JP6267009B2 - Separating agent for optical isomers - Google Patents

Description

  The present invention relates to a separating agent for optical isomers, and more particularly to a separating agent formed by supporting a protein on a carrier.

Chromatographic packing materials formed by supporting a protein on a carrier are known. For example, it is known that avidin or ovomucoid is used as a protein and is directly supported on a carrier or the like by chemical bonding through a carbon chain (see Patent Document 1).
In addition, a separating agent for optical isomers comprising a stationary phase obtained by supporting a bovine serum albumin (BSA) fragment or a BSA fragment having a partially modified molecular structure on a carrier is known (see Patent Document 2).
In addition to these techniques, optical isomer separation agents using acidic glycoproteins are also known.
α ₁ -acid glycoprotein (AGP) is known to have affinity with many drugs, and molecular pharmacological studies on the interaction between drugs and AGP have been conducted (non-patent literature). 1). In addition to AGP, molecular pharmacological studies on human serum albumin have also been conducted (see Non-Patent Document 1).
As specific acidic glycoproteins, proteins such as human α ₁ -acid glycoprotein (h-AGP) and chicken AGP (c-AGP) are known.
Regarding human α ₁ -acid glycoprotein (h-AGP), research on the elucidation of its structure has also been conducted (see Non-Patent Document 2). In addition, chicken α ₁ -acid glycoprotein (c-A
With regard to GP), research has been conducted to identify a portion that causes asymmetric recognition (see Non-Patent Document 3).

JP-A-5-87790 JP-A-8-67640

YAKUGAKU ZASSHI 129 (4) 413-425 2009 Biochem. & Biophys. Res. Comm. 300 41-46 2003 Biochem. & Biophys. Res. Comm. 295 587-590 2002 Comprehensive Chirality 8 153-176

Conventionally known human α ₁ -acid glycoprotein (h-AGP), chicken AGP (
Regarding the separation agent for optical isomers in which an α ₁ -acid glycoprotein such as c-AGP) is supported on a carrier, there is further room for improvement in separation performance.
Accordingly, an object of the present invention is to provide a separating agent for optical isomers in which α ₁ -acid glycoprotein is supported on a carrier, with further improved separation performance.

The present inventor paid attention to the particle size of a carrier for carrying α ₁ -acid glycoprotein or human serum albumin (hereinafter also referred to as HSA).
When a carrier having a particle size of 3 μm or less is used as a carrier for supporting these proteins, the optical resolution of a specific compound is superior to that of a separating agent that has been conventionally supported on a carrier. As a result, the present invention shown below was completed.
<1> A separation agent for optical isomers formed from a carrier and a protein supported on the carrier by chemical bonding, wherein the protein is α ₁ -acid glycoprotein or human serum albumin, and the carrier particles A separating agent for optical isomers having a diameter of 3 μm or less.
<2> The separating agent for optical isomers according to <1>, wherein the carrier has a particle size of 2.5 μm or less. <3> The α ₁ -acid glycoprotein is human α ₁ -acid glycoprotein or chicken α ₁
-The separation agent for optical isomers according to <1> or <2>, which is an acidic glycoprotein and the carrier is silica gel.
<4> The separating agent for optical isomers according to <3>, wherein the carrier is a surface-treated silica gel.
<5> The optical isomer separating agent according to <4>, wherein the surface treatment is by introduction of an aminoalkylsilyl group, a glycerylalkylsilyl group, or a glycidylalkylsilyl group.

  The separating agent for optical isomers of the present invention is formed by supporting a specific protein on a particle having a smaller particle size than that of a conventional carrier. According to the present invention, a separating agent for optical isomers having improved separation characteristics of a specific optical isomer is provided.

When optical isomers are separated in columns 1 to 3, when a 5 μm carrier is used (column 3), and Rs obtained with each particle size (columns 1 and 2) is used. It is a figure which shows the change rate of the obtained resolution (Rs). When optical isomers are separated in columns 4 to 6, when 5 μm is used as the carrier (column 6), and Rs obtained with each particle size (columns 4 and 5) is used. It is a figure which shows the change rate of obtained Rs. When optical isomers are separated in columns 7 to 9, when 5 μm is used as the carrier (column 9), and Rs obtained with each particle size (columns 7 and 8) is used. It is a figure which shows the change rate of obtained Rs. It is a figure which shows the chromatogram obtained by isolate | separating an optical isomer using the columns 2 and 3 with which c-AGP was carry | supported as protein. (A) A chromatogram obtained by separating Ketoprofen. (B) A chromatogram obtained by separating propranolol. It is a figure which shows the chromatogram obtained by isolate | separating an optical isomer using the columns 4 and 6 with which h-AGP was carry | supported as protein. (A) A chromatogram obtained by separating Ethotoin. (B) A chromatogram obtained by separating propranolol. It is a figure which shows the chromatogram obtained by isolate | separating an optical isomer using the columns 7 and 8 with which human serum albumin was carry | supported as protein. (A) A chromatogram obtained by separating Oxazepam. (B) A chromatogram obtained by separating Warfarin.

<Protein supported on separation agent of the present invention>
The separating agent for optical isomers of the present invention is formed by supporting α ₁ -acid glycoprotein or human serum albumin by chemical bonding on a carrier.

In the present invention, α ₁ -acid glycoprotein can be used as the protein. Examples of the origin of α ₁ -acid glycoprotein include mammals such as humans, cows, and rabbits, and birds such as chickens, swans, and turkeys. Among them, specific examples of preferable α ₁ -acid glycoprotein include human α ₁ -acid glycoprotein (hereinafter also simply referred to as h-AGP) and chicken AGP (hereinafter also simply referred to as c-AGP). Can do.
The specific structure of human α ₁ -acid glycoprotein (h-AGP) is described in Non-Patent Document 2 described above.
Human α ₁ -acid glycoprotein (h-AGP) used in the present invention is a commercially available product (eg, Sigma).
a-Aldrich), and may be purified by high performance liquid chromatography as necessary.

The chicken α ₁ -acid glycoprotein (c-AGP) used in the present invention is obtained by subjecting a crude chicken ovomucoid to liquid chromatography using a cation exchange carrier (for example, SP-Sepharose), and an ammonium acetate buffer (pH 4.6). It is obtained by separating ovomucoid and chicken α ₁ -acid glycoprotein (c-AGP) using a stepwise elution method.
Further, a fraction containing c-AGP may be collected and purified using a carrier for ion exchange chromatography such as SP-Sepharose.

The above human α ₁ -acid glycoprotein is a glycoprotein having a molecular weight of about 41000 to 43000, consisting of 183 amino acid residues and 5 sugar chains. Chicken α ₁ -acid glycoprotein has the same amino acid residues and sugar chains as human α ₁ -acid glycoprotein but has a molecular weight of about 30,000.

  A commercially available product can be used as the human serum albumin used in the present invention. Human serum albumin is a simple protein consisting of 585 amino acid residues and having a molecular weight of about 66500. When using human serum albumin, purification may be performed as necessary. The properties of human serum albumin are described in detail in Non-Patent Document 1.

<Carrier>
As the carrier used for the separation agent for optical isomers of the present invention, a carrier accommodated in a column tube and having chemical and physical durability in separation can be used. As such a carrier, a known carrier can be used, for example, an inorganic carrier such as silica gel, alumina, magnesia, glass, kaolin, titanium oxide, silicate, and hydroxyapatite, and polystyrene, polyacrylamide, And organic carriers such as polyacrylate.
The carrier is preferably porous from the viewpoint of increasing the separation ability for the target product. The carrier may be in the form of particles, or may be an integral carrier that is integrally accommodated in the column tube, but is in the form of particles from the viewpoint of the production of the separating agent and the ease of handling at that time. It is preferable. A specific example of such a carrier is silica gel.

The particle size of the carrier used for the optical isomer separating agent of the present invention is 3 μm or less. By supporting the above protein on a carrier having a particle size of 3 μm, the separation characteristics are remarkably improved depending on the type of optical isomers compared to those of a carrier having a particle size of 5 μm, which has been conventionally used.
When the particle size of the carrier is 2.5 μm or less, the improvement in separation characteristics becomes more remarkable. In particular, depending on the compound to be separated, it is obtained when the carrier is changed from 3 μm to 2.5 μm or less compared to the improvement in separation characteristics obtained when the particle size of the carrier is changed from 5 μm to 3 μm. In some cases, the improvement in separation characteristics obtained becomes more remarkable.
In addition, the minimum of the particle size of the support | carrier used by this invention is 1.0 micrometer or more normally.

The average pore size of the carrier that can be used in the present invention is 10 to 2,000 cm, preferably 50 to 1,000 mm.
This average pore diameter can be measured by a gas adsorption method. When using a commercially available carrier, the average pore diameter is a catalog value.

The particle diameter of the carrier used in the present invention refers to the median diameter (D50), and the measurement was performed with an average particle diameter D ₅₀ : a laser diffraction scattering type particle distribution measuring device (for example, model name: HORIBA LA-950). The median value of particle distribution (diameter) is measured three times, and this average value can be calculated.
The carrier having the above particle size can be obtained by purchasing a commercially available product, and the particle size of the commercially available carrier is a catalog value.

<Surface treatment of carrier>
The separating agent for optical isomers of the present invention is formed by supporting the protein on a carrier, and the carrier is preferably subjected to a surface treatment in advance in order to support the protein.
When silica gel is used as the carrier, a method for introducing a glycerylalkylsilyl group, an aminoalkylsilyl group, or a glycidylalkyl group can be used as the surface treatment.
In any of the above silyl groups, examples of the alkyl group include those having about 1 to 6 carbon atoms, and those that are propyl groups can be preferably used.
When a glycerylalkylsilyl group is introduced into a carrier, a glycerylalkylsilyl group such as a glycerylpropylsilyl group can be introduced into the carrier, for example, by the method described in JP-A-61-65159.
When the aminoalkylsilyl group is introduced into the carrier, the aminoalkylsilyl group can be introduced, for example, by reacting the aminoalkylalkoxysilane and the carrier by a known method. As the aminoalkylalkoxysilane, for example, a silane coupling agent having an amino group such as aminopropyltriethoxysilane can be used and reacted with a carrier such as silica gel by a known reaction method.
In the case of introducing a glycidylalkylsilyl group into the carrier, examples of the glycidylalkylalkoxysilane include a silane coupling agent having a glycidyl group such as 3-glycidylpropyltriethoxysilane. By reacting by a reaction method, a glycidylalkylsilyl group can be introduced into the carrier.

For example, the following method can be used to chemically bond the surface-treated carrier and the protein. The following method is described in detail in Non-Patent Document 4 above.
When reacting the amino group of a protein with a carrier, a carrier having a glycerylalkylsilyl group introduced as the carrier is used, and 1,1′-carbonyldiimidazole is reacted with the hydroxyl group of the glyceryl group, and then the protein An embodiment in which an amino group and a group derived from 1,1′-carbonyldiimidazole are reacted and bonded can be exemplified.
In addition, a carrier having an aminoalkylsilyl group introduced is used as a carrier and dicarbonate (N--
An example is a mode in which a group derived from di (N-succinimidyl) carbonate is reacted with a protein amino group and reacted with succinimidyl).
N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide is first reacted with the above protein, then the obtained reaction product is reacted with N-hydroxysuccinimide, and this is converted to an aminoalkylsilyl group. An embodiment in which the reaction is caused to react with the introduced carrier to bind thereto can be mentioned.
In addition, when reacting a protein thiol group with a carrier, a carrier into which an aminoalkylsilyl group has been introduced is used, and the amino group of this carrier and N-[(4-maleimidomethyl) cyclohexylcarbonyl Examples include a mode in which a group derived from N-[(4-maleimidomethyl) cyclohexylcarbonyloxy] succinimide is introduced by reacting with oxy] succinimide, and this is reacted with a thiol group of a protein to bind.

In the separating agent for optical isomers of the present invention, the amount of protein supported on the carrier is not particularly limited, but from the viewpoint of immobilization efficiency and steric hindrance, the weight ratio of 0.01 is 0.01 when the weight of the carrier is 1. It is about -0.5, Preferably it is about 0.03-0.2 by weight ratio.

After chemically binding the protein and the carrier, an operation of inactivating a functional group (for example, amino group or glycidyl group) remaining on the carrier may be performed as necessary.
For example, when using a carrier having an aminoalkylsilyl group introduced, the amino group can be blocked by reacting the amino group remaining on the carrier with glucosamine. Thereby, the fall of the separation performance by the remaining functional group can be suppressed. Functional groups other than amino groups can also be inactivated by known methods.

The separating agent for optical isomers of the present invention is one in which h-AGP is supported as the above-mentioned protein, such as neutral compounds (Benzoin, Ethotoin, etc.), acidic compounds (2-phenyl- n-butyric acid, 2-Phenoxypropionic acid, Warfarin, and the like) and basic compounds (Alprenolol, Oxprenolol, Propranolol, Bupivacaine, and the like) are excellent in separation characteristics.
Those carrying c-AGP as the above protein are particularly excellent in separation characteristics of acidic compounds (Ibuprofen, Ketoprofen) and basic compounds (Chlorophenylamine, Tolperisone, Alprenolol, Propranolol, Oxprenolol).
As the above-mentioned proteins, those loaded with human serum albumin have excellent separation characteristics for acidic compounds such as Oxazepam, Ibuprofen, Flurbiprofen and Warfarin, and neutral compounds such as Benzoin.

In the present invention, with regard to h-AGP, c-AGP, and human serum albumin that have been conventionally used as proteins to be carried on a separating agent for optical isomers, the particle diameter of the carrier on which they are carried is focused, It was found that the degree of separation was significantly improved with respect to a specific optical isomer only by devising that a material having a size of 3 μm or less was used.
In particular, it was found that when the particle size was further reduced, the separation characteristics were remarkably improved depending on the type of the separation target compound of the protein to be carried.

  Examples The present invention will be described more specifically with reference to examples. However, the present invention is not limited to the description of the following examples unless it exceeds the gist.

Preparation of Silica Particle Filler Immobilizing c-AGP <Example 1>
Chicken α ₁ -acid glycoprotein isolated from crude ovomucoid by activating silica gel with aminopropylsilyl group with a particle size of 3 μm activated with N, N'-disuccinimidyl carbonate
c-AGP) was carried out in 20 mM phosphate buffer (pH 6.6) at 4 ° C. for 20 hours. After washing, it was further reacted with glucosamine at room temperature for 1 hour and washed with water and methanol to obtain the filler 1 of the present invention. The obtained packing material 1 was packed into a stainless steel column having an inner diameter of 2.0 mm and a length of 100 mm to obtain a column 1.

<Example 2>
A filler 2 of the present invention was obtained by the same procedure as in Example 1 except that silica gel having a particle size of 2.1 μm was used. In the same manner as in Example 1, the obtained packing material 2 was packed into a stainless steel column having an inner diameter of 2.0 mm and a length of 100 mm to obtain a column 2.

<Comparative Example 1>
A filler 3 was obtained by the same procedure as in Example 1 except that silica gel having a particle size of 5 μm was used. In the same manner as in Example 1, the obtained packing material 3 was packed into a stainless steel column having an inner diameter of 2.0 mm and a length of 100 mm to obtain a column 3.

Production of Silica Particle Filler Immobilized with h-AGP <Example 3>
A filler 4 was obtained by the same material and procedure as in Example 1 except that human α ₁ -acid glycoprotein (h-AGP) obtained from Sigma-Aldrich was used as the protein to be supported on the carrier. In the same manner as in Example 1, the obtained packing material 4 was packed into a stainless steel column having an inner diameter of 2.0 mm and a length of 100 mm to obtain a column 4.
<Example 4>
A filler 5 of the present invention was obtained by the same procedure as in Example 3 except that silica gel having a particle size of 2.1 μm was used. In the same manner as in Example 1, the obtained packing material 5 was packed into a stainless steel column having an inner diameter of 2.0 mm and a length of 100 mm to obtain a column 5.

<Comparative example 2>
A filler 6 was obtained by the same procedure as in Example 3 except that silica gel having a particle size of 5 μm was used. In the same manner as in Example 3, the obtained packing material 6 was packed into a stainless steel column having an inner diameter of 2.0 mm and a length of 100 mm to obtain a column 6.

Production of Silica Particle Filler Immobilized with Human Serum Albumin <Example 5>
A filler 7 was obtained by the same material and procedure as in Example 2 except that human serum albumin was used as the protein. In the same manner as in Example 2, the obtained packing material 7 was packed into a stainless steel column having an inner diameter of 2.0 mm and a length of 100 mm to obtain a column 7.

<Example 6>
A filler 8 was obtained by the same material and procedure as in Example 1 except that human serum albumin was used as the protein. In the same manner as in Example 1, the obtained packing material 8 was packed into a stainless steel column having an inner diameter of 2.0 mm and a length of 100 mm to obtain a column 8.

<Comparative Example 3>
A filler 9 was obtained by the same material and procedure as in Example 5 except that a carrier having a particle size of 5 μm was used. In the same manner as in Example 5, the obtained filler 9 was packed into a stainless steel column having an inner diameter of 2.0 mm and a length of 100 mm to obtain a column 9.

<Separation performance confirmation test>
The separation test of optical isomers was performed using columns 1 to 3.
The analysis was performed by high performance liquid chromatography using a mixture of 20 mM phosphate buffer (pH 3.0 to 6.8) and ethanol or acetonitrile as a mobile phase, and the retention coefficients of various optical isomers shown in Table 1. (K ₁ ), separation factor (α), and degree of separation (Rs) were determined. The flow rate was 0.2 mL / min, and detection was performed at 210 nm. The results are shown in Table 1 and FIG. Moreover, the separation result (chromatogram) of some compounds is shown in FIG.

From the results shown in Table 1, in the separation agent for optical isomers supported with C-AGP as a protein, the degree of separation (Rs) particularly for acidic compounds and basic compounds decreases as the particle size of the carrier becomes smaller than 3 μm. Many compounds were improved. Among acidic compounds and basic compounds, some compounds with a particle size of 2.1 μm showed a significant increase in Rs compared to those with a particle size of 5 μm and 3 μm.

The separation test of optical isomers was performed using columns 4-6.
The analysis was carried out using the mobile phases shown in Table 2 (eluent A: 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (pH 5.1), eluent B: 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (pH 5.1) / 2-propanol = 96: 4 (v / v)) using a high-performance liquid chromatography (elution rate 0.2 mL / min; column temperature, 25 ° C; detection wavelength, 210 nm). Retention of various optical isomers shown in Table 2 A coefficient (k ₁ ), a separation coefficient (α), and a degree of separation (Rs) were obtained. The results are shown in Table 2 and FIG. Moreover, the separation results (chromatogram) of some compounds are shown in FIG.

From the results shown in Table 2, in the separation agent for optical isomers using h-AGP as the protein, when the carrier particle size is 3 μm or less, the neutral compounds and acidic compounds shown in Table 2 and FIG. In addition, many of the basic compounds had a significantly improved resolution (Rs). Looking at the degree of separation between the acidic compound and the neutral compound, the increase in Rs was remarkable when the particle size was 2.1 μm compared to when the particle size was 5 μm and 3 μm.

Separation tests of optical isomers were performed using columns 7-9.
The analysis was carried out by changing the mobile phase for each target substance, and using high performance liquid chromatography (elution rate 0.2 mL / min; column temperature, 25 ° C .; detection wavelength, 210 nm). ₁ ), the separation factor (α) and the degree of separation (Rs) were determined. The results are shown in Table 3 and FIG. FIG. 6 is a diagram (chromatogram) showing an example of the separation result.
The mobile phase used for the analysis is as follows.
Oxazepam: 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (pH 7.5) / 1-propanol. (96: 4, v / v)
Ibuprofen: 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (pH 6.6) / 1-propanol. (85:15, v / v) containing 4 mM octanoic acid
Flurbiprofen: 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (pH 5.1) / 1-propanol (85:15, v / v) containing 4 mM octanoic acid
Ketoprofen, Warfarin: 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (pH 5.1) / 1-propanol. (94: 6, v / v)
Benzoin: 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (pH 7.5) / 1-propanol. (98: 2, v / v)

  From the results shown in Table 3, in the separation agent for optical isomers using human serum albumin as the protein, when the carrier particle size is 3 μm or less, the neutral compound and acidic compound shown in Table 3 are separated. It was found that the degree (Rs) was remarkably improved.

  The separating agent for optical isomers of the present invention is expected to be very useful for separation of various optical isomers that have been difficult to separate up to now, particularly compounds acting on living bodies, specifically, drugs. From this, the column having the separation agent for optical isomers of the present invention not only discovers and improves new separation conditions for bioactive substances in the future, but also improves the convenience of identification and analysis of separated bioactive substances. There is expected.

Claims (4)

A separation agent for optical isomers formed from a carrier and a protein carried by chemical bonding to the carrier, wherein the protein is α ₁ -acid glycoprotein or human serum albumin, and the particle size of the carrier is Separating agent for optical isomers, which is 2.5 μm or less. The optical isomer separating agent according to claim 1, wherein the α ₁ -acid glycoprotein is human α ₁ -acid glycoprotein or chicken α ₁ -acid glycoprotein, and the carrier is silica gel. The separating agent for optical isomers according to claim 2 , wherein the carrier is a surface-treated silica gel. The separating agent for optical isomers according to claim 3 , wherein the surface treatment is performed by introducing an aminoalkylsilyl group, a glycerylalkylsilyl group, or a glycidylalkylsilyl group.