JP2019137635A - Dendrimer-using standard substance and method for calibrating analyzer using same - Google Patents

Abstract

To provide: a novel standard substance that is advantageous in terms of a long-term storage stability and a cost; and a method for calibrating analyzer using the same.SOLUTION: A standard substance of the present invention comprises a dendrimer having at least one selected from a phenylazomethine skeleton and a carbazole skeleton as a structural unit of a dendritic branch structure. Unlike a normal polymer, this dendrimer has a clear structure and a very small molecular weight distribution, and the standard substance of the present invention can be used for calibration or the like of an analyzer such as a mass spectrometry or a high performance liquid chromatography, etc.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a standard substance and a method for calibrating an analyzer using the standard substance.

  In general, analytical devices such as mass spectrometers do not have a constant response capability of the detector, and the response capability of the detector of the analytical device varies over time. Line creation is required.

  Conventionally, mass spectrometers have high molecular weight, are monodispersed, and are easily available, so proteins and peptides are commercially available as standard substances and have been used for calibration. However, these are expensive in terms of purification and the like, and they are easily decomposed by a chemical action and have a problem in storage stability.

  In addition, although it has been proposed to use the synthesized dendrimer for proofreading (Patent Document 1), since the structural unit of the dendritic branched structure has an ester bond, it is easily hydrolyzed and has long-term storage stability. There is room for further improvement.

  In gel permeation chromatography (GPC), polystyrene is used as a standard material for calibration. However, it is expensive because special purification is required to prepare a sample without dispersion.

Special table 2012-517000 gazette

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a new standard substance advantageous in terms of long-term storage stability and cost, and a method for calibrating an analyzer using the same. .

  In order to solve the above problems, according to the present invention, there is provided a standard substance composed of a dendrimer having at least one selected from a phenylazomethine skeleton and a carbazole skeleton as a constituent unit of a dendritic branched structure.

  This standard substance is preferably used for mass spectrometry. Moreover, it is preferably used for high performance liquid chromatography.

  The method of the present invention is characterized in that the analyzer is calibrated using the standard substance. In a preferred aspect of this method, a plurality of dendrimers of different generations are used to create a calibration curve for the value to be measured by the analyzer based on the measurement results of the plurality of dendrimers.

  A dendrimer having at least one selected from a phenylazomethine skeleton and a carbazole skeleton as a constitutional unit of a dendritic branched structure has a clear structure and a very small molecular weight distribution unlike ordinary polymers, and the standard material and method of the present invention According to this, it is possible to calibrate analyzers such as mass spectrometry and high performance liquid chromatography.

  In addition, the dendritic structure of the phenylazomethine skeleton or carbazole skeleton is excellent in long-term storage stability because chemical decomposition such as hydrolysis does not easily occur, and is advantageous in terms of cost compared to conventional products.

  According to the method of the present invention, each dendrimer having a different number of generations is suitable as a multi-point calibration standard because it exhibits a discrete molecular weight distribution.

2 is an MS spectrum of human Insulin measured by calibrating with the standard substance of Example 1. 2 is an MS spectrum of human Insulin measured by calibrating with the standard substance of Example 2. 3 is an MS spectrum of bovine Ubiquitin measured by calibrating with the standard substance of Example 3. It is a logarithmic plot of the retention time and molecular weight which measured the reference material of Example 4 by SEC.

  The present invention is described in detail below.

  The standard substance of the present invention comprises a dendrimer having at least one selected from a phenylazomethine skeleton and a carbazole skeleton as a constitutional unit of a dendritic structure.

  A dendrimer is a dendritic polymer having a structure that is regularly branched from the center, and is composed of a central molecule as a core and a dendron as a side chain portion. The number of branches in the dendron portion is also called a generation. A portion branched from the central molecule of the dendrimer by one step is called a first generation, and a portion branched by two steps is called a second generation. In general, dendrimers are regular and completely dendritic polymers from the core, have a spherical structure with a sparse and dense surface near the center, and the number of generations increases with each branch from the center. . In contrast, a hyperbranched polymer is a polymer having an incomplete dendritic branch, unlike a dendrimer having a complete dendritic structure. In the present invention, the dendrimer may be a hyperbranched polymer having such partial branch unit defects.

  The dendrimer can be produced by a divergent method, a convergent method, or the like. In the divergent method, a molecule having a plurality of functional groups is used as a core, and branches are extended from the center toward the outside. The convergent method is a method in which branches are extended from the outside to the inside, and finally bonded to the core to form a spherical polymer. The synthesis of dendron proceeds from the outer shell of the dendrimer toward the inside. Finally, bind some dendron to the core.

  As a dendrimer used for this invention, the compound represented by following formula (1) is mentioned, for example.

A in the above formula (1) is a core molecular group that becomes the core of the dendrimer, and the dendrimer molecule has a unit represented by B in the formula (1) toward the outside centering on this core molecular group. Has a chain. As a result, the dendrimer molecule has a structure in which the B is linked and centered on the A to grow radially. The number of times B is linked and reaches C is called a “generation”. The generation adjacent to the core molecular group A is the first generation, and the number of generations increases toward the outside. B directly bonded to the core molecular group A is the first generation, and C forms a bond to B as a terminal group and is positioned at the end of the radially extended structure of the dendrimer molecule. A in the formula (1) is an organic group R ¹ (R ^1- (N...) _R is bonded to B as an r-valent residue), and the organic group R ¹ is not particularly limited. Are monocyclic or polycyclic aromatic groups, heteroaromatic groups, porphyrin groups, phthalocyanine groups, cyclam groups and the like, and these may have a substituent. In the above formula (1), p represents the number of generations of B, q represents the number of end groups C of the dendrimer, and q = 2 ^pr . The formula R ¹ - In (N ...) core portion represented by _r, as the r, is not particularly limited, for example, be mentioned an integer of 1 to 4. Moreover, although p in the said Formula (1) is not specifically limited, It is preferable that it is 0-6.

  B and C in the above formula (1) are represented by the structure of the following formula (2-1) or formula (2-2).

In B in the formula (1), any one of R ^{a1 to} R ^a5 and any one of R ^{a6 to} R ^a10 are the next generation formulas (2-1) and (2-2) The part couple | bonded with the nitrogen atom N in the structure of) is shown. In preferred embodiments, the moieties are para positions R ^a3 and R ^a8 , or meta positions R ^a2 and R ^a9 . In B in the above formula (1), any one of R ^{b1 to} R ^b4 and any one of R ^{b5 to} R ^b8 are the next generation formulas (2-1), (2-2 The part couple | bonded with the nitrogen atom N of the structure of) is shown. In preferred embodiments, the moieties are para positions R ^b3 and R ^b6 . In B in the above formula (1), R ^{a1 to} R ^a10 and R ^{b1 to} R ^b8 other than the portion each independently represent a hydrogen atom or a substituent, and the substituent is not particularly limited. Halogen atoms such as chlorine atom, bromine atom and fluorine atom, alkyl groups such as methyl group and ethyl group, haloalkyl groups such as chloromethyl group and trifluoromethyl group, alkoxy groups such as methoxy group and ethoxy group, methoxyethyl group And alkoxyalkyl groups such as an alkylthio group, a carbonyl group, a cyano group, an amino group, a nitro group, and an aromatic group that may have these as a substituent. Among these, a hydrogen atom is preferable.

In C in the above formula (1), R ^{a1 to} R ^a10 and R ^{b1 to} R ^b8 each independently represent a hydrogen atom or a substituent, and the substituent is not particularly limited. Bromine atom, halogen atom such as fluorine atom, alkyl group such as methyl group and ethyl group, haloalkyl group such as chloromethyl group and trifluoromethyl group, alkoxy group such as methoxy group and ethoxy group, alkoxyalkyl such as methoxyethyl group Group, an alkylthio group, a carbonyl group, a cyano group, an amino group, a nitro group, and an aromatic group which may have these as a substituent.

  The basic structure of a typical example of a dendrimer used as a standard substance is shown below. The following shows the structure of a dendron (one branch) (n and m are integers greater than or equal to 0), and it becomes a dendrimer by binding it to a central molecule called the core.

  Examples of the phenylazomethine dendrimer having phenylazomethine as a structural unit of a dendritic branch structure include a para-position linking type of the following formula (I-1) and a meta-position linking type of the following formula (I-2). Further, examples include a structure in which a meta-position-linked structure and a para-position-linked structure are arranged in the outer layer and the inner layer, and a structure in which these are alternately connected.

Examples of the carbazole dendrimer having carbazole as a constituent unit of a dendritic branched structure include a para-position linked type of the following formula (II).

Furthermore, a structure in which phenylazomethine and carbazole are combined, for example, a carbazole / phenylazomethine dendrimer in which the inner layer is phenylazomethine and the outer layer is carbazole as shown in the following formula (III) may be used.

The dendrimer used in the present invention has at least one selected from a phenylazomethine skeleton and a carbazole skeleton as a constitutional unit of a dendritic branch structure, so that chemical decomposition such as hydrolysis hardly occurs, so long-term storage stability Excellent. For example, carbazole dendrimers are stable for over a year when stored at room temperature in the atmosphere.

  The dendrimer used in the present invention is more advantageous than conventional products in terms of cost. Although it is influenced by the amount used as a standard substance, in an example, it is about 1/100 of commercially available proteins and peptides.

  The dendrimer used in the present invention has high purity, which can be confirmed by mass spectrum and NMR data.

  The standard substance of the present invention can be used for calibration of an analyzer in various chemical analyzes for measuring the molecular weight, content, etc. of a substance to be analyzed.

  The substance to be analyzed is not particularly limited, and examples thereof include biomolecules (for example, proteins, peptides, sugars, oligonucleotides, etc.), large organic molecules (for example, polymers and other macromolecules), and the like.

  Examples of the chemical analysis include mass spectrometry and chromatography.

  Mass spectrometry (MS) is a technique for ionizing a sample to generate charged molecules (and fragments thereof) and measuring the mass-to-charge ratio. One of these is time-of-flight mass spectrometry (TOF-MS), which measures the mass of an object by measuring the time of flight of the accelerated ions, and the ions are accelerated by an electric field with a known electric field strength. Thus, kinetic energy proportional to the electric charge is obtained. The mass-to-charge ratio can be measured by measuring the time for which the accelerated ions fly a certain distance.

  Examples of ionization techniques used in mass spectrometry include substrate-assisted laser desorption ionization (MALDI), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), fast atom bombardment (FAB), and the like.

  As a mass spectrometer, MALDI-TOF-MS, ESI-TOF-MS, etc. can be used, for example. In the mass spectrometer, when analyzing a substance having a large molecular weight such as a biomolecule (eg, protein, peptide, sugar, oligonucleotide, etc.) or a large organic molecule (eg, polymer or other polymer), MALDI- TOF-MS is preferably used.

  Further, the analyzer may be a combination of liquid chromatography, gas chromatography, supercritical fluid chromatography, electrophoresis, inductively coupled plasma method and the like and mass spectrometry.

In calibration with a standard substance, it is a precondition that the molecular weight has monodispersity. However, the present inventors have prepared a zinc porphyrin core carbazole dendrimer or carbazole-phenylazomethine dendrimer (ZnPG1-1, ZnPG1-2, ZnPG2-). 2 shows the following structural formula), and in the MALDI-TOF-MS spectrum, the target molecular weight peak of the dendrimer itself appears clearly as follows, and it is reported that it has a single molecular weight (Macromolecules 2008, 41, 3793).
[ZnPG1-1 + H] ⁺
Calcd: 2715.9
Found: 2715.4
[ZnPG1-2 + H] ⁺
Calcd: 5357.9
Found: 5355.7
[ZnPG2-2 + H] ⁺
Calcd: 10753.9
Found: 10752.9

Examples of chromatography include gas chromatography using a gas as a mobile phase, liquid chromatography using a liquid as a mobile phase, and supercritical fluid chromatography using a supercritical fluid as a mobile phase.

  Among these, it can use suitably for a high performance liquid chromatography (HPLC). In HPLC, a liquid mobile phase is pressurized by a pump or the like and passed through a column, and an analyte is increased by utilizing a difference in interaction (adsorption, distribution, ion exchange, size exclusion, etc.) between the stationary phase and the mobile phase. Detect by separating performance.

  The standard substance of the present invention can be suitably used for size exclusion chromatography (SEC) and one type of gel permeation chromatography (GPC) among HPLC. SEC is a type of liquid chromatography that performs separation based on the difference in molecular size, and is a technique for measuring the molecular weight distribution and average molecular weight of a polymer substance. In calibration with a standard substance, it is a prerequisite that the molecular weight is monodisperse, but the present inventors have reported the following.

  GPC chart of 1st to 4th generation phenylazomethine dendrimers DPAGn-ZnP (n is an integer of 1 to 4: DPAG1-ZnP, DPAG2-ZnP, DPAG3-ZnP, DPAG4-ZnP) having tetraphenylzinc porphyrin as a core Are both PDI (polydispersity index, an index indicating the molecular weight distribution of a polymer, and is 1 if it is completely monodispersed, and the larger the dispersion, the larger the value. Number average molecular weight divided by weight average molecular weight) Has a monomodality of 1.02 or less, and it is reported that it is monodisperse including analysis errors (supporting J. AM. CHEM. SOC. 2005, 127, 13896). Among these, the structural formula of the fourth generation DPAG4-ZnP is shown below.

1st to 4th generation carbazole dendrimers having benzophenone as the core: GnB (n is an integer of 1 to 4: G1B, G2B, G3B, G4B) are also reported to have a single molecular weight (no dispersion) (Supporting J. Am. Chem. Soc. 2009, 131, 2244). Among these, the structural formula of the third generation G3B is shown below.

First- to third-generation carbazole / phenylazomethine dendrimer TPMG1-n (n is an integer of 1 to 3: TPMG1-1, TPMG1-2, TPMG1-3) having tetraphenylmethane as a core is also a single molecular weight (dispersed) (No support for Macromolecules 2012, 45, 1288). Among these, the structural formula of TPMG1-3 is shown below.

When the analyzer is calibrated with the standard substance of the present invention and the molecular weight is measured, a calibration curve is created with the standard substance of the present invention having a known molecular weight. For example, in mass spectrometry, the standard substance of the present invention is measured, and the mass number is calibrated based on the obtained mass spectrum. In SEC and GPC, a graph of retention time (elution time) and molecular weight is created and calibrated. Can do.

  In quantitative analysis, a calibration curve method is generally used in which a standard curve is prepared by analyzing a plurality of standard substances with measurement points of an analysis target compound in between. Examples of the quantitative analysis method using the calibration curve method include an external standard method and an internal standard method. In the external standard method, an external standard having a known concentration is used to obtain a correlation between a concentration and a signal (a signal, specifically, a peak area, a peak height, etc.) in advance, and information obtained therefrom (a calibration curve). ) To obtain the concentration in the specimen sample. The internal standard method is a method in which a fixed amount of a component called an internal standard substance is added to a standard sample and an unknown sample, and the horizontal axis of the calibration curve takes the concentration ratio of the internal standard substance and the target component instead of the concentration of the target component. is there. When the analyzer is calibrated with the standard substance of the present invention and the molecular weight or the like is measured, a technique corresponding to the external standard method, the internal standard method, or the like can be used.

  In a preferred embodiment of the present invention, a plurality of dendrimers of different generations are used as the standard substance of the present invention, and a calibration curve is created based on the measurement results of the plurality of dendrimers for values to be measured by an analyzer. To do. Each dendrimer having a different number of generations exhibits a discrete molecular weight distribution, so that a higher molecular weight compound can be adjusted in parallel and is suitable as a multi-point calibration standard.

  The standard substance of the present invention can be used for calibration, resolution adjustment, sensitivity adjustment and the like. For example, in mass spectrometry, based on the obtained mass spectrum, it can be used not only for calibration of mass number but also for adjustment of mass resolution and sensitivity.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
Example 1
Two types of dendrimers, TPMG3 (tetraphenylmethane core 3rd generation phenylazomethine dendrimer) and TPMG4 (tetraphenylmethane core 4th generation phenylazomethine dendrimer) are calibrated as standard materials (external standards) for MALDI-TOF-MASS equipment (calibration) )

  Next, using a MALDI-TOF-MASS apparatus, an MS spectrum of human insulin, which is a commercially available protein, was measured. The result is shown in FIG. A mass peak of 5806.86 was obtained with respect to the calculated value (M + H: mass obtained by adding one hydrogen atom to the maximum intensity mass) 5806.64, and the result obtained by matching the calculated value with the actually measured value was obtained.

  Structural formulas of TPMG3 and TPMG4 used as standard substances are shown below.

Example 2
Two types of dendrimers, G3TAZ (1,3,5-triazine core 3rd generation carbazole dendrimer) and G4TAZ (1,3,5-triazine core 4th generation carbazole dendrimer), are used as standard materials for MALDI-TOF-MASS equipment (external Calibration was performed as a standard).

  Next, using a MALDI-TOF-MASS apparatus, an MS spectrum of human insulin, which is a commercially available protein, was measured. The result is shown in FIG. A mass peak of 5807.23 was obtained for the calculated value (M + H: mass obtained by adding one hydrogen atom to the maximum intensity mass) 5806.64, and the calculated value and the actually measured value were in agreement.

  The structural formula of G3TAZ among the standard materials used is shown below.

Example 3
Two types of dendrimers, G3TAZ (1,3,5-triazine core 3rd generation carbazole dendrimer) and G4TAZ (1,3,5-triazine core 4th generation carbazole dendrimer), are used as standard materials for MALDI-TOF-MASS equipment (external Calibration was performed as a standard).

  Shimadzu / KRATOS time-of-flight mass spectrometer AXIMA-CFR Plus was used as an apparatus for measuring MS spectra.

  Samples of standard substances G3TAZ and G4TAZ were prepared as follows. A standard substance was dissolved in a solvent (chloroform), and a matrix reagent (disranol) was dissolved in a similar solvent (chloroform). Next, the standard substance solution and the matrix reagent solution were placed on the sample plate in this order, and allowed to stand at room temperature to dry and cocrystallize. The sample plate was set in the apparatus, measured in the positive ion / reflectron mode, and a calibration file was created using the theoretical mass number.

  Biomaterial bovine Ubiquitine sample was prepared as follows. First, a matrix reagent (CHCA: α-cyano-4-hydroxycinnamic acid) was dissolved in a solvent (water: acetonitrile: TFA = 50%: 50%: 0.1%). Next, the prepared matrix reagent solution and the biological material to be measured were mixed, and the mixed solution was placed on a sample plate and allowed to stand at room temperature to dry and cocrystallize.

  The sample plate was set in the apparatus, measured in the positive ion / reflectron mode, and the calibration file was applied (calibrated by the external standard method).

  As described above, FIG. 3 shows the results of measuring the MS spectrum of bovine Ubiquitine, which is a commercially available protein, using the MALDI-TOF-MASS apparatus. A mass peak of 8565.45 was obtained with respect to the calculated value (M + H: mass obtained by adding one hydrogen atom to the average mass) 8565.84, and a result in which the calculated value and the actually measured value coincided with each other was obtained.

Example 4
The following groups [1] to [3] were used as standard substances for SEC (GPC).
[1] First- to fourth-generation phenylazomethine dendrimer DPAGn-ZnP having tetraphenylzinc porphyrin as a core (n is an integer of 1 to 4: DPAG1-ZnP, DPAG2-ZnP, DPAG3-ZnP, DPAG4-ZnP)
[2] First to fourth generation carbazole dendrimers having benzophenone as a core: GnB (n is an integer of 1 to 4: G1B, G2B, G3B, G4B)
[3] First generation to third generation carbazole-phenylazomethine dendrimer TPMG1-n (n is an integer of 1 to 3: phenylazomethine: carbazole = 1: 1 first-generation phenylazomethine- having tetraphenylmethane as the core) First generation carbazole dendrimer TPMG1-1, phenylazomethine: carbazole = 1: 2 first generation phenylazomethine-second generation carbazole dendrimer TPMG1-2, phenylazomethine: carbazole = 1: 3 first generation phenylazomethine-3rd Generation carbazole dendrimer TPMG1-3)

Each of [1] to [3] was subjected to size exclusion chromatography analysis.
The size exclusion chromatography analysis was performed at 40 ° C. by connecting a TSK-GEL CMHXL column (Tosoh) to high performance liquid chromatography (Shimadzu, LC-10AP). The solvent used was tetrahydrofuran (THF), and the flow rate was 1 ml / min. The detector used was a three-way detector (Viscotec, TriSEC model 302) for viscosity, light scattering, and refractive index. For the measurement, a sample solution of about 5 mg / ml was prepared, and 100 μl was injected.

  The result is shown in FIG. In FIG. 4, DPAGn-ZnP of [1] is indicated by ◆, GnB of [2] is indicated by ▲, and TPMG1-n of [3] is indicated by ■. It was confirmed that the retention times and molecular weights of the above-mentioned various dendrimers had a clear linearity (linearity) in a logarithmic plot. Accordingly, the various dendrimers described above can be used as SEC standard substances. Specifically, an unknown substance is measured, and the dendrimer equivalent molecular weight of the unknown substance is derived from the data of FIG. 4 from the retention time of the SEC.

Claims (5)

  A standard substance comprising a dendrimer having at least one selected from a phenylazomethine skeleton and a carbazole skeleton as a constitutional unit of a dendritic branch structure.   The standard substance according to claim 1, which is used for mass spectrometry.   The standard substance according to claim 1, which is used for high performance liquid chromatography.   The method to calibrate an analyzer using the standard material as described in any one of Claims 1-3.   The method according to claim 4, wherein a calibration curve is created based on a measurement result of the plurality of dendrimers for a value to be measured by the analyzer using a plurality of dendrimers having different generation numbers.