders 56, 57 projecting horizontally in front of and behind the support substrate 55, a drive motor 58, a drive pulley 59, a driven pulley 60, and a drive pulley 59 and a follower fixed to the upper surface of the support plate 53. The moving operation mechanism includes a belt 61 wound around a pulley 60 and a set screw 62 that couples the belt 61 and the support substrate 55.

  63 is a lower layer (1) solvent bottle, 64 is a lower layer (2) reagent bottle (de-Fmoc agent), 65 is a lower layer (3) reagent bottle, 66 is an upper layer (1) solvent bottle, 67 is an upper layer (2) reagent bottle, 68 is a waste liquid bottle. These bottles are stored in a storage chamber 2 below the main body case 1. Reference numeral 69 denotes a cleaning tank, and 70 and 71 denote purge tanks.

  72 is a flow path connecting the dispensing nozzle 49, the dispensing nozzle 18, the lower layer (1) solvent bottle 63, the lower layer (2) reagent bottle 64, and the lower layer (3) reagent bottle 65, and the dispensing nozzle 49 and the lower layer ( 2) A flow path 72a connecting the reagent bottle 64, a flow path 72b to the dispensing nozzle 18 connected to the middle part of the flow path 72a via a three-way electromagnetic valve 73, and a middle part of the flow path 72a. The lower layer (1) connected to the upstream side of the flow path 72b via a three-way electromagnetic valve 74, the flow path 72c to the solvent bottle 63, and the three-way electromagnetic valve in the middle portion connecting the flow path 72b and the flow path 72c. A lower layer (3) connected via a three-way solenoid valve 77 between the flow path 72d in which the syringe pump 76 is disposed via 75 and the flow path 72b and the flow path 72c in the middle of the flow path 72a. ) The flow path 72e to the reagent bottle 65 and the middle part of the flow path 72a The flow path 72b is connected to the upstream and downstream ends of the flow path 72b, and the upstream end is connected to the flow path 72f via a three-way electromagnetic valve 78, and the flow path 72a is in the middle of the flow path 72a. Between the ceramic pump 79 disposed on the downstream side of the connection portion on the downstream side of the flow path 72f, and the flow path 72b in the middle portion of the flow path 72a and the connection portion on the upstream side of the flow path 72f. The ceramic pump 80 is provided.

  81 is a flow path connecting the dispensing nozzle 50, the dispensing nozzle 51, the upper layer (1) solvent bottle 66, and the upper layer (2) reagent bottle 67, and connects the dispensing nozzle 51 and the upper layer (1) solvent bottle 66. A flow path 81a, a flow path 81b to the dispensing nozzle 50 connected to the middle portion of the flow path 81a via a three-way solenoid valve 82, and upstream of the flow path 81b in the middle portion of the flow path 81a From the flow path 81c to the upper layer (2) reagent bottle 67 connected via the three-way solenoid valve 83, and the ceramic pump 84 disposed between the flow path 81b and the flow path 81c in the flow path 81a. It will be.

  Reference numeral 85 denotes a flow path connecting the nozzle 33 and the nozzles 46 and 47, and a flow path 85a connecting the nozzle 33 and the nozzle 46 and a portion connected to the middle portion of the flow path 85a via a three-way electromagnetic valve 86. The flow path 85b to the injection nozzle 47 and a ceramic pump 87 disposed upstream of the flow path 85b in the middle of the flow path 85a.

Next, the operation of the above embodiment will be described.
First, the amino acid is activated. The amino acid table 5 is moved to position the first row of amino acid containers 6 directly below the dispensing nozzle 18. At this time, the presence or absence of a container is confirmed by the container sensor 18A, and if there is no container, an error is displayed. Next, the lower layer solvent (DMF) in the lower layer (1) solvent bottle 63 is poured into the amino acid container 6 containing the amino acid. At this time, the injection amount of the DMF is 20 ml. Moreover, stirring is not performed at this time. Subsequently, the reagent in the reagent bottle 6A and the lower layer solvent (DMF) in the lower layer (1) solvent bottle 63 are injected. The reagent injection volume is 0.1 ml and DMF is 20 ml. At this time, the stirring is performed with the stirrer 14. Finally, stirring is performed by the stirrer 14 for a predetermined time. In addition, the amino acids in the adjacent amino acid container 6 are sequentially activated by the above procedure.

  On the other hand, the upper layer solution is prepared simultaneously with the activation of the amino acid. The upper layer solvent (MCH) in the upper layer (1) solvent bottle 66 is injected into the reaction vessel 31. The injection amount of MCH is 40 ml. Next, the reaction vessel 31 is heated to 40 to 80 ° C., preferably 60 ° C., by the temperature adjusting mechanism 36 and stirred with the stirrer 40 for 5 minutes. Next, the MCH in the upper MCH bottle 66 is injected until the liquid level sensor 34 detects and reaches a predetermined amount.

  Next, a de-Fmoc reaction is performed. A lower layer reagent (de-Fmoc agent: DBU reagent) in the lower layer (2) reagent bottle 64 is injected into the reaction vessel 31. The DBU reagent injection volume is 40 ml. At this time, the stirring is performed. Next, the reaction vessel 31 is heated to 40 to 80 ° C., preferably 60 ° C. by the temperature adjusting mechanism 36 and stirred with the stirrer 40 for 10 minutes. Next, it is cooled by the temperature adjusting mechanism 36 and then left to stand. The cooling time is 5 minutes and the standing time is 0.5 minutes. Next, it is detected by the interface sensor 35, and the lower DBU is extracted to the interface portion and sent to the collection container 44. Finally, the flow path is emptied.

  Next, recovery from the waste liquid is performed. A predetermined amount of the upper layer solvent (MCH) in the upper layer (1) solvent bottle 66 is injected into the collection container 44. The amount of MCH to be injected is 10 ml, and stirring is performed. Next, after stirring with the stirrer 48, it is left still. The stirring time is 1 minute and the standing time is 0.5 minutes. Then, the upper layer MCH in the collection container 44 is returned to the reaction container 31. The amount of the upper layer MCH in the reaction vessel 31 returned at this time is detected by the liquid level sensor 34, and the remainder exceeding the predetermined amount is regarded as waste liquid. Thereafter, the flow path is emptied. Further, it is detected by the liquid level sensor 34, and a predetermined amount of MCH in the upper layer (1) solvent bottle 66 is injected and replenished. This is limited to a maximum of 5 ml.

  Next, the de-Fmoc agent is washed. The lower layer solvent (DMF) in the lower layer (1) solvent bottle 63 is injected into the reaction vessel 31. The injection amount of DMF is 40 ml, and stirring is performed. Next, it stirs with the stirrer 40 for 1 minute. Next, it cools with the temperature control mechanism 36, and is left still after that. The cooling time is 1 minute and the standing time is 0.5 minutes. Next, it is detected by the interface sensor 35, and the lower layer DMF is extracted to the interface portion and sent to the collection container 44. Finally, the flow path is emptied.

  Next, recovery from the waste liquid is performed. A predetermined amount of the upper layer solvent (MCH) in the upper layer (1) solvent bottle 66 is injected into the collection container 44. The amount of MCH to be injected is 10 ml, and stirring is performed. Next, after stirring with the stirrer 48, it is left still. The stirring time is 1 minute and the standing time is 0.5 minutes. Then, the upper layer MCH in the collection container 44 is returned to the reaction container 31. The amount of the upper layer MCH in the reaction vessel 31 returned at this time is detected by the liquid level sensor 34, and when the amount exceeds a predetermined amount, the remaining is made waste. Thereafter, the flow path is emptied. Further, it is detected by the liquid level sensor 34, and a predetermined amount of MCH in the upper layer (1) solvent bottle 66 is injected and replenished.

  Next, a condensation reaction is performed. All the activated amino acids in the amino acid containers 6, 6, 6... Are fed to the reaction containers 31, 31, 31. This is also done with stirring. Next, the reaction vessel 31 is heated to 40 to 80 ° C., preferably 60 ° C., by the temperature adjusting mechanism 36 and stirred with the stirrer 40 for 30 minutes. Next, it cools with the temperature control mechanism 36, and is left still after that. The cooling time is 5 minutes and the standing time is 0.5 minutes. Next, it is detected by the interface sensor 35, and the lower layer DMF is extracted to the interface portion and sent to the collection container 44. Finally, the flow path is emptied.

  Next, recovery from the waste liquid is performed. A predetermined amount of the upper layer solvent (MCH) in the upper layer (1) solvent bottle 66 is injected into the collection container 44. The amount of MCH to be injected is 10 ml, and stirring is performed. Next, after stirring with the stirrer 48, it is left still. The stirring time is 1 minute and the standing time is 0.5 minutes. Then, the upper layer MCH in the collection container 44 is returned to the reaction container 31. The amount of the upper layer MCH in the reaction vessel 31 returned at this time is detected by the liquid level sensor 34, and when the amount exceeds a predetermined amount, the remaining is made waste. Thereafter, the flow path is emptied. Further, it is detected by the liquid level sensor 34, and a predetermined amount of MCH in the upper layer (1) solvent bottle 66 is injected and replenished. This is limited to a maximum of 5 ml.

  The condensation reaction is repeated by repeating the steps from the amino acid activation reaction to the replenishment of the upper layer MCH. Further, it is desirable that the above processes are automatically controlled by a computer. Then, the liquid carrier containing the peptide in the reaction vessel 31 after the completion of synthesis is taken out from the apparatus together with the reaction vessel 31, and is manually operated (cutout, evaluation, purification, etc.).

It is a schematic block diagram of the whole apparatus based on this invention. It is a front view of the upper part side of the device concerning the present invention. It is a front view of the lower part side of the apparatus which concerns on this invention. It is the right view which abbreviate | omitted and showed the lower part side of the apparatus which concerns on this invention. 1 is a plan view of an apparatus according to the present invention. It is a perspective view of a reaction container, a recovery container, and a dispensing nozzle part.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Main body case 4 Inner case 5 Amino acid table 6, 6 Amino acid container 14 Stirring stirrer 18 Dispensing nozzle 19 Dispensing nozzle movement operation mechanism 20 Horizontal movement operation part 28 Vertical movement operation part 31, 31 Reaction container 33 Nozzle 34 Liquid level sensor 35 Interface sensor 36 Temperature control mechanism 37 Jacket 38 Cooling water circulator 39 Heater 40 Stirring stirrer 44, 44 Recovery container 46 Nozzle 47 Nozzle 48 Stirring stirrer 49, 50 Dispensing nozzle 51 Dispensing nozzle 52 Dispensing nozzle moving operation mechanism 63 Lower layer (1) Solvent bottle 64 Lower layer (2) Reagent bottle (de-Fmoc agent)
65 Lower layer (3) Reagent bottle 66 Upper layer (1) Reagent bottle 67 Upper layer (2) Reagent bottle 68 Waste liquid bottle 69 Cleaning tank 70, 71 Purge tank 72 Channel 81 Channel 85 Channel

Claims (2)

  A plurality of amino acid containers held by an amino acid table that moves horizontally at a predetermined timing, and an amino acid container that sequentially moves onto each amino acid container and reagent bottle at a predetermined timing and moves up and down at a predetermined timing A plurality of reaction vessels that are held on a support table and are rapidly heated and cooled within a predetermined range by an appropriate temperature adjustment mechanism, and the liquid level and interface of the liquid in the reaction vessel A liquid level sensor and an interface sensor for detecting the above, a plurality of recovery containers held on a support table, a nozzle disposed in the reaction container, with a lower end facing the bottom of the reaction container, and disposed in the recovery container , A nozzle whose lower end faces the bottom of the recovery container and a nozzle that faces a predetermined height from the bottom, and moves to each reaction container sequentially at a predetermined timing Among the two dispensing nozzles for the reaction container, the dispensing nozzle for the collecting container that is sequentially moved onto each collecting container at a predetermined timing, and the two dispensing nozzles for the reaction container , A flow path connecting a dispensing nozzle for an amino acid container and a plurality of lower layer reagent bottles, another one of the two dispensing nozzles for the reaction container, and a dispensing container for a recovery container Each flow path comprising a nozzle and a flow path connecting a plurality of upper layer reagent bottles, and a flow path connecting the nozzle disposed in the reaction container and the two nozzles disposed in the recovery container. A continuous peptide automatic synthesizer characterized in that a pump and a three-way solenoid valve are arranged at an appropriate position in order to send liquid at an appropriate timing. 2. The peptide continuous automatic synthesizer according to claim 1, wherein an inner case is installed in the main body case to increase confidentiality and pressure and is filled with an inert gas, and a processing operation is performed in the inner case.

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