CN114773254B - Quaternary ammonium salt compound or salt thereof and application thereof, and method for distinguishing 2-hydroxyl-substituted fatty acid from 3-hydroxyl-substituted fatty acid - Google Patents

Quaternary ammonium salt compound or salt thereof and application thereof, and method for distinguishing 2-hydroxyl-substituted fatty acid from 3-hydroxyl-substituted fatty acid Download PDF

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CN114773254B
CN114773254B CN202210505512.8A CN202210505512A CN114773254B CN 114773254 B CN114773254 B CN 114773254B CN 202210505512 A CN202210505512 A CN 202210505512A CN 114773254 B CN114773254 B CN 114773254B
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fatty acid
acid
substituted fatty
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CN114773254A (en
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叶阳
刘佳
俞启扬
王凯
朱展成
唐春萍
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Shanghai Institute of Materia Medica of CAS
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Abstract

The invention provides a quaternary ammonium salt compound or a salt formed by pairing amino groups and acids, which can be used as a derivatization reagent of fatty acids and is used for distinguishing 2-hydroxyl-substituted fatty acids from 3-hydroxyl-substituted fatty acids in mass spectrometry detection. In mass spectrometry detection, the 2-hydroxyl-substituted fatty acid and the 3-hydroxyl-substituted fatty acid which are derivatized by the quaternary ammonium salt compound or the salt formed by pairing amino groups and the acid can respectively generate respective characteristic ion fragments under the induction of higher collision energy, and the 2-hydroxyl-substituted fatty acid and the 3-hydroxyl-substituted fatty acid can be specifically distinguished based on the two characteristic ions. The quaternary ammonium salt compound can also be used for detecting the existence of 2-hydroxyl substituted fatty acid or 3-hydroxyl substituted fatty acid in the mixture.

Description

Quaternary ammonium salt compound or salt thereof and application thereof, and method for distinguishing 2-hydroxyl-substituted fatty acid from 3-hydroxyl-substituted fatty acid
Technical Field
The invention relates to a quaternary ammonium salt compound or a salt formed by pairing amino groups with acids and application thereof, in particular to a method for distinguishing 2-hydroxyl-substituted fatty acid from 3-hydroxyl-substituted fatty acid by using the quaternary ammonium salt compound as a derivatization reagent and utilizing mass spectrometry.
Background
Fatty Acids (FA) have attracted scientists' interest for their critical role in many physiological processes. In recent years, polyunsaturated fatty acids (PUFAs) and hydroxyl-containing fatty acids (OHFAs) have been reported to be also important bioactive molecules, which exhibit significant bioactivity in many diseases including inflammation, diabetes, liver damage, and also some cancers, etc. Different PUFAs, OHFAs can exert completely different activities, with different chain lengths, numbers and positions of double bonds or hydroxyl groups. N-6PUFAs such as arachidonic acid (AA, C20:4) are important immune and pro-inflammatory factors in biological systems; eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6) with higher unsaturation are both n-3 PUFAs, an important participant in brain function; EPA and DHA also have anti-inflammatory activity, since they inhibit the biosynthesis of n-6PUFAs in vivo.
Hydroxy-substituted fatty acids at the 2-position (hereinafter also referred to as 2-OHFA) and hydroxy-substituted fatty acids at the 3-position (hereinafter also referred to as 3-OHFA) are two more specific classes of hydroxy-containing fatty acids, which are closely related to fatty acid α -oxidation and β -oxidation, which also suggests that 2/3-OHFA may play an important role in the in vivo energy metabolism process.
More importantly, it has been reported that (R) -2-OHA and unsaturated 2-OHA have excellent inhibitory effect on tumor cell growth (see non-patent documents: sun, L.; yang, X.; huang, X.; yao, Y.; wei, X.; yang, S.; zhou, D.; zhang, W.; long, Z.; xu, X.; zhu, X.; he, S.; su, X. Cancer Res 2021,81,289-302.).
However, how to distinguish and identify 2-OHFA and 3-OHFA has not yet been effective, both of which behave relatively similarly in terms of primary infrared, nuclear magnetic resonance, ultraviolet, etc. primary spectroscopy data, and are indistinguishable. This problem is more difficult to solve when there is an unsaturated bond whose position is unknown in 2-OHFA and 3-OHFA (i.e., unsaturated 2-OHFA and 3-OHFA, hereinafter also referred to as 2-OHUFA and 3-OHUFA, respectively), although in recent years, liquid chromatography-mass spectrometry (LC-MS) combined techniques have been widely used for structural identification of various types of compounds due to their good sensitivity, ability to accurately provide diagnostic information on mass-to-charge ratios, ion fragments, and retention times of the compounds, and the like. However, because of the problems of the 2/3-OHUFA standard product such as high synthesis difficulty and low ionization efficiency on mass spectrum, it is difficult to establish a corresponding standard product database by LC-MS so as to distinguish and identify the 2/3-OHUFA; in addition, it is difficult to directly determine the position of the OH group in 2/3-OFUFA by LC-MS because the ion fragments obtained by 2/3-OFUFA on mass spectrometry do not provide effective functional group information. Furthermore, the content of hydroxy fatty acids in organisms is far lower than other fatty acids, which also adds a lot of difficulty to the identification of hydroxy fatty acids. This presents great difficulty in the intensive studies of the physiological activities of 2/3-OHA and 2/3-OHIFA in organisms, and the studies of the biological activities of 2/3-OHIFA have been slow due to the lack of effective analytical methods.
In recent years, scientists have developed many approaches to try to solve the analysis problem of 2/3-OHA (including 2/3-OHIFA). Direct analysis of 2/3-OHFA by LC-MS is greatly limited due to the problems described above; to date, rapid, comprehensive analysis of OHFAs remains a significant challenge.
Disclosure of Invention
In the present invention, the inventors have found a derivatizing agent, which is a class of quaternary ammonium compounds, which can rapidly distinguish whether a sample is 2-OHFA or 3-OHFA by allowing it to react with a fatty acid sample, subjecting the derivatization product to mass spectrometry, and recognizing the position of 2/3-OH by characteristic ion fragments generated by fragmentation. The derivatizing agents of the invention can also be used to identify 2-OHA or 3-OFA from other isomers. In addition, isotopically-labeled quaternary ammonium compounds of the present invention (e.g., D 3 Markers) can be used to rapidly distinguish low levels of 2/3-OHFAs in complex samples.
Specifically disclosed is a quaternary ammonium salt compound represented by the formula (1) below or a salt thereof formed by pairing an amino group with an acid,
wherein,
R 1 、R 2 each independently is saturated or unsaturated C1 toAlkyl of C6, saturated or partially unsaturated C3-6 cycloalkyl, saturated or partially unsaturated 3-10 membered heterocyclyl or C6-10 aryl;
X - represents a counter anion;
a and B are each independently a straight-chain or branched C3-C10 alkylene chain, wherein the linear or branched C3-C10 alkylene chain is optionally interrupted by one or more groups selected from the group consisting of-O-; -CO-, -C (=o) O-, -CONH-, -NHCO-, -NHCONH-, -NH-, -NR 3 -、-C(R 3 ) 2 -, -S-, sulfinyl, sulfonyl, sulfinyloxy, sulfonyloxy, -aminosulfonylamino-; the divalent group in alkynylene, alkenylene, cycloalkylene, or any combination thereof is interrupted one or more times, wherein R is 3 H, C1C 6 alkyl, saturated or partially unsaturated C 3-6 Cycloalkyl, a saturated or partially unsaturated 3-to 10-membered heterocyclyl or a C6-to 10-aryl.
In a preferred embodiment of the present invention, the compound of formula (I) is a quaternary ammonium compound of formula (II),
wherein,
R 1 、R 2 each independently is a C1-C6 alkyl group;
X - represents a counter anion;
m and n each independently represent an integer of 1 to 6.
In a preferred embodiment of the invention, it is a salt of a compound of formula (I) formed by pairing an amino group with an acid, said acid being an inorganic acid selected from hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, formic acid, acetic acid, trifluoroacetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, trifluoromethanesulfonic acid, sulfamic acid, or an organic acid selected from maleic acid, citric acid, lactic acid, tartaric acid, succinic acid, X - X in (2) is a halogen atom.
In a preferred embodiment of the present invention, wherein R 1 、R 2 Each independently isA C1-C3 alkyl group,
further preferably, R 1 、R 2 Each independently of the other is a methyl group or an ethyl group,
still further preferably R 1 、R 2 Are all methyl groups.
In a preferred embodiment of the invention, X is chlorine, bromine or iodine.
In a preferred embodiment of the invention, m=1 and n=1, or m=2 and n=2.
In another aspect of the invention, the use of the quaternary ammonium salt compound or the salt thereof formed by pairing an amino group with an acid as a fatty acid derivatizing agent for distinguishing between a hydroxy-substituted fatty acid at the 2-position and a hydroxy-substituted fatty acid at the 3-position in mass spectrometry is provided.
In another aspect of the invention, there is provided the use of a quaternary ammonium salt compound according to the invention or a salt thereof formed by pairing an amino group with an acid as a fatty acid derivatizing agent for identifying a hydroxy-substituted fatty acid at the 2-position or a hydroxy-substituted fatty acid at the 3-position in mass spectrometry.
In another aspect of the present invention, there is provided a method for distinguishing between a 2-hydroxy-substituted fatty acid and a 3-hydroxy-substituted fatty acid based on mass spectrometry using the quaternary ammonium salt compound of the present invention or a salt thereof formed by pairing an amino group with an acid as a fatty acid derivatization reagent, distinguishing between a 2-hydroxy-substituted fatty acid and a 3-hydroxy-substituted fatty acid by the m/z value of fragments detected by mass spectrometry.
In a preferred embodiment of the present invention, the method for distinguishing between a 2-hydroxy-substituted fatty acid and a 3-hydroxy-substituted fatty acid comprises the steps of:
s1 derivatization procedure: in an alkaline environment, in the presence of a polypeptide condensing agent, enabling a sample to be detected to perform condensation reaction with the quaternary ammonium salt compound or salt formed by amino and acid pairing of the quaternary ammonium salt compound in claim 1 to obtain a derivative product;
s2 mass spectrum detection procedure: carrying out mass spectrum detection on the derivative product obtained in the step S1;
s3, mass spectrum data analysis procedure: among the ion fragments of the secondary mass spectrum, characteristic fragments were found, and it was judged that the 2-hydroxy-substituted fatty acid was present when the secondary fragment of the m/z value of [ molecular weight-X of formula (I) +26] was present, and that the 3-hydroxy-substituted fatty acid was present when the secondary fragment of the m/z value of [ molecular weight-X of formula (I) +43] was present.
2-OHFA or 3-OHFA derivatized with a compound of the invention are capable of producing characteristic ion fragments, respectively, under higher collision energy induction; the two characteristic ions can specifically distinguish whether the fatty acid is 2-substituted hydroxyl or 3-substituted hydroxyl, so that the 2/3-OHA which is completely independent of a standard substance can be rapidly and comprehensively identified, and the compound and the identification method are also suitable for distinguishing 2/3-hydroxyl substituted unsaturated fatty acid.
Drawings
FIG. 1 is a diagram showing structural formulas of Compound 1 and Compound 2;
FIG. 2 is a schematic diagram showing the results of mass spectrometry analysis of the derivatized product of example 1;
FIG. 3 is an explanatory diagram showing the results of mass spectrometry analysis of the derivative product in example 1;
FIG. 4 is a graph of the results of mass spectrometry analysis when Compound 1 was used for other hydroxy-position-substituted fatty acids;
FIG. 5 is a graph showing the results of mass spectrometry of comparative example 2;
FIG. 6 is a graph showing the results of mass spectrometry analysis after derivatization of tumor metastasis groups and blank groups of mice.
Detailed Description
The present invention is described in more detail below.
The invention provides a quaternary ammonium salt compound shown in a formula (1) or a salt thereof formed by pairing amino groups with acid,
wherein,
R 1 、R 2 each independently is a saturated or unsaturated C1-C6 alkyl, a saturated or partially unsaturated C3-6 cycloalkyl, or a saturatedAnd or a partially unsaturated 3-to 10-membered heterocyclyl or C6-to 10-aryl;
X - represents a counter anion;
a and B are each independently a straight-chain or branched C3-C10 alkylene chain, wherein the linear or branched C3-C10 alkylene chain is optionally interrupted by one or more groups selected from the group consisting of-O-; -CO-, -C (=o) O-, -CONH-, -NHCO-, -NHCONH-, -NH-, -NR 3 -、-C(R 3 ) 2 -, -S-, sulfinyl, sulfonyl, sulfinyloxy, sulfonyloxy, -aminosulfonylamino-; the divalent group in alkynylene, alkenylene, cycloalkylene, or any combination thereof is interrupted one or more times, wherein R is 3 H, C1C 6 alkyl, saturated or partially unsaturated C 3-6 Cycloalkyl, a saturated or partially unsaturated 3-to 10-membered heterocyclyl or a C6-to 10-aryl.
In the present invention, the expression of Ca to Cb means that the group has a carbon number of a to b, and unless otherwise specified, the carbon number generally excludes the carbon number of the substituent. In the present invention, unless otherwise specified, the expression of a chemical element generally includes the concept of an isotope having the same chemical properties, for example, the expression of "hydrogen" and also includes the concept of "deuterium" and "tritium" having the same chemical properties.
Thus, the C1-C6 alkyl group is exemplified by methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, 2-methylbutyl, n-pentyl, sec-pentyl, neopentyl, n-hexyl, neohexyl, n-heptyl, etc., but is not limited to these, and among them, C1-C5 alkyl groups are preferred, and examples of the preferred groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, 2-methylbutyl, n-pentyl, sec-pentyl, neopentyl, etc.
The alkylene group is a divalent group obtained by removing one hydrogen atom from the above alkyl group, and examples thereof include methylene, ethylene, propylene, and butylene.
Examples of the aryl group having 6 to 10 carbon atoms include phenyl, naphthyl, anthryl and phenanthryl groups, and phenyl is preferred.
Examples of the C6-C10 arylene group include divalent groups obtained by removing one hydrogen atom from the above-mentioned C6-C10 aryl group, and among these, phenylene and naphthylene are preferable.
The inventors of the present invention found that the quaternary ammonium salt structure having a cyclic structure is superior in stability in mass spectrometry cleavage, forms a peptide bond with fatty acid through amino group, and the cleavage rule in secondary mass spectrometry is traced, especially when the 2-position of fatty acid is substituted with hydroxyl group, or the 3-position is substituted with hydroxyl group, after it is linked with the quaternary ammonium salt structure derivatization reagent having a cyclic structure, the corresponding derivatization product by using the compound of the present invention can generate ion fragments of m/z [ molecular weight of molecular weight-x+26 ] of formula (I) in specific secondary mass spectrometry (MS 2), which are generated by bond cleavage between the carbon atom to which OH is attached and the adjacent carbonyl carbon atom, and thus can be directed to the position of 2-OH group, taking standard 2-hydroxyhexadecanoic acid (2-OH-C16) as an example; 3-hydroxyhexadecanoic acid (3-OH-C16) derivatized with the compounds of the invention is capable of producing m/z [ molecular weight of molecular weight-X of formula (I). ] ion fragments of molecular weight +43 in MS 2. It can thus be referred to as distinguishing whether the hydroxyl group is specifically substituted in the 2-or 3-position of the fatty acid.
If specified, when using the 4-amino-1, 1-dimethylpiperidine iodide (also referred to herein as ADMI) synthesized in Synthesis example 1, taking the standard 2-hydroxyhexadecanoic acid (2-OH-C16) as an example, the corresponding ADMI derivative product is capable of generating ion fragments of m/z 155.1 in a specific secondary mass spectrum (MS 2), which are generated by cleavage of the bond between the carbon atom to which OH is attached and the adjacent carbonyl carbon atom, and thus is capable of pointing to the position of the 2-OH group (see FIG. 2); ADMI-derivatized 3-hydroxyhexadecanoic acid (3-OH-C16) is capable of generating m/z 171.1 ion fragments in MS2, which result from bond cleavage between the OH-attached carbon atom and the adjacent carbon atom near the carboxyl group, and thus is capable of directing to the position of the 3-OH group (for specific cleavage mode analysis, see FIG. 2).
The quaternary ammonium salt cations in the compounds of the invention are useful as convenient final desired cations in mass spectrometry fragments, R being attached to N in the quaternary ammonium salt 1 、R 2 Can be full ofAnd or unsaturated C1-C6 alkyl, saturated or partially unsaturated C 3-6 Cycloalkyl, saturated or partially unsaturated 3-to 10-membered heterocyclyl or C6-to 10-aryl, but R is preferred from the viewpoint of ease of synthesis and good stability in cleavage 1 、R 2 Saturated C1-C6 alkyl,
the cyclic structure of the compounds of formula (I) is of critical importance. The inventors have also synthesized quaternary ammonium salts of acyclic structure having amino groups and quaternary ammonium salt cationic moieties as well.
However, when the 2/3 hydroxy-substituted fatty acid is discriminated and identified by the same method as the present invention, the characteristic ion fragments cannot be formed, and the discrimination and identification cannot be performed. The importance of the cyclic structure for the derivative to give a characteristic cleavage behavior can be deduced from this. The ring structure is essential in the present invention as determined by the regularity of the generation of characteristic secondary mass spectrometry ion fragments by the several ring structures of the examples.
X - The counter anion is a conjugate base of a protonic acid. Specific examples of the conjugate base of the protonic acid include a halogen ion, a sulfate ion, a nitrate ion, a carbonate ion, a perchlorate ion, a tetrafluoroborate ion, a tetrakis (pentafluorophenyl) borate ion, a hexafluorophosphate ion, a methanesulfonate ion, and a trifluoroacetate ion. Preferred are halide ions, i.e. X - X in (2) is a halogen atom.
The amino group may be stored in the form of a salt with various acids, so long as the derivatization reaction is not affected by the acid, for example, an inorganic acid selected from hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, formic acid, acetic acid, trifluoroacetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, trifluoromethanesulfonic acid, sulfamic acid, or an organic acid selected from maleic acid, citric acid, lactic acid, tartaric acid, succinic acid.
Amino groups are used for condensation with carboxyl groups of fatty acids to complete derivatization. The condensation of amino groups with fatty acids is equivalent to the peptide bond synthesis procedure in the polypeptide synthesis process. This step is generally carried out under alkaline conditions by catalysis with a polypeptide condensing agent. The derivatization reaction of the quaternary ammonium salt compound of the present invention with a fatty acid is a well-known technique, and the person skilled in the art can select the condensation reaction mode of the fatty acid with the quaternary ammonium salt compound of the present invention according to the need.
The polypeptide condensing agent is not particularly limited, and known polypeptide condensing agents can be used, and examples thereof include carbodiimide type, phosphorus positive ion type and urea positive ion type. Examples of the carbodiimide type condensing agent include N, N '-Dicyclohexylcarbodiimide (DCC), N' -Diisopropylcarbodiimide (DIC), a DCC-HOBt compound condensing agent, and a DCC-HOAt compound condensing agent, wherein HOBt and HOAt are condensing activators. Examples of the phosphorus positive ion condensing agent include a catter condensing agent BOP, pyBOP, AOP, pyAOP, brOP, pyClOP, pyBrOP. Examples of the urea positive ion condensing agent include TBTU, HBPyU, HATU, TOTU, pyCIU, TFFH, BTFFH, CIP. In the present invention, it is preferable to use a urea cation condensing agent having a high reaction rate, a small product racemization and a high yield, and it is more preferable to use HATU (compound name: O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate).
The condensation reaction is generally carried out in a weak alkaline environment, and organic bases such as DIPEA and triethylamine, and sodium hydrogen carbonate may be used. The solvent may be any solvent such as DMF, dichloromethane, acetonitrile, and acetonitrile is preferably used as the solvent from the viewpoint of convenience for direct use in mass spectrometry detection.
A and B in the compounds of the formula (I) are ring-forming linking groups which are each independently a straight-chain or branched C3-C10-alkylene chain, wherein the linear or branched C3-C10 alkylene chain is optionally interrupted by one or more groups selected from the group consisting of-O-; -CO-, -C (=o) O-, -CONH-, -NHCO-, -NHCONH-, -NH-, -NR 3 -、-C(R 3 ) 2 -, -S-, sulfinyl, sulfonyl, sulfinyloxy, sulfonyloxy, -aminosulfonylamino-, alkynylene, alkenylene, cycloalkanyleneThe divalent groups in the radicals, or any combination thereof, are interrupted one or more times, wherein R 3 H, C1C 6 alkyl, saturated or partially unsaturated C 3-6 Cycloalkyl, a saturated or partially unsaturated 3-to 10-membered heterocyclyl or a C6-to 10-aryl. Among them, the alkylene chain C1 to C3 is preferably saturated, and A and B may be the same or different.
As a further preferred compound of the present invention, the compound represented by the formula (I) is a quaternary ammonium compound represented by the formula (II),
wherein,
R 1 、R 2 each independently is a C1-C6 alkyl group;
X - represents a counter anion;
m and n each independently represent an integer of 1 to 6.
In a preferred embodiment of the present invention, wherein R 1 、R 2 Each independently is a C1-C3 alkyl group,
further preferably, R 1 、R 2 Each independently of the other is a methyl group or an ethyl group,
still further preferably R 1 、R 2 Are all methyl groups.
In a preferred embodiment of the invention, X is chlorine, bromine or iodine.
In a preferred embodiment of the invention, m=1 and n=1, or m=2 and n=2.
From the viewpoint of ease of synthesis and versatility, examples of the most preferable compounds of the present invention include the compounds having the following structures, but the present invention is not limited to the following compounds.
The quaternary ammonium salt compound or the salt formed by pairing amino and acid is used as a fatty acid derivatization reagent for distinguishing 2-hydroxy-substituted fatty acid from 3-hydroxy-substituted fatty acid in mass spectrometry.
In addition, the invention can also be used for detecting the existence of 2-hydroxyl substituted fatty acid or 3-hydroxyl substituted fatty acid in complex mixture samples. The quaternary ammonium salt compound or the salt formed by pairing amino groups with acid is used as a fatty acid derivatization reagent, the fatty acid derivatization reagent reacts with a complex sample, the product directly enters mass spectrum, or LC-MS is used for detection, if m/z ion fragments of [ molecular weight of formula (I) -X with molecular weight of +26] or ion fragments of [ molecular weight of formula (I) -X with molecular weight of +43] are detected, the existence of 2-hydroxyl substituted fatty acid and 3-hydroxyl substituted fatty acid in the complex mixture sample can be proved.
In summary, the present invention can provide a method for distinguishing between a 2-hydroxy-substituted fatty acid and a 3-hydroxy-substituted fatty acid based on mass spectrometry, which uses the quaternary ammonium salt compound or a salt thereof formed by pairing an amino group with an acid as a fatty acid derivatization reagent, and distinguishes between a 2-hydroxy-substituted fatty acid and a 3-hydroxy-substituted fatty acid based on the m/z value of fragments detected by mass spectrometry. The method of distinguishing between a 2-hydroxy-substituted fatty acid and a 3-hydroxy-substituted fatty acid according to the present invention may include, but is not limited to, the following steps:
s1 derivatization procedure: in an alkaline environment, in the presence of a polypeptide condensing agent, enabling a sample to be detected to perform condensation reaction with the quaternary ammonium salt compound or salt formed by amino and acid pairing of the quaternary ammonium salt compound in claim 1 to obtain a derivative product;
s2 mass spectrum detection procedure: carrying out mass spectrum detection on the derivative product obtained in the step S1;
s3, mass spectrum data analysis procedure: among the ion fragments of the secondary mass spectrum, characteristic fragments were found, and it was judged that the 2-hydroxy-substituted fatty acid was present when the secondary fragment of the m/z value of [ molecular weight-X of formula (I) +26] was present, and that the 3-hydroxy-substituted fatty acid was present when the secondary fragment of the m/z value of [ molecular weight-X of formula (I) +43] was present.
The mass spectrometry detection of the present invention can be any detection means that can perform secondary mass spectrometry detection. Because the resulting derivatized product contains a positive charge in its structure, the derivatized product can be ionized by a variety of commercially available ionization means, including electron bombardment ionization (EI), chemical Ionization (CI), desorption Ionization (DI), spray Ionization (SI), and the like. In the present invention, the preferred mass spectrometer may be an ultra-high performance liquid chromatography-quadrupole tandem time of flight (UPLC-Q/TOF) mass spectrometry system, but is not limited to this system, and any mass spectrometry system capable of performing secondary mass spectrometry detection may be used. The derivatization products of the invention can produce very definite characteristic cleavage fragments indicative of the hydroxyl position at the 2-or 3-position, and have very strong specificity, so that the hydroxyl position of unknown hydroxyl-containing fatty acids can be determined by directly comparing ion fragments without necessarily comparing the retention times of the corresponding standards by liquid chromatography. Therefore, in the analysis flow designed by the invention, the sample can be directly injected, or can be injected after high performance liquid chromatography.
The basic environment and the invention elements such as the polypeptide condensing agent are described in detail in the description of the compound of the present invention, and are not described in detail here.
Examples
The following examples illustrate the preparation of the present invention, but are not intended to limit the scope of the invention. The embodiment of the present invention is provided to more fully describe the present invention to those skilled in the art, and the following embodiment may be modified into various other configurations, and the scope of the present invention is as follows, but the present invention is not limited to this embodiment. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art. The term "and/or" includes any and all combinations of one or more of the listed items. In the following drawings, the thickness or size may be exaggerated for clarity and convenience.
Instrument and reagents: all experiments were performed on ultra high performance liquid chromatography-quadrupole tandem time of flight (UPLC-Q/TOF) mass spectrometry systems (H-class UPLC, synapt G2 Si MS, waters, USA). The separation was carried out using a Waters ACQUITY UPLC BEH C column (2.1X100 mm,1.8 μm); the column temperature was maintained at 45℃and the autosampler temperature was set at 15℃with a flow rate of 0.4mL/min. Mobile phases a and B were water containing 0.1% formic acid and Acetonitrile (ACN) containing 0.1% formic acid, respectively, with the gradient set as follows: 0-8 minutes, 5% -95% B;8-10 minutes, 95% B;10-12 minutes, 5% B. The mass spectrum was set as follows: the electrospray ion source voltage in the negative ion mode is-2800V, the electrospray ion source voltage in the positive ion mode is 3300V, the source temperature is 120 ℃, and the desolvation gas flow rate is 750L/h.
The reagents used in the examples were purchased from Aldrich Chemical Company, shanghai Pi De medical technologies Co., ltd., shanghai national medicine reagent Co., ltd., carboline reagent Co., ltd., shaoshan Yu technology (Shanghai) Co., ltd., ai Bai technology Co., etc.
Synthesis of derivatizing agents
Synthesis example 1: synthesis of 4-amino-1, 1-dimethylpiperidine-1-iodinated ammonium hydrochloride (ADMI)
Tert-butyl (1-methylpiperidin-4-yl) carbamate (399mg, 1.8 mmol) was dissolved in dichloromethane (4 ml), then methyl iodide (0.135 ml,2.16 mmol) was added and reacted at room temperature overnight. Evaporating under reduced pressure to obtain yellow gum, namely 4- ((tert-butoxycarbonyl) amino) -1, 1-dimethylpiperidine-1-ammonium iodide.
The above product was dissolved in methanol (4 mL), concentrated hydrochloric acid (0.3 mL) was added, and then the reaction was stirred at 50℃for 3 hours, and the progress of the reaction was monitored by LC-MS. After the reaction was completed, the solvent was evaporated under reduced pressure, and the crude product was recrystallized from absolute ethanol to obtain 370mg of pale yellow solid, which was the target compound example 4 (ADMI). 1 H NMR (600MHz,DMSO-d 6 )δ7.91(s,3H,-NH 2 HCl),3.49(m,2H),3.43(m,2H),3.31 (m,1H),3.14(s,3H),3.04(s,3H),2.04(m,2H),1.97(m,2H)； 13 C NMR(151 MHz,MeOD)δ60.03,54.91,46.91,44.75,23.95；MS(ESI):m/z＝129.3[M－I － － HCl] + 。
Synthesis example 2 Synthesis of Compound 2
The synthesis of compound 2 is similar to that of compound except that N, N-dimethyltrimethyldiamine is used to replace 1-methyl-4-piperidylamine. 1 H NMR(600MHz,DMSO-d 6 )δ3.2(s,9H,),3.5(m,2H),2.19 (m,2H),3.06(td,2H,J＝7.6,1.6)； 13 C NMR(151MHz,MeOD)δ53.9,64.4,22.6, 37.7；MS(ESI):m/z＝117.1[M－I － －HCl] + 。
The structures of the two derivatizing agents, compound 1 and compound 2, are shown in figure 1.
Synthesis example 3: synthesis of 4-amino-1, 1-dimethylpiperidine-1-ammonium bromide hydrobromide
4-amino-1-methylpiperidine (1 g,8.77 mmol) was dissolved in dichloromethane (10 mL), di-tert-butyl dicarbonate (2.32 mL,10.10 mmol) and triethylamine (1.58 mL,11.40 mmol) were added, and the mixture was stirred at room temperature to react for 8h. After the completion of the reaction of the raw materials was monitored by TLC, the reaction was quenched by adding a saturated aqueous sodium bicarbonate solution, the dichloromethane phase was separated, washed three times with a saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, then concentrated under reduced pressure, and purified by column chromatography (dichloromethane/methanol=10/1) to give 1.2g of a pale yellow solid, i.e., (1-methylpiperidin-4-yl) carbamic acid tert-butyl ester. 1 H NMR(500MHz, Chloroform-d)δ4.44(s,1H),3.44(m,1H),277(m,2H),2.27(s,3H),2.06(t,2H, J＝11.4),1.92(m,2H),1.47(m,2H),1.43(s,9H)；MS(ESI):m/z＝215.2[M+H] + 。
Tert-butyl (1-methylpiperidin-4-yl) carbamate (200 mg,0.93 mmol) was dissolved in dichloromethane (4 ml), and stirred at-20℃for 15 min, followed by bromomethylAlkane (0.16 mL,5.61 mmol) was added to the reaction system via syringe and allowed to react overnight with stirring at-20 ℃. The white solid is separated out, filtered, a filter cake is collected, washed by a small amount of dichloromethane, and vacuum dried for 3 hours at 40 ℃ to obtain 226mg of white solid, namely 4- ((tert-butoxycarbonyl) amino) -1, 1-dimethylpiperidine-1-ammonium bromide. 1 H NMR(500MHz, Methanol-d 4 )δ3.67(m,1H),3.58–3.40(m,4H),3.18(s,3H),3.15(s,3H),2.16– 2.02(m,2H),1.98–1.84(m,2H),1.45(s,9H)；MS(ESI):m/z＝229.2[M－Br － ] + 。
4- ((Boc) amino) -1, 1-dimethylpiperidine-1-ammonium bromide (100 mg,0.44 mmol) was dissolved in methanol (4 mL), hydrobromic acid (0.5 mL) was added, and the reaction was stirred at 50℃for 2h, and the progress of the reaction was monitored by LC-MS. After the completion of the reaction, 93mg of a white solid was obtained by spin-drying, which was the target compound example 1. 1 H NMR(500MHz,DMSO-d 6 )δ8.13(s,3H,-NH 2 HBr),3.54(m,2H),3.48(m, 2H),3.36(m,1H),3.14(s,3H),3.08(s,3H),2.10(m,4H)；MS(ESI):m/z＝129.3[M－Br － －HBr] + 。
Synthesis example 4: synthesis of 4-amino-1-ethyl-1-methylpiperidine-1-iodinated ammonium hydrochloride
The starting material (1-methylpiperidin-4-yl) carbamic acid tert-butyl ester (415 mg,1.94 mmol) was dissolved in dichloromethane (8 mL), iodoethane (0.64 mL,8.53 mmol) was added and stirred at room temperature overnight, and the progress of the reaction was monitored by LC-MS. After the reaction was completed, 710mg of yellow solid was obtained by spin-drying, which was 4- ((tert-butoxycarbonyl) amino) -1-ethyl-1-methylpiperidine-1-ammonium iodide. 1 H NMR(500MHz,Methanol-d 4 ) δ3.71(m,1H),3.52(m,4H),3.43(m,2H),3.09/3.09(2×s,3H),2.11(m,2H),1.95(m,2H),1.47(s,9H),1.42/1.39(2×t,3H,J＝7.0)；MS(ESI):m/z＝243.3[M－I － ] + 。
4- ((Boc) amino) -1-ethyl-1-methylpiperidine-1-ammonium iodide (300 mg,1.24 mmol) was dissolved in anhydrous methanol (4 mL), 1mL of a 2M hydrogen chloride/1, 4-dioxane solution was added, and the reaction was stirred at room temperature for 9 hours, and the progress of the reaction was monitored by LC-MS. After the reaction, the crude product was obtained by spin-drying, and was slurried with absolute ethanol, and then dried in vacuo at 50℃to give 124mg of a pale yellow solid, which was the objective compound example 2. 1 H NMR(500MHz,DMSO-d 6 )δ8.51(s,3H,-NH 2 HCl), 3.57(m,2H),3.49–3.28(m,5H),3.00(s,3H),2.25–1.97(m,4H),1.26/1.23(2×t, 3H,J＝7.2)；MS(ESI):m/z＝143.2[M－I － －HCl] + 。
Synthesis example 5: 3-amino-1, 1-dimethyl azetidine-1-iodoammonium hydrochloride
The starting azetidine-3-carbamic acid tert-butyl ester (100 mg,0.58 mmol) was dissolved in tetrahydrofuran (5 mL), potassium carbonate (321 g,2.32 mmol) and methyl iodide (0.145 mL,2.32 mmol) were added and reacted at room temperature for 3h, and the progress of the reaction was monitored by LC-MS. After the reaction is finished, insoluble matters are removed by filtration, and the crude product is obtained by spin drying. Then beating with mixed solution of methyl tertiary butyl ether and acetonitrile, and drying in vacuum at 50 ℃ to obtain light yellow solid 27mg, namely 3- ((tert-butoxycarbonyl) amino) -1, 1-dimethyl azetidine-1-ammonium iodide. 1 H NMR(500MHz,DMSO-d 6 )δ7.64(d,J＝7.7Hz,1H),4.63(m, 1H),4.45(m,2H),4.21(dd,J＝11.4,7.5Hz,2H),3.17(s,3H),3.16(s,3H),1.39 (s,9H)；MS(ESI):m/z＝201.2[M－I － ] + 。
3- ((tert-Butoxycarbonyl) amino)1, 1-Dimethylazetidine-1-ammonium iodide (150 mg, 0.75 mmol) was dissolved in methanol (3 mL), 1mL of a 2M hydrogen chloride/1, 4-dioxane solution was added, and the reaction was stirred at room temperature for 3 hours, and the progress of the reaction was monitored by LC-MS. After the reaction, the crude product was obtained by spin-drying, and the crude product was slurried with absolute ethanol and dried in vacuo to obtain 56mg of a pale yellow solid, which was the target compound example 3. 1 H NMR(500MHz,DMSO-d 6 )δ9.06(s,3H,-NH 2 HCl),4.67 (m,2H),4.43(m,3H),3.33(s,3H),3.23(s,3H)；MS(ESI):m/z＝101.2[M－I － － HCl] + 。
Synthesis example 6: 4-amino-1-methyl-1- (methyl-d 3) piperidine-1-iodinated ammonium hydrochloride (DADMI)
Preparation method is the same as in Synthesis example 1, except that CD is used 3 I replaces methyl iodide as a raw material to obtain a target compound example 5 as a light yellow solid. 1 H NMR(600MHz,DMSO-d 6 )δ9.09(s,3H,-NH 2 HCl),3.48(m, 2H),3.41(m,2H),3.30(m,1H),3.12/3.04(2×s,3H),2.04(m,2H),1.98(m,2H)； MS(ESI):m/z＝132.2[M－I － －HCl] + 。
Example 1 fatty acid derivative mass spectrometry experiments on the basis of the present invention representing compound ADMI utilize ADMI condensation reaction: the blow-dried 2-hydroxyhexadecanoic acid standard was dissolved in 200. Mu.L Acetonitrile (ACN), 10. Mu.L HATU (50. Mu.M in ACN) and 10. Mu.L sodium bicarbonate (NaHCO) 3 20. Mu.M in H2O) and allowed to react at room temperature for 5 minutes with shaking; then, 20. Mu.L of 1:1 Mixed ADMI derivatizing reagent [ 50. Mu.M dissolved in ACN/H2O (8:2, v/v)]Then reacted at 37℃for 30 minutes. At the end of the reaction, 200. Mu.L of Ethyl Acetate (EA) and 200. Mu.L of deionized water were added to the reaction solution to extract the product into the organic layer, and NaHCO, which had a large influence on the next oxidation reaction, was also removed 3 The method comprises the steps of carrying out a first treatment on the surface of the Transferring an upper organic layer after extraction is finished, and repeating the extraction step twice; finally merging the EA layers and drying by nitrogen; blow-dried sample is subjected to MeOH re-dissolution and sample loading UPLC-Q/TOF MS, analysis.
Analysis of mass spectrometry results:
ADMI derivatization identifies the 2/3-OH position: the derivatization strategy helps to improve the mass spectral response of the fatty acids. In this paper, the quaternary ammonium salt reagent ADMI can improve the mass spectrum response and the liquid chromatography separation efficiency of 2/3-OHA at the same time. More importantly, the product of ADMI derivatization of 2/3-OHFA can fragment out characteristic ion fragments upon higher collision energy induction, which can be used to identify the position of the 2/3-OH group (fig. 2 a). Taking the standard 2-hydroxyhexadecanoic acid (2-OH-C16) as an example, the corresponding ADMI derivative product is obtained by specific secondary mass spectrometry (MS 2 ) Can generate m/z 155.1 of ionic fragments resulting from bond cleavage between the carbon atom to which the OH is attached and the adjacent carbonyl carbon atom, and can therefore be directed to the position of the 2-OH group (fig. 2); ADMI-derived 3-hydroxyhexadecanoic acid (3-OH-C16) in MS 2 Can produce m/z 171.1 of ionic fragments resulting from bond cleavage between the carbon atom to which the OH is attached and the adjacent carbon atom near the carboxyl group, and can therefore be directed to the position of the 3-OH group (fig. 2).
Example 2ADMI derivatization was applied to other C8-C18 carbon chain length saturated 2/3-OHA standards
To further confirm that the production of these two diagnostic ions was not affected by carbon chain length, ADMI derivatization was applied to other C8-C18 carbon chain length saturated 2/3-OHFA standards (fig. 3) and both gave the expected results.
In the case of liquid chromatography-mass spectrometry equipment, good chromatographic separation effect can be achieved on liquid chromatography after the OHFA isomers with different carbon chain lengths are subjected to ADMI derivatization. The molecular formula, mass-to-charge ratio and retention time information of the isomeride derivative product of fatty acid can be analyzed together, and a rapid, effective and comprehensive analysis method for saturated 2/3-OHOFAs can be established by combining the information of accurate mass-to-charge ratio, fragment ion fragments of 155.1/171.1, retention time and the like.
Example 3 experiments on Mass Spectrometry of fatty acid derivatives based on 4-amino-1-ethyl-1-methylpiperidine-1-iodinated ammonium hydrochloride (Compound of Synthesis example 4)
Example 4 experiments on Mass Spectrometry of fatty acid derivatives based on 3-amino-1, 1-dimethyl azetidine-1-iodinated ammonium hydrochloride (Compound of Synthesis example 5)
Comparative example 1
It should be noted that the inventors have also attempted to use ADMI for OHFA isomers where the hydroxyl group is located elsewhere, and ADMI derivatization products failed to produce similar ion fragments that could indicate the OH position (fig. 4); this is probably due to the fact that the longer carbon chain between the hydroxyl function and the newly formed amide bond in the ADMI derivatization product limits the formation of similar ionic fragments. (FIG. 4)
Comparative example 2
The inventors performed derivatization of 2-hydroxyhexadecanoic acid standard with compound 2 in the same manner as in the example, and as a result, did not generate similar ion fragments that could indicate the OH position. No ion fragment capable of directing to the 2/3-OH position was found in the mass spectrum of the derivatization of the 2/3-OHFA product with compound 2 (fig. 5).
Example 5 identification of the presence or absence of 2/3-OH fatty acids in Complex tissue extracts
Mouse melanoma lung metastasis model construction and derivatization: the melanoma cell line B16F10 used in this study was purchased from the American type culture Collection and the cells were cultured in DMEM medium containing 10% bovine serum albumin, 100U/mL penicillin and 100. Mu.g/mL streptomycin (Gibco); the cells were incubated at 37℃with 5% CO 2 Is cultured in a humidified air environment. 18 male C57BL/6J mice were used for this study. Male mice of three weeks of age were purchased from Shanghai SLAC laboratory animal company. Mice were randomly divided into tumor and blank groups, and 100 μl of each was injected tail vein with 1x10 6 Culture medium of individual tumor cells or blank DMEM culture medium. Two weeks later mice were sacrificed and lung tissue was collected; tissue samples collected prior to the experiment were stored in a-80 ℃ refrigerator. Firstly, 200mg of mouse tissue is weighed and homogenized in 2mL of deionized water; then carrying out ultrasonic treatment on the homogenate for 30 minutes; after the ultrasonic treatment is finished, 50 mu L of sample homogenate is taken and transferred into a 1.5mL centrifuge tube; then 10 μl of phosphate buffered saline (dppbs) was added to the centrifuge tube and the sample was acidified with 0.1% HCl; then 100. Mu.L of ethyl acetate was added to the sample, andvortex 40s; after vortexing was completed, the upper organic layer was removed and 100 μl ethyl acetate was again added and the extraction procedure was repeated twice. Finally, the entire EA layer was combined and blow-dried with nitrogen.
The blow-dried mouse tissue fluid was dissolved in 200. Mu.L acetonitrile/tetrahydrofuran (ACN/THF, 1:1), 10. Mu.L HATU (50. Mu.M in ACN) and 10. Mu.L sodium bicarbonate (NaHCO) 3 20. Mu.M in H2O) and allowed to react at room temperature for 5 minutes with shaking; then, 20. Mu.L of ADMI derivatizing reagent [ 50. Mu.M ] dissolved in ACN/H2O (8:2, v/v) was added to the reaction solution]Then reacted at 37℃for 30 minutes. At the end of the reaction, 200. Mu.L of ethyl acetate and 200. Mu.L of deionized water were added to the reaction solution, and the product was extracted into the organic layer; transferring an upper organic layer after extraction is finished, and repeating the extraction step twice; finally merging the EA layers and drying by nitrogen; blow-dried samples were analyzed by MeOH reconstitution loading UPLC-Q/TOF MS.
Identification of 2/3-OFAs-like compounds in murine melanoma lung metastasis tissue: we further applied the established two-step derivatization method to analyze 2/3-OHFA-like compounds in mouse tumor tissues. We collected lung organs from both tumor and blank groups of 18 mice and performed the corresponding analysis in the same way. As previously mentioned, OHFA content is much lower than fatty acids without hydroxyl groups. Mass spectrometry using ADMI derivatization of the compounds of the invention, we identified eight relatively high levels of 2/3-OHFA from a range of isomers, 3-OHC12 (c=c), 3-OHC14 (c=c), 2/3-OHC16, 2/3-OHC18 (table 1), by screening for m/z 155.1 or 171.1 signature ion fragments; we then further identified by mcpba oxidation that the double bond positions of unsaturated acids 3-OHC12 (c=c), 3-OHC14 (c=c) are at Δ -9, Δ -5, respectively. The method for identifying the double bond position of unsaturated acid by mcpba oxidation reaction is a known technology, and specific method can be referred to Y.Feng; chen; yu; li. Animal Chem 2019, 91, 1791-1795.
TABLE 1 differential hydroxy fatty acids identified in mouse tumors
After identifying 2/3-OHFA compounds in mouse tissues, we further analyzed the content difference between 2-OHFAs and 3-OHFAs in melanoma metastasis lung tissue samples and common lung tissue samples by using a t-test method. As shown in FIG. 6, the content of the eight identified 2/3-OHFAs in the lung tissue of the tumor group was lower than that of the normal samples.
Experiments prove that the compound disclosed by the invention can be efficiently used for judging whether 2-OHA and 3-OHA exist in complex samples, and can be further used for other qualitative and quantitative experiments.
The technical features disclosed above are not limited to the combination with other features disclosed, and other combinations of the technical features can be performed by those skilled in the art according to the purpose of the invention to achieve the purpose of the invention, and various modifications made by those skilled in the art to the technical solution of the invention should fall within the scope of protection defined by the claims of the invention without departing from the design spirit of the invention.

Claims (7)

1. A quaternary ammonium salt compound represented by the formula (II) or a salt thereof formed by pairing an amino group with an acid,
（II）
wherein,
R 1 、R 2 each independently is a C1-C3 alkyl group;
X - represents a halogen atom;
m=1 and n=1, or m=2 and n=2,
which is a salt of a compound of formula (II) formed by amino pairing with an acid, X - X in (2) is a halogen atom.
2. The quaternary ammonium salt according to claim 1 or a salt thereof formed by pairing an amino group with an acid, wherein R 1 、R 2 Each independently is methyl or ethyl.
3. The quaternary ammonium salt compound according to claim 1 or 2 or a salt thereof formed by pairing an amino group with an acid, wherein X is chlorine, bromine or iodine.
4. Use of a quaternary ammonium salt compound according to claim 1 or a salt thereof formed by pairing an amino group with an acid as a fatty acid derivatizing agent for distinguishing between a hydroxy-substituted fatty acid at the 2-position and a hydroxy-substituted fatty acid at the 3-position in mass spectrometry.
5. Use of a quaternary ammonium salt compound according to claim 1 or a salt thereof formed by pairing an amino group with an acid as a fatty acid derivatizing agent for identifying a hydroxy-substituted fatty acid at the 2-position or a hydroxy-substituted fatty acid at the 3-position in mass spectrometry.
6. A method for distinguishing between a 2-hydroxy-substituted fatty acid and a 3-hydroxy-substituted fatty acid based on mass spectrometry, which uses the quaternary ammonium salt compound according to claim 1 or a salt thereof formed by pairing an amino group with an acid as a fatty acid derivatizing agent, and distinguishing between a 2-hydroxy-substituted fatty acid and a 3-hydroxy-substituted fatty acid by the m/z value of fragments detected by mass spectrometry.
7. The method for distinguishing between 2-hydroxy-substituted fatty acids and 3-hydroxy-substituted fatty acids according to claim 6, comprising the steps of:
s1 derivatization procedure: in an alkaline environment, in the presence of a polypeptide condensing agent, enabling a sample to be detected to perform condensation reaction with the quaternary ammonium salt compound or salt formed by amino and acid pairing of the quaternary ammonium salt compound to be detected to obtain a derivative product;
s2 mass spectrum detection procedure: carrying out mass spectrum detection on the derivative product obtained in the step S1;
s3, mass spectrum data analysis procedure: among the ion fragments of the secondary mass spectrum, characteristic fragments were found, and it was judged that the 2-hydroxy-substituted fatty acid was present when the secondary fragment of the m/z value of [ molecular weight-X of formula (II) +26] was present, and that the 3-hydroxy-substituted fatty acid was present when the secondary fragment of the m/z value of [ molecular weight-X of formula (II) +43] was present.
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WO2014044849A1 (en) * 2012-09-24 2014-03-27 Boehringer Ingelheim International Gmbh Heterocyclic compounds, medicaments containing said compounds, use thereof and processes for the preparation thereof
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