JP2015155884A - Surface active matrix for maldi mass spectrometry - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a surface active matrix for MALDI mass spectrometry, capable of sensitively measuring poorly soluble samples, such as membrane protein, peptide and polyphenol and the like without using a surfactant being an unnecessary additive even when the poorly soluble samples are measured.

SOLUTION: The surface active matrix for MALDI mass spectrometry consists of: a derivative of organic acid selected from a group consisting of ferulic acid, α-cyano 4-hydroxycinnamic acid, sinapic acid, caffeine acid and gentisic acid; and an amphiphilic organic acid derivative obtained by hydrophilizing or hydrophobizing a carboxyl and/or phenol group of the organic acid.

COPYRIGHT: (C)2015,JPO&INPIT

Description

  In the present invention, in MALDI mass spectrometry, an analyte (analyte) that is difficult to detect due to poor solubility in a solvent is solubilized to form a good co-crystal, thereby efficiently ionizing the analyte. The present invention relates to a surface active matrix that can be detected with high sensitivity, a MALDI mass spectrometry method using the surface active matrix, and a novel organic acid derivative that can be suitably used for the surface active matrix.

MALDI (Matrix Assisted Laser Desorption / Ionization) is a process for molecular ionization. Since its development in the 1980s, it has been shown to be particularly effective for mass spectrometry of biopolymers and synthetic polymers.
In MALDI mass spectrometry, an analyte is ionized by preparing a mixed crystal (co-crystal) of a target sample (analyte) and a matrix and irradiating it with laser light. The matrix absorbs the light energy of the irradiated laser and ionizes, and at the same time, rapidly heats and vaporizes. Although the analyte molecules are not directly vaporized by the laser irradiation, they are detached together with the matrix molecules surrounding the analyte molecules. Subsequently, the transfer of protons and electrons between the ionized matrix molecules and the analyte molecules causes the target sample to be ionized. Since a nitrogen laser (wavelength 337 nm) or a YAG laser (wavelength 355 nm) is generally used as the laser source, a substance having an absorption band in this wavelength region is used for the matrix.

Matrix can be applied to virtually all substances suitable for analysis by MALDI-MS (MALDI mass spectrometry), i.e. high or low molecular weight, non-volatile, thermally labile compounds, e.g. biopolymers For example, proteins and lipids, and low molecular weight organic and inorganic analytes such as drugs, plant metabolites and the like.
The best known matrix compounds are the most commonly used α-cyano-4-hydroxycinnamic acid (CHCA, see Patent Document 1) and its derivatives. Examples of other matrices used in MALDI-MS are sinapinic acid (4-hydroxy-3,5-dimethoxycinnamic acid), ferulic acid (4-hydroxy-3-methoxy cinnamic acid, see Non-Patent Document 1) or 2,5-dihydroxybenzoic acid (2,5-DHB).

US Patent Publication No. 2002/0142982 (see [0139]) Special table 2010-537206 gazette JP 2003-128632 A JP 2003-261443 A

Ronald C. Beavis et al. Rapid Communications in Mass Spectrometry, Vol. 3, No.12. 432-435 (1989) Santi M. Mandal et al. Eur. J. Mass Spectrom. Vol.16, 567-575 (2010) Fukuyama Y. et al. Anal. Chem. Vol.84, 4237-4243 (2012)

  However, even with these matrices, there are currently problems in measuring poorly soluble samples such as membrane proteins, peptides, and polyphenols. In order to solubilize such analytes, sodium dodecyl sulfate is used. A surfactant which is an extra additive such as (SDS) is used. On the other hand, the addition of a surfactant such as SDS has a problem that the measurement sensitivity of the analyte in MALDI-MS analysis is lowered (see Non-Patent Document 2). Therefore, even if a poorly soluble co-crystal of analyte and matrix is formed, a process for removing the surfactant from the co-crystal is necessary.

The present invention is based on the background of such a conventional technique, and is a surfactant that is an extra additive even when measuring hydrophobic and hardly soluble samples such as membrane proteins, peptides, and polyphenols. It is an object of the present invention to provide a surface active matrix for MALDI mass spectrometry capable of measuring those poorly soluble samples with high sensitivity without using any of the above.
In addition, the present invention is also capable of measuring poorly soluble samples with high sensitivity without using a surfactant, which is an additional additive, even when measuring poorly soluble samples such as membrane proteins, peptides, and polyphenols. It is an object of the present invention to provide a MALDI mass spectrometry method that can be used.
Another object of the present invention is to provide a novel organic acid derivative useful as a surface active matrix for MALDI mass spectrometry that can measure such a hardly soluble sample with high sensitivity.

The inventors of the present invention have made extensive studies to solve the above problems. As a result, an organic acid such as ferulic acid used as an existing matrix in MALDI mass spectrometry is derivatized and amphiphilized, so as to solubilize the analyte by making it a matrix having a surface active function, and efficiently As a result, it was found that an analyte that is difficult to detect with existing organic acids can be detected with high sensitivity by MALDI mass spectrometry, and the present invention has been completed.
A matrix for MALDI mass spectrometry generally has (a) the ability to absorb laser light, (b) the stability under high vacuum, (c) the solubilization ability of the sample to be detected, and (d) the coexistence with the sample to be detected. The function and properties of crystal forming ability are required, but by derivatizing existing organic acid for matrix and making it amphiphilic, (a) the ability to absorb laser light, (b) under high vacuum While maintaining the stability, various ideas such as examples of the inventors' original idea that (c) the solubilization ability of the sample to be detected and (d) the eutectic formation ability with the sample to be detected can be improved. The above-mentioned knowledge was obtained by confirming by a test.

In Patent Document 2, a cyanocinnamic acid derivative is used as a matrix for MALDI mass spectrometry in order to improve detection and sensitivity to cations, anions, and ions having double or higher charges. However, it is not described at all that the cyanocinnamic acid derivative is a surface active type, and the cyanocinnamic acid derivative has a group of position number 4 on the benzene ring as halogen or carbon 1 to Since it is an alkyl of 10, it cannot be said to be a derivative of α-cyano-4-hydroxycinnamic acid (CHCA).
Patent Document 3 describes a ferulic acid derivative having an alkyl group, and Patent Document 4 describes a ferulic acid amide derivative having an alkyl group, which are used as an ultraviolet absorber or a therapeutic agent for diabetes. However, the use of these ferulic acid derivatives as a matrix for MALDI mass spectrometry is not described at all.
In Non-Patent Document 3, an alkylated DHB derivative (ADHB) in which a hydrophobic alkyl chain is bonded to a hydroxyl group of 2,5-DHB is added to a CHCA matrix as an additive for a matrix for hydrophobic peptide analysis. However, it does not describe that ADHB itself has a function or property as a matrix, nor does it describe that ADHB is a surface active type.
Therefore, these patent documents 2, 3, 4 and non-patent document 3 do not suggest the present invention.

［一般式(１)中において、
Ｒ¹は、水素原子、又は、メトキシ基、
Ｒ²は、水素原子、ＯＨ、ＯＸ、Ｏ(ＣＨ₂)_kＳＯ₃Ｘ、又は、Ｏ(ＣＨ₂)₂(ＣＦ₂)_lＣＦ₃、
Ｒ³は、水素原子、ＯＨ、又は、メトキシ基、
Ｒ⁴は、水素原子、又は、ＣＮ、
Ｒ⁵は、ＯＨ、ＯＸ、ＮＨ(ＣＨ₂)_mＣＨ₃、又は、Ｏ(ＣＨ₂)_nＣＨ₃、
をそれぞれ表す(Ｒ²、Ｒ⁵において、Ｘは、Ｌｉ、Ｎａ、Ｋ、Ｒｂ、Ｃｓから選択されるアルカリ金属、ｋは2〜8の整数、ｌは3〜17の整数、ｍは3〜17の整数、ｎは3〜17の整数である。)。］
〈３〉Ｒ¹、Ｒ³の一方が水素原子、他方がメトキシ基であり、Ｒ²が、ＯＸ、又は、Ｏ(ＣＨ₂)_kＳＯ₃Ｘであり、Ｒ⁴が水素原子であり、Ｒ⁵がＮＨ(ＣＨ₂)_mＣＨ₃である、〈２〉に記載のMALDI質量分析用界面活性型マトリックス。
〈４〉〈１〉〜〈３〉のいずれか１項に記載の界面活性型マトリックスと、フェルラ酸、α-シアノ-4-ヒドロキシ桂皮酸、シナピン酸、カフェイン酸、及び、ゲンチジン酸からなる群から選択される１種又は２種以上の有機酸との混合物からなるMALDI質量分析用マトリックス。
〈５〉〈１〉〜〈４〉のいずれか１項に記載のマトリックスを用いるMALDI質量分析方法。
〈６〉〈５〉に記載のMALDI質量分析方法において、溶媒、分析対象試料、及び、前記マトリックスからなる混合物を調製する際に、前記界面活性型マトリックスを臨界ミセル濃度の10％以上とすることを特徴とする、MALDI質量分析方法。
〈７〉分析対象試料が、タンパク質、ペプチド、核酸、糖類、ポリマー、脂質、及び、有機化合物からなる群から選択されるものであることを特徴とする〈５〉又は〈６〉に記載のMALDI質量分析方法。
〈８〉下記一般式(１)で表される有機酸誘導体(ただし、Ｒ²が、水素原子、ＯＨ、ＯＸ、又は、Ｏ(ＣＨ₂)_kＳＯ₃Ｘのとき、Ｒ⁵は、ＮＨ(ＣＨ₂)_mＣＨ₃、又は、Ｏ(ＣＨ₂)_nＣＨ₃であり、Ｒ²がＯ(ＣＨ₂)₂(ＣＦ₂)_lＣＦ₃のとき、Ｒ⁵は、ＯＨ、又は、ＯＸである。Ｒ⁵がＮＨ(ＣＨ₂)_mＣＨ₃、又は、Ｏ(ＣＨ₂)_nＣＨ₃であって、かつ、Ｒ²が、水素原子、又は、ＯＨである場合の有機酸誘導体を除く。)。

［一般式(１)中において、
Ｒ¹は、水素原子、又は、メトキシ基、
Ｒ²は、水素原子、ＯＨ、ＯＸ、Ｏ(ＣＨ₂)_kＳＯ₃Ｘ、又は、Ｏ(ＣＨ₂)₂(ＣＦ₂)_lＣＦ₃、
Ｒ³は、水素原子、ＯＨ、又は、メトキシ基、
Ｒ⁴は、水素原子、又は、ＣＮ、
Ｒ⁵は、ＯＨ、ＯＸ、ＮＨ(ＣＨ₂)_mＣＨ₃、又は、Ｏ(ＣＨ₂)_nＣＨ₃、
をそれぞれ表す(Ｒ²、Ｒ⁵において、Ｘは、Ｌｉ、Ｎａ、Ｋ、Ｒｂ、Ｃｓから選択されるアルカリ金属、ｋは2〜8の整数、ｌは3〜17の整数、ｍは3〜17の整数、ｎは3〜17の整数である。)。］ The present invention has been made based on the above-described ideas and knowledge, and the following invention is provided in this application.
<1> A surface active matrix for MALDI mass spectrometry comprising a derivative of an organic acid selected from the group consisting of ferulic acid, α-cyano-4-hydroxycinnamic acid, sinapinic acid, caffeic acid, and gentisic acid. And a surface active matrix for MALDI mass spectrometry, comprising an amphiphilic organic acid derivative obtained by hydrophilizing or hydrophobizing the carboxyl group and / or phenol group of the organic acid.
<2> A surface active matrix for MALDI mass spectrometry comprising an amphiphilic organic acid derivative represented by the following general formula (1) (where R ² is a hydrogen atom, OH, OX or O (CH ₂ ) _{When k} SO ₃ X, R ⁵ is NH (CH ₂ ) _m CH ₃ or O (CH ₂ ) _n CH ₃ , and R ² is O (CH ₂ ) ₂ (CF ₂ ) _l CF ₃ When R ⁵ is OH or OX).

[In general formula (1),
R ¹ is a hydrogen atom or a methoxy group,
R ² represents a hydrogen atom, OH, OX, O (CH ₂ ) _k SO ₃ X, or O (CH ₂ ) ₂ (CF ₂ ) ₁ CF ₃ ,
R ³ is a hydrogen atom, OH, or a methoxy group,
R ⁴ is a hydrogen atom or CN,
R ⁵ is OH, OX, NH (CH ₂ ) _m CH ₃ , or O (CH ₂ ) _n CH ₃ ,
(In R ² and R ⁵ , X is an alkali metal selected from Li, Na, K, Rb and Cs, k is an integer of 2 to 8, l is an integer of 3 to 17, and m is 3 to 3) An integer of 17 and n is an integer of 3 to 17.) ]
<3> One of R ¹ and R ³ is a hydrogen atom, the other is a methoxy group, R ² is OX or O (CH ₂ ) _k SO ₃ X, R ⁴ is a hydrogen atom, R ^The surface active matrix for MALDI mass spectrometry according to <2>, wherein ⁵ is NH (CH ₂ ) _m CH ₃ .
<4> The surface active matrix according to any one of <1> to <3>, ferulic acid, α-cyano-4-hydroxycinnamic acid, sinapinic acid, caffeic acid, and gentisic acid. A matrix for MALDI mass spectrometry comprising a mixture with one or more organic acids selected from the group.
<5> A MALDI mass spectrometry method using the matrix according to any one of <1> to <4>.
<6> In the MALDI mass spectrometry method according to <5>, when preparing a mixture comprising a solvent, a sample to be analyzed, and the matrix, the surface active matrix should be 10% or more of the critical micelle concentration. MALDI mass spectrometry method characterized by the above.
<7> The MALDI according to <5> or <6>, wherein the sample to be analyzed is selected from the group consisting of proteins, peptides, nucleic acids, sugars, polymers, lipids, and organic compounds. Mass spectrometry method.
<8> An organic acid derivative represented by the following general formula (1) (wherein R ² is a hydrogen atom, OH, OX, or O (CH ₂ ) _k SO ₃ X, R ⁵ is NH ( When CH ₂ ) _m CH ₃ or O (CH ₂ ) _n CH ₃ and R ² is O (CH ₂ ) ₂ (CF ₂ ) ₁ CF ₃ , R ⁵ is OH or OX. (Excluding organic acid derivatives in which R ⁵ is NH (CH ₂ ) _m CH ₃ or O (CH ₂ ) _n CH ₃ and R ² is a hydrogen atom or OH.) .

[In general formula (1),
R ¹ is a hydrogen atom or a methoxy group,
R ² represents a hydrogen atom, OH, OX, O (CH ₂ ) _k SO ₃ X, or O (CH ₂ ) ₂ (CF ₂ ) ₁ CF ₃ ,
R ³ is a hydrogen atom, OH, or a methoxy group,
R ⁴ is a hydrogen atom or CN,
R ⁵ is OH, OX, NH (CH ₂ ) _m CH ₃ , or O (CH ₂ ) _n CH ₃ ,
(In R ² and R ⁵ , X is an alkali metal selected from Li, Na, K, Rb and Cs, k is an integer of 2 to 8, l is an integer of 3 to 17, and m is 3 to 3) An integer of 17 and n is an integer of 3 to 17.) ]

［式(２)中において、Ｒ²は、ＯＨ、又は、ＯＸを表し、Ｘは、Ｌｉ、Ｎａ、Ｋ、Ｒｂ、Ｃｓから選択されるアルカリ金属、ｍは3〜17の整数である。］
〈１７〉下記式(３)で表される有機酸誘導体。

［式(３)中において、Ｘは、Ｌｉ、Ｎａ、Ｋ、Ｒｂ、Ｃｓから選択されるアルカリ金属、ｋは2〜8の整数、ｍは3〜17の整数である。］
〈１８〉下記式(４)で表される有機酸誘導体。

［式(４)中において、Ｒ⁵は、ＯＨ、又は、ＯＸを表し、Ｘは、Ｌｉ、Ｎａ、Ｋ、Ｒｂ、Ｃｓから選択されるアルカリ金属、ｌは3〜17の整数である。］
〈１９〉フェルラ酸、α-シアノ-4-ヒドロキシ桂皮酸、シナピン酸、カフェイン酸、及び、ゲンチジン酸からなる群から選択される有機酸の誘導体からなるMALDI質量分析用界面活性型マトリックスの製造方法であって、該有機酸のカルボキシル基及び／又はフェノール基を親水化又は疎水化して両親媒性有機酸誘導体とすることを特徴とするMALDI質量分析用界面活性型マトリックスの製造方法。
〈２０〉前記有機酸のカルボキシル基とフェノール基の一方を親水化又は疎水化して両親媒性有機酸誘導体とすることを特徴とする〈１９〉に記載のMALDI質量分析用界面活性型マトリックスの製造方法。
〈２１〉前記有機酸のカルボキシル基とフェノール基の一方を親水化し、他方を疎水化して両親媒性有機酸誘導体とすることを特徴とする〈１９〉に記載のMALDI質量分析用界面活性型マトリックスの製造方法。 The present invention can include the following aspects.
<9> The surfactant for MALDI mass spectrometry according to <1>, comprising an amphiphilic organic acid derivative obtained by hydrophilizing or hydrophobizing one of a carboxyl group and a phenol group of the organic acid. Type matrix.
<10> The surfactant for MALDI mass spectrometry according to <1>, comprising an amphiphilic organic acid derivative obtained by hydrophilizing one of a carboxyl group and a phenol group of the organic acid and hydrophobizing the other. Type matrix.
<11> A matrix for MALDI mass spectrometry comprising a mixture of the surface active matrix according to any one of <1> to <3>, <9>, and <10> and ferulic acid.
<12> A MALDI mass spectrometry method using the matrix according to any one of <9> to <11>.
<13> In the MALDI mass spectrometry method according to <12>, the surface active matrix should be 10% or more of the critical micelle concentration when preparing a mixture of the solvent, the sample to be analyzed, and the matrix. MALDI mass spectrometry method characterized by
<14> The MALDI mass spectrometry method according to <6> or <13>, wherein the concentration of the surface active matrix is 20% or more and 50 times or less of the critical micelle concentration.
<15> The sample to be analyzed is selected from the group consisting of proteins, peptides, nucleic acids, saccharides, polymers, lipids, and organic compounds, and any one of <12> to <14> MALDI mass spectrometry method according to item.
<16> An organic acid derivative represented by the following formula (2).

[In the formula (2), R ² is, OH, or represents OX, X is an alkali metal selected Li, Na, K, Rb, from Cs, m is an integer from 3 to 17. ]
<17> An organic acid derivative represented by the following formula (3).

[In Formula (3), X is an alkali metal selected from Li, Na, K, Rb, and Cs, k is an integer of 2 to 8, and m is an integer of 3 to 17. ]
<18> An organic acid derivative represented by the following formula (4).

[In the formula (4) in, R ⁵ is, OH, or represents OX, X is an alkali metal selected Li, Na, K, Rb, from Cs, l is an integer of 3 to 17. ]
<19> Production of a surface active matrix for MALDI mass spectrometry comprising a derivative of an organic acid selected from the group consisting of ferulic acid, α-cyano-4-hydroxycinnamic acid, sinapinic acid, caffeic acid, and gentisic acid A method for producing a surface active matrix for MALDI mass spectrometry, wherein the carboxyl group and / or phenol group of the organic acid is hydrophilized or hydrophobized to obtain an amphiphilic organic acid derivative.
<20> Production of a surface active matrix for MALDI mass spectrometry according to <19>, wherein one of a carboxyl group and a phenol group of the organic acid is hydrophilized or hydrophobized to obtain an amphiphilic organic acid derivative. Method.
<21> The surface active matrix for MALDI mass spectrometry according to <19>, wherein one of a carboxyl group and a phenol group of the organic acid is hydrophilized and the other is hydrophobized to obtain an amphiphilic organic acid derivative. Manufacturing method.

The surface active matrix according to the present invention can solubilize a hardly soluble sample due to its surface active function. In particular, a surface active matrix having a critical micelle concentration or more can spontaneously form micelles in various solvents and freely solubilize various analytes therein. Therefore, it is possible to detect the analyte with high sensitivity without the need for addition of a surfactant, which is an extra additive that causes a decrease in detection sensitivity, or the process of removing it.
In addition, the surface active matrix can form a co-crystal with an analyte better than the existing organic acid matrix because it forms crystal nuclei by micelle formation and partially introduces an amide bond. . Moreover, favorable sensitivity can be given in MALDI mass spectrometry by selectively giving specific ions to the analyte solubilized inside by micelle formation. Therefore, this invention is very useful industrially as what can improve the characteristic of matrices, such as the existing organic acid, significantly.

FIG. 1 is a diagram showing a synthesis scheme of ferulic acid derivatives 1 and 2 (FAD1, FAD1-Na, and FAD2) according to the present invention. FIG. 2 is a diagram showing a synthesis scheme of ferulic acid derivative 3 (FAD3) according to the present invention. FIG. 3 is a diagram showing a synthesis scheme of an α-cyano-4-hydroxycinnamic acid derivative (CHCA4), a gentisic acid derivative (DHB5), and a sinapinic acid derivative (SA6) according to the present invention. FIG. 4 is a view showing the relationship between the surface tension and scattered light intensity of the aqueous FAD1-Na solution according to the present invention and the FAD1-Na concentration. FIG. 5 is a graph showing the relationship between the concentration of FAD1-Na according to the present invention and the amount of curcumin solubilized. FIG. 6 is a view showing an SEM observation image of a crystal on a MALDI substrate of FAD1-Na according to the present invention. (a) shows that prepared from 5 mM FAD1-Na, and (b) and (c) show those prepared from 25 mM FAD1-Na, respectively. FIG. 7 is a view showing an SEM observation image of sodium ferulate (prepared from a concentration of 25 mM) on a MALDI substrate. FIG. 8 shows SEM observation images of co-crystals of FAD1-Na and curcumin at various concentrations [(a) 10 mM, (b) 1 mM, (c) 0.1 mM] according to the present invention on a MALDI substrate. FIG. 9 is a diagram showing MALDI TOFMS spectra at various concentrations ((a) 50 mM, (b) 5 mM) of FAD1-Na according to the present invention. FIG. 10 is a graph showing the relationship between the intensity of various molecular ion peaks of FAD1-Na according to the present invention and the FAD1-Na concentration. FIG. 11 shows MALDI TOFMS spectra of curcumin at various concentrations of FAD1-Na according to the present invention [(a) 10 mM, (b) 1 mM, (c) 0.1 mM)]. FIG. 12 is a diagram showing MALDI TOFMS spectra of curcumin at various concentrations (0.1 mM to 10 mM) of ferulic acid (FA). FIG. 13 is a diagram showing the intensity of the molecular ion peak of angiotensin I when ferulic acid (FA) and ferulic acid derivative (FAD1) are mixed.

(Description of the surface active matrix for MALDI mass spectrometry of the present invention)
The surface active matrix for MALDI mass spectrometry of the present invention comprises an amphiphilic organic acid derivative obtained by hydrophilizing or hydrophobizing a carboxyl group and / or a phenol group of an existing matrix organic acid.

Examples of the existing matrix organic acid for obtaining the surface active matrix for MALDI mass spectrometry of the present invention include ferulic acid, α-cyano-4-hydroxycinnamic acid, sinapinic acid, caffeic acid, and gentisic acid. it can.
Among these organic acids, ferulic acid has been chemically synthesized so far from vanillin and malonic acid. However, this process takes about 3 weeks for the reaction, and the production cost is very high. It was more than 200,000 yen / kg). Therefore, although the use in each field is expected, the effective use has not progressed. On the other hand, in recent years, a method for producing γ-oryzanols by hydrolysis from rice bran pitch, which is a renewable resource, has been developed, and ferulic acid is very inexpensive (10,000 yen / kg or more) and its use is expected to be promoted. Yes. The present invention greatly improves the function of ferulic acid as a matrix for MALDI mass spectrometry.

When derivatizing an existing matrix organic acid with an amphiphilic organic acid, only one of the carboxyl group and the phenol group may be hydrophilized or hydrophobized, but one of the carboxyl group and the phenol group is hydrophilized and the other is hydrophobic. It can also be converted.
When the carboxyl group or the phenol group is hydrophilized, the hydroxyl group contained in them can be an alkali metal salt or an alkyl sulfonic acid alkali metal salt. In this case, the alkali metal is selected from Li, Na, K, Rb, and Cs, and Na, K, and Li are preferable. The alkyl chain of the alkylsulfonic acid alkali metal salt can have 2 to 8 carbon atoms (preferably 2 to 5 carbon atoms).
On the other hand, when a carboxyl group or a phenol group is hydrophobized, an alkyl chain or a (partially) fluorinated alkyl chain can be added, or an alkylamide bond can be introduced. The alkyl chain for hydrophobizing can have 4 to 18 carbon atoms (preferably 6 to 14 carbon atoms). The fluorination of the alkyl chain may be complete or partial.

［一般式(１)中において、
Ｒ¹は、水素原子、又は、メトキシ基、
Ｒ²は、水素原子、ＯＨ、ＯＸ、Ｏ(ＣＨ₂)_kＳＯ₃Ｘ、又は、Ｏ(ＣＨ₂)₂(ＣＦ₂)_lＣＦ₃、
Ｒ³は、水素原子、ＯＨ、又は、メトキシ基、
Ｒ⁴は、水素原子、又は、ＣＮ、
Ｒ⁵は、ＯＨ、ＯＸ、ＮＨ(ＣＨ₂)_mＣＨ₃、又は、Ｏ(ＣＨ₂)_nＣＨ₃、
をそれぞれ表す(Ｒ²、Ｒ⁵において、Ｘは、Ｌｉ、Ｎａ、Ｋ、Ｒｂ、Ｃｓから選択されるアルカリ金属、ｋは2〜8の整数、ｌは3〜17の整数、ｍは3〜17の整数、ｎは3〜17の整数である。)。ただし、Ｒ²が、水素原子、ＯＨ、ＯＸ、又は、Ｏ(ＣＨ₂)_kＳＯ₃Ｘのとき、Ｒ⁵は、ＮＨ(ＣＨ₂)_mＣＨ₃、又は、Ｏ(ＣＨ₂)_nＣＨ₃であり、Ｒ²がＯ(ＣＨ₂)₂(ＣＦ₂)_lＣＦ₃のとき、Ｒ⁵は、ＯＨ、又は、ＯＸである。］

［式(２)中において、Ｒ²は、ＯＨ、又は、ＯＸを表し、Ｘは、Ｌｉ、Ｎａ、Ｋ、Ｒｂ、Ｃｓから選択されるアルカリ金属、ｍは3〜17の整数である。］

［式(３)中において、Ｘは、Ｌｉ、Ｎａ、Ｋ、Ｒｂ、Ｃｓから選択されるアルカリ金属、ｋは2〜8の整数、ｍは3〜17の整数である。］

［式(４)中において、Ｒ⁵は、ＯＨ、又は、ＯＸを表し、Ｘは、Ｌｉ、Ｎａ、Ｋ、Ｒｂ、Ｃｓから選択されるアルカリ金属、ｌは3〜17の整数である。］ Specifically, the organic acid derivative in the surface active matrix comprising the amphiphilic organic acid derivative of the present invention can be represented by the following general formulas (1) to (4).

[In general formula (1),
R ¹ is a hydrogen atom or a methoxy group,
R ² represents a hydrogen atom, OH, OX, O (CH ₂ ) _k SO ₃ X, or O (CH ₂ ) ₂ (CF ₂ ) ₁ CF ₃ ,
R ³ is a hydrogen atom, OH, or a methoxy group,
R ⁴ is a hydrogen atom or CN,
R ⁵ is OH, OX, NH (CH ₂ ) _m CH ₃ , or O (CH ₂ ) _n CH ₃ ,
(In R ² and R ⁵ , X is an alkali metal selected from Li, Na, K, Rb and Cs, k is an integer of 2 to 8, l is an integer of 3 to 17, and m is 3 to 3) An integer of 17 and n is an integer of 3 to 17.) However, when R ² is a hydrogen atom, OH, OX, or O (CH ₂ ) _k SO ₃ X, R ⁵ is NH (CH ₂ ) _m CH ₃ or O (CH ₂ ) _n CH ₃ When R ² is O (CH ₂ ) ₂ (CF ₂ ) ₁ CF ₃ , R ⁵ is OH or OX. ]

[In the formula (2), R ² is, OH, or represents OX, X is an alkali metal selected Li, Na, K, Rb, from Cs, m is an integer from 3 to 17. ]

[In Formula (3), X is an alkali metal selected from Li, Na, K, Rb, and Cs, k is an integer of 2 to 8, and m is an integer of 3 to 17. ]

[In the formula (4) in, R ⁵ is, OH, or represents OX, X is an alkali metal selected Li, Na, K, Rb, from Cs, l is an integer of 3 to 17. ]

  The fact that the matrix for MALDI mass spectrometry of the present invention is a surface active type is due to a decrease in surface tension when the matrix concentration in the solvent described later is increased and a substantially constant surface tension above a predetermined concentration (critical micelle concentration). This can be confirmed (see FIG. 4). The fact that the matrix for MALDI mass spectrometry of the present invention is a surface active type can also be confirmed by directly examining that it has micelle forming ability. The presence or absence of micelle formation at that time can be confirmed, for example, by measuring the light scattering intensity of solvents with various matrix concentrations.

The surface active matrix composed of the amphiphilic organic acid derivative of the present invention exhibits surface tension reducing ability and micelle forming ability in various solvents, and can solubilize various analytes including hardly soluble samples. In particular, a surface-active matrix having a CMC value or higher can freely solubilize the analyte in the micelle. Therefore, even for a poorly soluble analyte, it is possible to detect the analyte with high throughput at high throughput without the need for addition of a surfactant, which is an additional additive that causes a decrease in detection sensitivity, and the process of removing it. As described in Example 3 below, the surface active matrix in the solvent has a concentration of 10% or more of the CMC value (preferably having a CMC value of from the viewpoint of forming a co-crystal with the analyte. 20% or more), but from the viewpoint of solubilization of the analyte inside the micelles and formation of a good co-crystal based on the solubilization, the critical micelle concentration (CMC) value is preferable or more preferable. Is 110% or more of the CMC value, more preferably 120% or more of the CMC value, and still more preferably 150% or more of the CMC value. The upper limit of the concentration of the surface active matrix is not particularly limited, but from an economic viewpoint, it is 50 times or less of the CMC value, preferably 20 times or less of the CMC value, more preferably 10 times or less of the CMC value. can do.
In addition, by forming crystal nuclei by forming micelles in the matrix, or by partially introducing amide groups, it is possible to form better co-crystals with analytes than existing organic acid matrices. Further, by applying specific ions to the analyte solubilized inside by binding of counter ions to the micelle surface accompanying micelle formation, good sensitivity can be given in MALDI mass spectrometry.

  The surface active matrix composed of the amphiphilic organic acid derivative of the present invention may be used alone as a matrix, or a plurality of types may be mixed and used. In addition, by mixing with one or more organic acids of ferulic acid, α-cyano-4-hydroxycinnamic acid, sinapinic acid, caffeic acid, gentisic acid, which are existing organic acids for matrix, In some cases, the amphiphilic organic acid derivative of the present invention and the existing organic acid for a matrix can be improved in detection sensitivity compared to the case where each is used alone as a matrix. Examples of such a preferable mixed matrix include a mixture of an organic acid derivative represented by the above formula (2) and ferulic acid. The mixing ratio (molar ratio) of the amphiphilic organic acid derivative of the present invention to the existing matrix organic acid is, for example, 3: 7 to 9: 1, preferably 4: 6 to 8: 2.

(Description of solvent)
In MALDI mass spectrometry, a sample to be analyzed and a matrix are mixed with a solvent, and then the solvent is removed by drying or the like to prepare a co-crystal of the sample to be analyzed and the matrix. As a solvent used in such a case, water, acetonitrile, and them were mixed at an arbitrary volume ratio [for example, water: acetonitrile = 10: 1 to 1:10 (preferably 4: 1 to 1: 2)]. However, the surface active matrix is not limited to this because it exhibits surface activity even in various media, but it is not limited to methanol, ethanol, acetone, tetrahydrofuran (THF), trifluoroacetic acid (TFA). , Toluene, chloroform, methylene chloride, or the like, or any mixture thereof.

(Analyst description)
The analyte (sample to be analyzed) in the MALDI mass spectrometry of the present invention is not particularly limited, but polyphenols such as curcumin that is unstable under acid and alkaline conditions, pyrroloquinoline quinone (PQQ), coenzyme Q10, fat Soluble vitamins, proteins, angiotensin I, β-amyloid peptide, peptides such as humanin, nucleic acids, saccharides, polymers, lipids and organic compounds.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention.

(Example 1) <Synthesis of various surface active matrices>
(Example 1-1) Synthesis of ferulic acid derivative 1 (FAD1, FAD1-Na) (see FIG. 1)
Synthesis of (E) -N-octyl-3- (4-hydroxy-3-methoxyphenyl) -2-propenamide (FAD1) In a three-necked flask, ferulic acid (FA) (10.0 g, 51.7 mmol) Was dissolved in dichloromethane (DCM) (200 mL) and stirred at room temperature for 45 minutes. The reaction solution was cooled in an ice bath, where octylamine (7.32 g, 56.7 mmol), triethylamine (NEt ₃ ) (3.69 g, 36.4 mmol), 1-hydroxybenzotriazole (HOBt) (8.36 g, 61.9 mmol) Diisopropylcarbodiimide (DIC) (11.9 g, 94.0 mmol) was added and stirred at room temperature for 17 hours. Chloroform and hydrochloric acid (a few drops) were added to the reaction solution, and the organic phase was washed three times with a saturated aqueous sodium chloride solution. The aqueous layer was extracted with chloroform, and the combined organic layer was washed with water and dried over anhydrous magnesium sulfate. Magnesium sulfate was removed by filtration, the solvent was distilled off, and the residue was purified by SiO ₂ column chromatography (chloroform: ethanol = 98: 2, Rf = 0.15), and the resulting crude product was washed with diethyl ether. As a result, the desired (E) -N-octyl-3- (4-hydroxy-3-methoxyphenyl) -2-propenamide (FAD1) was obtained (yield: 8.58 g, 54%).
¹ H NMR (400 MHz, DMSO-d ₆ , rt) δ: 9.39 (s, 1 H, OH), 7.90 (t, J (H, H) = 4 Hz, 1 H, NH), 7.28 (d, J (H, H) = 16Hz, 1H, ArCH = CH), 7.09 (s, 1H), 6.96 (d, J (H, H) = 8Hz, 1H, ArH), 6.77 (d, J (H, H) = 8Hz, 1H, ArH), 6.42 (d , J (H, H) = 16Hz, 1H, ArCH = CH), 3.79 (s, 3H, OCH 3), 3.13 (dd, J (H, H) = 4,8Hz, _{2H, NHCH 2), 1.42 (} t, J (H, H) = 8Hz, 2H, CH 2), 1.15-1.30 (10H, CH 2), 0.84 (t, J (H, H) = 4Hz, 3H, CH ₃ ).
¹³ C { ¹ H} NMR (100 MHz, DMSO-d ₆ , rt) δ: 165.2 (C = O), 156.8 (C ₆ H ₃ ), 147.8 (C ₆ H ₃ ), 138.7 (ArCH), 126.4 (C ₆ H ₃ ), 121.4 (C ₆ H ₃ ), 119.1 (ArCH = CH), 115.6 (C ₆ H ₃ ), 110.6 (C ₆ H ₃ ), 55.5 (OCH ₃ ), 38.6 (NCH ₂ ), 31.2 ( _{CH 2), 28.7 (CH 2} ), 28.6 (2CH 2), 26.5 (CH 2), 22.1 (CH 2), 13.9 (CH 3).
MS (MALDI TOFMS) m / z: 306 (M + H) ⁺ ; calcd for C ₁₈ H ₂₇ NO ₃ : 306.1 (M + H) ⁺ .
IR (ATR, rt): ν (cm ⁻¹ ) 3337 (NH), 1563, 1463 (CONH), 1253, 1035 (OCH ₃ ).

Synthesis of (E) -N-octyl-3- (3-methoxy-4-sodium phenoxide) -2-propenamide (FAD1-Na) In a vial, (E) -N-octyl-3- (4 -Hydroxy-3-methoxyphenyl) -2-propenamide (200 mg, 0.65 mmol) was dissolved in water, and 1N aqueous sodium hydroxide solution (0.65 mL, 0.65 mmol) was slowly added dropwise thereto and stirred at room temperature. . The solvent was distilled off by freeze-drying the reaction solution to obtain the desired (E) -N-octyl-3- (3-methoxy-4-sodium phenoxide) -2-propenamide (FAD1-Na). It was.
¹ H NMR (400 MHz, CD ₃ OD, rt) δ: 7.38 (d, J (H, H) = 16 Hz, 1H, ArCH = CH-), 6.94 (d, J (H, H) = 2 Hz, 1H, ArH), 6.90 (dd, J (H, H) = 2 and 8Hz, 1H, ArH), 6.57 (d, J (H, H) = 8Hz, 1H, ArH), 6.20 (d, J (H, H ) = 16Hz, 1H, ArCH = CH-), 3.79 (s, 3H, OCH ₃ ), 3.78 (t, J (H, H) = 8Hz, 2H, NHCH ₂ ), 1.55 (m, 2H, NHCH ₂ CH _{2), 1.3-1.7 (10H, CH} 2), 0.90 (t, J (H, H) = 8Hz, 3H, CH 3).
¹³ C NMR (100 MHz, CD ₃ OD, rt) δ: 170.1 (C═O), 159.9 (C ₆ H ₃ CH═CH), 152.1 (C ₆ H ₃ ), 143.4 (C ₆ H ₃ ), 124.9 ( C ₆ H ₃ ), 122.5 (C ₆ H ₃ ), 118.8 (C ₆ H ₃ ), 114.5 (C ₆ H ₃ CH = CH), 110.6 (C ₆ H ₃ ), 55.8 (CH ₂ ), 42.6 (OCH _{3), 40.5 (CH 2)} , 33.0 (CH 2), 30.6 (CH 2), 30.4 (CH 2), 30.3 (CH 2), 28.1 (CH 2), 14.4 (CH 3).
MS (MALDI TOFMS) m / z: 328.1 (M + H) ⁺ ; calcd for C ₁₈ H ₂₆ NNaO ₃ : 328.2 (M + H) ⁺ .
IR (ATR, rt): ν (cm ⁻¹ ) 3338 (NH), 1575, 1437 (CONH), 1229, 1029 (OCH ₃ ).

(Example 1-2) Synthesis of ferulic acid derivative 2 (FAD2) (see FIG. 1)
Synthesis of (E) -N-octyl-3- {3-methoxy-4- (sodium-3-propoxy-1-sulfonate)}-2-propenamide (FAD2) FAD1 (205 mg, 0.67 mmol in an eggplant flask ) Was dissolved in ultrapure water (5 mL), an aqueous sodium hydroxide solution (1N, 0.8 mL, 0.8 mmol) was added thereto, and the mixture was stirred at room temperature. Thereafter, 1.3-propane sultone (101 mg, 0.83 mmol) dissolved in THF (1 mL) was added dropwise to the reaction solution, and the mixture was stirred at room temperature for 16 hours. The reaction solution was lyophilized to distill off the solvent, and the crude product was washed with acetone to give the desired (E) -N-octyl-3- {3-methoxy-4- (sodium-3- Propoxy-1-sulfonate)}-2-propenamide (FAD2) was obtained (yield: 205 mg, 68%).
¹ H NMR (400 MHz, CD ₃ OD, rt) δ: 7.45 (d, J (H, H) = 6 Hz, 1H, ArCH = CH-), 7.15 (d, J (H, H) = 2 Hz, 1H, ArH), 7.11 (dd, J (H, H) = 2 and 8Hz, 1H, ArH), 6.98 (d, J (H, H) = 8Hz, 2H, ArH), 6.47 (d, J (H, H ) = 16Hz, 1H, ArCH = CH -), 4.17 (t, J (H, H) = 8Hz, CH 2), 3.87 (s, 3H, OCH 3), 3.86 (t, J (H, H) = _{8Hz, 2H, CH 2),} 3.00 (t, J (H, H) = 8Hz, 2H, CH 2), 2.27 (m, 2H, CH 2), 1.56 (m, 2H, NHCH 2 CH 2), 1.24 _{-1.44 (10H, CH 2),} 0.90 (t, J (H, H) = 8Hz, 3H, CH 3).
¹³ C NMR (100 MHz, CD ₃ OD, rt) δ: 169.0 (C = O), 151.5 (C ₆ H ₃ ), 151.1 (C ₆ H ₃ ), 141.5 (ArCH = CH-), 129.6 (C ₆ H ₃ ), 123.0 (C ₆ H ₃ ), 119.8 (C ₆ H ₃ ), 114.5 (ArCH = CH), 111.9 (C ₆ H ₃ ), 61.9 (CH ₂ ), 56.5 (CH ₂ ), 49.4 (CH ₂ ), 40.6 (OCH ₃ ), 33.0 (CH ₂ ), 30.5 (CH ₂ ), 30.4 (2CH ₂ ), 29.3 (CH ₂ ), 28.1 (CH ₂ ), 23.7 (CH ₂ ), 14.4 (CH ₃ ).
MS (MALDI TOFMS) m / z: 450.0 (M + H) ⁺ ; calcd for C ₂₁ H ₃₃ NNaSO ₆ : 450.12 (M + H) ⁺ .
IR (ATR, rt): ν (cm ⁻¹ ) 3296 (NH), 1613, 1467 (CONH), 1255, 1027 (OCH ₃ ), 1191 (SO ₃ Na).

(Example 1-3) Synthesis of ferulic acid derivative 3 (FAD3) (see FIG. 2)
Synthesis of 1H, 1H, 2H, 2H-perfluoro-n-octyl iodide 1H, 1H, 2H, 2H-tridecafluoro-1-n-octanol (13.9 g, 30.0 mmol) was added to diethyl ether (Et _It was dissolved in a mixed solvent of ₂ O) (75 mL) / acetonitrile (25 mL) and stirred at room temperature. To this, imidazole (6.13 g, 90.0 mmol), triphenylphosphine (PPh ₃ ) (11.8 g, 45.0 mmol) were added, and iodine (11.4 g, 45.0 mmol) was added while cooling in an ice bath. For 19 hours. Dichloromethane was added to the reaction solution, and the organic phase was washed four times with an aqueous sodium thiosulfate solution and further washed once with a saturated aqueous sodium chloride solution. The aqueous layer was extracted by adding dichloromethane, and the combined organic layer was washed with water and then dried over anhydrous magnesium sulfate. After magnesium sulfate is removed by filtration, the solvent is distilled off, and the residue is purified by SiO ₂ column chromatography (hexane, Rf = 0.76) to obtain the desired 1H, 1H, 2H, 2H-perfluoro-n-octyl iodide. (Yield: 1.18 g, 69%) was obtained.
¹ H NMR (400 MHz, CDCl ₃ , rt) δ: 2.74 (m, 2H, OCH ₂ CH ₂ -CF ₂ ), 3.24 (m, 2H, HOCH ₂ ).
_{^{19 F NMR (377MHz, CDCl 3}} , rt) δ: -80.8 (CF 3), - 114.9 (CH 2 CF 2), - 121.9 (CF 2), - 122.7 (CF 2), - 123.4 (CF 2), -126.1 (CF ₂ CF ₃ ).

Synthesis of (E) -3- {3-methoxy-4- (1H, 1H, 2H, 2H-perfluoro-n-octoxyphenyl)}-2-propenoic acid (FAD3) ferulic acid (847 mg , 4.36 mmol) in N, N-dimethylformamide (DMF) (10 mL), where tetrabutylammonium bromide (TBAB) (1.40 g, 8.71 mmol), 1H, 1H, 2H, 2H-perfluoro-n -Octyl iodide (5.00 g, 8.71 mmol) was added and stirred at 50 ° C for 10 minutes. To the reaction solution was added potassium hydroxide (733 mg, 13.1 mmol), and the mixture was further stirred at 70 ° C. for 30 minutes. Chloroform and dilute hydrochloric acid (a few drops) were added to the solution after the reaction, and the organic phase was washed 3 times with a saturated aqueous sodium chloride solution. After drying the organic layer over anhydrous magnesium sulfate and removing the magnesium sulfate by filtration, the solvent is distilled off and the residue is purified by SiO ₂ column chromatography (chloroform: hexane = 2: 1, Rf = 0.11). (E) -3- {3-methoxy-4- (1H, 1H, 2H, 2H-perfluoro-n-octoxyphenyl)}-2-propenoic acid (FAD3) was obtained (yield: 467 mg, 17 %).
¹ H NMR (400 MHz, DMSO-d ₆ , rt) δ: 9.61 (s, 1 H, COOH), 7.56 (d, J (H, H) = 16 Hz, 1 H, ArCH = CH—), 7.32 (d, J (H, H) = 2Hz, 1H, ArH), 7.10 (dd, J (H, H) = 2 and 8Hz, 1H, ArH), 6.78 (d, J (H, H) = 8Hz, 1H, ArH) , 6.46 (d, J (H, H) = 16Hz, 1H, ArCH = HCCO ₂ H), 4.42 (t, J (H, H) = 4Hz, OCH ₂ ), 3.80 (s, 3H, OCH ₃ ), 2.73 (m, 2H, OCH ₂ CH ₂ ).
^{19 F NMR (377Hz, DMSO-} d 6, rt) δ: -80.2 (CF 3), - 112.6 (CH 2 CF 2), - 121.7 (CF 2), - 122.4 (CF 2), - 123.1 (CF 2 ), -125.7 (CF ₂ ).
¹³ C NMR (100 MHz, DMSO-d ₆ , rt) δ: 166.1 (C = O), 149.5 (C ₆ H ₃ ), 147.9 (C ₆ H ₃ ), 145.6 (ArCH = CH), 125.3 (C ₆ H ₃ ), 123.0 (C ₆ H ₃ ), 115.4 (C ₆ H ₃ ), 113.4 (ArCH = CH), 111.0 (C ₆ H ₃ ), 55.6 (CH ₂ ), 55.4 (OCH ₃ ), 29.5 (CH ₂ ).
IR (ATR, rt): ν (cm ⁻¹ ) 3524, 1670 (COOH), 1263, 1060 (OCH ₃ ), 1241-1114 (CF).

Example 1-4 Synthesis of α-cyano-4-hydroxycinnamic acid derivative 4 (CHCA4) (see FIG. 3)
Synthesis of (E) -N-octyl-2-cyano-3- (4-hydroxyphenyl) -2-propenamide (CHCA4) α-cyano-4-hydroxycinnamic acid (CHCA) in a three-necked flask (4.7 g, 25.0 mmol) and octylamine (3.6 g, 27.9 mmol) were dissolved in dichloromethane (DCM) (300 mL) and stirred at room temperature. The reaction solution was cooled in an ice bath, where triethylamine (NEt ₃ ) (1.8 g, 17.5 mmol), 1-hydroxybenzotriazole (HOBt) (4.1 g, 30.0 mmol), diisopropylcarbodiimide (DIC) (6.9 g, 55.0 mmol) was added and stirred at room temperature for 1 day. Chloroform and hydrochloric acid (a few drops) were added to the reaction solution, and the organic phase was washed three times with a saturated aqueous sodium chloride solution. The aqueous layer was extracted with chloroform, and the combined organic layer was washed with water and dried over anhydrous magnesium sulfate. After removing magnesium sulfate by filtration, the solvent was distilled off and the residue was purified by SiO ₂ column chromatography (chloroform: ethanol = 98: 2, Rf = 0.19). Further, the obtained crude product was dissolved in ethanol, 3 N HCl aqueous solution was added, and the mixture was stirred at room temperature. After the solvent was distilled off, the desired (E) -N-octyl-2-cyano-3- (4-hydroxyphenyl) -2-propenamide (CHCA4) was obtained by washing the solid with water. (Yield: 3.45 g, 11.5 mmol, 41%).
¹ H NMR (400MHz, DMSO-d ₆ , rt) δ: 10.54 (s, 1H, OH), 8.25 (t, J (H, H) = 8Hz, 1H, NH), 8.00 (s, 1H, ArCH = C), 7.85 (d, J (H, H) = 12Hz, 2H, ArH), 6.92 (d, J (H, H) = 12Hz, 2H, ArH), 3.17 (m, 2H, NHCH ₂ ), 1.47 (t, J (H, H) = 8Hz, 2H, CH ₂ ), 1.15-1.30 (10H, CH ₂ ), 0.85 (t, J (H, H) = 4Hz, 3H, CH ₃ ).
¹³ C { ¹ H} NMR (100MHz, DMSO-d ₆ , rt) δ: 161.6 (C = O or HO-C), 161.3 (C = O or HO-C), 150.1 (C ₆ H ₄ CH = C ), 132.8 (C ₆ H ₄ ), 123.0 (C ₆ H ₄ ), 117.2 (CN), 116.2 (C ₆ H ₄ ), 101.4 (CH = CRCN), 39.6 (NCH ₂ ), 31.2 (CH ₂ ), 28.9 (CH ₂ ), 28.7 (CH ₂ ), 28.6 (CH ₂ ), 26.3 (CH ₂ ), 22.1 (CH ₂ ), 13.9 (CH ₃ ).
MS (MALDI TOFMS) m / z: 301.5 (M + H) ⁺ ; calcd for C ₁₈ H ₂₅ N ₂ O ₂ : 301.9 (M + H) ⁺ .

(Example 1-5) Synthesis of gentisic acid derivative 5 (DHB5) (see FIG. 3)
Synthesis of 2,5-dihydroxybenzoylamide octane (DHB5)
In a 1000 mL three-necked flask, gentisic acid (DHB) (5.4 g, 35.0 mmol) and octylamine (5.0 g, 38.5 mmol) were dissolved in dichloromethane (DCM) (500 mL) and stirred at room temperature. The reaction solution was cooled in an ice bath, where triethylamine (NEt ₃ ) (2.5 g, 24.5 mmol), 1-hydroxybenzotriazole (HOBt) (5.7 g, 42.0 mmol), diisopropylcarbodiimide (DIC) (9.7 g, 77.0 mmol) was added and stirred at room temperature for 1 day. Chloroform and hydrochloric acid (a few drops) were added to the reaction solution, and the organic phase was washed three times with a saturated aqueous sodium chloride solution. The aqueous layer was extracted with chloroform, and the combined organic layer was washed with water and dried over anhydrous magnesium sulfate. Magnesium sulfate was removed by filtration, the solvent was distilled off, and the residue was purified by SiO ₂ column chromatography (chloroform: ethanol = 96: 4, Rf = 0.43). Further, the obtained crude product was dissolved in ethanol, 3 N HCl aqueous solution was added, and the mixture was stirred at room temperature. After the solvent was distilled off, the intended 2,5-dihydroxybenzoylamidooctane (DHB5) was obtained by washing the solid with water (yield: 2.67 g, 10.1 mmol, 29%).
¹ H NMR (400 MHz, DMSO-d ₆ , rt) δ: 11.8 (s, 1H, OH), 8.96 (s, 1H, OH), 8.64 (t, J (H, H) = 8Hz, 4H, NH ), 7.21 (d, J (H, H) = 8Hz, 1H, ArH), 6.84 (dd, J (H, H) = 8 and 2Hz, 1H, ArH), 6.70, (d, J (H, H ) = 8Hz, 1H, ArH), 3.23 (m, 2H, NHCH ₂ ), 1.50 (t, J (H, H) = 8 Hz, 2H, CH ₂ ), 1.15-1.30 (10H, CH ₂ ), 0.84 (t, J (H, H) = 4Hz, 3H, CH ₃ ).
¹³ C { ¹ H} NMR (100 MHz, DMSO-d ₆ , rt) δ: 168.4 (C = O), 152.4 (C ₆ H ₃ ), 149.1 (C ₆ H ₃ ), 121.1 (C ₆ H ₃ ), 117.7 (C ₆ H ₃ ), 115.6 (C ₆ H ₃ ), 113.2 (C ₆ H ₃ ), 40.6 (NCH ₂ ), 31.2 (CH ₂ ), 28.8 (CH ₂ ), 28.7 (CH ₂ ), 28.6 ( CH ₂ ), 26.4 (CH ₂ ), 22.1 (CH ₂ ), 13.9 (CH ₃ ).
MS (MALDI TOFMS) m / z: 266.0 (M + H) ⁺ ; calcd for C ₁₅ H ₂₄ NO ₃ : 266.2 (M + H) ⁺ .

Example 1-6 Synthesis of sinapinic acid derivative 6 (SA6) (see FIG. 3)
Synthesis of (E) -N-octyl-3- (4-hydroxy-3,5-dimethoxyphenyl) -2-propenamide (SA6) sinapinic acid (SA) (488 mg, 2.2 mmol) in a three-necked flask And octylamine (282 mg, 2.2 mmol) were dissolved in dichloromethane (DCM) (100 mL) and stirred at room temperature. The reaction solution was cooled in an ice bath, where triethylamine (NEt ₃ ) (141 mg, 1.4 mmol), 1-hydroxybenzotriazole (HOBt) (324 mg, 2.4 mmol), diisopropylcarbodiimide (DIC) (555 mg, 4.4 mmol) And stirred at room temperature for 1 day. Chloroform and hydrochloric acid (a few drops) were added to the reaction solution, and the organic phase was washed three times with a saturated aqueous sodium chloride solution. The aqueous layer was extracted with chloroform, and the combined organic layer was washed with water and dried over anhydrous magnesium sulfate. After removing magnesium sulfate by filtration, the solvent was distilled off and the residue was purified by SiO ₂ column chromatography (chloroform: ethanol = 98: 2, Rf = 0.19). Further, the obtained crude product was dissolved in ethanol, 3 N HCl aqueous solution was added, and the mixture was stirred at room temperature. After the solvent is distilled off, the desired solid ((E) -N-octyl-3- (4-hydroxy-3,5-dimethoxyphenyl) -2-propenamide (SA6) is washed with water. (Yield: 418 mg, 1.2 mmol, 55%).
¹ H NMR (400 MHz, DMSO-d ₆ , rt) δ: 8.75 (s, 1H, OH), 7.90 (t, J (H, H) = 4Hz, 1H, NH), 7.38 (d, J (H , H) = 16Hz, 1H, ArCH = CH), 7.29, (d, J (H, H) = 16Hz, 1H, ArCH = CH), 6.83 (s 2H, ArH), 6.45 (d, J (H, H) = 16Hz, 1H, ArCH = CH), 3.78 (s, 6H, OCH ₃ ), 3.12 (m, 2H, NHCH ₂ ), 1.42 (t, J (H, H) = 8Hz, 2H, CH ₂ ) , 1.15-1.30 (10H, CH ₂ ), 0.85 (t, J (H, H) = 4Hz, 3H, CH ₃ ).
¹³ C { ¹ H} NMR (100 MHz, DMSO-d ₆ , rt) δ: 165.1 (C = O), 148.0 (C ₆ H ₂ ), 139.0 (ArCH = CH), 137.2 (C ₆ H ₂ ), 125.3 (C ₆ H ₂ ), 119.5 (ArCH = CH), 105.1 (C ₆ H ₃ ), 56.0 (OCH ₃ ), 38.6 (NCH ₂ ), 31.2 (CH ₂ ), 29.2 (CH ₂ ), 28.7 (2CH ₂ ), 26.5 (CH ₂ ), 22.1 (CH ₂ ), 13.9 (CH ₃ ).
MS (MALDI TOFMS) m / z: 336.0 (M + H) ⁺ ; calcd for C ₁₉ H ₃₀ NO ₄ : 336.0 (M + H) ⁺ .

(Example 2) <Surface activity of synthesized matrix>
Water: acetonitrile = 4: 1, FAD1-Na was added to the vial to adjust the concentration to 100 mM. After the solution was stirred, the solution was diluted to 75, 50, 25, 10, 7.5, 5, 2.5, and 1 mM. The surface tension of each concentration of FAD1-Na with respect to water: acetonitrile = 4: 1 was measured using a pendant drop surface tension measuring device (product name “Drop Master-500” manufactured by Kyowa Interface Science Co., Ltd.). The needle used was a Teflon (registered trademark) coated needle PD 22G, and Young Laplace's formula was used for the analysis. The result is shown in FIG.
FAD1-Na exhibits interfacial activity because the surface tension of the mixed solvent is reduced, and the critical micelle concentration (CMC) value is 7.2 mM because the surface tension value in FIG. 4 is constant. It was found that the tension value γCMC value was 29.9 mN / m. Furthermore, surface tension was measured for FAD2 and FAD3. As the solvent, water was used for FAD2 and water: acetonitrile = 1: 1 for FAD3. As a result, the CMC value of FAD2 was 1.49 mM, the γCMC value was 36.5 mN / m, the CMC value of FAD3 was 0.197 mM, and γCMC was 16.2 mN / m. Therefore, it was revealed that all of the obtained new matrices were surface active.
In addition, using a dynamic light scattering device (product name “DLS-8000”, manufactured by Otsuka Electronics Co., Ltd.), the scattering intensity at each concentration was measured. At that time, an Ar laser (λ = 488 nm) was used as the light source, and the scattering angle was set to 90 degrees. The solution was passed through a filter (Merck Millipore, 0.45 μm, PVDF) three times. The results are also shown in FIG. FAD1-Na showed a sharp increase in scattering intensity above CMC, indicating that FAD1-Na forms micelles at concentrations above CMC.
In addition, curcumin polyphenols (manufactured by Wako Pure Chemical Industries, Ltd.) were used as hydrophobic compounds, and the solubilizing ability to FAD micelles was examined. Weigh curcumin (5 mg, 14 mM) into a microtube, add 1 mL of each concentration of FAD1-Na solution (prepared as above), incubate for 24 hours (25 ° C, 150 r / min), then centrifuge for 5 minutes (9000 rpm) was performed. The absorbance of the upper layer solution at 429 nm (manufactured by JASCO Corporation, product name “V-560”) was measured, and the amount of curcumin solubilized in FAD1 micelles was calculated. The result is shown in FIG. Above CMC, the amount of curcumin solubilized in FAD1-Na micelles increased rapidly, indicating that curcumin was solubilized in micelles. Furthermore, the solubilization amount was improved about 100 times or more by the formation of micelles (see FIG. 5. The curcumin solubilization amount was about 0.03 mM when the matrix concentration was 1 mM of the monomer region, whereas the matrix in the micelle region was The concentration is about 3.5 mM). When the amount of curcumin solubilized by FAD2 was also measured, the amount of solubilization improved by about 10 times or more due to micelle formation.

(Example 3) <Crystallinity of novel matrix> (See FIGS. 6 to 8)
Water: acetonitrile = 4: 1, FAD1-Na was added to the vial, and the concentration was adjusted to 5, 25 mM. After 1 μL of these solutions were dropped on a MALDI substrate and dried, SEM observation (manufactured by JOEL, product name “JSM-6010LA”) was performed. The result is shown in FIG. Crystals that were not observed at concentrations below CMC of FAD1-Na were observed at concentrations above CMC. Next, FIG. 7 shows the result of SEM observation at 25 mM for ferulic acid sodium salt that has not been amphiphilized. The crystals observed with FAD1-Na were not observed with the sodium salt of ferulic acid, suggesting that micelle formation and the introduction of amide bonds into the structure contributed to improved crystallinity.
Also, weigh curcumin (5 mg, 14 mM) in a microtube, add 1 mL of 0.1, 1, 10 mM FAD1-Na solution, incubate for 24 hours (25 ° C, 150 r / min), then centrifuge for 5 minutes (9000 rpm) ) This upper layer solution in which the analyte was solubilized in the matrix micelle was also subjected to SEM observation in the same manner as described above. The result is shown in FIG. Even with normal surfactants, it is known that CMC decreases when a hydrophobic substance is added, but crystals were not observed at 0.1 mM, whereas at 1 mM, which is about 10% of the CMC value. As a result, cocrystals were obtained, and better cocrystals were observed at 10 mM above the CMC value. From this, at a concentration of 10% or more of the CMC value, the co-crystal can be obtained by solubilizing the analyte in the matrix, and at a concentration above the CMC value, by solubilizing the analyte in the matrix micelle, It has been found that better co-crystals can be obtained.

  In addition, although the above Examples 2 and 3 are related to the surface activity and crystallinity of the derivative of ferulic acid, α-cyano-4-hydroxycinnamic acid, sinapinic acid, which are existing MALDI mass spectrometry matrices, As for caffeic acid and gentisic acid, as in the case of the ferulic acid derivative, when derivatizing without changing the basic structure of these organic acid compounds to be amphiphilized and surface active, (a) laser light (C) Solubility of sample to be detected and (d) Sample to be detected through acquisition of surface activity ability and micellar performance while maintaining (b) stability under high vacuum It is thought that the eutectic formation ability of the can be improved.

(Example 4) <MALDITOFMS spectrum of novel matrix> (See FIGS. 9 and 10)
Water: acetonitrile = 4: 1, FAD1-Na was added to the microtube to adjust the concentration to 5, 50 mM. 1 μL of a solution of each concentration was dropped on a MALDI substrate and dried, and then measured using a MALDI TOF-MS (time-of-flight mass spectrometry) apparatus (manufactured by Bruker, apparatus name “Autoflex speed TOF / TOF”). At that time, using a YAG laser (355 nm), measurement was performed in a reflector positive mode (acceleration voltage: 20 kV, extraction delay time: 130 ns, laser power: 80%), and number of integrations: 5 times. The result is shown in FIG. At any concentration, a molecular ion peak of FAD1-Na was observed, and further, a peak of H ⁺ adduct was observed at 5 mM, and a peak of Na ⁺ adduct was observed at 50 mM. In order to conduct a more detailed study, the concentration of FAD1-Na was adjusted to 50, 25, 10, 7.5, 5, 2.5, 1 mM, MALDI TOF-MS measurement was performed, and H ⁺ and Na ⁺ adducts were measured. The relationship between each peak intensity and the concentration of FAD1-Na was examined. The result is shown in FIG. As is clear from the figure, the peak intensity of the Na ⁺ adduct increased rapidly above CMC. This is thought to be because Na ⁺ is bound to the micelle surface due to micelle formation of FAD1-Na.

(Example 5) <MALDITOFMS spectrum of an analyte using a novel matrix> (see FIGS. 11 and 13)
Water: acetonitrile = 4: 1, FAD1 was added to the microtube to adjust the concentration to 0.1, 1, 10 mM. In addition, weigh curcumin (5 mg, 14 mM) into another microtube, add 1 mL of each concentration of FAD1 solution, incubate for 24 hours (25 ° C, 150 r / min), then centrifuge for 5 minutes (5 min, 9000 rpm) Went. 1 μL of the upper layer solution was dropped onto a MALDI substrate and dried, and then MALDI TOF-MS measurement of curcumin was performed using FAD1-Na as a matrix. The molar ratio of the matrix (FAD1-Na) micelle and the solubilized analyte (curcumin) at this time was as follows: matrix: analyte = 3: 1, 8: 1, 14: 1. The result is shown in FIG. The molecular ion peak of curcumin was hardly observed at FAD1-Na concentration of 0.1 mM, while it was observed at 1 and 10 mM. Furthermore, as in the case of FAD1-Na, in the molecular ion peak of curcumin, the peak of H ⁺ adduct is below CMC (1 mM) of FAD1-Na, and the peak of Na ⁺ adduct is mainly above CMC (10 mM). was detected. This is thought to be because solubilized curcumin added Na ⁺ bound to matrix micelles.
By the same operation as above, MALDI TOF-MS measurement of curcumin was performed using ferulic acid as a matrix. The result is shown in FIG. When ferulic acid was used as a matrix, no molecular ion peak of curcumin was observed at matrix concentrations of 0.1 and 1 mM, while a weak molecular ion peak was observed at 10 mM, and its detection sensitivity was FAD1- Compared to the case where Na was used as a matrix, it was about 1/30.
Next, water: acetonitrile = 1: 1 (0.1% TFA), FAD1 protonated with acid or ferulic acid were added to the microtube to prepare a concentration of 25 mM, respectively. Furthermore, angiotensin I (0.154 nmol), FAD1 and ferulic acid solution were added to another tube. At that time, FAD1: ferulic acid was added in a volume ratio of 0:10, 2: 8, 4: 6, 6: 4, 8: 2, 9: 1, 9.9: 1.1, 10: 0, and the total amount Was adjusted to 100 μL. Using a mixed system of FAD1 and ferulic acid as a matrix, MALDI TOF-MS measurement of angiotensin I was performed in the same manner as described above. FIG. 13 shows the relationship between the observed peak intensity of the molecular ion peak of angiotensin I and the molar fractions of FAD1 and ferulic acid. When FAD1: ferulic acid = 4: 6, the molecular ion peak of angiotensin I was observed with the highest sensitivity. This indicates that the ferulic acid derivative improves the detection sensitivity of the analyte in the MALDITOFMS spectrum when mixed with the precursor ferulic acid.
Further, water: acetonitrile = 1: 1 (0.1% TFA), protonated FAD1 or ferulic acid was added to the microtube to prepare a concentration of 25 mM. Further, poorly soluble β-amyloid (7.55 pmol) or humanin (1.04 pmol) was added to another microtube, and FAD1 solution: ferulic acid solution = 1: 1 (50 μL) was further added. Using a mixed system of FAD1 and ferulic acid (1: 1) as a matrix, MALDI TOF-MS measurement of β-amyloid and humanin was performed in the same manner as described above. As a result, molecular ion peaks of β-amyloid and humanin were observed, and it was also clarified that the peak intensities of both samples were improved compared to the case where FAD1 or ferulic acid was used alone.

(Example 6) <MALDITOFMS spectra of various analytes using a novel matrix>
Water: acetonitrile = 4: 1, ferulic acid derivative (FAD1) or CHCA derivative (CHCA4) was added to the microtube to adjust the concentration to 10 mg / mL. The sinapinic acid derivative (SA6) was adjusted to a concentration of 2.5 mg / mL. Furthermore, water: acetonitrile = 4: 1, curcumin, α-cyclodextrin, TritonX-100, isoflavone, pyrroloquinoline quinone disodium, rutin, coenzyme Q10, or catechin is added to another microtube at a concentration of 10 mg / Adjusted to mL. Various prepared surfactant matrix solutions (5 mL) and various analyte solutions (1 mL) were mixed, 1 μL, dropped on a MALDI substrate, dried, and then subjected to MALDI TOF-MS measurement.
As a result, for ferulic acid derivative (FAD1), α-cyclodextrin, TritonX-100 and isoflavone were well detected in addition to curcumin. As for the CHCA derivative (CHCA4), α-cyclodextrin, TritonX-100 and rutin were well detected. Regarding the sinapinic acid derivative (SA6), it was found that α-cyclodextrin, TritonX-100, isoflavone and rutin were well detected.

  The novel organic acid derivative of the present invention is an amphiphilic and surface active type, and spontaneously forms micelles in various solvents. Therefore, it is difficult to start with membrane proteins, peptides, polyphenols, etc. Soluble analytes can also be solubilized and can form good co-crystals with analytes, so it can be effectively used as a matrix for MALDI mass spectrometry of various analytes including the above-mentioned poorly soluble samples. it can.

Claims (8)

  A surface active matrix for MALDI mass spectrometry comprising a derivative of an organic acid selected from the group consisting of ferulic acid, α-cyano-4-hydroxycinnamic acid, sinapinic acid, caffeic acid, and gentisic acid, A surface active matrix for MALDI mass spectrometry, comprising an amphiphilic organic acid derivative obtained by hydrophilizing or hydrophobizing a carboxyl group and / or a phenol group of an organic acid. Surface active matrix for MALDI mass spectrometry comprising an amphiphilic organic acid derivative represented by the following general formula (1) (where R ² is a hydrogen atom, OH, OX, or O (CH ₂ ) _k SO ₃ When X is R ⁵ is NH (CH ₂ ) _m CH ₃ or O (CH ₂ ) _n CH ₃ , and R ² is O (CH ₂ ) ₂ (CF ₂ ) _l CF ₃ , R 5 ⁵ is OH or OX.)

[In general formula (1),
R ¹ is a hydrogen atom or a methoxy group,
R ² represents a hydrogen atom, OH, OX, O (CH ₂ ) _k SO ₃ X, or O (CH ₂ ) ₂ (CF ₂ ) ₁ CF ₃ ,
R ³ is a hydrogen atom, OH, or a methoxy group,
R ⁴ is a hydrogen atom or CN,
R ⁵ is OH, OX, NH (CH ₂ ) _m CH ₃ , or O (CH ₂ ) _n CH ₃ ,
(In R ² and R ⁵ , X is an alkali metal selected from Li, Na, K, Rb and Cs, k is an integer of 2 to 8, l is an integer of 3 to 17, and m is 3 to 3) An integer of 17 and n is an integer of 3 to 17.) ] One of R ¹ and R ³ is a hydrogen atom, the other is a methoxy group, R ² is OX or O (CH ₂ ) _k SO ₃ X, R ⁴ is a hydrogen atom, and R ⁵ is NH The surface active matrix for MALDI mass spectrometry according to claim 2, which is (CH ₂ ) _m CH ₃ .   It is selected from the group consisting of the surface active matrix according to any one of claims 1 to 3, ferulic acid, α-cyano-4-hydroxycinnamic acid, sinapinic acid, caffeic acid, and gentisic acid. A matrix for MALDI mass spectrometry comprising a mixture of one or more organic acids.   The MALDI mass spectrometry method using the matrix of any one of Claims 1-4.   6. The MALDI mass spectrometry method according to claim 5, wherein the surface active matrix is set to 10% or more of the critical micelle concentration when preparing a mixture comprising a solvent, a sample to be analyzed, and the matrix. MALDI mass spectrometry method.   The MALDI mass spectrometry method according to claim 5 or 6, wherein the sample to be analyzed is selected from the group consisting of proteins, peptides, nucleic acids, saccharides, polymers, lipids, and organic compounds. An organic acid derivative represented by the following general formula (1) (where R ² is a hydrogen atom, OH, OX, or O (CH ₂ ) _k SO ₃ X, R ⁵ is NH (CH ₂ ) _m CH _3, or a _{O (CH 2) n CH 3} , when R ² is _{O (CH 2) 2 (CF} 2) l CF 3, R 5 is, OH, or an OX .R ⁵ Except for an organic acid derivative in which is NH (CH ₂ ) _m CH ₃ or O (CH ₂ ) _n CH ₃ and R ² is a hydrogen atom or OH.

[In general formula (1),
R ¹ is a hydrogen atom or a methoxy group,
R ² represents a hydrogen atom, OH, OX, O (CH ₂ ) _k SO ₃ X, or O (CH ₂ ) ₂ (CF ₂ ) ₁ CF ₃ ,
R ³ is a hydrogen atom, OH, or a methoxy group,
R ⁴ is a hydrogen atom or CN,
R ⁵ is OH, OX, NH (CH ₂ ) _m CH ₃ , or O (CH ₂ ) _n CH ₃ ,
(In R ² and R ⁵ , X is an alkali metal selected from Li, Na, K, Rb and Cs, k is an integer of 2 to 8, l is an integer of 3 to 17, and m is 3 to 3) An integer of 17 and n is an integer of 3 to 17.) ]

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