JP2006036693A - Compound for labeling aldehyde compound, method for analyzing aldehyde compound, and labeled sugar - Google Patents

Abstract

（式中、Ｒ₁ 、Ｒ₂及びＲ₃はそれぞれ独立に置換されていても良い炭化水素基を表し、Ｒ₁ 、Ｒ₂及びＲ₃の少なくとも２ヶが結合して含窒素複素環を形成していても良い。Ｘ―は対アニオンを表す。ベンゼン環の２−，４−，５−，及び６−位はそれぞれ独立に置換基で置換されていても良い。）
【選択図】 なしPROBLEM TO BE SOLVED: To provide an aldehyde compound labeling compound capable of increasing the sensitivity of mass analysis of an aldehyde compound, and a method for analyzing an aldehyde compound capable of detecting and quantifying a trace amount of aldehyde compound, particularly a trace amount of sugar chain.
A labeling compound for an aldehyde compound comprising a 3-aminobenzylammonium salt represented by the following general formula (I).

(In the formula, R ₁ , R ₂ and R ₃ each independently represents a hydrocarbon group which may be substituted, and at least two of R ₁ , R ₂ and R ₃ are combined to form a nitrogen-containing heterocycle. X- represents a counter anion, and the 2-, 4-, 5-, and 6-positions of the benzene ring may be each independently substituted with a substituent.)
[Selection figure] None

Description

  The present invention relates to a compound for labeling an aldehyde compound, a method for analyzing an aldehyde compound using the compound, and a labeled sugar. More specifically, the present invention relates to in vivo aldehyde compounds in the diagnostic field, particularly aldehyde compound labeling compounds suitable for detecting saccharides with high sensitivity, aldehyde compound analysis methods using the same, and labeled sugars. is there.

  Glycoprotein sugar chains (aldehyde compounds) are used as important markers in the diagnostic field, particularly in cancer diagnosis. Tumor markers such as sialyl-Lewis X (SLX), sialyl Tn, CA19-9, and CA50 have already been clinically used. Applied in the field. However, these tumor markers may be present in a small amount or sometimes at a high concentration even in normal persons, and it has been difficult to determine cancer using only these test agents.

In recent years, in order to solve this problem, an attempt by an analytical chemistry method for separating and detecting a sugar chain itself, rather than an antibody detection method that recognizes only a part of the sugar chain functional groups, has been spreading.
The analysis method of glycoprotein sugar chains is to analyze the glycoproteins that are excised from glycoproteins, labeled with fluorescent compounds, separated by normal phase liquid chromatography and reverse phase liquid chromatography, and detected by a fluorescence detector. The method is general (Non-Patent Document 1).

However, in the method combining the normal phase liquid chromatography and the reverse phase liquid chromatography, it is difficult to completely separate a plurality of sugar chains, and there is a problem in quantitativeness.
In recent years, with the development of mass spectrometers, the effectiveness of mass spectrometry has been recognized in the analysis of biopolymers, particularly proteins. A mass spectrometer can separate a plurality of molecular groups with a slight difference in mass number, and is an excellent analysis method particularly for separating a plurality of mixtures such as biopolymers. However, sugar chains have difficulty in ionization, and high sensitivity cannot be obtained like proteins.

  Under such circumstances, some labeling compounds as sugar chain derivative reagents have been developed for the purpose of improving the mass spectrometry sensitivity of sugar chains, and their application to mass spectrometry is progressing. 2-Aminopyridine is known as the most widely used labeling compound. Besides, 2-aminobenzamide, 4-aminobenzoic acid ethyl ester (ABEE), 4-aminobenzoic acid n-butyl ester (ABBE). ), Trimethylaminophenylammonium (TMAPA) and the like are known (Non-Patent Document 2). In particular, it is known that TMAPA significantly improves the sensitivity with respect to unlabeled sugar chains or other labeling compounds in the electrospray ionization method (Non-patent Document 3).

However, the amount of sugar chains in vivo is very small, and there are still many sugar chains that cannot be detected even using TMAPA, which has the highest sensitivity increasing effect. Also, TMAPA is very expensive and very difficult to synthesize, so that this reagent was not suitable for direct application to diagnosis and the like.
Hase S. Ikenaka T .; , Matsushima Y .; J .; Biochem. , 90, 407-414 (1981) Harvey D.H. J. et al. J .; Am. Soc. Mass. Spectrom. , 11, 900-915 (2000) Okamoto M.M. , Takahashi K .; , Doi T. Rapid Commun. Mass Spectrom. , 9, 641-643 (1995)

The present invention has been made in view of the above problems, and provides a labeling compound that enables highly sensitive analysis of aldehyde compounds such as sugar chains derived from glycoproteins. It aims at providing the analytical method which detects aldehyde compounds, such as.
It is another object of the present invention to provide a sugar labeled with the labeling compound, which is useful as a standard substance for mass spectrometry of a sugar that is a kind of aldehyde compound.

  As a result of investigations to solve the above-mentioned problems, the present inventors have made quaternary ammonium salts having a specific structure a labeling compound for aldehyde compound analysis, thereby significantly improving the analytical sensitivity of aldehyde compounds such as sugar chains. The present invention has been reached by finding higher. Furthermore, the present inventors have found that a saccharide labeled with the above-described labeling compound is useful as a standard substance for saccharide mass spectrometry, and reached the present invention.

  That is, the gist of the present invention resides in a labeling compound for an aldehyde compound comprising a 3-aminobenzylammonium salt represented by the following general formula (I).

(In the formula, R ₁ , R ₂ and R ₃ each independently represents a hydrocarbon group which may be substituted, and at least two of R ₁ , R ₂ and R ₃ are combined to form a nitrogen-containing heterocycle. X- represents a counter anion, and the 2-, 4-, 5-, and 6-positions of the benzene ring may be each independently substituted with a substituent.)
In another aspect, the 3-aminobenzylammonium salt represented by the general formula (I) is added to an aldehyde compound by a reductive amination reaction, and the obtained reaction product is analyzed by mass spectrometry. And an analysis method for an aldehyde compound.
Still another gist lies in a labeled saccharide characterized in that 3-aminobenzylammonium salt represented by the general formula (I) is added to the reducing end of the saccharide.

The 3-aminobenzylammonium salt of the present invention is effective as a labeling compound for an aldehyde compound, and it is possible to increase the sensitivity of mass analysis of the aldehyde compound by binding it to the aldehyde compound by a reductive amination method. As a result, it is possible to detect and quantify trace aldehyde compounds, particularly trace sugar chains.
Further, the labeled sugar of the present invention can facilitate identification or quantitative analysis of sugars such as unknown sugar chains.

<Labeling compound>
In the present invention, the labeling compound for the aldehyde compound is a compound having reactivity with an aldehyde used for the purpose of facilitating detection of a trace amount of aldehyde compound by a mass spectrometer by reacting with the aldehyde compound.
The compound for labeling aldehyde compounds of the present invention comprises 3-aminobenzylammonium salts represented by the following general formula (I).

(In the formula, R ₁ , R ₂ and R ₃ each independently represents a hydrocarbon group which may be substituted, but at least two of R ₁ , R ₂ and R ₃ are bonded to form a nitrogen-containing heterocyclic ring. X- represents a counter anion, and the 2-, 4-, 5-, and 6-positions of the benzene ring may be each independently substituted with a substituent.)
Examples of the hydrocarbon group represented by R ₁ , R ₂ and R ₃ include a straight or branched alkyl group having 1 to 6 carbon atoms such as a methyl group, an ethyl group and a propyl group, a cyclopentyl group, a cyclohexyl group and a cycloheptyl group. Examples thereof include cycloalkyl groups having 5 to 7 carbon atoms such as groups, and aryls having 6 to 12 carbon atoms such as phenyl groups.

Further, at least two of R ₁ , R ₂ and R ₃ may be bonded to form a nitrogen-containing heterocycle, and examples of such nitrogen-containing heterocycle include pyridyl group, piperidyl group, pyrazine group, pyrimidine group, And aromatic heterocyclic groups such as a pyridazine group and a triazine group.
Since the aldehydes labeled with the labeling compound of the present invention are ionized and detected in mass spectrometry, the labeling compound represented by the general formula (I) has a quaternary ammonium structure moiety. is there.

Among the above, R ₁ , R ₂ and R ₃ are preferably alkyl groups, particularly preferably methyl groups and ethyl groups, and most preferably methyl groups from the viewpoint of the stability of the labeling compound. R ₁ , R ₂ and R ₃ may be the same or different, but are preferably the same from the viewpoint of synthesis.
In Formula (I), the 2-, 4-, 5-, and 6-positions of the benzene ring to which the amino group is bonded may be independently substituted with a substituent. The substituent is a substituent that does not interfere with the reaction between the labeling compound represented by the general formula (I) described later and the aldehyde compound, and does not reduce the sensitivity of mass spectrometry, such as an alkyl group, a hydroxyl group, An aryl group, halogen, etc. are mentioned.
Further, the counter anion represented by X- is not particularly limited, and examples thereof include halogen ions such as chloro ion, brom ion and iodo ion, carboxylate ions such as acetate ion, and the like. Halogen ions such as bromo ions and iodo ions are desirable.
In addition, the compound represented by the general formula (I) is prepared according to a known method, for example, by reacting 3-nitrobenzyl chloride with an equimolar amine compound such as trimethylamine, and the like such as 3-nitrobenzyltrimethylammonium salt. A compound in which the amino group in formula (I) is a nitro group is obtained, and this can be obtained by a method such as heating in hydrochloric acid in the presence of a reducing catalyst such as iron.

<Aldehyde compound>
The labeling compound comprising the 3-aminobenzylammonium salt represented by the general formula (I) of the present invention obtains an aldehyde compound labeled by a reductive amination reaction of the aldehyde compound and provides it for mass spectrometry Is possible. Accordingly, the aldehyde compound is not particularly limited as long as it is an organic compound having an aldehyde group, but saccharides are preferable, and saccharides may be either monosaccharides or polysaccharides. Examples of monosaccharides include glucose, galactose, fucose, mannose, xylose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, N-glycolylneuraminic acid, and polysaccharides include N -Linked Biantenary, Triantenary, Tetrantenary sugar chain and the like. The labeling compound comprising 3-aminobenzylammonium salts represented by the general formula (I) is particularly effective for analysis of N-linked sugar chains or O-linked sugar chains bound to glycoproteins. Therefore, glycoproteins contained in blood derived from the human body, glycoproteins expressed in organs, and the like can be analyzed. It is also effective for sugar chain analysis in biopharmaceuticals such as hormones and antibodies.

When the aldehyde compound is a saccharide derived from glycoprotein, for example, Hase S. Ikenaka T .; , Matsushima Y .; J .; Biochem. , 90, 407-414 (1981), and Sugiyama K. et al. , Ishihara H. , Takahashi N .; , Biochem. Biophis. Res. Commun. , 112 (1), 155-160 (1983), etc., free glycans obtained from glycoproteins by hydrazine decomposition or enzymatic digestion can be used.
<Method for analyzing aldehyde compound>
In the method for analyzing an aldehyde compound of the present invention, 3-aminobenzylammonium salt represented by the general formula (I) is added to the aldehyde compound by a reductive amination reaction, and the resulting reaction product is analyzed by mass spectrometry. To analyze.

  The reductive amination reaction is represented by, for example, the following reaction formula (II) to obtain a reductive amination product of an aldehyde.

(In the above formula, Q represents an organic group constituting an aldehyde compound together with a formyl group, and R ₁ , R ₂ , R ₃ and X- have the same meanings as in the formula (I).)
The use ratio of the 3-aminobenzylammonium salt represented by the general formula (I) is 1 mol or more, preferably 5 mol or more with respect to 1 mol of the aldehyde compound, and the upper limit is not particularly limited. In this case, the amount is usually 10 mol because it is separated and removed without contributing to the reductive amination reaction. In addition, the above reductive amination reaction is usually preferably performed in the presence of a reducing agent such as sodium cyanoborohydride and dimethylamine borane from the viewpoint of the equilibrium of the reaction. The ratio of the reducing agent to be used is usually 1 mol or more, preferably 5 mol or more with respect to 1 mol of the aldehyde compound, and although the upper limit is not particularly limited, it is usually about 10 mol.

The reaction temperature is usually 70 ° C. or higher and 100 ° C. or lower, preferably 90 ° C. or higher. The reaction time is usually 20 minutes or more and 3 hours or less, but if the reaction is continued for a long time, the sugar chain may be decomposed, preferably 30 minutes or more, while 1 hour is preferred.
The reaction is usually performed in a solvent that dissolves the aldehyde compound and the 3-aminobenzylammonium salt represented by the general formula (I), and is not particularly limited. However, a mixed solvent of water and acetic acid is preferable. On the other hand, acetic acid is preferably 2% by volume or more and 60% by volume or less, and particularly preferably 5% by volume or more and 20% by volume or less. The concentration of the aldehyde compound relative to the solvent is not particularly limited, but is usually 1 nanomol / liter or more and 100 mmol / liter or less, particularly preferably 10 mmol / liter or less.

<Analysis sample purification and mass spectrometry>
A reaction solution containing a reductive amination product (labeled sugar or the like) of an aldehyde is usually purified to remove unreacted products prior to analysis. Specifically, for example, an unreacted reducing agent and 3-aminobenzylammonium represented by the above general formula (I) are obtained by adding the reaction liquid to a graphite carbon column and washing the column with an ammonium acetate buffer. Remove salt. The reductive aminated product adsorbed on the column can be recovered, for example, by passing a buffer solution containing acetonitrile. Next, the obtained reductive amination compound (labeled sugar, etc.) of the aldehyde is subjected to sialic acid digestion with neuraminidase enzyme as it is or according to a known method, and separated by mass spectrometry according to a conventional method. Quantify. The mass spectrometry may be a liquid chromatography / mass spectrometer (LC-MS), a capillary electrophoresis / mass spectrometer, or simply a mass spectrometer, but is preferably a liquid chromatography / mass spectrometer. The mass spectrometry measurement method may be either a scanning method or a SIM (Select Ion Monitoring) method.

  By performing the mass spectrometry, if it is an unknown aldehyde compound, its molecular weight is revealed by the scanning method, and the structure can be estimated. If it is a known compound, its abundance can be quantified by the SIM method. In particular, in the analysis of saccharides, identification can be performed by comparing a standard compound (standard sugar chain) measured in advance with a retention time of liquid phase chromatography and a spectrum of mass spectrometry.

<Labeled saccharide>
The labeled saccharide of the present invention is obtained by adding 3-aminobenzylammonium salts represented by the above general formula (I) (aldehyde compound labeling compound) to the reducing end of the saccharide. As shown in the above reaction formula, “impossible at the reducing end of a saccharide” has a structure in which 3-aminobenzylammonium salts are added by an amino group to a structure in which the aldehyde group at the end of the saccharide is reduced. By identifying the structure of the labeled saccharide in advance, it can be used as a standard substance for saccharide mass spectrometry, and can be used for structure identification or quantitative analysis of an unknown sugar chain.

EXAMPLES Next, although an Example demonstrates this invention still in detail, this invention is not limited to a following example, unless the summary is exceeded.
[Example 1]
Take 1 mg of glucose oligomer (manufactured by Hornen) in a test tube, add 12 μmol of 3-aminobenzyl-trimethylammonium hydrochloride, 90 μL of water and 10 μL of acetic acid, heat at 90 ° C. for 5 minutes, and further add 25 μmol of 3-aminobenzyl-trimethylammonium hydrochloride, 15 mg of sodium cyanotrihydroborate, 450 μL of water and 50 μL of acetic acid were added and heated at 90 ° C. for 1 hour. After the reaction, water was added to make the total volume 1 ml, and LC-MS was measured. Table 2 shows the peak area ratio of the glucose oligomer hexamer.

Table 1 shows LC-MS conditions. The liquid A and liquid B in Table 1 are liquids constituting mobile phases, respectively, and the liquid A and liquid B are mixed to adjust the polarity of the mobile phase. In Table 1, “B: a% (T ₁ min) → B: b% (T ₂ min)” indicates that the concentration of the B solution is changed from a% to b% in T ₂ -T ₁ min. It means that it was changed. However, T ₁
, T ₂ , a, b each represent a real number. In Tables 1 and 2,% represents a percentage based on volume.
In addition, the 3-aminobenzyl-trimethylammonium hydrochloride used as the labeling compound was heated in heavy water at 90 ° C. for 1 hour, and the decomposition rate (residual rate) after heating was determined by 1H-NMR. I investigated. The results are shown in Table 3.

[Comparative Example 1]
In the same manner as in Example 1, the labeled glucose oligomer was analyzed by LC-MS.
However, equimolar 2-aminopyridine was used instead of 3-aminobenzyl-trimethylammonium hydrochloride as the labeling compound.
[Comparative Example 2]
In the same manner as in Example 1, the labeled glucose oligomer was analyzed by LC-MS.
However, equimolar amount of TMAPA (trimethylaminophenylammonium hydrochloride) was used as the labeling compound instead of 3-aminobenzyl-trimethylammonium hydrochloride.

[Comparative Example 3]
Take 1 mg of glucose oligomer in a test tube and dissolve in 25 μL of water.
8 μmol of P reagent (manufactured by Tokyo Chemical Industry) and 25 μL of water were added. Further, 2.5 μL of acetic acid was added and heated at 75 ° C. for 3 hours. After the reaction, water was added to make the total volume 1 ml, and LC-MS was measured. Table 2 shows the peak area ratio of the glucose oligomer hexamer.

［比較例４］
４−アミノベンジル−トリメチルアンモニウム・塩酸塩の熱安定性を実施例１と同様に調べた。結果を表３に示す。

[Comparative Example 4]
The thermal stability of 4-aminobenzyl-trimethylammonium hydrochloride was examined in the same manner as in Example 1. The results are shown in Table 3.

Claims (6)

A compound for labeling an aldehyde compound comprising a 3-aminobenzylammonium salt represented by the following general formula (I).

(In the formula, R ₁ , R ₂ and R ₃ each independently represents a hydrocarbon group which may be substituted, and at least two of R ₁ , R ₂ and R ₃ are combined to form a nitrogen-containing heterocycle. X- represents a counter anion, and the 2-, 4-, 5-, and 6-positions of the benzene ring may be each independently substituted with a substituent.) The labeling compound according to claim ₁ , wherein R ₁ , R ₂ and R ₃ in the general formula (I) are each independently an alkyl group. A 3-aminobenzylammonium salt represented by the following general formula (I) is added to an aldehyde compound by a reductive amination reaction, and the obtained reaction product is analyzed by mass spectrometry. Analysis method.

(In the formula, R ₁ , R ₂ and R ₃ each independently represents a hydrocarbon group which may be substituted, and at least two of R ₁ , R ₂ and R ₃ are combined to form a nitrogen-containing heterocycle. X- represents a counter anion, and the 2-, 4-, 5-, and 6-positions of the benzene ring may be each independently substituted with a substituent.) The method for analyzing an aldehyde compound according to claim 3, wherein R ₁ , R ₂ and R ₃ in the general formula (I) are each independently an alkyl group.   The analysis method according to claim 3 or 4, wherein the aldehyde compound is a saccharide. A labeled saccharide of 3-aminobenzylammonium salt, wherein 3-aminobenzylammonium salt represented by the following general formula (I) is added to the reducing end of the saccharide.

(In the formula, R ₁ , R ₂ and R ₃ each independently represents a hydrocarbon group which may be substituted;
_At least two of ₁ , R ₂ and R ₃ may be bonded to form a nitrogen-containing heterocyclic ring. X- represents a counter anion. The 2-, 4-, 5-, and 6-positions of the benzene ring may each independently be substituted with a substituent. )