JP2006151948A - Method for synthesizing sugar chain and automatic sugar chain synthesis apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for synthesizing a sugar chain, suitable for an automatic apparatus, and to provide an automatic sugar chain synthesis apparatus. <P>SOLUTION: This method for synthesizing the sugar chain comprises a glycosylation reaction process for binding a sugar donor to a sugar acceptor having low mol. wt. polyethylene glycol as a support carrier, a process for capping the hydroxy groups of the unreacted sugar acceptor in the glycosylation reaction product, a process for removing the temporary protecting group of the sugar acceptor bound to the sugar donor by the glycosylation reaction to produce the sugar acceptor for the next glycosylation reaction, wherein each of these processes includes a purification treatment for passing the reaction product through a silica gel column to recover only the product bound to the low mol. wt. polyethylene glycol after each reaction, and each process is sequentially repeated prescribed times to perform the chemical synthesis of the sugar chain at prescribed steps. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for chemically synthesizing sugar chains by linking sugars and sugars, and an automatic synthesizer that automatically performs the synthesis method.

  With respect to the synthesis of sugar chains that link sugars to sugars, a method of chemically reacting using a solid phase as a carrier has been widely studied, starting with the technique developed by Danishefsky et al. (See Non-Patent Document 1) in 1993.

  Research is also being conducted on a method of chemically reacting with low molecular weight polyethylene glycol (hereinafter abbreviated as PEG) as a carrier.

  As a technique different from the above, a sugar chain synthesis technique using an enzyme has also been proposed. This method is well known by Mr. Shinichiro Nishimura of AIST and Hokkaido University. The applicant has no knowledge of patent documents or papers related to this.

  As for automatic synthesizers, methods for automatically synthesizing compounds such as peptide synthesis and combinatorial chemistry have been put into practical use. In the form of developing these automatic synthesis techniques, Seeberger et al. Of MIT have already announced an automatic sugar chain synthesis apparatus by solid phase reaction as shown in Non-Patent Document 1 (Non-Patent Document 2, Patent Document 1). reference).

  An automatic synthesizer using a sugar chain synthesis method using the above-mentioned enzyme has also been announced by Mr. Shinichiro Nishimura and others.

Science, 1993, 260, 1370. Danishefsky Science, 2001, 291, 1523. Seeberger Seeberger, Peter H .; Plante Obadiash J. Apparatus and methods for the automated synthesisi of oligosaccharides PCT Int Appl (2002) CODEN PIXXD2 WO 2002016384 A2 20020228

  The sugar synthesis method disclosed in Non-Patent Document 1 uses a solid phase resin. The technique using a solid phase resin has a problem that the reactivity is low, and it is difficult to track the progress of the reaction.

  Moreover, since the sugar chain synthesis method using an enzyme uses an enzyme, there remains a problem that only a part of the structure of a naturally occurring sugar chain can be synthesized.

  In addition, there is currently no known example in which an automatic synthesis method using a method of chemically reacting PEG as a carrier is applied to an automatic synthesis apparatus. This is because, in general, organic synthesis reactions use solid reagents and use processes such as liquid separation and filtration. However, the handling of solid reagents is not suitable for automation. Since temperature control is required, the conditions differ between manual operation and automatic operation using a mechanical device, such as environmental conditions such as reagents must be prepared in the device in advance and kept at room temperature for a long time. It is because it is difficult to make it.

  Accordingly, an object of the present invention is to solve the above-described problems and provide a sugar chain synthesis method and an automatic sugar chain synthesis apparatus suitable for automation.

  In order to solve the above-mentioned object, the sugar chain synthesis method of the present invention comprises a glycosylation reaction step in which a sugar donor is bound to a sugar acceptor having a low molecular weight polyethylene glycol as a support carrier, and a glycosylation reaction product. Capping the hydroxyl group of the unreacted saccharide acceptor and deprotecting the protecting group of the saccharide acceptor to which the sugar donor is bound in the glycosylation reaction to produce a sugar acceptor for the next glycosylation reaction These steps include a purification treatment for recovering only the product to which the low molecular weight polyethylene glycol is bound by passing through a silica gel column after each reaction, and each step is sequentially repeated a predetermined number of times. As a result, a predetermined number of sugar chains are chemically synthesized.

  The conventional solid-phase reaction has the problem of low reactivity because the reaction system becomes heterogeneous, but low molecular weight polyethylene glycol gives a homogeneous reaction solution under the reaction conditions, and also has low molecular weight and is reactive. Can be suppressed. Further, since the molecular weight is small, it is possible to scale up the reaction, which is difficult in the solid phase reaction. In addition, the structure and purity of the compound bonded to the polymer can be confirmed by a usual measurement method. For example, take out a very small amount of reaction solution with a capillary, check the progress of the reaction by mass spectrometer, nuclear magnetic resonance apparatus or color reaction, check for the presence of by-products, etc., and check the temperature, amount of reagent, reaction Reaction conditions such as time can be fed back.

  In each step, a compound bonded to a low molecular weight polyethylene glycol and a compound not bonded can be easily separated with a silica gel column by using the high polarity characteristics of the low molecular weight polyethylene glycol.

More specifically, the method for synthesizing a sugar chain of the present invention can be obtained by binding low-molecular-weight polyethylene glycol (CH ₃ (OCH ₂ CH ₂ ) _n OH) to a sugar as a support carrier, removing a temporary protecting group at the reaction site, The sugar acceptor is used as a sugar acceptor, and at least one of imidate sugar, fluoride sugar, chlorosugar, bromosugar, and thioglycoside is used as a sugar donor, and each of the glycosylation, capping reaction, and deprotection reaction It is a sugar chain synthesis method in which a chemical reaction is sequentially repeated a predetermined number of times to generate a predetermined number of sugar chains, and the low molecular weight polyethylene glycol is separated from the product to synthesize sugar chains in which sugars and sugars are linked, The glycosylation reaction, capping reaction, and deprotection reaction include the following steps.

  The glycosylation reaction is a step of binding a sugar donor to a sugar acceptor. The sugar acceptor and the sugar donor are mixed, a small amount of solvent is added, and boron trifluoride ether complex salt is added as a reaction activator. Mix and stir at a predetermined temperature condition, and inject the reaction solution and the mixed solvent of ethyl acetate and hexane in order into a silica gel column swollen in advance with a mixed solvent of a small amount of ethyl acetate and hexane, and discard the waste liquid. A mixed solvent of ethyl acetate and methanol is poured into the solution to recover the liquid, and the recovered liquid is concentrated under reduced pressure to distill off the solvent.

  The capping reaction is a process of capping the unreacted hydroxyl group of the sugar acceptor. A small amount of solvent is added to the product of the glycosylation reaction, isocyanad benzoyl is mixed and stirred at room temperature, and a very small amount of methanol is added. The reaction solution and the mixed solvent of ethyl acetate and hexane are poured in order onto a silica gel column swollen in advance with a small amount of a mixed solvent of ethyl acetate and hexane, and the waste solution is discarded. The solvent is poured to recover the liquid, and the recovered liquid is concentrated under reduced pressure to distill off the solvent.

  The deprotection reaction is a step of deprotecting a temporary protecting group of a sugar acceptor to which a sugar donor is bound by a glycosylation reaction, and a mixed solvent of pyridine and methanol is added to the product of the glycosylation reaction. The mixture is stirred under temperature conditions, the solvent is distilled off by concentration under reduced pressure, and the reaction solution and the mixed solvent of ethyl acetate and hexane are sequentially poured into a silica gel column swollen with a small amount of a mixed solvent of ethyl acetate and hexane, and the waste liquid is discarded. The liquid is recovered by pouring a mixed solvent of ethyl acetate and methanol into the column, and the solvent is distilled off by concentrating the recovered liquid under reduced pressure.

  Each reaction of each glycosylation reaction, capping reaction, and deprotection reaction is carried out by pouring the reaction solution, a mixed solvent of ethyl acetate and hexane into a silica gel column swollen in advance with a mixed solvent of a small amount of ethyl acetate and hexane, and the waste solution. Dispose of and collect the liquid by pouring a mixed solvent of ethyl acetate and methanol into the same column, and collect the solvent by distilling the recovered liquid under reduced pressure. Can be purified.

  The method for synthesizing a sugar chain of the present invention can be carried out without using a solid reagent as a reagent to be used, and can be used with a reagent that can be kept at room temperature.

  Moreover, the sugar chain automatic synthesizing apparatus of the present invention has a configuration suitable for realizing the sugar chain synthesizing method of the present invention with an automatic apparatus, and the solvent in the container can be distilled off by decompression and heating in the container. A reaction vessel, a reagent bottle having a reagent used for each reaction, a temperature setting mechanism for controlling the temperature of the reaction vessel over a wide range, a shaking mechanism for shaking the reaction vessel or its internal liquid, and each reaction A silica gel column through which the product of the silica gel is passed with a solvent, a recovery flask for recovering the falling part of the silica gel column, a switching mechanism for switching the falling part from the silica gel column to a drain or a recovery flask, a recovery flask, a reaction vessel, a reagent bottle, and silica gel A liquid movement mechanism for moving the liquid between the columns, a pressure control mechanism for controlling the pressure in the reaction vessel and / or the silica gel column, and the operation of each mechanism. It can be configured to include a Gosuru control means.

  In the above configuration, the control means controls each mechanism to perform a glycosylation reaction for binding a sugar donor to a sugar acceptor in a reaction vessel, a capping reaction for capping an unreacted hydroxyl group of the sugar acceptor, and the glycosylation reaction. The deprotection reaction for deprotecting the temporary protecting group of the sugar acceptor bound with the sugar donor is sequentially performed, and the product of each reaction is purified on a silica gel column, and the product recovered in the recovery flask is put into a reaction vessel. Perform post-processing to return.

  According to the present invention, it is possible to synthesize a sugar chain using only a stable reagent that can be maintained at room temperature without requiring the use of a solid reagent, a liquid separation operation, filtration, and the like.

  In the above reaction, the sugar donor is a substance from which the sugar group is removed by the reaction activator, and is an imidate sugar, a fluorinated sugar, a chlorosugar, a bromosugar, a thioglycoside (a thioglycoside having an SR group, and an SPh group). In the case of imidate sugar, prepare 2.5 to 3.5 equivalents (based on the amount of sugar acceptor), and use a silica gel column with a capacity of about 20 times or more that of sugar acceptor, for example. Use.

  Moreover, the reaction conditions in a glycosylation reaction, a capping reaction, and a deprotection reaction can be set as follows as an example.

  In the glycosylation reaction, the sugar acceptor and sugar donor are mixed, a small amount of solvent is added, and 2.5 to 3.5 equivalents (based on the amount of sugar acceptor) of boron trifluoride ether complex is used as the reaction activator. For example, for about 45 minutes or more at a temperature of 5 ° C. or lower. In the subsequent silica gel column treatment, the mixture is swollen in advance with a small amount of a mixed solvent of ethyl acetate and hexane having a mixing ratio in the range of 3: 1 to 5: 1. Subsequently, the reaction solution, a mixed solvent of ethyl acetate and hexane (mixed) The ratio is the same as before, and the waste liquid is discarded. A mixed solvent of ethyl acetate and methanol (with a mixing ratio of 3: 1 to 5: 1) is poured into the same column to recover the dropped liquid, and the solvent is stored by concentration under reduced pressure. Leave.

  In the capping reaction, a small amount of solvent is added to the product of the glycosylation reaction, 1.5 to 2.5 equivalents (based on the amount of sugar acceptor) isocyanad benzoyl are mixed, and stirred at room temperature, for example, for about 45 minutes or more. Add a small amount of methanol. In the subsequent silica gel column treatment, the mixture is swollen in advance with a small amount of a mixed solvent of ethyl acetate and hexane having a mixing ratio in the range of 3: 1 to 5: 1. Subsequently, the reaction solution, a mixed solvent of ethyl acetate and hexane (mixed) The ratio is the same as before, and the waste liquid is discarded. A mixed solvent of ethyl acetate and methanol (with a mixing ratio of 3: 1 to 5: 1) is poured into the same column to recover the dropped liquid, and the solvent is stored by concentration under reduced pressure. Leave.

  In addition, in the deprotection reaction, a mixed solvent of pyridine and methanol (mixing ratio in the range of 1: 5 to 5: 1) is added to the product of the capping reaction (no equivalent ratio condition), and for example at a temperature of 45 to 60 ° C. Stir for about 4 hours or more, and remove the solvent by concentration under reduced pressure. In the subsequent silica gel column treatment, the mixture is swollen in advance with a small amount of a mixed solvent of ethyl acetate and hexane having a mixing ratio in the range of 3: 1 to 5: 1. Subsequently, the reaction solution, a mixed solvent of ethyl acetate and hexane (mixed) The ratio is the same as before, and the waste liquid is discarded. A mixed solvent of ethyl acetate and methanol (with a mixing ratio of 3: 1 to 5: 1) is poured into the same column to recover the dropped liquid, and the solvent is stored by concentration under reduced pressure. Leave.

  In another aspect of the sugar chain synthesis method and automatic synthesizer of the present invention, a plurality of sugar chain synthesis chemical reactions including the above-described steps are provided, and stirring is performed in any one of the plurality of sugar chain synthesis chemical reactions. During the treatment, a plurality of sugar chain synthesis chemical reactions are performed independently and in parallel by performing a treatment other than shaking in other sugar chain synthesis chemical reactions.

  The automatic synthesizer according to this aspect has a plurality of reaction vessels, the shaking mechanism shakes the plurality of reaction vessels at once, and the control means controls the shaking mechanism and the liquid transfer mechanism to synthesize any one sugar chain. While the stirring process is performed in the chemical reaction, a process other than shaking is performed in another sugar chain synthesis chemical reaction, so that a plurality of sugar chain synthesis chemical reactions are performed independently and in parallel.

  According to this aspect, a plurality of chemical reactions can be performed with one apparatus. According to this aspect, in the case of comparing the results by proceeding a plurality of the same chemical reaction, or in the case of simultaneously proceeding the chemical reaction performed by changing the reagent or sugar donor to be used, without requiring a plurality of devices, Moreover, it can carry out without requiring a large installation area.

  In an actual chemical reaction, the total time required to move the reagent / reaction solution is a few tens of minutes. On the other hand, the shaking time including mixing of the reagents and the progress of the reaction is on the order of one hour, which is a long time. . This aspect makes it possible to perform a plurality of chemical reactions with a single device by utilizing the long shaking time.

  According to the sugar chain synthesis method and the sugar chain automatic synthesizer of the present invention, it is not necessary to use a solid reagent, or to perform a process such as liquid separation operation and filtration. Synthesis can be performed.

  In addition, a sugar chain automatic synthesis device using low molecular weight polyethylene glycol (PEG) as a carrier, which has not been put into practical use, can be realized. The limitation of the structure of the synthesizable sugar chain can be solved.

  In addition, the sugar chain synthesis method of the present invention is a mechanization of a sugar chain synthesis method using a low molecular weight polyethylene glycol (PEG) as a carrier in that a solid reagent is not used or a liquid separation operation is not performed. It can be easily realized.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  First, a schematic procedure of the sugar chain synthesis method of the present invention will be described with reference to FIG.

  In general, the sugar chain synthesis method of the present invention can be carried out by the following procedure.

  In FIG. 1, the sugar chain synthesis method is carried out by repeating the steps of glycosylation (1), capping reaction (2), and deprotection reaction (3) counterclockwise around the circle shown in the figure.

  The glycosylation reaction is a process of transition from the part of reference numerals 1 and 2 shown in the upper left part of the figure to the part of reference numerals 3 and 4 shown in the lower left part. Here, reference numeral 1 is a sugar with a low molecular weight polyethylene glycol (PEG), which becomes a sugar acceptor. PEG serves as a carrier here, and HO (hydroxyl group) serves as a reaction site. Reference numeral 2 denotes a sugar donor. The ClAcO moiety does not react, and the L moiety is detached by the reaction activator and binds to the sugar acceptor reaction site. Reference numeral 3 is a product resulting from coupling of a glycosylation reaction.

  The capping reaction is a process of transition from the parts 3 and 4 shown in the lower left part of the figure to the parts 5 and 6 shown in the lower right part.

  Reference numeral 4 denotes a sugar acceptor that remains without being used in the reaction in the course of the glycosylation reaction in which the portion indicated by reference numeral 3 is generated. Therefore, reference numeral 1 and reference numeral 4 are the same compound. Since the HO (hydroxyl group) in the portion 4 is a reaction point, there is a possibility that it will react in subsequent processing. Therefore, by performing a capping reaction, another group is bonded to this reaction point so that it does not react in the subsequent processing. The part of the code | symbol 6 has shown the result of the capping reaction. In this capping reaction, there is no change in the sugar acceptor to which the sugar donors of code 3 and code 5 are bound.

  The deprotection reaction is a process of transition from the parts 5 and 6 shown in the lower right part of the figure to the parts 7 and 8 shown in the upper right part. In this deprotection reaction, the ClAcO part is changed to the reaction site in order to make the part of symbol 5 a sugar acceptor for the next glycosylation reaction. For this reason, the group of this part is removed and it changes into a hydroxyl group. The part of the code | symbol 7 is a result of a deprotection reaction. In this deprotection reaction, the capped sugar receptors of reference numbers 6 and 8 do not change.

  As a result, the reference numeral 7 generated in the above-described process is obtained by linking the reference numeral 1 to the reference numeral 2 sugar donor.

  Next, the next glycosylation reaction can be started by considering the generated part 7 as a new part 1, and the processes of the glycosylation reaction, the capping reaction, and the deprotection reaction are performed. By repeating, the sugar donor of the code | symbol 2 can be couple | bonded further.

  A predetermined number of sugar donors can be bound by repeating the above steps a predetermined number of times.

  Hereinafter, chemical conditions for each reaction will be described.

  In any of the glycosylation reaction, the capping reaction, and the deprotection reaction, it is necessary to extract only the target product by removing the reagents as a post-treatment after completion of each reaction. When it is usually performed in a laboratory, etc., liquid separation operation and column separation are combined. However, here, the post-treatment means is only a separation using a silica gel column in order to obtain optimum conditions for the apparatus.

  Specifically, in the post-treatment, first, reagents and by-products are poured out using a mixed solvent of ethyl acetate and hexane (mixing ratio 4: 1), and then mixed solvent of ethyl acetate and methanol (mixing ratio 4: 1). Recover the PEG-attached sugar. This recovery utilizes the polarity of PEG. This is a fixed condition.

  As chemical conditions, first, selection of a sugar donor and selection of a reaction activator in a glycosylation reaction will be described.

As the sugar receptor, a sugar receptor with PEG as shown in FIG. 2 is used.
On the other hand, sugars as shown in FIG. 4 can be generally used as sugar donors, but here, two of FIG. 3 were examined.

  When a glycosylation reaction was attempted under various conditions using the sugar donor shown in FIG. 3 (a), the results shown in Table 1 below were obtained.

  On the other hand, when a glycosylation reaction was attempted under various conditions using the sugar donor shown in FIG. 3B, the results shown in Table 2 below were obtained.

  In any of the results shown in Tables 1 and 2, “Entry” is an evaluation entry, “eq” is the amount indicated by equivalent, “Donor” is a donor, and “Glycosylate” is a yield (experimental result). .

  Regarding the reaction activator, “NIS” and “TfOH” are shown for the sugar donor shown in FIG. 3A, and the sugar donor shown in FIG.

  When these results are examined, since the NIS used as an activator of the sugar donor originally shown in FIG. 3A is solid and has low solubility, the yield is poor. As for the sugar donor shown in FIG. 3 (b), Entry8 has a problem of TMS formation of hydroxyl group, and Entry9 has a problem that decomposition of the sugar donor occurs.

  From the remaining entries, Entry 7 with good yield is judged as the optimum condition.

  Therefore, the imidate sugar which is a sugar donor shown in FIG. 3B is suitable as the sugar donor.

In addition, boron trifluoride etherate (BF ₃ OEt ₂ ) is used as a reaction activator. These equivalent ratios are sugar acceptor 1: sugar donor 3: reaction activator 3.

  This is the optimum condition for the glycosylation reaction.

  Next, selection of the capping agent will be described.

  When the capping reaction was tried under various conditions, the results shown in Table 3 below were obtained.

  In Table 3, the capping agent is also written in the Entry column, and yield indicates the capping rate. From the results in Table 3, Entry1 is determined as a capping agent.

  Next, selection of deprotection conditions will be described.

  When the deprotection reaction was tried under various conditions, the results shown in Table 4 below were obtained.

  In the Entry column, the deprotecting agent is also shown, solv indicates the solvent, and yield indicates the deprotection rate. Further, Hexamethylenetetramine of 3 in the Entry column is shown in FIG. 5A, 4 and 5 outside the frame in the Entry column are shown in FIG. 5B, and 6 is shown in FIG. 5C.

  In Table 4, those with “column elution” of “x” are not suitable as candidates because unintended substances are mixed in the separation by the silica gel column. In addition, it is pointed out that Entry4 may break PEG. Considering the reaction time, Entry 8 is determined as the optimum condition.

  As a capping agent, 2.4 equivalent of isocyanadobenzoyl is used. Further, a mixed solvent of pyridine and methanol (mixing ratio 7: 3) is used as a deprotecting agent. This is the optimum condition for the deprotection reaction.

  It can be said that the optimum conditions described above can be expected to produce a synthesis with good yield even under the condition that no solid reagent is used, the condition that no liquid separation operation is performed, and the condition that the reagent is stored at room temperature.

  Next, the sugar chain automatic synthesizer of the present invention will be described with reference to FIG. FIG. 6 is a configuration example of an automatic synthesizer that automatically realizes the sugar chain synthesis method of the present invention. In addition, the structural example shown here has shown about the case where there is one reaction container.

  In FIG. 6, each component provided in the automatic synthesis apparatus of the present invention will be described.

  Reference numeral 1 denotes a reaction vessel, which has, for example, a bifurcated shape made of glass and can be used for injection and extraction of gas and liquid, respectively, and a liquid port is hermetically sealed. The volume can be, for example, 25 mL. The reaction vessel 1 is accommodated in an aluminum block 2 having a shape covering and enclosing the vessel, and is fixed by an abutment 3.

  A temperature setting mechanism 4 is provided inside the aluminum block 2, and the temperature of the entire inside of the aluminum block 2 can be changed from 0 ° C. to 50 ° C., and the temperature can be kept stable. The temperature setting mechanism 4 can also perform cooling or heating. The aluminum block 2 and the temperature setting mechanism 4 that control the temperature of the reaction vessel 1 are not limited to the above-described configuration, and may be configured to be immersed in a liquid flow.

  A driving mechanism 6 driven by a motor 5 such as a stepping motor is connected to the abutment 3, and linearly reciprocates by the rotational driving of the motor 5 to shake the reaction vessel 1.

  The silica gel column 13 is held on the silica gel column cradle 7. In the silica gel column 13 held by the silica gel column cradle 7, it is recognized that the liquid level has come to these portions at several millimeters above the packing and several millimeters below the full column position. In order to do so, an optical sensor 12 is installed.

  The sealed case 8 can be depressurized with a pump, and the recovery flask 15 can be accommodated therein. The recovery flask 15 sucks the silica gel column 13 by reducing the pressure of the sealed case 8 and sucks the reaction solution injected into the silica gel column 13. The sealed case 8 can be configured to have the same function as a normal suction manifold.

  The silica gel column can be exchanged with the silica gel column cradle 7 by the column moving mechanism 11. The column moving mechanism 11 moves the silica gel column arranged in the unused column stock space 14a from the silica gel column cradle 7 to set it, and also uses the silica gel column 13 from the silica gel column cradle 7 to use the used column discard space. Move to 14b. As a result, the silica gel column can be automatically replaced.

  The recovery flask 15 is a container for recovering the reaction solution purified by passing through the silica gel column 13, and the reaction solution recovered in the recovery flask 15 is returned to the reaction container 1 and the reaction is performed next. As the collection flask 15, for example, a size of 50 mL or more is appropriate. A drain 16 is disposed below the silica gel column 13, and anything dropped from the silica gel column 13 is discarded.

  The recovery flask 15 and the drain 16 are arranged at a lower position in the closed case 8 of the silica gel column 13 or taken out by the recovery flask / drain moving mechanism 17 as necessary. When taken out of the sealed case 8, the recovery flask 15 can be injected and extracted by the needle 20. Moreover, if it is the drain 16, it will become a receiving tray of the discharge | emission from the needle 20. FIG.

  The column moving mechanism 11 and the collection flask / drain moving mechanism 17 can be controlled simultaneously as necessary, or can be controlled independently.

  Although not shown in the figure, the recovery flask 15 can be shaken and controlled by shaking. The shaking mechanism can be constituted by the motor 5 and the driving mechanism 6 described above.

  The sealed case 8 is provided with a sealed case internal pressure control mechanism 18 for controlling the pressure in the sealed case, and performs pressure control together with the silica gel column holder 7 and the sealed case 8. As pressure control, air is communicated inside and outside the case by removing the sealing of the sealed case 8, and then, after sealing, maintaining the atmospheric pressure, the inside is decompressed after sealing. The decompression in the sealed case 8 can be performed by, for example, sucking with the decompression pump 36.

  A plurality of reagent bottles 19 can be prepared. Each of the reagent bottles 19 is filled with nitrogen gas after a required reagent is put in the bottle, and a septum cap (Teflon (registered trademark) + silicon) is applied. The reagent bottle can be prepared manually by the operator prior to the operation of the apparatus.

  Examples of reagent bottles to be prepared include dichloromethane, boron trifluoride etherate (BF3 / OEt2), isocyanatobenzoyl, mixed solvent of pyridine and methanol (mixing ratio 7: 3), mixed solvent of ethyl acetate and hexane (mixed) 4: 1), mixed solvent of ethyl acetate and methanol (mixing ratio 4: 1 or 3: 1), sugar donor solution, methanol, dichloromethane (for washing only), toluene, ethyl acetate.

  As a liquid moving mechanism for moving a liquid such as a reagent or a reaction solution between the recovery flask 15, the reaction container 1, the reagent bottle 19, the silica gel column 13, etc., the needle 20 and the needle parallel moving means 25 for moving the needle 20, the needle Vertical movement means 26 is provided.

  In the configuration of FIG. 6, three needles 20 are provided. One of the needles 20 is for gas and is connected to the gas tube 29, and the other two are for liquid and is connected to the liquid tubes 27 and 28. The inner diameters of the liquid tubes 27 and 28 are set according to the scale of the syringes 21 and 22 to be connected. In the figure, although there are two types of needles 20, syringes 21 and 22, and syringe operation mechanisms 23 and 24 for injecting and extracting syringes according to the scale (μL or mL) of the liquid volume used, , Single type or multiple types. When switching between three or more types of scales, the number of needles, liquid tubes, syringes, and the number of syringe operation mechanisms are set according to the number of scales.

  In addition, when inserting a needle into a reagent bottle, it is set as the structure which inserts one each for gas and a liquid simultaneously, and the space | interval of two needles is set narrowly according to a reagent bottle.

  The syringe operation mechanisms 23 and 24 are provided with bubble sensors (not shown) for recognizing that gas has entered the syringes 21 and 22 to notify that the amount of liquid in the container to be sucked is small. it can.

  The needle parallel moving means 25 is a mechanism for moving the needle 20 in the horizontal (or plane) direction, and can be set at the position of each reagent bottle 19, reaction container 1, recovery flask 15, and silica gel column 13. The needle vertical movement means 26 is a mechanism for moving the needles 20 independently in the vertical direction. The needles 20 are moved downward to pierce the needle 20 into the septum of the reagent bottle 19, or the needle 20 is moved from the septum of the reagent bottle 19. It can be pulled off.

  The liquid tubes 27 and 28 are tubes that connect the liquid needle and the syringes 21 and 22 in the needle 20, and each includes one tube and two in total. The needle 20 and the syringes 21 and 22 are connected by the liquid tubes 27 and 28 regardless of the needle movement by the needle parallel moving means 25 and the needle vertical moving means 26.

  The gas tube 29 connects the gas needle and the nitrogen gas cylinder 30 through the manifold 35 in the needle 20. The needle 20 and the manifold 35 are connected by the gas tube 29 regardless of the needle movement by the needle parallel moving means 25 or the needle vertical moving means 26.

  The needle 20 is cleaned including the outside of the needle in the needle cleaning space 38. Further, the micro container 39b is held in the micro container holding means 39a.

  As a mechanism for suctioning and discharging by the needle 20 and adjusting the pressure in the reaction vessel 1, a nitrogen gas cylinder 30, a manifold 35, a decompression pump 36, and the like are provided.

  The nitrogen gas cylinder 30 is opened and closed by the control of a control means 42 such as a PC, and after opening, a constant pressure is applied. Reference numeral 31 denotes a safety valve which is acid-resistant. Reference numeral 33 denotes an electromagnetic valve, which is provided on the gas tube side and the pump side of the manifold 35 to be acid resistant. The inside of the manifold 35 is decompressed by a decompression pump 36 via an electromagnetic valve 33, and the pressure in the manifold 35 can be measured by a barometer 34.

  The gas inlet of the reaction vessel 1 and the manifold 35 are closely connected by a pipe 37. Since the reaction vessel 1 is reciprocated by a shaking mechanism constituted by the motor 5 and the drive mechanism 6, for example, the pipe 37 is made flexible so that it is closely connected regardless of the reciprocation of the reaction vessel. The mechanism.

  The manifold 35 has acid resistance, and is connected to the gas tube 29 through the electromagnetic valve 32 and is connected to the decompression pump 36 through the electromagnetic valve 33. A gas trap 41 is connected to the other end of the decompression pump 36. The gas trap 41 is a processing device for the escaped gas (vaporized organic solvent).

  The control device 42 can be configured by a personal computer (PC), and a processing program is executed by an operator. The control device 42 performs control by communicating with various mechanisms via the interface 43 according to the processing program. The control device 42 may be used in combination with a sequencer or may be substituted by a sequencer.

  The interface 43 includes the cooling element 4, the motor 5, the column moving means 11, the recovery flask moving means 17, the syringe operating mechanisms 23 and 24, the needle moving means 25 and 26, the cock operation of the nitrogen gas cylinder 30, the electromagnetic valves 32 and 33, A control signal is sent from the control means 42 to the decompression pump 36, the barometer 34, and other necessary devices.

  In addition to the operation start and operation interruption, various operation buttons such as solution extraction during reaction and micro container update are prepared for the operation unit (not shown), and an operation is instructed to the control means 42.

  Next, the basic operation of each component described above will be described.

  First, basic operations related to the syringe will be described. In the following, each operation is indicated by (operation (number)).

(Operation 1) Collecting reagent into syringe:
In the operation of sucking the reagent into the syringe, the reagent bottle and the sucking amount parameters are designated. Note that the liquid needle / syringe to be used is determined in advance according to the scale of the suction amount.

  Move the needle over the designated reagent bottle. If necessary, simultaneously move the designated reagent bottle to the needle position. Next, a needle is inserted into the reagent bottle. At this time, one liquid needle is inserted into the liquid. At this time, it is desirable to insert up to the bottom surface. On the other hand, the needle for gas is pierced to the upper part (gas part) of the liquid. Thereafter, the valve is opened and nitrogen gas is sent to the needle. After causing the syringe to “suck up” a specified amount, the valve is closed to stop sending the nitrogen gas needle. Remove the needle from the reagent bottle.

(Operation 2) Transferring syringe contents to reaction container:
In the movement operation of the syringe, a parameter of the liquid moving speed (amount of liquid to be injected at once / injection time interval) is designated. The amount of movement is the entire volume of the syringe, and control is performed on the syringe and liquid needle containing the contents.

  The needle is moved over the liquid inlet of the reaction vessel, and the needle is inserted into the liquid inlet (septum lid) of the reaction vessel. Open the nitrogen gas cylinder and open the manifold solenoid valve that matches the reaction vessel.

  After that, continue to observe atmospheric pressure in the manifold until the nitrogen gas cylinder is closed. When the atmospheric pressure exceeds the atmospheric pressure (for example, 1.2 times or more), the safety valve is forcibly opened for a predetermined time (for example, 0.1 sec) and immediately close.

  Causes the syringe to “push out” at a speed based on the specified parameters. As an actual operation, for example, an operation of setting a designated time based on a designated injection volume is repeated. An operation without specifying the speed may be performed. After moving the contents of the syringe to the reaction vessel, the solenoid valve is closed, the nitrogen gas cylinder is closed, and the needle is removed from the reaction vessel.

(Operation 3) Absorption operation of the contents of the reaction vessel into the syringe:
In the contents sucking operation, specify the sucking amount parameter. Note that the liquid needle / syringe to be used is determined in advance according to the scale of the suction amount.

  The needle is moved over the liquid inlet of the reaction vessel, and the needle is inserted into the liquid inlet (septum lid) of the reaction vessel. At this time, the needle pierces to the bottom surface of the container.

  Thereafter, the nitrogen gas cylinder is opened, and the manifold solenoid valve corresponding to the reaction vessel is opened. Cause the syringe to “suck up” the specified amount. The total amount in the container can be set as the amount designation. In this case, the end of siphoning may be determined by the bubble sensor.

  After sucking the contents of the reaction vessel into the syringe, the solenoid valve is closed, the nitrogen gas cylinder is closed, and the needle is withdrawn from the reaction vessel.

(Operation 4) Discarding the contents of the syringe to the drain:
In the operation of discarding the contents of the syringe to the drain, the entire contents in the syringe are discarded. Control is performed on the syringe and liquid needle containing the contents.

  Move the needle over the drain. If necessary, such as after use under the column, move the drain to a position where the needle can be used and allow the syringe to “push”.

(Operation 5) Movement of syringe contents to column:
In the movement operation of the contents of the syringe to the column, a parameter of the liquid moving speed (amount of liquid to be injected at once / injection time interval) is designated. Control is performed on the syringe and liquid needle containing the contents.

  The needle is moved over the silica gel column, causing the syringe to “push” at a rate based on the specified parameters. As an actual operation, for example, an operation of setting a specified time based on a specified injection volume is repeated.

(Operation 6) Absorption operation of the contents of the collection flask into the syringe:
In the operation of sucking the contents of the collection flask into the syringe, the sucking amount is designated as a parameter. The liquid needle / syringe to be used is determined in advance according to the scale of the amount to be sucked.

  Move the needle over the collection flask.

  If necessary, such as after using the recovery flask under the column, move the recovery flask to a position where the needle can be used and allow the syringe to “suck up” a specified amount. The total amount in the container can be set as the amount designation. In this case, the end of siphoning may be determined by the bubble sensor.

(Operation 7) Movement of syringe contents to collection flask:
In the movement operation of the contents of the syringe to the collection flask, all of the syringe contents are moved. The syringe and liquid needle containing the contents shall be controlled.

  Move the needle over the collection flask. If necessary, such as after using the collection flask under the column, move the collection flask to an available position on the needle and “push” the syringe.

  Next, basic operations related to the reaction container, reagent bottle, and recovery flask will be described.

(Operation 8) Temperature control of the reaction vessel:
In the temperature control of the reaction vessel, the temperature is specified as a parameter. A temperature control command is issued to the temperature control element in the aluminum block, and the temperature is monitored and controlled to a specified temperature or an allowable range.

(Operation 9) Shaking motion of reaction vessel and recovery flask:
In shaking operation, the container, shaking intensity, and time are set as parameters.
Turn the specified container side motor. The rotation speed is set according to the specified shaking intensity. After the specified time has elapsed, command the motor to stop and wait for the motor to stop completely.

(Operation 10) Pressure control operation in the reaction vessel:
In the atmospheric pressure control operation, the control atmospheric pressure is set as a parameter.

  Open the solenoid valve on the pump side of the manifold and set to the specified pressure with the air pump. After the set atmospheric pressure is reached, if the set atmospheric pressure is atmospheric pressure, the solenoid valve is closed and the pump is stopped. Note that the above processing is not performed when the set atmospheric pressure is the reduced pressure setting.

(Operation 11) Movement operation of the contents of the reagent bottle to the reaction container:
In the movement operation of the contents of the reagent bottle to the reaction container, the reagent bottle and the movement amount are set as parameters.

  The contents of the reagent bottle are transferred to the syringe according to the operation of collecting the reagent in the syringe in (Operation 1). If the specified amount exceeds the syringe volume, transfer only the syringe volume. Next, the contents of the syringe are transferred to the reaction container in accordance with the movement of the contents of the syringe in (Operation 2) to the reaction container. When the capacity moved so far does not reach the specified amount, the operations (operation 1) and (operation 2) are repeated for the remaining amount.

(Operation 12) Movement of reagent bottle contents to silica gel column:
In the operation of moving the contents of the reagent bottle to the silica gel column, the reagent bottle and the moving amount are set as parameters.

  The contents of the reagent bottle are transferred to the syringe according to the operation of collecting the reagent in the syringe in (Operation 1). At this time, if the specified amount exceeds the syringe capacity, only the syringe volume is transferred. Next, the contents of the syringe are transferred to the column in accordance with the operation of moving the contents of the syringe to the column in (Operation 5). In the actual operation, a drain or recovery flask setting operation below the column, a pressure setting operation for the column dropping part, an injection amount control operation by limiting the column capacity, and the like are performed.

(Operation 13) Transferring the contents of the reaction vessel to the silica gel column:
In the movement operation of the contents of the reaction vessel to the silica gel column, the movement amount is set as a parameter.

  In accordance with the operation of sucking the contents of the reaction container into the syringe in (Operation 3), the contents of the reaction container are transferred to the syringe. If the specified amount exceeds the syringe volume, transfer only the syringe volume. Next, the contents of the syringe are transferred to the column in accordance with the operation of moving the contents of the syringe to the column in (Operation 5).

(Operation 14) Movement of the contents of the recovery flask to the reaction vessel:
In the operation of transferring the contents of the recovery flask to the reaction vessel, the amount of movement is set as a parameter. The contents of the flask are transferred to the syringe in accordance with the operation of sucking the contents of the collection flask into the syringe in (Operation 6). At this time, if the specified amount exceeds the syringe capacity, only the syringe volume is transferred. Next, according to the movement of the contents of the syringe of (Operation 2) to the reaction container, the contents of the syringe are transferred to the reaction container. If the moved capacity has not reached the specified amount until the processing, the processing is repeated for the remaining amount.

(Operation 15) Evaporation operation of reaction vessel contents:
In the evaporation operation of the contents of the reaction vessel, temperature, time, and solvent type are set as parameters. Further, the temperature is set according to the temperature control of the reaction vessel in (Operation 8), and the atmospheric pressure is set in the reaction vessel in (Operation 10) according to the atmospheric pressure control operation. Set the temperature and pressure at the same time and slowly over time. The set value and time of the atmospheric pressure are set according to the type of solvent.

  For example, in the case of a solvent of dichloromethane, the pressure is changed from atmospheric pressure (760 mmHg) to (750 mmHg) in a short time and then over 2 minutes (430 mmHg). In the case of methanol, the pressure is changed from atmospheric pressure (760 mmHg) to a short time (280 mmHg), and then over 1 minute (160 mmHg). In the case of ethyl acetate, the pressure is changed from atmospheric pressure (760 mmHg) to (200 mmHg) in a short time, and then over 1 minute (100 mmHg). In the case of toluene or pyridine, the pressure is reduced from atmospheric pressure (760 mmHg) to (90 mmHg) in a short time and then over 2 minutes (22 mmHg).

  After the specified time has elapsed, the pressure is returned to atmospheric pressure in accordance with the pressure control operation in the reaction vessel in (Operation 10), and is returned to room temperature in accordance with the temperature control of the reaction vessel in (Action 8).

(Operation 16) Discarding the contents of the reaction vessel:
When discarding the contents of the reaction vessel, the discard amount is set as a parameter.

  In accordance with the operation of sucking the contents of the reaction container into the syringe in (Operation 3), the contents of the reaction container are transferred to the syringe. If the specified amount exceeds the syringe volume, transfer only the syringe volume. Next, the contents of the syringe are transferred to the drain in accordance with the operation of discarding the contents of the syringe to the drain in (Operation 4). If the transferred capacity has not reached the specified amount until the processing, the processing is repeated for the remaining amount.

(Operation 17) Movement operation of the contents of the reagent bottle to the recovery flask:
In the movement operation of the contents of the reagent bottle to the collection flask, the movement amount is set as a parameter. The contents of the reagent bottle are transferred to the syringe according to the operation of collecting the reagent in the syringe in (Operation 1). If the specified amount exceeds the syringe volume, transfer only the syringe volume. Next, the content of the syringe is transferred to the recovery flask in accordance with the operation of moving the content of the syringe to the recovery flask in (Operation 7). If the transferred capacity has not reached the specified amount until the processing, the processing is repeated for the remaining amount.

(Operation 18) Cleaning operation of reaction vessel and recovery flask:
In the washing operation of the reaction vessel and the recovery flask, the vessel to be washed is set as a parameter.

  The dichloromethane is transferred from the reagent bottle to the reaction container or the recovery flask in accordance with the movement operation of the contents of the reagent bottle in (Operation 11) to the reaction container or the movement operation of the contents of the reagent bottle to the recovery flask in (Action 17). . As the amount of movement, an appropriate amount for cleaning is determined in advance. Next, the container is shaken according to the shaking operation of the reaction vessel and the recovery flask in the above (Operation 9). For the shaking time and strength, an appropriate amount for washing is determined in advance.

  In accordance with the operation of discarding the contents of the reaction container in (Operation 16) (or the operation of discarding the contents of the collection flask in (Operation 20) below), all the contents of the container are discarded.

  The above operations are repeated again.

(Operation 19) Cleaning operation of needle and syringe:
In the washing operation of the needle and syringe, dichloromethane is transferred from the reagent bottle to the syringe in accordance with the operation of collecting the reagent in the syringe in (Operation 1). The amount of movement is the syringe capacity. Next, according to the operation of discarding the contents of the syringe to the drain in (Operation 4), the contents of the syringe are discarded to the drain. After each of the above treatments is further repeated twice, the outside of the needle is washed.

(Operation 20) Discarding the contents of the collection flask:
In the discarding operation of the contents of the collection flask, the discard amount is set as a parameter. In accordance with the operation of sucking the contents of the collection flask into the syringe in (Operation 6), the contents of the collection flask are transferred to the syringe. If the specified amount exceeds the syringe volume, transfer only the syringe volume. Next, the contents of the syringe are transferred to the drain in accordance with the operation of discarding the contents of the syringe to the drain in (Operation 4). If the transferred capacity has not reached the specified amount until the processing, the above processing is repeated for the remaining amount.

(Operation 21) Progress wait operation:
In the operation of waiting for the elapse of a predetermined time, the time is set as a parameter.

  Next, the basic operation regarding the silica gel column will be described.

(Operation 22) Silica gel column setting operation:
In the setting operation of the silica gel column, one silica gel column is set from the stock space of the unused column stock space 14 a to the installation position of the silica gel column 13 on the silica gel column cradle 7 by the column moving mechanism 11.

(Operation 23) Disposal of used silica gel column:
In the disposal operation of the used silica gel column, the silica gel column is moved from the installation position of the silica gel column to the used column discard space 14b by the column moving mechanism 11.

(Operation 24) Sealing and pressure control operation on the column drop side:
In the sealing and pressure control operation on the column drop side, either atmospheric pressure is set on opening or sealing, and reduced pressure is set on sealing.

  The recovery flask / drain moving mechanism 17 is set to either a state in which the sealing is removed and air inside and outside the case is allowed to pass, a state in which the air is kept sealed and the atmospheric pressure is maintained, or a state in which the inside is decompressed after being sealed. If necessary, the vacuum pump is controlled simultaneously.

(Operation 25) Extraction operation into a micro container:
In the operation of extracting a trace amount from the contents of the reaction vessel or the recovery flask into a trace vessel, if the vessel is shaking, the shaking is temporarily stopped.

  In accordance with the operation of sucking the contents of the reaction container into the syringe in (Operation 3), the contents of the reaction container are transferred to the syringe. The amount of movement is a predetermined minute amount (for example, about 10 μL). The contents of the syringe are discharged onto the tube at the specified position. If shaking is interrupted, resume shaking the container. Wash the syringe and needle.

  The operation example of the present invention used for the above-described configuration and each functional operation will be described below.

  In the operation of the sugar chain automatic synthesizer of the present invention, the sugar chain is chemically synthesized by repeating the first to third rounds after preparation. The first round is a glycosylation reaction, the second round is a capping reaction, and the third round is a deprotection reaction. Further, after the reaction in each round, post-treatment is performed, and when returning from the third round to the first round, pretreatment for the next glycosylation reaction is performed as the fourth round.

  Hereinafter, preparation and processing of each round will be described.

(A) Preparation First, preparation will be described. In the following description, the order is indicated by (preparation (number)) for each process.

  The following preparation process is performed manually in advance.

  An appropriate reagent is prepared in each reagent bottle (Preparation 1), and a predetermined amount of sugar acceptor and sugar donor are put in a reaction vessel. The sugar acceptor is, for example, 18.8 mg, and the sugar donor is, for example, 27 mg. When these are set, they can be dissolved in dichloromethane and poured, but the amount of solvent used at that time is, for example, within 5 mL in total (Preparation 2).

  All the reagent bottles are stoppered with a septum cap, and the gas inside is replaced with nitrogen. In addition, since nitrogen substitution of reaction container is performed by an apparatus mechanism, it can be abbreviate | omitted (preparation 3).

  Set the reagent bottle and reaction container in the predetermined position (Preparation 4), set the unused silica gel column in the unused column stock space (Preparation 5), and set the unused trace container in the predetermined position ( Preparation 6).

(B) First Round Glycosylation Reaction Next, the first round glycosylation reaction will be described. In the following description, the order is indicated by (first round (number)) for each process.

  First, the reaction vessel is evaporated. The evaporation is performed according to the evaporation operation of the contents of the reaction vessel in the above (operation 15). The operating conditions of the evaporation operation are, for example, time 10 minutes, temperature 30 ° C., and solvent type dichloromethane. After the ON evaporation is completed, nitrogen is fed and the gas region in the reaction vessel is filled with nitrogen (first round 1).

  Next, dichloromethane is injected into the reaction vessel. The injection of dichloromethane is performed by transferring the contents of the reagent bottle from the reagent bottle of dichloromethane to the reaction container according to the operation of moving the contents of the reagent bottle to the reaction container in (Operation 11). An example of the injection amount can be 0.3 mL. After the injection, the reaction vessel 1 is shaken according to the shaking operation of the reaction vessel in (Operation 9). An example of the shaking time is, for example, 1 minute (first round 2).

  Next, according to the temperature control of the reaction vessel in (Operation 8), the reaction vessel is set to a low temperature (for example, temperature 0 ° C. (˜−10 ° C.)), and the reaction is performed according to the shaking operation of the reaction vessel in (Operation 9). Acclimate the reaction vessel to low temperature by continuing to shake vessel 1 and waiting for the low temperature to adjust (first round 3).

Next, the boron trifluoride etherate complex (BF ₃ · OEt ₂ ) is injected into the reaction vessel. In accordance with the operation of transferring the contents of the reagent bottle in (Operation 11) to the reaction vessel, the injection process is transferred from the reagent bottle of boron trifluoride etherate (BF ₃ .OEt ₂ ) solution to the reaction vessel 1, and the above (Operation According to 19), the needle and syringe are washed according to the washing operation of the needle and syringe. The amount of reagent movement is, for example, 16 μL (first round 4).

  Next, the glycosylation reaction proceeds. In the progress of the glycosylation reaction, the reaction vessel is shaken according to the shaking operation of the reaction vessel in the above (operation 9). The shaking time is, for example, 24 hours (first round 5).

  Next, post-processing of the first round is performed. The following (first round post-processing 1) is started without waiting for the end of the (first round 5). For example, as a target, start 2 minutes before the end). In addition, after (first round post-processing 2), the process is started after the end of (first round 5).

(C) Post-Processing of the First Round Next, post-processing of the first round will be described. In the following description, the order of each process is indicated by (first round post-process (number)).

  First, the silica gel column is swollen. For the swelling of the silica gel column, the silica gel column is set according to the setting operation of the silica gel column described in (Operation 22), and the drain is set below the column. According to the above-mentioned (operation 24) column drop side sealing and pressure control operation, the column dropping portion is set to “sealing, atmospheric pressure”. The mixed solvent of ethyl acetate and hexane is transferred from the reagent bottle to the silica gel column in accordance with the movement operation of the contents of the reagent bottle in (Operation 12) to the silica gel column. The amount of liquid is 3 mL, for example. It is assumed that the liquid to be injected does not overflow from the column so far.

  Next, according to the sealing operation on the column dropping side in (Operation 24), the column dropping portion is set to “sealing release”. The detection by the optical sensor 12 waits for the water surface to be recognized near the column packing. As soon as the above recognition is made, the column dropping portion is set to “sealed, atmospheric pressure” in accordance with the pressure control operation on the column dropping side in (Operation 24). The needle / syringe is washed in accordance with the needle / syringe washing operation in (Operation 19) (first round post-processing 1).

  Next, the operation of transferring the contents of the reaction vessel to a silica gel column is performed. In this transfer operation, the content of the reaction vessel is transferred to the silica gel column according to the transfer operation of the content of the reaction vessel in (Operation 13) to the silica gel column. At this time, the amount of liquid is the total liquid in the reaction vessel. The needle and syringe are washed in accordance with the washing operation of the needle and syringe in (Operation 19), and the reaction vessel is returned to room temperature in accordance with the temperature control of the reaction vessel in (Operation 8) (first round post-treatment 2).

  Next, the reaction vessel is washed with dichloromethane, and the liquid so far is passed through the column. In this process, the contents of the reagent bottle of dichloromethane are transferred to the reaction container in accordance with the operation of moving the contents of the reagent bottle to the reaction container in (Operation 11). The amount of liquid is, for example, 0.2 mL. The reaction vessel is shaken according to the shaking operation of the reaction vessel in (Operation 9). The shaking time is 1 minute.

  The content of the reaction vessel is transferred to the silica gel column in accordance with the operation of moving the content of the reaction vessel to the silica gel column (operation 13). The amount of liquid that moves is the total liquid in the reaction vessel.

  Next, according to the pressure control operation on the column drop side in (Operation 24), the column drop portion is set to “sealed pressure reduction”, and the optical sensor 12 detects that the water surface is recognized in the vicinity of the column packing.

  As soon as it is recognized by the above detection, the column drop portion is set to “sealed, atmospheric pressure” according to the pressure control operation on the column drop side in (Operation 24).

  The needle and syringe are washed in accordance with the needle and syringe washing operation in (Operation 19), and the reaction container is washed in accordance with the reaction vessel washing operation in (Operation 18). In this state, the next “post-processing” is performed (first round post-processing 3).

  Next, the mixed solvent of ethyl acetate and hexane is transferred to the column. In this transfer process, the contents of the reagent bottles of ethyl acetate and hexane are transferred to the silica gel column in accordance with the movement operation of the contents of the reagent bottle to the silica gel column in (Operation 12). The total amount of liquid can be 50 mL, for example. When the water surface is recognized near the full position of the column by the optical sensor 12, the liquid injection is stopped.

  According to the column closing side pressure and pressure control operation in (Operation 24), the column dropping portion is set to “sealing reduced pressure”. The optical sensor 12 detects that the water surface is recognized near the column packing. If recognized by the above detection, the column dropping portion is immediately set to “sealed, atmospheric pressure” according to the sealing and pressure control operation on the column dropping side in (Operation 24).

  When the amount of liquid transferred to the column is less than the specified amount until the above processing, the above-described processing after the first round post-processing 3 is repeated.

  The needle / syringe is cleaned in accordance with the needle / syringe cleaning operation (operation 19) (first round post-processing 4).

  Next, the process which transfers the mixed solvent of ethyl acetate and methanol to a column is performed. This transfer process turns the bottom of the column into a collection flask. At this time, the set liquid in the drain is disposed (discarded) as necessary. The contents of the reagent bottles of ethyl acetate and methanol are transferred to the silica gel column in accordance with the movement operation of the contents of the reagent bottle to the silica gel column in (Operation 12). The total amount of liquid is, for example, 50 mL. When the water surface is recognized in the vicinity of the column full position by the detection of the optical sensor 12, the liquid injection is stopped.

  According to the sealing and pressure control operation on the column dropping side in (Operation 24), the column dropping portion is set to “sealing pressure reduction”, and the optical sensor 12 detects that the water surface is recognized in the vicinity of the column packing. When recognized by the above detection, the column dropping portion is immediately set to “sealing, atmospheric pressure” according to the sealing and pressure control operation on the column dropping side in (Operation 24).

  If the amount of liquid transferred to the column is less than the specified amount until the processing, the processing from the reagent is repeated.

  Finally, once again, the column dropping part is set to “closed pressure reduction” for about 30 seconds, and then returned to “sealed, atmospheric pressure” to completely drop the liquid in the column. Thereafter, the needle / syringe is washed in accordance with the needle / syringe washing operation in (Operation 19) (first round post-treatment 5).

  Next, an evaporation process is performed while transferring the contents of the recovery flask to the reaction vessel. In the evaporation process, each position is set so that the needle can be extracted from the recovery flask.

  The contents of the recovery flask are transferred to the reaction container in accordance with the operation of moving the contents of the recovery flask to the reaction container in (Operation 14). For example, 10 mL. Here, the entire liquid in the collection flask is finally moved. However, when there is a restriction on the reaction vessel size, the liquid is repeatedly transferred several times, for example, as an amount of 10 mL.

  The recovery flask is shaken according to the shaking operation of the reaction vessel and the recovery flask in (Operation 9). The shaking time is, for example, 1 minute. In addition, this shaking is performed only after moving to the said reaction container, when there is a liquid residue in the following collection flask.

  The reaction vessel is evaporated in accordance with the evaporation operation for the contents of the reaction vessel in (Operation 15). The operating conditions of the evaporation operation are, for example, time 10 minutes, temperature 45 ° C., and solvent type ethyl acetate.

  Up to the above, when there is a liquid residue in the recovery flask, the above processing from the transfer processing of the content of the recovery flask to the reaction vessel to the above processing is repeated.

  Thereafter, the contents of the reagent bottle of ethyl acetate are transferred to the recovery flask in accordance with the operation of moving the contents of the reagent bottle to the reaction container in (Operation 11). The amount of liquid can be 10 mL, for example.

  The recovery flask is shaken according to the shaking operation of the reaction vessel and the recovery flask in (Operation 9). The shaking time can be, for example, 1 minute.

  Next, the contents of the recovery flask are again transferred to the reaction container in accordance with the operation of moving the contents of the recovery flask to the reaction container in (Operation 14). The amount of liquid to be transferred is the total liquid in the collection flask.

  Evaporation is performed according to the evaporation operation of the contents of the reaction vessel in (Operation 15). The operating conditions of the evaporation operation are, for example, time 10 minutes, temperature 45 ° C., and solvent type ethyl acetate.

  The recovery flask is washed in accordance with the operation for washing the reaction vessel and the recovery flask in (Operation 18) (first round post-treatment 6).

  Next, the evaporation operation of the contents of the reaction vessel is performed again. In this re-evaporation operation, toluene is transferred from the reagent bottle to the reaction container in accordance with the movement operation of the contents of the reagent bottle (operation 11) to the reaction container. The amount of liquid can be, for example, 10 mL. The reaction vessel is shaken in accordance with the shaking operation of the reaction vessel and the recovery flask in (Operation 9). The shaking time is, for example, 1 minute.

  Evaporation is performed according to the evaporation operation of the contents of the reaction vessel in (Operation 15). As operating conditions of the evaporation operation, for example, time 10 minutes, temperature 45 ° C., solvent type toluene (first round post-processing 7).

(D) Second Round Capping Reaction Next, the second round capping reaction will be described. In the following description, the order is indicated by (second round (number)) for each process.

  First, an injection process for injecting dichloromethane into the reaction vessel is performed. In this dichloromethane injection process, dichloromethane is transferred from the reagent bottle to the reaction container in accordance with the operation of moving the contents of the reagent bottle to the reaction container in (Operation 11). The amount of liquid is 0.6 mL, for example. The reaction vessel 1 is shaken according to the shaking operation of the reaction vessel and the recovery flask in the above (operation 9). The shaking time is, for example, 1 minute (second round 1).

  Next, an injection process for injecting isocyanatobenzoyl into the reaction vessel is performed. In this isocyanad benzoyl injection process, it is confirmed in advance that the reaction vessel 1 is at room temperature, and the reagent bottle of the isocyanad benzoyl solution is moved according to the movement operation of the contents of the reagent bottle in (Operation 11) to the reaction vessel. Transfer isocyanad benzoyl from to the reaction vessel. The amount of liquid is, for example, 40 μL. Thereafter, the needle / syringe is washed according to the washing operation of the needle / syringe in (Operation 19) (second round 2).

  Next, a progress process of the capping reaction is performed. In the progress process of the capping reaction, the reaction vessel is shaken according to the shaking operation of the reaction vessel and the recovery flask in the above (operation 9). The shaking time is, for example, 2 hours (second round 3).

  Next, an injection process for injecting methanol into the reaction vessel is performed. In the methanol injection process, methanol is transferred from the reagent bottle to the reaction container in accordance with the operation of moving the contents of the reagent bottle to the reaction container in (Operation 11). The amount of liquid is, for example, 10 μL. The reaction vessel is shaken in accordance with the shaking operation of the reaction vessel and the recovery flask in (Operation 9). The shaking time is, for example, 5 minutes. The needle and syringe are cleaned in accordance with the operation of cleaning the needle and syringe in (Operation 19) (second round 4).

  Next, post-processing of the second round is performed. The post-processing of the second round can be the same as the post-processing of the first round described above, and is started without waiting for the end of (second round 4). For example, as a target, it is started 2 minutes before the end. In addition, after the (second round post-processing 2), the process is started after the end of (second round 4).

  Note that the post-processing of the second round is the same as the post-processing of the first round, and thus description thereof is omitted here.

(E) Third-round deprotection reaction Next, the third-round deprotection reaction will be described. In the following description, the order is indicated by (third round (number)) for each process.

  First, a mixed solvent of pyridine and methanol is injected into the reaction vessel. In the mixed solvent injection process, the mixed solvent is transferred from the reagent bottle of pyridine and methanol to the reaction container in accordance with the operation of moving the contents of the reagent bottle to the reaction container in (Operation 11). The amount of liquid is, for example, 1.0 mL. The reaction vessel is shaken in accordance with the shaking operation of the reaction vessel and the recovery flask in (Operation 9). The shaking time is, for example, 1 minute (third round 1).

  Next, the process which makes a reaction container acclimatize to high temperature is performed. In this high temperature treatment, the reaction vessel is set to a high temperature in accordance with the temperature control of the reaction vessel in (Operation 8). The set temperature is, for example, 50 ° C. The needle and syringe are washed in accordance with the needle and syringe washing operation (operation 19) (third round 2).

  Next, the process which advances a deprotection reaction is performed. In the process of proceeding with the deprotection reaction, the reaction vessel is shaken in accordance with the shaking operation of the reaction vessel and recovery flask described in (Operation 9). The shaking time is, for example, 36 hours (third round 3).

  Next, an evaporation process for the contents of the reaction vessel is performed. In the evaporation process, after the reaction vessel is once returned to room temperature, evaporation is performed according to the evaporation operation of the contents of the reaction vessel in (Operation 15). The operating conditions of the evaporation operation are, for example, time 10 minutes, temperature 45 ° C., and solvent type methanol. Furthermore, evaporation is performed under different conditions. The operating conditions at this time are, for example, time 10 minutes, temperature 45 ° C., and solvent type pyridine.

  As described above, the reason why the evaporation process is performed twice under different conditions is that it is a mixed solvent. In the first evaporation, the pressure is set for a solvent having a low boiling point, and in the subsequent evaporation, the pressure is set for another solvent (third round 4).

  Next, the reaction material in the reaction vessel is dissolved with dichloromethane. In the dissolution process, the contents of the reagent bottle of dichloromethane are transferred to the reaction container in accordance with the operation of moving the contents of the reagent bottle to the reaction container in (Operation 11). The amount of liquid is 0.5 mL, for example. The reaction vessel is shaken in accordance with the shaking operation of the reaction vessel and the recovery flask in (Operation 9). The shaking time is 1 minute (3rd round 5).

  Next, post-processing of the third round is performed. The post-process of the third round can be the same as the post-process of the first round described above, and is started without waiting for the end of (third round 5). For example, as a target, it is started 2 minutes before the end. In addition, after (third round post-processing 2) and after, (third round 5) is started.

  Note that the post-processing of the third round is the same as the post-processing of the first round, and a description thereof is omitted here.

  Thereafter, returning to the first round of (B), the first to third rounds described above are repeated to chemically synthesize sugar chains. When returning to the first round, pre-processing is performed as the fourth round.

(F) Fourth Round Preprocessing Next, the fourth round preprocessing will be described. This pretreatment is performed before the next glycosylation reaction. In the following description, the order is indicated by (fourth round (number)) for each process.

  In the pretreatment, the sugar donor solution is injected into the reaction vessel. In this sugar donor injection process, the content of the reagent bottle in (Operation 11) is transferred from the reagent bottle of the sugar donor solution to the reaction container (fourth round 1). The amount of the liquid is an amount in which, for example, 27 mg of the sugar donor is dissolved. The reaction vessel is shaken in accordance with the shaking operation of the reaction vessel and the recovery flask in (Operation 9). The shaking time is, for example, 1 minute (4th round 2).

  Thereafter, the process returns to the first round described above and is repeated.

  In the above operation example, (A) preparation, (B) glycosylation reaction in the first round, (C) post-treatment in the first round, (D) capping reaction in the second round, (E) third round The deprotection reaction of (F) and the pretreatment of the fourth round are repeated for one reaction vessel.

  The present invention applies the repetition of the above series of treatments not only to one reaction vessel but also to a plurality of reaction vessels, and during the stirring process in the sugar chain synthesis chemical reaction in any one reaction vessel, By performing a process other than shaking in the sugar chain synthesis chemical reaction in the container, a plurality of sugar chain synthesis chemical reactions are performed independently and in parallel.

  Hereinafter, a configuration example of the automatic synthesizing apparatus according to the second aspect will be described with reference to FIG. FIG. 7 is substantially the same as the configuration example shown in FIG. 6 except that a plurality of reaction vessels are mainly provided. FIG. 7 illustrates the case of two reaction vessels.

  In the configuration example shown in FIG. 7, the difference from the configuration example shown in FIG. 6 is that a plurality of reaction vessels 1, 1 ′ and reaction vessels 1, 1 ′ that perform sugar chain synthesis chemical reactions independently and in parallel are provided. Covering aluminum blocks 2, 2 ', a plurality of reaction vessels 1, 1' and abutment 3 for simultaneously fixing the aluminum blocks 2, 2 ', temperature setting mechanisms 4, 4' provided in the respective aluminum blocks 2, 2 ' , Manifolds 35 and 35 ′ connected to the reaction vessels 1 and 1 ′ through pipes 37 and 37 ′, a plurality of sets of safety valves 31 and 31 ′ provided in the manifolds 35 and 35 ′, and a plurality of solenoid valves 32. , 32 ', 33, 33' and a plurality of barometers 34, 34 '. The control means 42 such as a PC controls the opening and closing of these cocks so that the sugar chain synthesis chemical reaction is independently performed in parallel by the plurality of reaction vessels 1 and 1 ′.

  In addition, in order to perform a sugar chain synthesis chemical reaction in the plurality of reaction vessels 1 and 1 ′, each operation described above has the following functions.

(Operation 2) Transferring syringe contents to reaction container:
In this operation, in addition to the liquid moving speed (the amount of liquid to be injected at once / injection time interval) as a parameter to be designated, the selection of the reaction vessel is added. In addition, the movement amount itself is all the contents of the syringe.

(Operation 3) Absorption operation of the contents of the reaction vessel into the syringe:
In this operation, the selection of the reaction vessel is added in addition to the suction amount as a parameter to be designated.

(Operation 8) Temperature control of the reaction vessel:
In this operation, selection of the reaction vessel is added in addition to the control temperature as a parameter to be specified.

(Operation 9) Shaking motion of reaction vessel and recovery flask:
The parameters to be specified for this operation include selection of reaction vessel or recovery flask, shaking intensity, and shaking time. In addition, when shaking a reaction container, since several reaction containers are shaken simultaneously, reaction containers cannot be selected.

(Operation 10) Pressure control operation in the reaction vessel:
In this operation, selection of the reaction vessel is added in addition to the set atmospheric pressure as a parameter to be specified.

(Operation 11) Movement operation of the contents of the reagent bottle to the reaction container:
In this operation, selection of a reaction container is added in addition to a reagent bottle and a reagent amount as parameters to be designated.

(Operation 13) Transferring the contents of the reaction vessel to the silica gel column:
This operation adds the selection of the reaction vessel in addition to the quantity as a parameter to be specified.

(Operation 14) Movement of the contents of the recovery flask to the reaction vessel:
This operation adds the selection of the reaction vessel in addition to the quantity as a parameter to be specified.

(Operation 15) Evaporation operation of reaction vessel contents:
In this operation, selection of a reaction vessel is added to parameters to be designated in addition to temperature, time, solvent type, and the like.

(Operation 16) Discarding the contents of the reaction vessel:
This operation adds the selection of the reaction vessel in addition to the quantity as a parameter to be specified.

(Operation 18) Cleaning operation of reaction vessel and recovery flask:
This operation has the choice of reaction vessel or recovery flask as a parameter to be specified. In the case of a reaction vessel, the selection of the reaction vessel is added.

(Operation 25) Extraction operation into a micro container:
This operation has the choice of reaction vessel or recovery flask as a parameter to be specified. In the case of a reaction vessel, the selection of the reaction vessel is added.

    Tables 1 to 9 show the relationship between the steps when two sugar chain synthesis chemical reactions are performed independently and in parallel. The steps in each reaction are the same as the treatments (A) to (F) described above.

  In a plurality of sugar chain synthesis chemical reactions, by using a long-time shaking (for example, 2 hours) included in one reaction, and by proceeding with a process other than “shaking” of other reactions during this shaking, Perform the reactions in parallel. These treatments are shifted in time when viewed at the process level, but can be said to be simultaneous progress when viewed as a whole sugar chain synthesis chemical reaction.

  The number of the plurality of sugar chain synthesis chemistries is not limited to the two described above, and may be three or more. For example, if three sugar chain synthesis chemical reactions are allowed to proceed, while one reaction system is performing a process other than shaking, the other two reaction systems are shaken for a long time (for example, 2 hours) It will control the relationship of the temporal operating state.

  Since the shaking mechanisms of a plurality of reaction vessels are common, it is necessary to interrupt shaking for a short time, for example, for needle operation of other reaction vessels, during a shaking operation for a long time. Nonetheless, this brief shaking interruption does not affect the effect of long shaking.

  In the above example, an example in which the same chemical reaction is performed a plurality of times is shown, but not limited to the above example, a reaction using another sugar donor or a reaction using another reaction activator may be used. In this case, necessary reagents (sugar donor / reaction activator) are prepared in the reagent bottle.

  Since the configuration of the present invention can be configured simply by increasing the number of reaction vessels, without changing many other configurations, a configuration in which a plurality of apparatuses having the same number as the number of reactions is prepared. In comparison, space can be saved.

  INDUSTRIAL APPLICABILITY The present invention can be applied mainly to the field of biochemical experiments, in preparation of preparations for sugar chain analyzers, research using synthesized sugar chains, or in the process of synthesizing organic compounds as well as sugar chains. Can be used or used.

It is a figure for demonstrating the schematic procedure of the sugar chain synthesis method of this invention. It is a figure for demonstrating the sugar receptor with PEG. It is a figure for demonstrating the example of the sugar donor used for the sugar_chain | carbohydrate synthesis | combination of this invention. It is a figure for demonstrating the other example of the sugar donor used for the sugar_chain | carbohydrate synthesis | combination of this invention. It is a figure for demonstrating the example of the deprotection agent used for the sugar_chain | carbohydrate synthesis | combination of this invention. It is a figure for demonstrating one structural example of the automatic synthesizer which implement | achieves automatically the synthesis method of the sugar_chain | carbohydrate of this invention. It is a figure for demonstrating the other structural example of the automatic synthesizer which implement | achieves automatically the synthesis method of the sugar_chain | carbohydrate of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1,1 '... Reaction vessel, 2, 2' ... Aluminum block, 3 ... Abutment, 4, 4 '... Temperature setting mechanism, 5 ... Motor, 6 ... Drive mechanism, 7 ... Silica gel column cradle, 8 ... Sealed case , 11 ... Column moving mechanism, 12 ... Optical sensor, 13 ... Silica gel column, 14a ... Unused column stock space, 14b ... Used column discarding space, 15 ... Recovery flask, 16 ... Drain, 17 ... Recovery flask / drain moving mechanism 18 ... Sealed case internal pressure control mechanism, 19 ... Reagent bottle, 20 ... Needle, 21, 22 ... Syringe, 23, 24 ... Syringe operating mechanism, 25 ... Needle parallel movement means, 26 ... Needle up / down movement means, 27, 28 ... Liquid tube, 29 ... Gas tube, 30 ... Nitrogen gas cylinder, 31, 31 '... Safety valve, 32, 32', 33 ... Solenoid valve, 34, 34 ' Barometer, 35, 35 '... manifold, 36 ... vacuum pump, 37, 37' ... pipe, 39a ... trace container holding means, 39 b ... trace container, 41 ... gas trap, 42 ... control unit, 43 ... interface.

Claims (6)

A glycosylation reaction step of binding a sugar donor to a sugar acceptor using low molecular weight polyethylene glycol as a support carrier;
Capping the hydroxyl group of the unreacted sugar acceptor in the product of the glycosylation reaction;
Deprotecting the protecting group of the sugar acceptor bound with the sugar donor in the glycosylation reaction to produce a sugar acceptor for the next glycosylation reaction,
Each step includes a purification process for recovering only the product to which the low molecular weight polyethylene glycol is bound by passing through a silica gel column after each reaction,
A method for synthesizing a sugar chain, comprising chemically synthesizing a predetermined number of sugar chains by sequentially repeating the above steps a predetermined number of times. A plurality of sugar chain synthesis chemical reactions including the above steps are provided,
While performing a stirring process in any one sugar chain synthesis chemical reaction, by performing a process other than shaking in another sugar chain synthesis chemical reaction,
The sugar chain synthesis method according to claim 1, wherein the plurality of sugar chain synthesis chemical reactions are performed independently and in parallel. A sugar acceptor is obtained by binding low-molecular-weight polyethylene glycol (CH ₃ (OCH ₂ CH ₂ ) _n OH) to a sugar as a support carrier, removing the temporary protecting group at the reaction site to form a hydroxyl group,
At least one of imidate sugar, fluorinated sugar, chloro sugar, bromo sugar, and thioglycoside is used as a sugar donor,
Each pure chemical reaction of glycosylation, capping, and deprotection is sequentially repeated a predetermined number of times to form a predetermined number of sugar chains, and the low molecular weight polyethylene glycol is separated from the product to produce a sugar and a sugar. A sugar chain synthesis method for synthesizing linked sugar chains,
The glycosylation reaction is
Linking a sugar donor to a sugar acceptor,
Mix the sugar acceptor and sugar donor and add a small amount of solvent;
Mixing boron trifluoride etherate complex as a reaction activator and stirring at a predetermined temperature condition,
The reaction solution and a mixed solvent of ethyl acetate and hexane are sequentially poured into a silica gel column swollen in advance with a mixed solvent of a small amount of ethyl acetate and hexane, and the waste solution is discarded.
Pour a mixed solvent of ethyl acetate and methanol into the same column to recover the liquid,
Each step of distilling off the solvent by vacuum concentration of the recovered liquid,
The capping reaction is
Capping the unreacted hydroxyl group of the sugar acceptor,
Adding a small amount of solvent to the product of the glycosylation reaction,
Isocyanadobenzoyl is mixed and stirred at room temperature,
Add a small amount of methanol,
Into a silica gel column swollen in advance with a mixed solvent of a small amount of ethyl acetate and hexane, the reaction solution, a mixed solvent of ethyl acetate and hexane are poured in order, and the waste solution is discarded.
Pour a mixed solvent of ethyl acetate and methanol into the same column to recover the liquid,
Each step of distilling off the solvent by vacuum concentration of the recovered liquid,
The deprotection reaction is
Deprotecting a temporary protecting group of a sugar acceptor bound with a sugar donor in the glycosylation reaction,
A mixed solvent of pyridine and methanol is added to the product of the glycosylation reaction and stirred at a predetermined temperature condition,
Distill off the solvent by vacuum concentration,
Into a silica gel column swollen in advance with a mixed solvent of a small amount of ethyl acetate and hexane, the reaction solution, a mixed solvent of ethyl acetate and hexane are poured in order, and the waste solution is discarded.
Pour a mixed solvent of ethyl acetate and methanol into the same column to recover the liquid,
Each step of distilling off the solvent by vacuum concentration of the recovered liquid,
A method for synthesizing sugar chains. A plurality of sugar chain synthesis chemical reactions including a pure chemical reaction of the glycosylation reaction, capping reaction, and deprotection reaction,
Each sugar chain synthesis chemical reaction is characterized by performing the plurality of sugar chain synthesis chemical reactions independently and in parallel by performing a process other than shaking during execution of the stirring process in the other sugar chain synthesis chemical reaction. The method for synthesizing sugar chains according to claim 3. A reaction vessel capable of removing the solvent in the vessel by decompression and heating in the vessel; and
A reagent bottle having a reagent used for each of the reactions;
A temperature setting mechanism for controlling the temperature of the reaction vessel over a wide range;
A shaking mechanism that gives shaking to the reaction vessel or its internal liquid;
A silica gel column for passing the product of each reaction together with a solvent;
A recovery flask for recovering the falling part of the silica gel column;
A switching mechanism that switches the falling part from the silica gel column to a drain or a recovery flask;
A liquid transfer mechanism for transferring liquid between the recovery flask, reaction vessel, reagent bottle, and silica gel column;
A pressure control mechanism for controlling the pressure in the reaction vessel and / or silica gel column;
Control means for controlling the operation of each mechanism,
The control means controls each mechanism, and in the reaction vessel, a glycosylation reaction for binding a sugar donor to a sugar acceptor, a capping reaction for capping an unreacted hydroxyl group of the sugar acceptor, and a sugar donor in the glycosylation reaction And sequentially performing a deprotection reaction to deprotect the temporary protecting group of the sugar receptor bound with
An apparatus for automatically synthesizing sugar chains, comprising purifying the product of each reaction in the silica gel column and performing post-treatment to return the product recovered in a recovery flask to a reaction vessel. A plurality of reaction containers capable of removing the solvent in the container by decompression and heating in the container;
A reagent bottle having a reagent used for each of the reactions;
A temperature setting mechanism that performs wide-range temperature control on the plurality of reaction vessels;
A shaking mechanism that collectively shakes the plurality of reaction vessels or the internal liquid thereof;
A silica gel column for passing the product of each reaction together with a solvent;
A recovery flask for recovering the falling part of the silica gel column;
A switching mechanism that switches the falling part from the silica gel column to a drain or a recovery flask;
A liquid transfer mechanism for transferring liquid between the recovery flask, reaction vessel, reagent bottle, and silica gel column;
A pressure control mechanism for controlling the pressure in the reaction vessel and / or silica gel column;
Control means for controlling the operation of each mechanism,
The control means controls each mechanism, and a glycosylation reaction for binding a sugar donor to a sugar acceptor in each reaction vessel, a capping reaction for capping an unreacted hydroxyl group of the sugar acceptor, and a sugar donation in the glycosylation reaction The deprotection reaction for deprotecting the temporary protecting group of the sugar receptor bound to the body is sequentially performed, and the shaking mechanism and the liquid transfer mechanism are controlled, and the stirring process is performed by any one of the sugar chain synthesis chemical reactions. In the meantime, in other sugar chain synthesis chemical reactions, processing other than shaking is performed,
Purify the product of each reaction in the silica gel column, perform a post-treatment to return the product recovered in the recovery flask to the reaction vessel,
An apparatus for automatically synthesizing sugar chains, wherein the plurality of sugar chain synthesis chemical reactions are performed independently and in parallel.

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