JPWO2006104166A1 - Crystallization separation carrier and compound separation method - Google Patents

Abstract

A support for crystallization separation, which is a compound that reversibly changes from a liquid phase state to a solid phase state with a change in solution composition and / or solution temperature, and has a reaction site that binds to another compound, The reaction site has one or more atoms selected from a carbon atom, an oxygen atom, a sulfur atom, and a nitrogen atom, and the other reacts via any of the carbon atom, the nitrogen atom, the sulfur atom, and the nitrogen atom. According to the carrier for crystallization separation that binds to the above compound, the chemical reaction can be performed in the liquid phase, and a specific substance can be selectively solidified (crystallized) easily from the liquid phase after the reaction is completed. In addition, the compound can be identified by structural analysis or the like while the separated compound remains bound to the carrier.

Description

  The present invention relates to a carrier for crystallization separation and a method for separating a compound, and in particular, a carrier for crystallization separation that is a compound that rapidly changes from a liquid phase state to a solid phase state due to a change in solution composition and / or solution temperature. And a method for separating a compound using the carrier for crystallization separation.

  Conventionally, in a chemical process, a method of separating a specific component dissolved in a liquid as a solid has been widely used. This is because solidification (crystallization) of only a specific component facilitates separation and purification after the reaction. In particular, in sequential multi-step synthesis such as compound library synthesis used in pharmaceutical development research in recent years, a necessary or unnecessary compound is solidified (crystallized) at the end of each reaction. Separation and purification of the crystallized (crystallized) substance is facilitated, and the process can be prevented from becoming complicated.

  Such solidification (crystallization) of the specific component dissolved in the solution is realized by satisfying certain conditions in relation to the chemical properties, physical properties, and solvent of the compound.

  However, in many cases, the conditions for solidification (crystallization) must be empirically searched through trial and error. In particular, in sequential multi-step synthesis, it is necessary to study the solidification (crystallization) conditions based on the properties unique to the compounds synthesized in each step, which requires significant cost and time for process development. Was.

  Therefore, a carrier molecule having a linker has been proposed in which a change in the solvent composition is sensitively detected and the dissolved state and the insolubilized (crystallized) state are reversibly changed. Various compounds can be bound to such a carrier molecule via a linker, and the bound compound can be easily changed from a dissolved state to an insolubilized (crystallized) state or vice versa with the carrier molecule. Can change state. In addition, such a compound bound to a carrier can reversibly repeat a dissolved state and an insolubilized (crystallized) state under substantially the same conditions even when the chemical structure is changed by successive chemical reactions. .

  If such a carrier molecule that reversibly changes between a dissolved state and an insolubilized (crystallized) state is used, a compound to be separated from a uniform solution state while utilizing the knowledge of the liquid phase reaction of organic chemistry as it is Can be selectively insolubilized (crystallized). That is, after the liquid phase reaction, it was possible to separate a specific compound while leaving other soluble components in the solution.

As a carrier capable of reversibly repeating a dissolved state and an insolubilized state, for example, a method using a polymer soluble in a solvent such as polyethylene glycol is known (see Non-Patent Document 1).
“Liquid-phase combinatorial synthesis” Hyunsoo Han, Mary M. et al. Wolfe, Sydney Brenner, and Kim D.W. Janda, Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 6419-6423, July 1995 Chemistry

  However, when a polymer such as polyethylene glycol described in Non-Patent Document 1 is used as a carrier, the compound remains in a state in which the compound remains bonded to the carrier due to the non-uniformity of the polymer. It was difficult to identify. In addition, since polyethylene glycol is hydrophilic, there is a problem in that handling is complicated due to difficulties in carrying out an anhydrous reaction.

  The present invention has been made in view of the problems as described above, and a chemical reaction can be performed in a liquid phase, and a specific substance is selectively solidified (crystallized) from the liquid phase after completion of the reaction. Thus, a carrier for crystallization separation and a method for separating a compound that facilitates separation of a specific substance, and further enables identification of the compound by structural analysis or the like while the separated compound remains bound to the carrier. It is to provide.

  The inventors of the present invention have made extensive studies to solve the above problems. As a result, if a carrier for crystallization separation having a reaction site that binds to another compound and reversibly changing between a liquid phase state and a solid phase state with a change in solution composition and / or solution temperature is used, The present inventors have found that the problem can be solved and have completed the present invention. More specifically, the present invention provides the following.

  (1) A carrier for crystallization separation, which is a compound that reversibly changes from a liquid phase state to a solid phase state with a change in solution composition and / or solution temperature, and has a reaction site that binds to another compound. The reaction site has one or more atoms selected from a carbon atom, an oxygen atom, a sulfur atom, and a nitrogen atom, and any one of a carbon atom, a nitrogen atom, a sulfur atom, and a nitrogen atom is substituted. A carrier for crystallization separation which binds to other compounds via

  The carrier for crystallization separation of (1) binds to another compound at the reaction site and remains in the state of being bound to the other compound, and is solidified from the liquid phase state as the solution composition and / or the solution temperature changes. Reversibly changes to the phase state. For this reason, it becomes possible to selectively crystallize other compounds that react with the reaction site.

  In addition, the other compound bonded to the crystallization separation support of (1) can be reversibly changed between a liquid phase state and a solid phase state under substantially the same conditions even when the chemical structure is changed by a sequential chemical reaction. Can be repeated. For this reason, it is not necessary to examine the crystallization conditions based on the properties unique to each compound.

  Therefore, according to the carrier for crystallization separation of (1), not only process development is facilitated, but also research and development of pharmaceuticals and the like by, for example, compound library synthesis can be promoted. And can contribute to technological innovation in the chemical industry.

  (2) The crystallization separation support according to (1), wherein the crystallization separation support has a saccharide skeleton.

  The carrier for crystallization separation of (2) has a saccharide skeleton. Here, “having a saccharide skeleton” means a structure including a skeleton derived from a saccharide in part. For example, a structure in which a saccharide is used as a starting material and at least one hydroxyl group of the saccharide, or a hydrogen atom of the hydroxyl group is substituted at the reaction site can be exemplified.

  Since the carrier for crystallization separation of (2) has a saccharide skeleton, it is easy to introduce a large number of hydrocarbon groups. For this reason, the hydrophobicity of the carrier for crystallization separation can be improved.

  (3) The carrier for crystallization separation according to (2), wherein the saccharide is pentose or hexose.

  The carrier for crystallization separation of (3) has pentose or hexose as a saccharide skeleton. Among saccharides, by using pentose or hexose as a skeleton, it is easy to obtain and introduction of hydrocarbon groups is facilitated.

（式中、
Ｘは、炭素、酸素、硫黄、及び、窒素原子から選ばれる１以上の原子であり、
Ｒ_１〜Ｒ_２７は、同一でも異なっていてもよく、置換基を有してもよい炭素数１〜６０の炭化水素基、置換基を有してもよい炭素数１〜６０のアシル基、及び、水素、の群から選ばれるいずれかであり、
且つ、それぞれ、Ｒ_１〜Ｒ_５の少なくとも１つ、Ｒ_６〜Ｒ_１０の少なくとも１つ、Ｒ_１１〜Ｒ_１６の少なくとも１つ、Ｒ_１７〜Ｒ_２２の少なくとも１つ、Ｒ_２３〜Ｒ_２７の少なくとも１つ、及び、Ｒ_２８〜Ｒ_３３の少なくとも１つは、置換基を有してもよい炭素数８以上の炭化水素基、又は、置換基を有してもよい炭素数８以上のアシル基を示す。）(4) The crystallization separation support according to any one of (1) to (3), wherein the crystallization separation support is represented by the following chemical formulas (A) to (F).

(Where
X is one or more atoms selected from carbon, oxygen, sulfur and nitrogen atoms,
R _{1 to} R ₂₇ may be the same or different, and may have a substituent, a C 1-60 hydrocarbon group, a C 1-60 acyl group that may have a substituent, And any one selected from the group of hydrogen,
And at least one of R _{1 to} R ₅ , at least one of R _{6 to} R ₁₀ , at least one of R _{11 to} R ₁₆ , at least one of R _{17 to} R ₂₂ , and R _{23 to} R ₂₇ , respectively. At least one and at least one of R _{28 to} R ₃₃ is a hydrocarbon group having 8 or more carbon atoms which may have a substituent, or an acyl having 8 or more carbon atoms which may have a substituent. Indicates a group. )

  The carrier for crystallization separation of (4) is a derivative having a pentose or hexose skeleton. Among these, when it has a furanose or pyranose skeleton having a cyclic structure, the crystallization effect tends to increase.

R _{1 to} R ₂₇ in the formula may be the same or different, and may be a hydrocarbon group having 1 to 60 carbon atoms which may have a substituent, or 1 to 1 carbon atoms which may have a substituent. 60 acyl groups. If the carbon number is within this range, it is easy to obtain and crystallization can be facilitated.

  Further, the support for crystallization separation of (4) has at least one hydrocarbon group which may have a substituent having 8 or more carbon atoms, or at least one substituent which has 8 or more carbon atoms. It also has a good acyl group. By having a long-chain hydrocarbon group or acyl group, the carrier for crystallization separation of (4) exhibits hydrophobicity, and therefore, by adding a highly polar solvent after the reaction is completed, crystallization is facilitated. be able to. The number of carbon atoms is preferably 12 or more, more preferably 18 or more.

（式中、
Ｘは、炭素、酸素、硫黄、又は、窒素原子であり、
Ｙは、炭素、酸素、硫黄、及び、窒素原子から選ばれる１以上の原子を有する反応部位であり、
Ｒ_３４は、炭素数１〜６０の炭化水素基であり、
ｎは、１〜５の整数を示す。）(5) The crystallization separation support according to (1), wherein the crystallization separation support is represented by the following chemical formula (G).

(Where
X is a carbon, oxygen, sulfur or nitrogen atom,
Y is a reaction site having one or more atoms selected from carbon, oxygen, sulfur, and nitrogen atoms;
R ₃₄ is a hydrocarbon group having 1 to 60 carbon atoms,
n shows the integer of 1-5. )

  The carrier for crystallization separation of (5) has a reactive site that binds to another compound and a hydrocarbon group having 1 to 60 carbon atoms bonded via carbon, oxygen, sulfur, or a nitrogen atom. . By having a long-chain hydrocarbon group having 1 to 60 carbon atoms, the carrier for crystallization separation of (5) exhibits hydrophobicity, and can therefore be dissolved at a high concentration in many organic solvents. 12-30 are preferable and, as for carbon number, More preferably, it is 20-30.

  (6) A method for separating a compound, wherein the crystallization separation carrier according to any one of (1) to (5) is dissolved in a soluble solvent to prepare a carrier solution, and the crystallization separation carrier The separation method of the compound which includes the coupling | bonding process which couple | bonds another compound with the reaction site | part, and the crystallization process of crystallizing the support | carrier which the said other compound couple | bonded.

  The compound separation method (6) is a separation method using the crystallization separation carrier of the present invention. According to the compound separation method of (6), it becomes possible to selectively crystallize a specific substance. Therefore, it is possible to separate only a specific substance while leaving a soluble component in the liquid phase, thereby avoiding complicating the separation process.

  In addition, the compound accompanying the crystallization separation carrier crystallized and separated in the crystallization step can be subjected to structural analysis or the like as it is without being separated from the carrier. Therefore, since the confirmation of the obtained compound can be carried out without going through the process of separating the compound after crystallization, the time required for research and development etc. can be shortened and the research can be promoted. Can be realized.

  Therefore, the method for separating compounds of (6) can be an innovative technique in the separation and purification of biochemical substances, the search for drug candidate substances, the construction of new chemical synthesis reaction methods, continuous peptide synthesis methods, and the like.

  (7) The method for separating a compound according to (6), further comprising a separation step of separating another compound from the crystallized carrier after the crystallization step.

  The method for separating a compound of (7) includes a step of separating the compound accompanying the crystallization separation carrier of the present invention from the crystallization carrier after crystallization. Thereby, the target compound obtained by synthesis or the like can be obtained as a single compound. Further, the carrier for crystallization separation after separation can be reused thereafter.

  (8) The method for separating a compound according to (6) or (7), further comprising an impurity removal step of removing impurities from the solution before the crystallization step.

  The method for separating a compound of (8) includes a step of removing impurities from a solution before crystallization from the solution of the crystallization separation support of the present invention and the compound. This makes it possible to increase the purity of a substance that is subsequently crystallized (a complex of a carrier for crystallization separation and a compound).

  (9) The method for separating a compound according to any one of (6) to (8), wherein in the crystallization step, the support is precipitated by means for changing the solution composition and / or means for changing the solution temperature.

  In the method for separating a compound (9), crystallization is carried out by changing the solution composition and / or the solution temperature. The carrier for crystallization separation of the present invention reacts sensitively to changes in solution composition and / or solution temperature. Therefore, by using means for changing the composition and / or temperature of the solution, it becomes possible to crystallize the crystallization separation carrier with the compound, and the soluble substance not bound to the crystallization separation carrier. Can easily separate certain compounds while remaining in solution.

  (10) The method for separating a compound according to (9), wherein the solution composition is changed by adding a solvent having a high affinity for the soluble solvent.

  In the method for separating a compound of (10), a solvent having high affinity is added to a solution in which a composite in which the compound and the carrier for crystallization separation are dissolved is dissolved. Since the solution composition can be changed by adding a solvent having high affinity, it is possible to crystallize the carrier for crystallization separation accompanied with the compound.

  (11) The method for separating a compound according to (9), wherein the means for changing the solution composition concentrates the soluble solvent.

  In the method for separating a compound of (11), a solution in which a complex in which a compound and a carrier for crystallization separation are bound is dissolved is concentrated. By concentrating the solution, the concentration of the compound in which the compound present in the solution and the crystallization separation support are combined is increased. For this reason, a solution composition can be changed and it becomes possible to crystallize the support | carrier for crystallization separation accompanying a compound.

  (12) The method for separating a compound according to (9), wherein the means for changing the solution temperature is to cool the solution.

  The method for separating the compound of (12) has a crystallization step of cooling the solution. By cooling the solution, the solution temperature can be changed, and it becomes possible to crystallize the carrier for crystallization separation accompanied with the compound.

  According to the crystallization separation carrier of the present invention, the chemical reaction can be performed in the liquid phase, and the specific substance is selectively solidified (crystallized) from the liquid phase after the completion of the reaction. Can be easily separated. In addition, the compound can be identified by structural analysis or the like while the separated compound remains bound to the carrier. For this reason, it is not necessary to study the crystallization conditions based on the properties unique to each compound, so that not only the process development is facilitated, but also the research and development of pharmaceuticals etc. by, for example, compound library synthesis is promoted. As a result, it can contribute to technological innovation in the biochemical and chemical industries.

  Moreover, according to the separation method of the compound of this invention, it becomes possible to selectively crystallize a specific substance. Therefore, it is possible to separate only a specific substance while leaving a soluble component in the liquid phase, thereby avoiding complicating the separation process. For this reason, the method for separating a compound of the present invention can be an innovative technique in the separation and purification of biochemical substances, the search for drug candidate substances, the construction of new chemical synthesis reaction methods, continuous peptide synthesis methods, and the like.

BEST MODE FOR CARRYING OUT THE INVENTION

  Hereinafter, embodiments of the present invention will be described in detail.

<Carrier for crystallization separation>
The carrier for crystallization separation of the present invention is a carrier for crystallization separation that is a compound that reversibly changes from a liquid phase state to a solid phase state with changes in solution composition and / or solution temperature, The reaction site has one or more atoms selected from a carbon atom, an oxygen atom, a sulfur atom, and a nitrogen atom, and a carbon atom, a nitrogen atom, a sulfur atom, and And a carrier for crystallization separation bonded to another compound via any one of nitrogen atoms.

[Reaction site]
The reaction site for binding to the other compound of the carrier for crystallization separation of the present invention has one or more atoms selected from a carbon atom, an oxygen atom, a sulfur atom, and a nitrogen atom, A plurality may be included.

  In addition, the size of the reaction site is not particularly limited as long as it has a portion that becomes a reaction site for binding to another compound in a part of the reaction site. In addition, the position at the reaction site where the reaction site for binding to another compound exists is not particularly limited, but it may be present at the end of the reaction site for the purpose of facilitating the reaction. preferable.

  In the reaction site, it is bonded to another compound via any of a carbon atom, a nitrogen atom, a sulfur atom, and a nitrogen atom. The structure of the reaction site having any one of a carbon atom, a nitrogen atom, a sulfur atom, and a nitrogen atom bonded to another compound at the reaction site is not particularly limited, but examples thereof include a hydroxyl group and an amino group. And carboxyl groups.

  The number of reaction sites is not particularly limited, and a plurality of reaction sites may be provided. In the present invention, since subsequent structural analysis and the like are facilitated, for example, it is preferable to bond 1: 1 with other compounds to be subjected to crystallization separation. In this case, the crystallization of the present invention is preferable. The number of reaction sites with other compounds in the separation carrier is preferably one.

  As the carrier for crystallization separation in the present invention, those having a saccharide skeleton are preferable. Here, “having a saccharide skeleton” means a structure including a skeleton derived from a saccharide in part. The saccharide includes polyalcohol itself, polyalcohol aldehyde, ketone, acid, derivatives thereof, condensates, and the like. In the present invention, when it has a saccharide skeleton, it may be a monosaccharide or a oligosaccharide or a polysaccharide obtained by condensing a plurality of these.

  The saccharide skeleton in the crystallization separation carrier of the present invention is not particularly limited, but is preferably pentose or hexose.

  When the carrier for crystallization separation of the present invention has a saccharide skeleton, for example, the carrier for crystallization separation can be obtained by introducing a reaction site into the saccharide using saccharide as a starting material. The introduction position of the reaction site is not particularly limited. Further, the method for introducing the reaction site is not particularly limited. For example, a method of substituting at least one hydroxyl group of a saccharide or a hydrogen atom of the hydroxyl group with a reaction site can be mentioned.

（式中、
Ｘは、炭素、酸素、硫黄、及び、窒素原子から選ばれる１以上の原子であり、
Ｒ_１〜Ｒ_２７は、同一でも異なっていてもよく、置換基を有してもよい炭素数１〜６０の炭化水素基、置換基を有してもよい炭素数１〜６０のアシル基、及び、水素、の群から選ばれるいずれかであり、
且つ、それぞれ、Ｒ_１〜Ｒ_５の少なくとも１つ、Ｒ_６〜Ｒ_１０の少なくとも１つ、Ｒ_１１〜Ｒ_１６の少なくとも１つ、Ｒ_１７〜Ｒ_２２の少なくとも１つ、Ｒ_２３〜Ｒ_２７の少なくとも１つ、及び、Ｒ_２８〜Ｒ_３３の少なくとも１つは、置換基を有してもよい炭素数８以上の炭化水素基、又は、置換基を有してもよい炭素数８以上のアシル基を示す。）As the crystallization separation carrier of the present invention, more preferred examples include those represented by the following chemical formulas (A) to (F).

(Where
X is one or more atoms selected from carbon, oxygen, sulfur and nitrogen atoms,
R _{1 to} R ₂₇ may be the same or different, and may have a substituent, a C 1-60 hydrocarbon group, a C 1-60 acyl group that may have a substituent, And any one selected from the group of hydrogen,
And at least one of R _{1 to} R ₅ , at least one of R _{6 to} R ₁₀ , at least one of R _{11 to} R ₁₆ , at least one of R _{17 to} R ₂₂ , and R _{23 to} R ₂₇ , respectively. At least one and at least one of R _{28 to} R ₃₃ is a hydrocarbon group having 8 or more carbon atoms which may have a substituent, or an acyl having 8 or more carbon atoms which may have a substituent. Indicates a group. )

The carriers for crystallization separation shown in the above (A) to (F) are derivatives having a pentose or hexose skeleton. The number of carbon atoms of the carbon chain of _R 1 _{to R 33} in the formula is usually 1 to 60. Among these, when it has a furanose or pyranose skeleton having a cyclic structure, the crystallization effect tends to increase.

  Moreover, the carrier for crystallization separation shown in (A) to (F) is a hydrocarbon group which may have at least one substituent having 8 or more carbon atoms, or at least one substituent having 8 or more carbon atoms. It has an acyl group which may have a group. The presence of long-chain hydrocarbon groups or long-chain acyl groups renders the crystallization separation support hydrophobic. The carbon number of the long chain portion is usually 8 or more, preferably 12 or more, and more preferably 18 or more.

  The method for producing the compounds (A) to (F) is not particularly limited. For example, a method of introducing an appropriate hydrocarbon into a compound having a saccharide skeleton via an ether bond by reacting a compound having a saccharide skeleton with a halogenated hydrocarbon under basic conditions. At this time, if a protecting group such as trityl chloride is used, it is possible to introduce a hydrocarbon chain to other hydroxyl groups while leaving a hydroxyl group at an arbitrary position.

（式中、
Ｘは、炭素、酸素、硫黄、又は、窒素原子であり、
Ｙは、炭素、酸素、硫黄、及び、窒素原子から選ばれる１以上の原子を有する反応部位であり、
Ｒ_３４は、炭素数１〜６０の炭化水素基であり、
ｎは、１〜５の整数を示す。）As another example of the carrier for crystallization separation of the present invention, one represented by the following chemical formula (G) can be given.

(Where
X is a carbon, oxygen, sulfur or nitrogen atom,
Y is a reaction site having one or more atoms selected from carbon, oxygen, sulfur, and nitrogen atoms;
R ₃₄ is a hydrocarbon group having 1 to 60 carbon atoms,
n shows the integer of 1-5. )

  The carrier for crystallization separation shown in (G) has a reactive site that binds to another compound, and is a hydrocarbon group having 1 to 60 carbon atoms bound via carbon, oxygen, sulfur, or a nitrogen atom. Have The presence of a long-chain hydrocarbon group having 1 to 60 carbon atoms makes the crystallization separation carrier hydrophobic. For this reason, the carrier for crystallization separation having a long-chain hydrocarbon group can be dissolved at a high concentration in many organic solvents. Although carbon number of a long chain part is 1-60 or more normally, 12-30 are preferable, More preferably, it is 20-30.

  It does not specifically limit as a method to manufacture a compound (G). For example, a phenol derivative having a functional group that becomes a binding site is reacted with a halogenated hydrocarbon under basic conditions to obtain an alkoxyphenyl derivative, and then the binding site and the binding site are obtained by a known functional group conversion method. The carrier for crystallization separation (G) can be synthesized by converting the functional group.

<Method for separating compounds>
The compound separation method of the present invention comprises a dissolution step in which the carrier for crystallization separation of the present invention is dissolved in a soluble solvent to prepare a carrier solution, and another compound is bonded to the reaction site of the carrier for crystallization separation. And a crystallization step of crystallizing the carrier to which the other compound is bonded.

[Dissolution process]
The soluble solvent used in the dissolution step in the present invention is not particularly limited as long as it can dissolve the crystallization separation carrier of the present invention. Examples thereof include halogenated hydrocarbons, chain ethers, cyclic ethers, cyclic hydrocarbons having 4 to 40 carbon atoms, or chain hydrocarbons. More specifically, for example, dichloromethane, tetrahydrofuran and the like can be mentioned. These may be used alone, or these solvents may be used in combination with cyclohexane, methylcyclohexane, decalin or the like.

  The concentration when the crystallization separation carrier of the present invention is dissolved in the soluble solvent can be appropriately selected according to the solvent used, the crystallization separation carrier, the properties of the compound to be bound to the crystallization separation carrier, and the like. Is usually 0.01 to 0.1 g / ml.

[Bonding process]
As a bonding method in the bonding step in the present invention, as long as it is a method of bonding another compound having a part that reacts with the reaction site to the reaction site of the crystallization separation carrier dissolved in the soluble solvent in the previous step, It is not limited, and various chemical reactions in the liquid phase can be used. For example, the method of couple | bonding by forming an ester bond and an amide bond can be mentioned.

[Crystalling process]
The crystallization step in the present invention is a step of crystallizing the compound bonded to the crystallization separation carrier in the bonding step, which is the previous step, while keeping the state accompanied with the carrier. The crystallization step in the present invention is not particularly limited as long as it can be crystallized while maintaining the state in which the compound to be crystallized is bonded to the support for crystallization separation. For example, the solution composition A means for changing the temperature and / or a means for changing the solution temperature can be preferably used.

[Means for changing the solution composition]
In the crystallization process of the present invention, the means for changing the solution composition preferably used is a means capable of changing the composition of the solution in which the crystallization separation carrier to which the compound to be crystallized is bonded is dissolved. There is no particular limitation.

  In the present invention, preferred means for changing the solution composition include, for example, means for further adding a solvent having a high affinity for the soluble solvent used for dissolving the crystallization separation carrier in the dissolution step. . The solvent having high affinity may be the same solvent as the solvent used as the soluble solvent or a different solvent. For example, when a solvent such as dichloromethane or tetrahydrofuran is used alone as a soluble solvent, or when these solvents are used in combination with cyclohexane, methylcyclohexane, decalin, or the like, acetonitrile, dimethylformamide, methanol, or the like as a high affinity solvent. Can be used.

  Another preferred means for changing the solution composition is, for example, a means for concentrating a solvent of a solution in which a crystallization separation carrier in a state where a compound to be crystallized is bound is dissolved. Here, the concentration means that a part or all of the solvent is distilled off. In addition, when the crystallization separation support to which the compound to be crystallized is crystallized by distilling off the whole, impurities contained in the solution may be precipitated together. It is preferable to include a step of removing impurities before performing the crystallization step.

[Means for changing the solution temperature]
In the crystallization step of the present invention, the means for changing the solution temperature that is preferably used is a means that can change the temperature of the solution in which the crystallization separation carrier to which the compound to be crystallized is dissolved is dissolved. There is no particular limitation. In the present invention, for example, a means for cooling the solution can be mentioned. For example, when cyclohexane is used as a soluble solvent for dissolving the carrier for crystallization separation, crystallization can be achieved by cooling to 5 ° C. In addition, when a means for cooling the solvent is used as a means for changing the solution temperature, ODS particles (silica gel having an octadecyl group bonded to the surface), a substance that becomes a crystallization nucleus, such as carrasbys, is added. Growth can be facilitated.

[Separation process]
In the method for separating a compound of the present invention, the crystallization separation support in a state in which the compound to be crystallized obtained in the crystallization step is bound is separated into a compound and a crystallization separation support. It is preferable that the process to include is included.

  The method for separating the compound from the crystallization separation carrier is not particularly limited as long as it can break the bond between the compound and the crystallization separation carrier. For example, when a paraalkoxybenzyl bond is formed, the bond can be dissociated by acid treatment.

[Impurity removal process]
In the method for separating a compound of the present invention, it is preferable to include an impurity removal step for removing impurities before carrying out the crystallization step. In the crystallization process, impurities contained in the solution may be precipitated together. In particular, when a means for distilling off all of the solvent is used as the crystallization step, impurities are precipitated together with the crystals of the carrier for crystallization separation to which the compound to be crystallized is bonded. Therefore, by removing impurities in advance before carrying out the crystallization step, the purity of the crystal of the crystallization separation support to which the compound to be crystallized obtained thereafter is bonded can be increased.

  The method for removing impurities is not particularly limited. For example, a method in which the entire solution in which the carrier for crystallization separation in which the compound to be crystallized is dissolved is washed with a solvent is used. Can be mentioned.

  Next, the present invention will be described in more detail based on examples, but the present invention is not limited thereto.

<Example 1>
[Synthesis of carrier for crystallization separation]
The process diagram of the carrier for crystallization separation is shown below. In addition, the number in a figure shows a compound number.

  100 g of trityl chloride, 89 g (1.3 eq.) Of methyl α-D-glucoside (Compound 1), 4.6 g of dimethylaminopyridine (DMAP) (0.1 eq.) Was placed in a 1 liter eggplant flask. To this were added 400 ml of dehydrated dichloromethane and 190 ml (4 eq.) Of triethylamine (TEA), and the flask was further washed with 100 ml of dichloromethane (DCM) and stirred well. Water was added to the resulting reaction solution, and the organic phase was washed with a separatory funnel to remove unreacted Methyl-α-D-glucoside. Thereafter, the organic phase was dried with about 50 g of anhydrous magnesium sulfate for 15 minutes, subsequently filtered and concentrated. This was dissolved in 350 ml of methanol while heating to about 70 ° C., and stirred as it was at room temperature for recrystallization. The resulting crystals were filtered, washed with about 100 ml each of methanol and hexane (n-hexane) on filter paper, and then the methanol was removed under reduced pressure to give the target compound 6-O-trityl- α-D-Methyl glucoside (Compound 2) was obtained. The yield was 70%.

  The recrystallized supernatant was further concentrated again and dissolved again in methanol for recrystallization. 26.2 g of the obtained compound was placed in a 1 liter eggplant flask, dissolved in 400 ml of tetrahydrofuran (THF) or dichloromethane (DCM), 230 ml (28 eq.) Of triethylamine (TEA), 2.2 g of dimethylaminopyridine (DMAP). (0.1 eq.) Was added and stirred for 5 minutes while cooling with ice. Using a dropping funnel, 111.4 ml of stearic acid chloride was slowly added over 10 minutes, and then stirred well for 4 hours. The reaction solution initially had a white precipitate (TEA hydrochloric acid) and then gradually turned orange. After confirming the completion of the reaction by TLC (silica gel thin layer chromatography), the salt was removed by filtration. The white compound on the obtained filter paper was washed with about 100 ml of hexane (n-hexane), and the liquid phase was concentrated. To this, 100 ml of hexane (n-hexane) and 100 ml of 2-propanol (2-propanol) were added and dissolved by heating at about 70 ° C., followed by recrystallization at room temperature to obtain the intended compound 3. The yield was 80%.

40.9 g of the obtained compound was placed in a 1 liter eggplant flask, dissolved in 250 ml of dichloromethane (DCM), 4 ml of trifluoroacetic acid (TFA) was added, and 10 ml of 3-mercaptopropionic acid was added little by little. Thereafter, the mixture was stirred in an ice bath for 60 minutes. Subsequently, 8 ml of triethylamine (TEA) was slowly added with ice cooling. The obtained reaction solution was concentrated, dissolved by adding 100 ml of hexane (n-hexane), and washed with a separating funnel with 50 ml × 3 of dimethylformamide (DMF) and 3 × 50 ml of acetonitrile. After washing, 150 ml of 2-propanol was added to perform recrystallization, and the target compound 4 (octadecanoic acid 2-hydroxymethyl-6-methoxy-4,5-bis-octadecanoyloxy was obtained. -Tetrahydro-pyran-3-yl ester). The obtained compound 4 was 31 g, and the yield was 95%.
¹ H-NMR (300 MHz; CDCl ₃ ) 5.58 (1H, t, J = 9.90 Hz), 5.01 (1H, t, J = 9.90 Hz), 4.97 (1H, d, J = 3.67 Hz), 4.87 (1 H, dd, J = 9.90, 3.67 Hz), 3.76 (1 H, m), 3.68 (1 H, m), 3.57 (1 H, m) , 3.40 (3H, s), 2.41-2.17 (6H, m), 1.64-1.43 (6H, m), 1.34-1.14 (84H, m), 1 .01 (9H, s) and 0.88 (9H, t, J = 6.60 Hz); HRMS calcdcd. _{for C 61 H 116 O 9 N} a m / z1015.8517, found 1015.85170

  81 g of the obtained compound 4 was placed in a 1 liter eggplant flask and dissolved in 300 ml of dichloromethane (DCM). Separately, 33.7 g (2 eq.) Of compound 5 synthesized by the method described below and 1 g of dimethylaminopyridine (DMAP) were added and stirred, followed by further heating and melting dicyclohexylcarbodiimide (DCC) 88 ml. (4 eq.) Was added and reacted at room temperature for 1 hour. The solution after the reaction was concentrated under reduced pressure and subsequently washed to obtain Compound 6.

4 g of sodium borohydride was dissolved in 100 ml of methanol, added to a hexane (n-hexane) solution of Compound 6, and reacted at room temperature for 2 hours. Furthermore, 10 ml of water was added to the reaction solution and stirred for 5 minutes. The lower layer was discarded using a separatory funnel, and the resulting solution phase was washed and dried over 50 g of anhydrous magnesium sulfate. Thereafter, the resultant was concentrated by filtration, and the target crystallization separation carrier of the present invention, compound 7 (octadecanoic acid 6- [4- (4-hydroxymethyl-phenoxy) -butyryloxymethyl] -2- Methoxy-4,5-bis-octadecanoyloxy-tetrahydro-pyran-3-yl ester) was obtained. The obtained compound 7 was 81 g, and the yield was 85%.
¹ H-NMR (600 MHz; CDCl ₃ ) δ: 7.29 (2H, d, J = 8.54 Hz), 6.883 (2H, d, J = 8.54 Hz), 5.49 (1H, t, J = 9.77 Hz), 5.07 (1H, t, J = 9.77 Hz), 4.932 (1H, dd, J = 10.3, 3.66 Hz) 4.84 (1H, dd, J = 10.3, 3.66 Hz), 4.62 (2H, d, J = 5.86), 4.24 (1H, dd, J = 12.2, 4.88 Hz), 4.15-4.10 (1H, m), 4.02 (2H, t, J = 6.11 Hz), 3.99-3.94 (1H, m), 3.37 (3H, s), 2.58 (2H, t , J = 7.08 Hz), 2.32-2.09 (8H, m), 1.64-1.58 (6H, m), 1.26 (84H, s), 0.89 (9 , T, J = 7.08Hz)
¹³ C-NMR (150 MHz; CDCl ₃ ) δ: 172.9, 172.69, 172.53, 172.22, 158.37, 133.16, 128.53, 114.5, 96.73, 70. 67, 69.54, 68.12, 67.19, 66.56, 64.97, 61.89, 55.32, 51.32, 42.15, 34.10, 34.02, 33.98, 33.94, 31.83, 30.47, 29.61, 29.58, 29.49, 29.43, 29.40, 29.38, 29.27, 29.23, 29.17, 29. 1, 29.0, 28.9, 24.8, 24.7, 24.6, 24.5, 23.4, 22.6, 14.0

[Synthesis of Compound 5]
470 g (5 eq.) Of potassium carbonate, 500 ml of acetonitrile, 81.82 g of parahydroxybenzaldehyde (p-hydroxybenzaldehyde), and 144 ml (1.5 eq.) Of ethyl 4-bromobutyrate are mixed and heated to reflux for 24 hours while stirring with a mechanical stirrer. Went. The solution after the reaction was filtered, potassium carbonate on the filter paper was washed with 100 ml of acetonitrile, and the solution was concentrated. The resulting yellow oily substance was dissolved in 200 ml of methanol, and 39 g of sodium hydroxide previously dissolved in 300 ml of water was added. After reacting with stirring at room temperature for 1 hour, 80 ml of 12M hydrochloric acid was carefully and slowly added with ice cooling. Subsequently, the mixture was extracted 3 times with 100 ml of diethyl ether, washed, and dried with 50 g of anhydrous magnesium sulfate. Then, filtration concentration was implemented and the target compound 5 was obtained. The obtained compound 5 was 130 g, and the yield was 95%.

<Example 2>
[Bonding process and crystallization process]
[Synthesis of Carrier-Phe-Fmoc (Compound 8)]
Octadecanoic acid 6- [4- (4-hydroxymethyl-phenoxy) -butyryloxymethyl] -2-methoxy-4,5-bis, which is the carrier for crystallization separation of the present invention obtained in Example 1. -0.174 g (0.3 mmol) of octadecanoyloxy-tetrahydro-pyran-3-yl ester (compound 7) was dissolved in 20 ml of dichloromethane (DCM). To this, 0.387 g (0.45 mmol) of Fmoc-Phe-OH, 0.114 g (0.9 mmol) of diisopropylcarbodiimide (DIPCI) and 0.1 eq of dimethylaminopyridine (DMAP) were added, and the mixture was stirred at room temperature for 1 hour. Stirring was performed. Subsequently, after the solvent was distilled off, 30 ml of acetonitrile was slowly added with stirring to cause crystallization. The substance that was filtered off with suction and deposited on the filter paper was washed with 30 ml of acetonitrile to give octadecanoic acid 6- (4- {4- [2- (9H-fluoren-9-ylmethoxycarbonylamino) -3. -Phenyl-propionyloxymethyl] -phenoxy} -butyryloxymethyl) -2-methoxy-4,5-bis-octadecanoyloxy-tetrahydro-pyran-3-yl ester (Compound 8) was obtained. The yield was 98%.
¹ H-NMR (400 MHz; CDCl ₃ ) δ: 7.77 (2H, d, J = 7.3 Hz), 7.55 (2H, d, J = 7.3 Hz), 7.40 (2H, t, J = 7.3 Hz), 7.33-7.27 (3H, m), 7.24-7.20 (4H, m), 7.02-6.98 (2H, m), 6.85 ( 2H, d, J = 8.5 Hz), 5.51 (1H, dd, J = 10.0, 9.5 Hz), 5.27-5.10 (2H, m), 5.08 (1H, dd) , J = 10.0, 9.5 Hz), 4.93 (1H, d, J = 3.7 Hz), 4.88 (1H, dd, J = 10.0, 3.7 Hz), 4.71- 4.65 (1H, m), 4.4-4.3 (2H, m), 4.25 (1H, dd, J = 12.2, 2.7 Hz), 4.21-4.17 (1H , M), 4.13 ( H, dd, J = 12.2, 2.7 Hz), 4.00 (2H, t, J = 5.6 Hz), 3.97-3.95 (1H, m), 3.38 (3H, s ), 3.10 (2H, dd, J = 6.8, 6.3 Hz), 2.58 (2H, t, J = 6.8 Hz) 2.34-2.19 (6H, m), 2. 12 (2H, qui, J = 6.8 Hz), 1.25 (90 H. brs), 0.88 (9 H, t, J = 7.08 Hz)
¹³ C-NMR (150 MHz; CDCl ₃ ) δ: 172.9, 172.7, 172.6, 159.1, 155.5, 143.8, 143.5, 141.3, 135.6, 130. 5,129.4,128.5,127.7,127.2,127.0,125.1,119.9,114.5,96.9,70.7,69.6,68.1, 67.3, 67.1, 66.966.6, 61.9, 55.4, 54.8, 47.1, 38.14.1, 34.0, 340.3, 31.9, 30. 5, 29.7, 29.6, 29.61, 29.5, 29.4, 29.3, 29.28, 29.23, 29.14, 29.1, 29.0, 24.9, 24.522.7, 14.1

[Synthesis of Carrier-Val-Pro-Fmoc (Compound 10)]
To the dried eggplant flask, octadecanoic acid 6- [4- (4-hydroxymethyl-phenoxy) -butyryloxymethyl] -2-2, which is the carrier for crystallization separation of the present invention obtained in Example 1, was used. 593 mg (0.5 mmol) of methoxy-4,5-bis-octadecanoyloxy-tetrahydro-pyran-3-yl ester (compound 7) and 340 mg (2 eq) of Fmoc-Val-OH were added to dichloromethane (DCM). ) 15 ml was added and dissolved. Further, 154 μl (2 eq) of diisopropylcarbodiimide (DIPCI) and 6.1 mg (0.1 eq) of dimethylaminopyridine (DMAP) were added and stirred at room temperature for about 1 hour. After the completion of the reaction was confirmed by TLC (silica gel thin layer chromatography), 1 ml of a 5% DBU acetonitrile solution was added and stirred for 10 minutes. After the completion of the reaction was confirmed by TLC (silica gel thin layer chromatography), the solvent was distilled off, and 30 ml of acetonitrile was slowly added with stirring to cause crystallization. By suction filtration and washing the substance deposited on the filter paper with 30 ml of acetonitrile, the target product octadecanoic acid 2- {4- [4- (2-amino-3-methyl-butyryloxymethyl)] - phenoxy] - butyrate Lilo carboxymethyl} -6-methoxy-4,5-bis - octadecanoate acetoxyphenyl - tetrahydro - pyran-3-yl ester (carrier _-Val-NH 2) (compound 9). The yield was 92%.

Further, 462 mg (0.36 mmol) of the obtained carrier-Val-NH ₂ (compound 9), 154 μl ( ₂ eq) of diisopropylcarbodiimide (DIPCI) and 135 mg of HOBt (2 eq) were dissolved in 1 ml of dimethylformamide (DMF) in advance, A solution obtained by activating 337 mg (2 eq) of Fmoc-Pro-OH was dissolved in 15 ml of dichloromethane and stirred at room temperature for 1 hour. After the completion of the reaction was confirmed by TLC (silica gel thin layer chromatography), the solvent was distilled off, and 30 ml of acetonitrile was slowly added with stirring to cause crystallization. By suction filtration and washing the substance deposited on the filter paper with 30 ml of acetonitrile, the target carrier-Val-Pro-Fmoc, that is, octadecanoic acid 6-methoxy-2- [4- (4- {3-Methyl-2-[(pyrrolidine-2-carbonyl) -amino] -butyryloxymethyl} -phenoxy) -butyryloxymethyl] -4,5-bis-octadecanoyloxy-tetrahydro-pyran-3-yl An ester (compound 10) was obtained. The obtained compound 10 was 473 mg (0.30 mmol), and the yield was 59%.
¹ H-NMR (CDCl ₃ , 600 MHz) δ: 7.79-7.73 (4H, m), 7.43-7.35 (4H, m), 7.34-7.28 (2H, dd, J = 13.9, 7.0 Hz), 6.87-6.79 (2 H, m), 5.50 (1 H, t, J = 9.9 Hz), 5.08 (1 H, t, J = 9) 0.9 Hz), 4.93 (1 H, d, J = 3.6 Hz), 4.88 (1 H, dd, J = 9.9, 3.7 Hz), 4.53-4.33 (3 H, m) 4.24 (1H, dd, J = 12.1, 4.8 Hz), 4.13 (1H, dd, J = 12.1, 2.2 Hz), 4.07-3.88 (3H, m ), 3.87-3.79 (2H, m), 3.67-3.42 (2H, m), 3.38 (3H, s), 2.56 (2H, t, J = 6.2 Hz) ), 2.38-2. 8 (8H, m), 2.01-1.86 (2H, m), 1.64-1.47 (8H, m), 1.31-1.21 (84H, m), 1.14 ( 9H, d, J = 6.6 Hz), 0.88 (9H, t, J = 7.0 Hz)
¹³ C-NMR (CDCl ₃ , 150 MHz) δ: 172.9, 172.7, 172.6, 172.3, 158.9, 141.3, 130.1, 127.7, 127.1, 127. 0, 125.1, 120.0, 114.4, 96.9, 70.7, 69.6, 68.2, 67.3, 66.7, 66.6, 62.0, 55.4 47.2, 42.2, 34.2, 34.1, 34.0, 31.9, 30.5, 29.7, 29.6, 29.5, 29.3, 29.2, 29. 1, 25.0, 24.9, 24.8, 24.5, 24.4, 23.5, 22.7, 19.0, 17.5, 14.1.

[Synthesis of Carrier-D-Ala- (2-pyridyl) -D-Phe-4-chloro-NH ₂ (Compound 11)]
To the dried eggplant flask, octadecanoic acid 6- [4- (4-hydroxymethyl-phenoxy) -butyryloxymethyl] -2-2, which is the carrier for crystallization separation of the present invention obtained in Example 1, was used. 948 mg (0.8 mmol) of methoxy-4,5-bis-octadecanoyloxy-tetrahydro-pyran-3-yl ester (Compound 7), 373 mg of Fmoc- (2-pyridyl) -D-Ala-OH ( 1.2 eq) and dissolved in 12 ml dichloromethane (DCM). Furthermore, 460 mg (3 eq) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (1-Ethyl-3- (3-dimethylaminopropyl) carbohydrate), 6.1 mg of dimethylaminopyridine (DMAP) (0.1 eq) was added and stirred at room temperature for 1 hour. After the completion of the reaction was confirmed by TLC (silica gel thin layer chromatography), 15 ml of 5% DBU acetonitrile solution was added and stirred for 10 minutes. After the completion of the reaction was confirmed by TLC (silica gel thin layer chromatography), the solvent was distilled off, and 30 ml of acetonitrile was slowly added with stirring to cause crystallization. The target substance, carrier-D-Ala- (2-pyrylyl) -NH _2, was obtained by suction filtration and washing the substance deposited on the filter paper with 30 ml of acetonitrile. The obtained carrier-D-Ala- (2-pyridyl) -NH ₂ was 1150 mg (0.76 mmol), and the yield was 95%.

Further, 1150 mg (0.76 mmol) of the obtained carrier-D-Ala- (2-pyridyl) -NH ₂ , 8 ml of dimethylformamide (DMF) in advance, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydro 405 mg of Fmoc-4-chloro-D-Phe-OH in a mixed solution of 460 mg (3 eq) of chloride (1-Ethyl-3- (3-dimethylaminopropyl) carbohydrate) and 216 mg (2 eq) of HOBt .2 eq) The activated material was taken into a dried eggplant flask, dissolved in 12 ml of dichloromethane (DCM), and stirred at room temperature for 1 hour. After the completion of the reaction was confirmed by TLC (silica gel thin layer chromatography), 15 ml of 5% DBU acetonitrile solution was added and stirred for 1 hour. After the completion of the reaction was confirmed by TLC (silica gel thin layer chromatography), the solvent was distilled off, and 30 ml of acetonitrile was slowly added with stirring to cause crystallization. The target substance, carrier-D-Ala- (2-pyrylyl) -D-Phe-4-chloro-NH ₂ (compound 11), was obtained by suction filtration and washing the substance deposited on the filter paper with 30 ml of acetonitrile. Obtained. The obtained compound 11 was 1170 mg (0.67 mmol), and the yield was 84%.
¹ H-NMR (CDCl ₃ , 400 MHz) δ: 8.44-8.39 (1H, m), 8.27-8.21 (1H, m), 7.81-7.73 (2H, m) 7.57-7.50 (2H, m), 7.44-7.37 (2H, m), 7.35-7.30 (2H, m), 7.29-7.25 (3H, m), 7.30-7.18 (3H, m), 7.06-7.02 (2H, m), 6.89-6.84 (2H, m), 5.50 (1H, t, J = 9.5 Hz), 5.32-5.22 (1 H, m), 5.09 (1 H, t, J = 9.5 Hz), 5.06-5.02 (1 H, m), 4. 95-4.92 (1H, d, J = 4.2 Hz), 4.90-4.85 (1H, dd, J = 9.5, 4.2 Hz), 4.82-4.72 (1H, m), 4.48-4.40 (1 M), 4.40-4.30 (2H, m), 4.25 (1H, dd, J = 12.5, 4.2 Hz), 4.21-4.16 (1H, m), 4 .13 (1H, dd, J = 12.5, 4.2 Hz), 4.00 (2H, t, J = 6.4 Hz), 3.98-3.93 (1H, m), 3.38 ( 3H, s), 3.13-2.93 (4H, m), 2.58 (2H, t, J = 6.8 Hz), 2.38-2.17 (6H, m), 2.12 ( 2H, q, J = 6.8 Hz), 1.76-1.47 (6H, m), 1.33-1.21 (84H, m), 0.90-0.84 (9H, t, J = 7.1Hz)
¹³ C-NMR (CDCl ₃ , 150 MHz) δ: 174.6, 173.0, 172.3, 162.5, 161.9, 141.2, 140.6, 137.1, 136.3, 134. 8, 134.0, 132.5, 130.4, 128.6, 128.4, 128.1, 127.7, 127.6, 127.0, 127.1, 126.3, 125.8 125.0, 120.0, 119.9, 114.6, 96.9, 74.3, 74.1, 70.7, 69.6, 68.2, 67.3, 67.2, 66 7, 62.7, 55.4, 53.0, 47.0, 36.5, 34.2, 34.1, 34.0, 31.9, 31.4, 30.5, 29.7 29.3, 24.9, 24.8, 24.4, 22.7, 14.1.

<Example 3>
[Separation process]
Compound 8 synthesized in Example 2 (octadecanoic acid 6- (4- {4- [2- (9H-fluoren-9-ylmethoxycarbonylamino) -3-phenyl-propionyloxymethyl] -phenoxy}) -Butylyloxymethyl) -2-methoxy-4,5-bis-octadecanoyloxy-tetrahydro-pyran-3-yl ester) was added to 2 ml of dichloromethane (DCM) and 2 ml of trifluoroacetic acid. When stirred overnight, Fmoc-Phe-OH and compound 7 (octadecanoic acid 6- [4- (4-hydroxymethyl-phenoxy) -butyryloxymethyl] -2-methoxy-, which is a carrier for crystallization separation, were obtained. 4,5-bis-octadecanoyloxy-tetrahydro-pyran-3-yl ester). After distilling off dichloromethane (DCM) and trifluoroacetic acid, the residue was analyzed by ¹ H-NMR and confirmed to be a 1: 1 mixture of Fmoc-Phe-OH and compound 7.

<Example 4>
[Crystalling process due to temperature change]
A solution prepared by mixing 30 ml of cyclohexane and 30 ml of dimethylformamide (DMF) was heated to 50 ° C., and then 2-amino-3-methyl-butyric acid 3,4,5-tris-octadecyloxy- 0.1 mmol of benzyl ester was dissolved. To this, 20 ml of a dimethylformamide (DMF) solution containing 0.3 mmol of Fmoc-G1y-OBt and 0.5 mmol of diisopropylcarbodiimide (DIPCD) was added and stirred for 90 minutes. The reaction system was then cooled to 5 ° C. with stirring. After cooling, crystals were precipitated, the cyclohexane-dimethylformamide (DMF) two-phase separation solution was removed by suction filtration, the solid was washed 3 times with 20 ml of acetonitrile cooled to 5 ° C. or lower, and 2- [2- ( 9H-Fluoren-9-ylmethoxycarbonylamino) -acetylamino] -3-methyl-butyric acid 3,4,5-tris-octadecyloxybenzyl ester was obtained. The yield was 96%.
¹ H-NMR (400 MHz) δ: 7.77 (2H, d, J = 7.3 Hz), 7.59 (2H, d, J = 7.3 Hz), 7.40 (2H, t, J = 7) .3 Hz), 7.31 (2H, dt, J = 0.7, 7.3 Hz), 6.52 (2H, s), 6.38 (1H, d, J = 8.4 Hz), 5.44 −5.37 (1H, br), 5.10 (1H, d, J = 12.1 Hz), 5.02 (1H, d, J = 12.1 Hz), 4.62 (2H, dd, J = 8.4, 4.8 Hz), 4.42 (2H, d, J = 7.0 Hz), 4.24 (1H, t, J = 7.0 Hz), 3.96-3.92 (8H, m ), 2.21-2.16 (1H, m), 1.81-1.76 (4H, m), 1.75-1.70 (2H, m), 1.48-1.43 (6H) , M), 1.37-1. 1 (84H, br), 0.91 (3 days, d, J = 7.0 Hz), 0.88 (9H, t, J = 7 OHz), 0.86 (3H, d, J = 7. 0Hz)
¹³ C-NMR (150 MHz) δ: 171.5, 168.7, 156.5, 153.1, 143.6, 141.2, 138.3, 130.0, 127.7, 127.0, 125 0.0, 120.0, 107.0, 73.4, 69.2, 67.5, 67.4, 57.1, 47.1, 32.0, 31.4, 30.4, 29.8 29.7, 29.5, 29.4, 26.1, 22.8, 19.0, 17.7, 14.2; MALITOF-MS (pos) calcford C83H138N208 [M + Na] + 1314, found1314.

<Example 5>
[Crystalling process by changing solvent composition]
1 mmol of 2-amino-3-methyl-butyric acid 3,4,5-tris-octadecyloxy-benzyl ester was dissolved in 100 ml of dichloromethane (DCM). 50 ml of a dichloromethane (DCM) solution containing 3 mmol of Fmoc-Gly-OBt and 5 mmol of diisopropylcarbodiimide (DIPCD) was added thereto, and the mixture was stirred for 90 minutes. Next, the reaction system was concentrated to 5 ml at 30 ° C. with stirring, cooled, and 100 ml of acetonitrile was slowly added with stirring. When this solution was allowed to stand at 20 ° C., the product crystallized. The crystals were fractionated by suction filtration to give 2- [2- (9H-fluoren-9-ylmethoxycarbonylamino) -acetylamino] -3-methyl-butyric acid 3,4,5-tris-octadecyloxy. -The benzyl ester was obtained. The yield was 97%.

<Example 6>
[Synthesis of carrier for crystallization separation]
20 ml of dehydrated DMF was added to 0.9 g of methyl gallate, 10 g of 1-Br docosane, and 9 g of potassium carbonate, and the mixture was reacted at 80 ° C. for 10 hours in a nitrogen stream. After confirming the completion of the reaction by TLC, 20 ml of toluene and 10 ml of water were added, and the mixture was further stirred at 80 ° C. for 5 minutes. The toluene layer was separated and the solvent was distilled off under reduced pressure, and then 100 ml of methanol was added. The precipitated crystals were filtered with a Kiriyama funnel to obtain crude crystals. The crude crystals were recrystallized with 200 ml of hexane to obtain 5.2 g of Compound 13 (yield 96%).
¹ H-NMR (CDCl ₃ , 300 MHz) δ: 7.25 (2H, s), 4.00 (6H, dd, J = 6.6, 1.8 Hz), 3.88 (3H, s), 1 .89-1.69 (6H, m), 1.52-1.18 (114H, m), 0.88 (9H, t, J = 6.6 Hz)

Compound 13 (5.2 g) was dissolved in THF (60 ml) at 40 ° C., and 270 mg of lithium aluminum hydride was slowly added with stirring to react for 1 hour. After confirming the completion of the reaction by TLC, 30 ml of 1N HCl aqueous solution was added to separate the organic layer. The organic layer was further washed twice with 30 ml of 1N aqueous HCl, once with 30 ml of saturated aqueous sodium bicarbonate, twice with 30 ml of brine, and dried over magnesium sulfate. The solvent was distilled off under reduced pressure to obtain 4.8 g (yield 95%) of Compound 14.
¹ H-NMR (CDCl ₃ , 300 MHz) δ: 6.56 (2H, s), 4.58 (2H, d, J = 5.9 Hz), 3.97 (4H, t, J = 6.6 Hz) 3.93 (2H, t, J = 6.6 Hz), 1.85-1.66 (6H, m), 1.51-1.40 (6H, m), 1.38-1.15 ( 114H, m), 0.88 (9H, t, J = 6.6 Hz)

<Example 7>
[Bonding process and crystallization process]
Compound 145 mg (0.5 mmol), Fmoc-Phe-OH 290 mg (0.75 mmol), dimethylaminopyridine (DMAP) 6.5 mg (0.05 mmol) and dichloromethane 10 ml were dissolved in diisopropylcarbodiimide (DIPCI). 112 μl (0.75 mmol) was added and stirred for 2 hours. After confirming the completion of the reaction by TLC, 50 ml of methanol was added, and dichloromethane was distilled off at room temperature under reduced pressure. The crystals were suction filtered with a Kiriyama funnel, and the crystals were washed with 50 ml of methanol. Drying yielded 720 mg of compound 15 (99% yield).
¹ H-NMR (CDCl ₃ , 300 MHz) δ: 7.76 (2H, d, J = 7.52 Hz), 7.55 (2H, d, J = 7.34 Hz), 7.39 (2H, t, J = 7.52 Hz), 7.29 (2H, m), 7.21 (3H, m), 6.97 (2H, m), 6.51 (2H, m), 5.27 (1H, d) , J = 8.26 Hz), 5.09 (1H, d, J = 11.93 Hz), 5.00 (1H, d, J = 11.93 Hz), 4.70 (1H, m), 4.44 (1H, m), 4.33 (1H, m), 4.20 (1H, t, J = 7.52 Hz), 3.98-3.82 (6H, m), 3.11 (2H, m ), 1.83-1.69 (6H, m), 1.51-1.40 (6H, m), 1.37-1.18 (108H, m), 0.88 (9H, t, J = 6. Hz)

  The carrier for crystallization separation and the method for separating a compound of the present invention can promote research and development of pharmaceuticals and the like by synthesis of a compound library and can contribute to technological innovation in the biochemical industry and the chemical industry. . In addition, it can be an innovative technique in the separation and purification of biochemical substances, the search for drug candidate substances, the construction of new chemical synthesis reaction methods, continuous peptide synthesis methods, and the like.

Claims (12)

A support for crystallization separation, which is a compound that reversibly changes from a liquid phase state to a solid phase state with a change in solution composition and / or solution temperature,
Having reactive sites that bind to other compounds;
The reaction site has one or more atoms selected from a carbon atom, an oxygen atom, a sulfur atom, and a nitrogen atom,
A carrier for crystallization separation that binds to another compound via any one of a carbon atom, a nitrogen atom, a sulfur atom, and a nitrogen atom.   The carrier for crystallization separation according to claim 1, wherein the carrier for crystallization separation has a saccharide skeleton.   The carrier for crystallization separation according to claim 2, wherein the saccharide is pentose or hexose. The carrier for crystallization separation according to any one of claims 1 to 3, wherein the carrier for crystallization separation is represented by the following chemical formulas (A) to (F).

(Where
X is one or more atoms selected from carbon, oxygen, sulfur and nitrogen atoms,
R _{1 to} R ₂₇ may be the same or different, and may have a substituent, a C 1-60 hydrocarbon group, a C 1-60 acyl group that may have a substituent, And any one selected from the group of hydrogen,
And at least one of R _{1 to} R ₅ , at least one of R _{6 to} R ₁₀ , at least one of R _{11 to} R ₁₆ , at least one of R _{17 to} R ₂₂ , and R _{23 to} R ₂₇ , respectively. At least one and at least one of R _{28 to} R ₃₃ is a hydrocarbon group having 8 or more carbon atoms which may have a substituent, or an acyl having 8 or more carbon atoms which may have a substituent. Indicates a group. ) The carrier for crystallization separation according to claim 1, wherein the carrier for crystallization separation is represented by the following chemical formula (G).

(Where
X is a carbon, oxygen, sulfur or nitrogen atom,
Y is a reaction site having one or more atoms selected from carbon, oxygen, sulfur, and nitrogen atoms;
R ₃₄ is a hydrocarbon group having 1 to 60 carbon atoms,
n shows the integer of 1-5. ) A method for separating compounds comprising:
A dissolution step of preparing the carrier solution by dissolving the carrier for crystallization separation according to any one of claims 1 to 5 in a soluble solvent;
A binding step of binding another compound to the reaction site of the crystallization separation carrier;
A crystallization step of crystallizing the carrier to which the other compound is bound;
A method for separating a compound comprising   The method for separating a compound according to claim 6, further comprising a separation step of separating another compound from the crystallized carrier after the crystallization step.   The method for separating a compound according to claim 6 or 7, further comprising an impurity removal step of removing impurities from the solution before the crystallization step.   The method for separating a compound according to any one of claims 6 to 8, wherein in the crystallization step, the carrier is precipitated by means for changing the solution composition and / or means for changing the solution temperature.   The method for separating a compound according to claim 9, wherein the means for changing the solution composition comprises adding a solvent having a high affinity for the soluble solvent.   The method for separating a compound according to claim 9, wherein the means for changing the solution composition concentrates the soluble solvent.   The method for separating a compound according to claim 9, wherein the means for changing the solution temperature cools the solution.