WO2018016645A1 - Novel phospholipid, uses thereof and development of phospholipid separation and measurement method - Google Patents

Abstract

The present invention provides a lysophosphatidylinositol phosphate that is a novel phospholipid, and uses thereof. The present invention also provides a method for isolating and analyzing the binding site of the pohosphate group of the phospholipid. Specifically provided is a method for measuring, detecting or identifying a phospholipid in a sample, the method comprising: (A) a step for subjecting the sample to liquid column chromatography-mass spectrometry (LC-MS) or ion mobility spectrometry-mass spectrometry (IMS-MS); and (B) a step for identifying the position of the phopsphate group of the phospholipid on the basis of the peak elution position in the LC-MS.

Description

Development of new phospholipids and their utilization and phospholipid separation measurement method

The present invention relates to a novel phospholipid and use thereof. Furthermore, the present invention relates to a phospholipid separation measurement method. More specifically, the present invention relates to a technique for specifying the position of a phosphate group of phosphatidylicitol phosphate and / or lysophosphatidylinositol phosphate while keeping an acyl group intact. Specifically, the present invention provides a phospholipid separation measurement method using mass spectrometry (MS), optionally in combination with chromatography or ion mobility separation (IMS).

Lipids are essential components of life as membrane constituents, energy sources, and signal transduction molecules. Among lipids, phospholipids are an important group of molecules that regulates various cellular functions as spatiotemporal regulators that target intracellular proteins to cell membranes and organelles and control their activity at those locations. . However, since it is sparingly soluble in water and not directly under the control of genes, it has limited analysis and has become a biochemical research field where research has not progressed.

The present inventors challenged such an undeveloped field by using a new technique of producing a genetically modified mouse having a phospholipid metabolizing enzyme or a phospholipid binding domain. The research of the present inventors has become a pioneer in the world, and elucidation of the biological function regulation mechanism by intracellular phospholipid has been developed so that it can be regarded as a new research field. In other words, various medical conditions such as various cancers, allergies, hepatic steatosis / fibrosis, cardiac dysfunction, neurodegeneration, enterocolitis, etc. were found one after another, and abnormal metabolic enzymes of individual phospholipids were found one after another. As a result of basic research that leads to future development, future development is highly expected (Non-patent Document 1).

On the other hand, in the research so far, metabolic enzymes to which genetic techniques can be applied have been the subject of analysis, and research on changes in phospholipid itself has hardly progressed (Non-patent Document 1). This is because lipid detection, identification and quantification methods have not been developed yet. Unlike genes, the type and number of lipids that make up living organisms are still uncertain, and many unknown lipids are thought to be sleeping without being discovered.

Moreover, it is known that abnormal expression of phospholipid metabolizing enzymes is related to pathological conditions such as cancer and metabolic syndrome, but details of the molecular mechanism are unknown. Phosphoinositide (also referred to as phosphatidylinositol phosphate, phosphatidylinositol phosphate (salt), etc.) involved in many diseases includes 8 classes (phosphatidylinositol, phosphatidylinositol-3-monophosphate, phosphatidylinositol-4-monophosphate) Acid, phosphatidylinositol-5-monophosphate, phosphatidylinositol-3,4-diphosphate, phosphatidylinositol-3,5-diphosphate, phosphatidylinositol-4,5-diphosphate, phosphatidylinositol-3,4 , 5-triphosphate). These lipids or metabolites may have different roles and functions in each class, and similarly their metabolic enzymes and receptors have significance as targets for various therapeutic drugs and as disease markers. There is a possibility.

Sasaki T. Et al. , Mammarian phosphoinosideide kinases and phosphates. Prog Lipid Res. 2009 Nov; 48 (6): 307-43, doi: 10.016 / j. pripres. 2009.6.001 Kielkouska, A.K. , Et al. A new approach to measuring phosphoinositides in cells by mass spectrometry. Adv Biol Request 54, 131-141 (2014).

In view of the above, the present inventors have begun to develop a new lipid analysis method based on mass spectrometry. The merit of using mass spectrometry is that it is possible to analyze biological samples (human clinical specimens, etc.) that could not be analyzed without using the radioisotope label to be analyzed. The composition of the lysophospholipid according to the present invention can be found. Established a new measurement method for the phosphatidylinositol phosphate group (also referred to as “PIPs” in this specification), which is extremely difficult to measure due to the presence of a small amount in the living body and multivalent phosphorylation. . By using this technique, an unknown lysophosphatidylinositol phosphate group (also referred to as “LPIPs” in the present specification) that could not be identified so far has been discovered. Since LPIPs did not exist until the disclosure of the present invention, neither the identification method, the detection method nor the synthesis method, the disclosure according to the present invention is the first report as a substance of LPIPs.

In the measurement method of the present invention, phosphoinositide and lysophosphoinositide (also referred to as lysophosphatidylinositol phosphate, lysophosphatidylinositol phosphate (salt), etc.) having an acyl group are obtained by chromatography-mass spectrometry. (It is also referred to as “intact state” in the present specification), and was completed by unexpectedly finding that the position of the phosphate group can be specified, measured, detected, and identified. In particular, the present invention relates to all 8 classes of phosphoinositides and all 8 classes of lysophosphoinositides with information on acyl groups (fatty acid side chains) by liquid column chromatography / mass spectrometry (LC-MS) or ion mobility separation. -Provide for the first time a method detected and identified by mass spectrometry (IMS-MS). Examples of the column used here include a chiral column and a hydrophobic column, but are not limited thereto, and any chromatography that separates a hydrophilic group and a hydrophobic group may be used.

Accordingly, the present invention provides the following.
(Item 1A)
A compound which is lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate.
(Item 2A)
1-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol monophosphate, 1-acyl-lysophosphatidylinositol diphosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl-lysophosphatidyl The compound according to item 1A, which is inositol triphosphate or 2-acyl-lysophosphatidylinositol triphosphate.
(Item 3A)
1-acyl-lysophosphatidylinositol-3-monophosphate, 1-acyl-lysophosphatidylinositol-4-monophosphate, 1-acyl-lysophosphatidylinositol-5-monophosphate, 2-acyl-lysophosphatidylinositol- 3-monophosphate, 2-acyl-lysophosphatidylinositol-4-monophosphate, 2-acyl-lysophosphatidylinositol-5-monophosphate, 1-acyl-lysophosphatidylinositol-3.4-diphosphate, 1-acyl-lysophosphatidylinositol-3.5-diphosphate, 1-acyl-lysophosphatidylinositol-4.5-diphosphate, 2-acyl-lysophosphatidylinositol-3,4-diphosphate, 2- Acyl-lysophosphatidylinositol-3,5-diphosphate, 2-acyl -With lysophosphatidylinositol-4,5-diphosphate, 1-acyl-lysophosphatidylinositol-3,4,5-triphosphate, or 2-acyl-lysophosphatidylinositol-3,4,5-triphosphate A compound according to item 1A or 2A.
(Item 4A)
Item 1A-3A which is 2-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl-lysophosphatidylinositol triphosphate or 2-acyl-lysophosphatidylinositol triphosphate The compound according to any one of the above.
(Item 5A)
1-butanyl-lysophosphatidylinositol 4-monophosphate;
1-hexanyl-lysophosphatidylinositol 4-monophosphate;
1-octanyl-lysophosphatidylinositol 4-monophosphate;
1-hexadecanyl-lysophosphatidylinositol 4-monophosphate;
1-9Z-hexadecenyl-lysophosphatidylinositol 4-monophosphate;
1-heptadecanyl-lysophosphatidylinositol 4-monophosphate;
1-octadecanyl-lysophosphatidylinositol 4-monophosphate;
1-9Z-octadecenyl-lysophosphatidylinositol 4-monophosphate;
1-9Z, 12Z-octadecandienyl-lysophosphatidylinositol 4-monophosphate;
1-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 4-monophosphate;
1-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 4-monophosphate;
1-7Z, 10Z, 13Z, 16Z-docosatetraenyl-lysophosphatidylinositol 4-monophosphate;
1-7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenyl-lysophosphatidylinositol 4-monophosphate;
1-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol 4-monophosphate;
2-butanyl-lysophosphatidylinositol 4-monophosphate;
2-hexanyl-lysophosphatidylinositol 4-monophosphate;
2-octanyl-lysophosphatidylinositol 4-monophosphate;
2-hexadecanyl-lysophosphatidylinositol 4-monophosphate;
2-9Z-hexadecenyl-lysophosphatidylinositol 4-monophosphate;
2-heptadecanyl-lysophosphatidylinositol 4-monophosphate;
2-octadecanyl-lysophosphatidylinositol 4-monophosphate;
2-9Z-octadecenyl-lysophosphatidylinositol 4-monophosphate;
2-9Z, 12Z-octadecanedienyl-lysophosphatidylinositol 4-monophosphate;
2-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 4-monophosphate;
2-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 4-monophosphate;
2-7Z, 10Z, 13Z, 16Z-docosatetraenyl-lysophosphatidylinositol 4-monophosphate;
2-7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenyl-lysophosphatidylinositol 4-monophosphate;
2-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol 4-monophosphate;
1-butanyl-lysophosphatidylinositol 4,5-diphosphate;
1-hexanyl-lysophosphatidylinositol 4,5-diphosphate;
1-octanyl-lysophosphatidylinositol 4,5-diphosphate;
1-hexadecanyl-lysophosphatidylinositol 4,5-diphosphate;
1-9Z-hexadecenyl-lysophosphatidylinositol 4,5-diphosphate;
1-heptadecanyl-lysophosphatidylinositol 4,5-diphosphate;
1-octadecanyl-lysophosphatidylinositol 4,5-diphosphate;
1-9Z-octadecenyl-lysophosphatidylinositol 4,5-diphosphate;
1-9Z, 12Z-octadecandienyl-lysophosphatidylinositol 4,5-diphosphate;
1-6Z, 9Z, 12Z-octadecanedienyl-lysophosphatidylinositol 4,5-diphosphate;
1-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 4,5-diphosphate;
1-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 4,5-diphosphate;
2-butanyl-lysophosphatidylinositol 4,5-diphosphate;
2-hexanyl-lysophosphatidylinositol 4,5-diphosphate;
2-octanyl-lysophosphatidylinositol 4,5-diphosphate;
2-hexadecanyl-lysophosphatidylinositol 4,5-diphosphate;
2-9Z-hexadecenyl-lysophosphatidylinositol 4,5-diphosphate;
2-heptadecanyl-lysophosphatidylinositol 4,5-diphosphate;
2-octadecanyl-lysophosphatidylinositol 4,5-diphosphate;
2-9Z-octadecenyl-lysophosphatidylinositol 4,5-diphosphate;
2-9Z, 12Z-octadecandienyl-lysophosphatidylinositol 4,5-diphosphate;
2-6Z, 9Z, 12Z-octadecanedienyl-lysophosphatidylinositol 4,5-diphosphate;
2-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 4,5-diphosphate;
2-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 4,5-diphosphate;
1-butanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-octanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-hexadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-9Z-hexadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-heptadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-octadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-9Z-octadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-butanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-octanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-hexadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-9Z-hexadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-heptadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-octadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-9Z-octadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate; and 2-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol Selected from the group consisting of 3,4,5-triphosphate,
The compound according to any one of items 1A to 4A.
(Item 6A)
A composition comprising the compound according to any one of items 1A to 5A for use as a disease marker.
(Item 7A)
Item 6. The composition according to Item 6A, wherein the disease marker is an inflammation marker or a cancer marker.
(Item 8A)
The composition of item 7A, wherein the cancer comprises prostate cancer.
(Item 9A)
Item 6. The method for producing a compound according to any one of Items 1A to 5A, comprising a step of deacylating phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate by contacting with alkylamine. .
(Item 10A)
Item 9. The production method according to Item 9A, wherein the alkylamine is methylamine.
(Item 11A)
Item 10. The production method according to Item 9A or Item 10A, wherein the deacylation is a milder condition than a condition for dediacylation.
(Item 12A)
The method according to Item 11A, wherein the mild condition is achieved by shortening a reaction time, reducing a concentration of the alkylamine, a reaction temperature, or a combination thereof among the conditions for dediacylation.
(Item 13A)
The mild condition is that methylamine is used as the alkylamine, the reaction time is 10 minutes or less, or the concentration of the methylamine is about 11% or less, the reaction temperature is 53 ° C. or less, or the reaction time, the concentration, and the reaction temperature. The method according to item 11A or 12A, which is a combination.
(Item 14A)
(A) a step of providing phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate having different mass acyl groups at the 1-position and the 2-position, wherein the acyl group having a desired mass is Existing in a desired position selected from the 1st or 2nd position;
(B) deacylating the phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate by contacting with an alkylamine, and (C) an acyl having a desired mass, if necessary. Removing lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate having a group, lysophosphatidylinositol monophosphate having a desired acyl group at a desired position, lysophosphatidylinositol A process for producing diphosphate or lysophosphatidylinositol triphosphate.
(Item 15A)
Concentrating the phospholipid by contacting the sample with an anion exchange resin;
Protecting a phosphate group in the acidic phospholipid with a protecting group; and detecting, identifying or quantifying lysophosphatidylinositol phosphate in the phospholipid by mass spectrometry;
For detecting, identifying or quantifying lysophosphatidylinositol phosphate.
(Item 16A)
The method according to Item 15A, further comprising detecting, identifying or quantifying phosphatidylinositol phosphate in the phospholipid.
(Item 17A)
The method of item 15A or 16A, wherein the protecting group comprises an alkyl group.
(Item 18A)
The method according to any one of Items 15A to 17A, wherein the protecting group comprises a methyl group.
(Item 19A)
The method according to any one of Items 15A to 18A, wherein the mass spectrometry includes a selective reaction monitoring method (SRM) using a triple quadrupole mass spectrometer.
(Item 20A)
Further, any one of items 15A to 19A, further comprising a step of detecting, identifying or quantifying the fatty acid side chain and / or phosphate group of the lysophosphatidylinositol phosphate using liquid column chromatography or ion mobility separation. 2. The method according to item 1.
(Item 21A)
Item 20. The method according to any one of Items 15A to 20A, which further specifies the position of a phosphate group.
(Item 22A)
Item 21A to Item 21A, wherein in the mass spectrometry, acylglycerol is selected as a product ion (fragment ion) to detect, identify or quantify the fatty acid side chain and / or phosphate group. Method.
(Item 23A)
A method for diagnosing cancer by detection, identification or quantification according to any one of items 15A to 22A.
(Item 24A)
A method for measuring, detecting or identifying phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample, the method comprising: A) applying the sample to mass spectrometry (MS); and B) a peak in the MS And locating the phosphate group of the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate according to the elution position of the phosphatidylinositol phosphate.
(Item 25A)
The method according to Item 24A, further comprising applying the sample to chromatography in the step A).
(Item 26A)
The mass spectrometry is liquid column chromatography-mass spectrometry (LC-MS);
The method according to item 24A or 25A, wherein the specifying step B) is a step of specifying the position of the phosphate group of the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate by the elution position of the peak in the LC-MS. .
(Item 27A)
Item 26. The method according to any one of Items 24A to 26A, wherein the total molecular weight of the acyl groups contained in the phosphatidylinositol phosphate is also specified in the specifying step.
(Item 28A)
The method according to any one of Items 24A to 27A, wherein in the identifying step, the kind of acyl group contained in the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is also identified.
(Item 29A)
The method according to any one of items 24A to 28A, wherein the position of the phosphate group of said phosphatidylinositol phosphate and lysophosphatidylinositol phosphate is specified in the same run.
(Item 30A)
30. The method according to any one of items 24A to 29A, wherein the measurement, detection or identification is performed by distinguishing phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate from other inositol-containing phospholipids.
(Item 31A)
31. The method according to any one of items 24A to 30A, wherein the sample is prepared under a condition that does not decompose phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate.
(Item 32A)
The method according to Item 31A, wherein the condition includes not performing a deacylation treatment.
(Item 33A)
The method according to any one of items 26A to 32A, wherein the liquid column chromatography is selected from the group consisting of a chiral column, a reverse phase column, and a column chromatography that separates a hydrophilic group and a hydrophobic group.
(Item 34A)
The method of any one of items 24A-33A, wherein the liquid column chromatography is combined with ion mobility separation.
(Item 35A)
The method of any one of items 24A-34A, further comprising quantifying the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate.
(Item 36A)
The method of any one of items 24A-35A, wherein the sample is an intact sample.
(Item 37A)
The method according to any one of Items 24A to 36A, wherein the identification is performed without labeling the sample.
(Item 38A)
The mass spectrometry is ion mobility separation-mass spectrometry (IMS-MS);
The specifying step B) is a step of specifying the position of the phosphate group of the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate by the elution position of the peak in the IMS-MS.
The method according to any one of items 24A to 37A.
(Item 39A)
The method of item 38A, comprising any one or more of the items 25A-33A, 35A-37A.
(Item 40A)
An apparatus for measuring, detecting or identifying the position of acyl group and phosphate group of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample under conditions where mass spectrometry (MS) and phosphatidylinositol phosphate are not decomposed And an application unit that applies the sample to the MS.
(Item 41A)
The device according to Item 40A, further comprising a chromatography device.
(Item 42A)
Item 40A, wherein the mass spectrometry is liquid column chromatography-mass spectrometry (LC-MS), and the application unit is an application unit that applies the sample to the LC-MS under conditions in which phosphatidylinositol phosphate is not decomposed. Or the apparatus of 41A.
(Item 43A)
42. The apparatus according to Item 42A, wherein the liquid column chromatography is selected from the group consisting of all column chromatography that separates hydrophilic groups and hydrophobic groups such as chiral columns and reverse phase columns.
(Item 44A)
The apparatus according to any one of items 40A-43A, further comprising means for detecting or quantifying said phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate.
(Item 45A)
The mass spectrometry is ion mobility separation-mass spectrometry (IMS-MS), and the application section is an application section that applies the sample to the IMS-MS under the condition that phosphatidylinositol phosphate is not decomposed. The apparatus of any one of 44A.
(Item 46A)
A method for separating or purifying phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample according to the position of a phosphate group while retaining an acyl group, the method comprising: A) Steps applied to graphy-mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS); and B) the elution position of the peak from the eluate of LC or IMS in the LC-MS or IMS-MS Collecting phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate having a phosphate group at the position of interest specified by.
(Item 47A)
From a mixture of two or more of the phosphate group positions of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate, from the number of types of phosphate groups of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate contained in the mixture A method for producing a sample comprising a phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate having a phosphate group at a small number of positions, comprising:
A) applying the sample to liquid column chromatography-mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS); and B) LC or IMS in the LC-MS or IMS-MS Collecting a fraction containing phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate having a phosphate group at a target position specified by the elution position of the peak from the eluate.
(Item 48A)
47. The method according to Item 47A, wherein the number of the phosphatidylinositol phosphate and / or the lysophosphatidylinositol phosphate contained in the mixture is less than the number of types of the phosphate group.

Furthermore, the present invention provides the following.
(1B) a method for measuring, detecting or identifying phosphatidylinositol phosphate in a sample, the method comprising: A) applying the sample to liquid column chromatography-mass spectrometry (LC-MS); and B) A method comprising the step of locating the phosphate group of the phosphatidylinositol phosphate by the elution position of the peak in LC-MS.
(2B) The method according to Item 1B, wherein the presence or absence of an acyl group contained in the phosphatidylinositol phosphate is also specified in the specifying step.
(3B) The method according to item 1B or 2B, wherein the total molecular weight of the acyl groups contained in the phosphatidylinositol phosphate is also specified in the specifying step.
(4B) The method according to any one of items 1B to 3B, wherein the type of acyl group contained in the phosphatidylinositol phosphate is also specified in the specifying step.
(5B) The method according to any one of Items 1B to 4B, wherein the measurement, detection or identification is performed by distinguishing phosphatidylinositol phosphate from other inositol-containing phospholipids.
(6B) The method according to any one of items 1B to 5B, wherein the sample is prepared under a condition in which phosphatidylinositol phosphate is not decomposed.
(7B) The method according to Item 6B, wherein the condition includes not performing a deacylation treatment.
(8B) The method according to any one of Items 1B to 7B, wherein the liquid column chromatography is selected from the group consisting of a chiral column, a reverse phase column, and column chromatography for separating a hydrophilic group and a hydrophobic group.
(9B) The method according to any one of items 1B to 8B, wherein the liquid column chromatography is combined with ion mobility separation.
(10B) The method according to any one of items 1B to 9B, further comprising the step of quantifying the phosphatidylinositol phosphate.
(11B) The method according to any one of items 1B to 10B, wherein the sample is an intact sample.
(12B) The method according to any one of items 1B to 11B, wherein the specifying is performed without labeling the sample.
(13B) a method for measuring, detecting or identifying phosphatidylinositol phosphate in a sample, the method comprising: A) applying the sample to ion mobility separation-mass spectrometry (IMS-MS); and B) the IMS A method comprising locating the phosphate group of the phosphatidylinositol phosphate by the elution position of the peak in MS.
(14B) The method according to item 13B, comprising any one or more of items 2B to 8B, 10B to 12B.
(15B) An apparatus for measuring, detecting or identifying the positions of acyl groups and phosphate groups of phosphatidylinositol phosphate in a sample, wherein liquid column chromatography-mass spectrometry (LC-MS) and decomposition of phosphatidylinositol phosphate are performed. And an application unit that applies the sample to the LC-MS under conditions that are not performed.
(16B) The apparatus according to item 15B, wherein the liquid column chromatography is selected from the group consisting of all column chromatography that separates hydrophilic groups and hydrophobic groups, such as chiral columns and reversed phase columns.
(17B) The apparatus according to item 15B or 16B, further comprising means for detecting or quantifying the phosphatidylinositol phosphate.
(17B2) The apparatus according to any one of items 15B to 17B, comprising any one or a plurality of features of items 2B to 12B.
(18B) An apparatus for measuring, detecting or identifying the position of acyl group and phosphate group of phosphatidylinositol phosphate in a sample, wherein ion mobility separation-mass spectrometry (IMS-MS) and phosphatidylinositol phosphate are not decomposed. And an application unit that applies the sample to the IMS-MS under non-operating conditions.
(18B2) The apparatus according to item 18B, comprising any one or more of the items 2B to 12B.
(19B) A method for separating or purifying phosphatidylinositol phosphate in a sample according to the position of a phosphate group while retaining an acyl group, the method comprising: A) liquid column chromatography-mass spectrometry of the sample (LC-MS) or a step applied to ion mobility separation-mass spectrometry (IMS-MS); and B) from the eluate of the LC-MS or IMS-MS to the target position specified by the elution position of the peak Collecting a phosphatidylinositol phosphate having a phosphate group.
(19B2) The method according to item 19B, comprising any one or more of the items 2B to 12B.
(20B) A phosphatidyl group having a phosphate group at a kind of position less than the number of kinds of the phosphate group of the phosphatidylinositol phosphate included in the mixture from a mixture of two or more phosphate groups of the phosphatidylinositol phosphate A method for producing a sample comprising inositol phosphate, comprising:
A) applying the sample to liquid column chromatography-mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS); and B) from the eluate of the LC-MS or IMS-MS, Collecting a fraction containing a phosphatidylinositol phosphate having a phosphate group at a target position specified by an elution position of a peak.
(21B) The method according to item 20B, wherein the number of the phosphoric acid group positions of the phosphatidylinositol phosphate contained in the mixture is less than the number of kinds.
(21B2) The method according to item 20B or 21B, comprising any one or more of the items 2B to 12B.

Previous analytical methods were unable to separate the regioisomers of phosphorylation and were measured by the sum of the three isomers, but mass spectrometry (MS) was performed by chromatography or ion mobility separation (IMS) as required. ) Has been developed to separate and measure isomers. In one embodiment, the present invention relates to an analytical method that can separate and measure eight PIPs and LPIPs isomers intact using a chiral column. An exemplary analysis uses MRM / SRM with triple quadrole mass spectrometer. Q1 sets an intact precursor ion, and Q3 sets DAG or MAG from which inositol phosphate is eliminated as a product ion.

The present invention also provides the following.
(1C) A compound which is lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate.
(2C) 1-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol monophosphate, 1-acyl-lysophosphatidylinositol diphosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl The compound according to item 1C, which is lysophosphatidylinositol triphosphate or 2-acyl-lysophosphatidylinositol triphosphate.
(3C) 1-acyl-lysophosphatidylinositol-3-monophosphate, 1-acyl-lysophosphatidylinositol-4-monophosphate, 1-acyl-lysophosphatidylinositol-5-monophosphate, 2-acyl-lyso Phosphatidylinositol-3-monophosphate, 2-acyl-lysophosphatidylinositol-4-monophosphate, 2-acyl-lysophosphatidylinositol-5-monophosphate, 1-acyl-lysophosphatidylinositol-3-4-2 Phosphoric acid, 1-acyl-lysophosphatidylinositol-3.5-diphosphate, 1-acyl-lysophosphatidylinositol-4.5-diphosphate, 2-acyl-lysophosphatidylinositol-3,4-diphosphate 2-acyl-lysophosphatidylinositol-3,5-diphosphate, -Acyl-lysophosphatidylinositol-4,5-diphosphate, 1-acyl-lysophosphatidylinositol-3,4,5-triphosphate, or 2-acyl-lysophosphatidylinositol-3,4,5-triphosphate The compound according to item 1C or 2C, which is an acid.
(4C) Item 1C which is 2-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl-lysophosphatidylinositol triphosphate or 2-acyl-lysophosphatidylinositol triphosphate The compound according to any one of ˜3C.
(5C) 1-butanyl-lysophosphatidylinositol 4-monophosphate;
1-hexanyl-lysophosphatidylinositol 4-monophosphate;
1-octanyl-lysophosphatidylinositol 4-monophosphate;
1-hexadecanyl-lysophosphatidylinositol 4-monophosphate;
1-9Z-hexadecenyl-lysophosphatidylinositol 4-monophosphate;
1-heptadecanyl-lysophosphatidylinositol 4-monophosphate;
1-octadecanyl-lysophosphatidylinositol 4-monophosphate;
1-9Z-octadecenyl-lysophosphatidylinositol 4-monophosphate;
1-9Z, 12Z-octadecandienyl-lysophosphatidylinositol 4-monophosphate;
1-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 4-monophosphate;
1-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 4-monophosphate;
1-7Z, 10Z, 13Z, 16Z-docosatetraenyl-lysophosphatidylinositol 4-monophosphate;
1-7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenyl-lysophosphatidylinositol 4-monophosphate;
1-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol 4-monophosphate;
2-butanyl-lysophosphatidylinositol 4-monophosphate;
2-hexanyl-lysophosphatidylinositol 4-monophosphate;
2-octanyl-lysophosphatidylinositol 4-monophosphate;
2-hexadecanyl-lysophosphatidylinositol 4-monophosphate;
2-9Z-hexadecenyl-lysophosphatidylinositol 4-monophosphate;
2-heptadecanyl-lysophosphatidylinositol 4-monophosphate;
2-octadecanyl-lysophosphatidylinositol 4-monophosphate;
2-9Z-octadecenyl-lysophosphatidylinositol 4-monophosphate;
2-9Z, 12Z-octadecanedienyl-lysophosphatidylinositol 4-monophosphate;
2-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 4-monophosphate;
2-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 4-monophosphate;
2-7Z, 10Z, 13Z, 16Z-docosatetraenyl-lysophosphatidylinositol 4-monophosphate;
2-7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenyl-lysophosphatidylinositol 4-monophosphate;
2-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol 4-monophosphate;
1-butanyl-lysophosphatidylinositol 4,5-diphosphate;
1-hexanyl-lysophosphatidylinositol 4,5-diphosphate;
1-octanyl-lysophosphatidylinositol 4,5-diphosphate;
1-hexadecanyl-lysophosphatidylinositol 4,5-diphosphate;
1-9Z-hexadecenyl-lysophosphatidylinositol 4,5-diphosphate;
1-heptadecanyl-lysophosphatidylinositol 4,5-diphosphate;
1-octadecanyl-lysophosphatidylinositol 4,5-diphosphate;
1-9Z-octadecenyl-lysophosphatidylinositol 4,5-diphosphate;
1-9Z, 12Z-octadecandienyl-lysophosphatidylinositol 4,5-diphosphate;
1-6Z, 9Z, 12Z-octadecanedienyl-lysophosphatidylinositol 4,5-diphosphate;
1-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 4,5-diphosphate;
1-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 4,5-diphosphate;
2-butanyl-lysophosphatidylinositol 4,5-diphosphate;
2-hexanyl-lysophosphatidylinositol 4,5-diphosphate;
2-octanyl-lysophosphatidylinositol 4,5-diphosphate;
2-hexadecanyl-lysophosphatidylinositol 4,5-diphosphate;
2-9Z-hexadecenyl-lysophosphatidylinositol 4,5-diphosphate;
2-heptadecanyl-lysophosphatidylinositol 4,5-diphosphate;
2-octadecanyl-lysophosphatidylinositol 4,5-diphosphate;
2-9Z-octadecenyl-lysophosphatidylinositol 4,5-diphosphate;
2-9Z, 12Z-octadecandienyl-lysophosphatidylinositol 4,5-diphosphate;
2-6Z, 9Z, 12Z-octadecanedienyl-lysophosphatidylinositol 4,5-diphosphate;
2-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 4,5-diphosphate;
2-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 4,5-diphosphate;
1-butanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-octanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-hexadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-9Z-hexadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-heptadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-octadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-9Z-octadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-butanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-octanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-hexadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-9Z-hexadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-heptadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-octadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-9Z-octadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate; and 2-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol Selected from the group consisting of 3,4,5-triphosphate,
The compound according to any one of Items 1C to 4C.
(6C) A composition comprising the compound according to any one of items 1C to 5C for use as a cancer marker.
(7C) The composition according to item 6C, wherein the cancer comprises prostate cancer.
(8C) The compound according to any one of items 1C to 5C, comprising a step of deacylating phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate by contacting with alkylamine. Or the manufacturing method of the composition of item 6C or 7C.
(9C) The method according to Item 8C, wherein the alkylamine is methylamine.
(10C) The production method according to item 8C or 9C, wherein the deacylation is a milder condition than a condition for dediacylation.
(11C) The method according to item 10C, wherein the mild condition is achieved by shortening a reaction time, reducing a concentration of the alkylamine, a reaction temperature, or a combination thereof among the conditions for dediacylation.
(12C) The mild condition is that methylamine is used as the alkylamine, the reaction time is 10 minutes or less, or the methylamine concentration is about 11% or less, the reaction temperature is 53 ° C. or less, or the reaction time, the concentration, and the concentration The method according to item 10C or 11C, which is a combination of reaction temperatures.
(13C) (A) A step of providing phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate having acyl groups with different masses at the 1-position and the 2-position, and having a desired mass An acyl group is present at a desired position selected from the 1-position or 2-position; (B) contacting the phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate with an alkylamine; And (C) optionally removing lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate having a desired mass of acyl group. At the desired position How to the production of lyso-phosphatidyl inositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate having an acyl group.
(14C) (A) a step of concentrating the phospholipid by contacting the sample with an anionic resin; (B) a step of protecting a phosphate group in the acidic phospholipid with a protecting group; and (C) a mass. A method for detecting, identifying or quantifying lysophosphatidylinositol phosphate, comprising the step of detecting, identifying or quantifying lysophosphatidylinositol phosphate in said phospholipid by an analytical method.
(15C) The method according to item 14C, wherein the protecting group comprises an alkyl group.
(16C) The method according to item 14C or 15C, wherein the protecting group comprises a methyl group.
(17C) The method according to any one of items 14C to 16C, wherein the mass spectrometry includes a selective reaction monitoring method (SRM) using a triple quadrupole mass spectrometer.
(18C) Any of items 14C to 17C, further comprising the step of detecting, identifying or quantifying the fatty acid side chain and / or phosphate group of the lysophosphatidylinositol phosphate using reverse phase column chromatography. The method according to one item.
(19C) The method according to item 18C, wherein in the reverse phase column chromatography, diacylglycerol is selected as a product ion (fragment ion) to detect, identify or quantify the fatty acid side chain and / or phosphate group.
(20C) A method for diagnosing cancer by detection, identification or quantification according to any one of items 14C to 19C.

In the present invention, it is contemplated that the one or more features described above may be provided in further combinations in addition to the explicit combinations. Still further embodiments and advantages of the invention will be recognized by those of ordinary skill in the art upon reading and understanding the following detailed description as needed.

The present invention provides new lipids to elucidate the vital functions of these lipids and can be applied to medical applications. In addition, new drug discovery targets can be presented. In addition, the present invention can provide a new diagnosis and detection method by providing a new measurement method.

In addition, when the present invention is used, phosphoinositide and / or lysophosphoinositide can be separated at the position of a phosphate group and measured, detected or identified in an intact state. Analysis can be performed. Such information is useful in biological information, pharmacological information, drug development, treatment, diagnosis, and the like.

FIG. 1A is a schematic diagram for explaining the principle of “measurement and detection method by SRM in a triple quadrupole mass spectrometer”. FIG. 1B shows the experimental results for studying the conditions for the synthesis of the compounds of the present invention. In this example, LPIP1 is not produced under the conventional deacylation reaction conditions, but LPIP1 can be obtained by shortening the reaction time (upper stage) or decreasing the methylamine concentration (lower stage). The vertical axis represents the yield (%) of the reaction product, and the horizontal axis represents time (minutes). FIG. 1C shows the results of examining the reaction temperature. 17: 0/20: 4 PI4P was used as a raw material. The products are 17: 0 LPIP1 and 20: 4 LPIP1. The vertical axis shows the yield (%) of LPIP1 product. 2A to 2G show that seven types of LPIPs having different phosphorylation patterns can be synthesized by a deacylation reaction under mild conditions shown in FIG. In either case, the ability to synthesize LPIPs with different acyl groups is illustrated by the generation of LPIPs with 17: 0, 20: 4. FIG. 2A shows the identification data of a synthetic product of the compound of the present invention. Raw material: 17: 0/20: 4 PI3P, target product: 17: 0 LPI3P (top): ms1 = 754.390, ms2 = 327.290; 20: 4 LPI3P (bottom): ms1 = 788.370, ms2 = 361.270. The vertical axis represents relative abundance, and the horizontal axis represents time (minutes). FIG. 2B shows the identification data of a synthetic product of the compound of the present invention. Raw material: 17: 0/20: 4 PI4P, target product: 17: 0 LPI4P (top): ms1 = 754.390, ms2 = 327.290; 20: 4 LPI4P (bottom): ms1 = 788.370, ms2 = 361.270. The vertical axis represents relative abundance, and the horizontal axis represents time (minutes). FIG. 2C shows identification data for a synthetic product of the compounds of the present invention. Raw material: 17: 0/20: 4 PI5P, target product: 17: 0 LPI5P (top): ms1 = 754.390, ms2 = 327.290; 20: 4 LPI5P (bottom): ms1 = 788.370, ms2 = 361.270. The vertical axis represents relative abundance, and the horizontal axis represents time (minutes). FIG. 2D shows identification data for a synthetic product of the compounds of the present invention. Raw material: 17: 0/20: 4 PI (3,4) P2, target product: 17: 0 LPI (3,4) P2 (top): ms1 = 862.390, ms2 = 327.290; 20: 4 LPI (3 4) P2 (bottom): ms1 = 896.370, ms2 = 361.270. The vertical axis represents relative abundance, and the horizontal axis represents time (minutes). FIG. 2E shows identification data for a synthetic product of the compounds of the present invention. Raw material: 17: 0/20: 4 PI (3,5) P2, target product: 17: 0 LPI (3,5) P2 (top): ms1 = 862.390, ms2 = 327.290; 20: 4 LPI (3 5) P2 (bottom): ms1 = 896.370, ms2 = 361.270. The vertical axis represents relative abundance, and the horizontal axis represents time (minutes). FIG. 2F shows identification data of a synthetic product of the compound of the present invention. Raw material: 17: 0/20: 4 PI (4,5) P2, target product: L17: 0 PI (4,5) P2 (top): ms1 = 862.390, ms2 = 327.290; 20: 4 LPI (4 5) P2 (bottom): ms1 = 896.370, ms2 = 361.270. The vertical axis represents relative abundance, and the horizontal axis represents time (minutes). FIG. 2G shows identification data for a synthetic product of the compounds of the invention. Raw material: 17: 0/20: 4 PI (3,4,5) P3, target product: 17: 0 LPI (3,4,5) P3 (top): ms1 = 970.390, ms2 = 327.290; 20: 4 LPI (3,4,5) P3 (bottom): ms1 = 1004.370, ms2 = 361.270. The vertical axis represents relative abundance, and the horizontal axis represents time (minutes). In Figure ^{3A ~ 3F, Thermo Fisher Scientific TM} Q Exactive TM plus ( hybrid quadrupole - Orbitrap mass spectrometer) by measuring accurate mass using the identification of the structure of the present invention (LPIP1, LPIP2, and LPIP3 The result of the confirmation of the structure is shown. FIG. 3A shows two types of LPI produced by a mild deacylation reaction (see FIG. 1) of 37: 4 PI (4) P (sn-1 17: 0, sn-2 20: 4 PI (4) P). (4) About P, the result of having performed the exact mass spectrometry of the fragment derived from LPI (4) P and LPI (4) P is shown. The vertical axis represents relative abundance, and the horizontal axis represents time (minutes). FIG. 3B shows the structural formula of 37: 4 PI (4) P, the product after the deacylation reaction, and fragments thereof. FIG. 3C shows two types of LPIs produced by a mild deacylation reaction of 37: 4 PI (4,5) P2 (sn-1 17: 0, sn-2 20: 4 PI (4,5) P2). 4,5) P2 shows the results of accurate mass analysis of LPI (4,5) P2 and fragments derived from LPI (4,5) P2. The vertical axis represents relative abundance, and the horizontal axis represents time (minutes). FIG. 3D shows the structural formula of 37: 4 PI (4,5) P2, the product after the deacylation reaction, and its fragment. FIG. 3E shows a two-dimensional reaction resulting from a mild deacylation reaction of 37: 4 PI (3,4,5) P3 (sn-1 17: 0, sn-2 20: 4 PI (3,4,5) P3). The result of carrying out the accurate mass spectrometry of the fragment derived from LPI (3,4,5) P3 and LPI (3,4,5) P3 is shown for the species LPI (3,4,5) P3. The vertical axis represents relative abundance, and the horizontal axis represents time (minutes). FIG. 3F shows the structural formula of 37: 4 PI (3,4,5) P3, the product after the deacylation reaction, and its fragment. FIG. 4A represents an overview of the spin system of inositol phospholipids. A portion surrounded by a solid line indicates a group of ¹ H nuclei connected by continuous ³ J coupling, and a portion surrounded by a broken line indicates a portion where ¹ H nuclei and ³¹ P nuclei are ³ J coupled. FIG. 4B shows the chemical structure observed from the NMR spectrum of Brain PI4P (Avanti Polar Lipids, Alabama, USA). The chemical structure of 1-stearoyl-2-arachidonyl-sn-glycero-3-phospho- (1′-myo-inositol-4′-monophosphate) and the chemical structure observed from the signals of ¹ H-1D and TOCSY spectra Show. A series of fatty acid, glycerol and inositol signals were observed. CH ₂ of C1 was magnetically non-equivalent. Arachidonic acids aCH ₂ , bCH ₂ and gCH ₂ were observed separately. FIG. 4C shows the chemical structure observed from the ¹ H-1D, TOCSY spectrum of Sample 1 of Example 1 (16: 0 LPI4P). The chemical structure observed from the structural formula of 16: 0 LPI4P and ¹ H-1D and TOCSY spectra is shown. Fatty acid and glycerol skeleton C1 and C3 signals were observed. In the region around 4 ppm, a signal considered to be an inositol ring was weakly observed. No signal of polyunsaturated fatty acids was observed. FIG. 4D shows the chemical structure observed from the ¹ H-1D, TOCSY spectrum of Sample 2 of Example 1 (17: 0 LPI (4,5) P2). 17: 0 shows the chemical structure observed from the structural formula of LPI (4,5) P2 and the signals of ¹ H-1D and TOCSY spectra. Fatty acid and glycerol skeleton C1 and C3 signals were observed. Minor signals of C1, C3, aCH ₂ (and bCH ₂ ) were observed. In the region around 4 ppm, a signal considered to be an inositol ring was weakly observed. FIG. 4E shows the chemical structure observed from the ¹ H-1D, TOCSY spectrum of sample 3 of Example 1 (18: 0 LPI (4,5) P2). 18: 0 shows the chemical structure observed from the structural formula of LPI (4,5) P2 and the signals of ¹ H-1D and TOCSY spectra. Fatty acid and glycerol skeleton C1 and C3 signals were observed. A minor signal of C1, C3, aCH ₂ (and bCH ₂ ) was observed, which was weaker than samples 1 and 2. In the region around 4 ppm, a signal considered to be an inositol ring was weakly observed. FIG. 4F shows the chemical structure observed from the ¹ H-1D, TOCSY spectrum of Sample 5 of Example 1 (17: 0/20: 4 PI (4,5) P2). 17: 0/20: 4 This shows the chemical structure observed from the structural formula of PI (4,5) P2 and the signals of ¹ H-1D and TOCSY spectra. Fatty acid and glycerol skeleton C1, C2, and C3 signals were observed. Arachidonic acids aCH ₂ , bCH ₂ , gCH ₂ were observed separately. Signals of polyunsaturated fatty acids (—CH═CH—CH ₂ —CH═CH—, —CH═CH—) were hardly observed. 5A-5D show data relating to the presence of a compound of the invention in a biological sample (cultured cancer cell line). FIG. 5A shows the presence of LPIPs in HEK293T cells. The vertical axis represents the abundance (pmol) of the compound relative to 1 nmol of phosphatidylserine (PS), and the horizontal axis represents the molecular species. FIG. 5B shows the presence of LPIPs in Jurkat cells. The vertical axis represents the abundance (pmol) of the compound relative to 1 nmol of phosphatidylserine (PS), and the horizontal axis represents the molecular species. FIG. 5C shows the results of accurate mass measurement of LPIP3 and fragments thereof present in HEK293T cells. The vertical axis represents relative abundance, and the horizontal axis represents m / z (mass / number of charges). FIG. 5D shows the coincidence of elution times of LPIP3 present in HEK293T cells and fragments thereof. The vertical axis represents relative abundance, and the horizontal axis represents time (minutes). 6A-6D show data relating to the presence in biological samples (mouse, human) for the compounds of the present invention. FIG. 6A shows that LPIPs are present in the mouse heart, large intestine, liver, brain (whole brain), and spleen. The vertical axis represents the abundance (pmol) of the compound relative to 1 nmol of phosphatidylserine (PS), and the horizontal axis represents the cell type. FIG. 6B shows the presence of LPIPs in mouse serum. The vertical axis represents the abundance (fmol) of the compound relative to 1 μL of serum, and the horizontal axis represents the molecular species. FIG. 6C shows the presence of LPIPs in mouse plasma. The vertical axis represents the abundance (fmol) of the compound relative to 1 μL of plasma, and the horizontal axis represents the molecular species. FIG. 6D shows the presence of LPIPs in human serum. The vertical axis represents the abundance (fmol) of the compound relative to 1 μL of serum, and the horizontal axis represents the molecular species. FIG. 7A shows mouse prostate weight in the upper row (vertical axis indicates weight (mg), horizontal axis indicates tissue type), and lower row shows prostate HE-stained image. Indicates that cancer develops. FIG. 7B shows that LPIPs levels in the prostate increase with the development of prostate cancer. The vertical axis represents LPIPs level (AU (arbitrary unit)) for 1 nmol of phosphatidylserine (PS), the horizontal axis Ctrl indicates normal mice, and PTEN KO expresses the tumor suppressor gene Pten in a prostate-specific manner. Refers to a deficient mouse. FIG. 8 shows that LPIP3 is generated in myelocytes upon stimulation with complement component 5a, which is a inflammatory substance. The vertical axis indicates the abundance (pmol) of LPIP3 with respect to 10 ⁶ cells, the horizontal axis “−” indicates that stimulation by the complement component 5a is not performed, and “+” indicates the complement component This means that stimulation by 5a is being performed. 9A and 9B are diagrams showing the separation of 17: 0/20: 4 phosphoinositides on a chiral column. Each graph is a chromatogram when the phosphoric acid group phosphoinositide sample displayed is measured with a mass spectrometer, the horizontal axis represents retention time (minutes), and the vertical axis represents peak-top signal intensity (cps). Represents the relative signal intensity with respect to 100%. The left column of FIG. 9A shows PI, the center column shows PI (3) P, PI (4) P and PI (5) P from the top, respectively, and the right column shows their mixture (PIP1mix). The second column from the right shows PI (3,5) P, PI (3,4) P2PI (4,5) P2 and mixtures thereof, respectively. The left column of FIG. 9B shows PI (3,5) P2, PI (3,4) P2 and PI (4,5) P2 from the top, the center shows their mixture (PIP2mix), and the right column shows PI (3,4,5) P3 is shown. 9A and 9B are diagrams showing the separation of 17: 0/20: 4 phosphoinositides on a chiral column. Each graph is a chromatogram when the phosphoric acid group phosphoinositide sample displayed is measured with a mass spectrometer, the horizontal axis represents retention time (minutes), and the vertical axis represents peak-top signal intensity (cps). Represents the relative signal intensity with respect to 100%. The left column of FIG. 9A shows PI, the center column shows PI (3) P, PI (4) P and PI (5) P from the top, respectively, and the right column shows their mixture (PIP1mix). The second column from the right shows PI (3,5) P, PI (3,4) P2PI (4,5) P2 and mixtures thereof, respectively. The left column of FIG. 9B shows PI (3,5) P2, PI (3,4) P2 and PI (4,5) P2 from the top, the center shows their mixture (PIP2mix), and the right column shows PI (3,4,5) P3 is shown. FIG. 10 shows seven different amounts (0.02, 0.05, 0.1, 0.2, 5, 10, 50 pmol) of the indicated phosphate group 17: 0/20: 4 phosphoinositide. The calibration curve prepared from the signal intensity (peak area) when measured with a mass spectrometer (repeatedly measured four times for each injection amount) is shown. The average of 4 repeated measurements is represented by black dots, and the standard deviation is represented by error bars. The upper row shows PI, PI (3) P, and PI (4) P from the left, the middle row shows PI (5) P, PI (3,5) P, and PI (3,4) P2 from the left, and the lower row. From the left, PI (4,5) P2, PI (3,4,5), P3 are shown. FIG. 11 shows fluctuations in signal intensity (cps) when 10 pmol of 17: 0/20: 4 phosphoinositide of the displayed phosphate group was measured 50 times with a mass spectrometer. The vertical axis represents signal intensity (cps), and the horizontal axis represents the number of injections. In the 10th injection, PI (3) P gives the first strongest signal strength, PI (4) P gives the second strongest signal strength, and PI (5) P gives the third strongest signal strength. , PI (3,4) P2 gives the fourth strongest signal strength, PI (3,5) P2 gives the fifth strongest signal strength, PI gives the sixth strongest signal strength, PI (4, 5) P2 gave the seventh strongest signal intensity and PI (3,4,5) P3 gave the weakest signal intensity. FIG. 12 shows the separation of 17: 0/20: 4 phosphoinositides (PI (3,4) P2, PI (3,5) P2, PI (4,5) P2) by ion mobility. The horizontal axis represents compensation voltage (COV, V), and the vertical axis represents signal intensity (cps). The measurement results of each phosphoinositide are shown superimposed. FIG. 13 shows the change in phosphoinositide composition of cells when the cells were treated with H ₂ O ₂ as a result of measurement using a combination of a chiral column and a mass spectrometer. In each graph, the horizontal axis represents the retention time, and the vertical axis represents the relative signal intensity when the peak top signal intensity (cps) is 100%. The graph shows the results of the H ₂ O ₂ treatment at 0 minute, 2 minutes, 5 minutes, and 15 minutes from the top, and the results for the mass corresponding to PI, PIP, PIP2 and PIP3 from the left. . FIGS. 14A to 14D show changes in the phosphoinositide composition for each diacylglycerol of the cells and the total amount of each phosphoinositide when the cells were treated with H ₂ O ₂ and quantification calculated from the measurement results of the chiral column-mass spectrometer Shown as a value. In the graph of the left column, the horizontal axis represents the type of diacylglycerol in phosphoinositide Tide, the amount of each phosphoinositide tide vertical axis contained in the cell ¹⁰⁶ which is calculated as the average of four measurements a (pmol) Expressed with standard deviation. For each type of diacylglycerol, the results at the 0 minute point, the 2 minute point, the 5 minute point, and the 15 minute point of H ₂ O ₂ treatment are shown from the left. In the graph in the right column, the vertical axis represents the total amount of phosphoinositide Tide with the same phosphate substitutions contained in the cell ¹⁰⁶ which is calculated as the average of four measurements a (pmol) with standard deviation, the horizontal axis represents the left To H ₂ O ₂ treatment at 0 min, 2 min, 5 min, and 15 min. It is a result of four repeated measurements, and the average of repeated measurements is shown together with a standard deviation (error bar). ANOVA, when statistical processing using the multiple comparison test of ^Tukey, that ^*** is p ^<0.001, that ^** is p ^{<0.01, *,} p < Each represents 0.05. FIGS. 14A to 14D show changes in the phosphoinositide composition for each diacylglycerol of the cells and the total amount of each phosphoinositide when the cells were treated with H ₂ O ₂ and quantification calculated from the measurement results of the chiral column-mass spectrometer Shown as a value. In the graph of the left column, the horizontal axis represents the type of diacylglycerol in phosphoinositide Tide, the amount of each phosphoinositide tide vertical axis contained in the cell ¹⁰⁶ which is calculated as the average of four measurements a (pmol) Expressed with standard deviation. For each type of diacylglycerol, the results at the 0 minute point, the 2 minute point, the 5 minute point, and the 15 minute point of H ₂ O ₂ treatment are shown from the left. In the graph in the right column, the vertical axis represents the total amount of phosphoinositide Tide with the same phosphate substitutions contained in the cell ¹⁰⁶ which is calculated as the average of four measurements a (pmol) with standard deviation, the horizontal axis represents the left To H ₂ O ₂ treatment at 0 min, 2 min, 5 min, and 15 min. It is a result of four repeated measurements, and the average of repeated measurements is shown together with a standard deviation (error bar). ANOVA, when statistical processing using the multiple comparison test of ^Tukey, that ^*** is p ^<0.001, that ^** is p ^{<0.01, *,} p < Each represents 0.05. FIGS. 14A to 14D show changes in the phosphoinositide composition for each diacylglycerol of the cells and the total amount of each phosphoinositide when the cells were treated with H ₂ O ₂ and quantification calculated from the measurement results of the chiral column-mass spectrometer Shown as a value. In the graph of the left column, the horizontal axis represents the type of diacylglycerol in phosphoinositide Tide, the amount of each phosphoinositide tide vertical axis contained in the cell ¹⁰⁶ which is calculated as the average of four measurements a (pmol) Expressed with standard deviation. For each type of diacylglycerol, the results at the 0 minute point, the 2 minute point, the 5 minute point, and the 15 minute point of H ₂ O ₂ treatment are shown from the left. In the graph in the right column, the vertical axis represents the total amount of phosphoinositide Tide with the same phosphate substitutions contained in the cell ¹⁰⁶ which is calculated as the average of four measurements a (pmol) with standard deviation, the horizontal axis represents the left To H ₂ O ₂ treatment at 0 min, 2 min, 5 min, and 15 min. It is a result of four repeated measurements, and the average of repeated measurements is shown together with a standard deviation (error bar). ANOVA, when statistical processing using the multiple comparison test of ^Tukey, that ^*** is p ^<0.001, that ^** is p ^{<0.01, *,} p < Each represents 0.05. FIGS. 14A to 14D show changes in the phosphoinositide composition for each diacylglycerol of the cells and the total amount of each phosphoinositide when the cells were treated with H ₂ O ₂ and quantification calculated from the measurement results of the chiral column-mass spectrometer Shown as a value. In the graph of the left column, the horizontal axis represents the type of diacylglycerol in phosphoinositide Tide, the amount of each phosphoinositide tide vertical axis contained in the cell ¹⁰⁶ which is calculated as the average of four measurements a (pmol) Expressed with standard deviation. For each type of diacylglycerol, the results at the 0 minute point, the 2 minute point, the 5 minute point, and the 15 minute point of H ₂ O ₂ treatment are shown from the left. In the graph in the right column, the vertical axis represents the total amount of phosphoinositide Tide with the same phosphate substitutions contained in the cell ¹⁰⁶ which is calculated as the average of four measurements a (pmol) with standard deviation, the horizontal axis represents the left To H ₂ O ₂ treatment at 0 min, 2 min, 5 min, and 15 min. It is a result of four repeated measurements, and the average of repeated measurements is shown together with a standard deviation (error bar). ANOVA, when statistical processing using the multiple comparison test of ^Tukey, that ^*** is p ^<0.001, that ^** is p ^{<0.01, *,} p < Each represents 0.05. FIG. 15 shows changes in the phosphoinositide composition in the thyroid gland (organ) of the mouse when the mouse is genetically modified as a result of measurement using a combination of a chiral column and a mass spectrometer. In each graph, the horizontal axis represents the retention time, and the vertical axis represents the relative signal intensity when the peak top signal intensity (cps) is 100%. The graph shows the results for wild type, INPP4B (− / −), PTEN (+/−), and INPP4B (− / −) PTEN (+/−) mice from the top, and from the left, PI, PIP , The results for masses corresponding to PIP2 and PIP3. FIGS. 16A to 16C show the change in phosphoinositide composition for each diacylglycerol and the total amount of each phosphoinositide in the thyroid gland (organ) of the mouse when the mouse is genetically modified from the measurement results of the chiral column-mass spectrometer. It is shown as a quantitative value. In the graphs of FIGS. 16A and B, the horizontal axis represents the type of diacylglycerol in phosphoinositide, and the vertical axis represents 3 times (wild type), 4 times (INPP4B (− / −), PTEN (+/−), And the amount (pmol) of each phosphoinositide contained in 1 mg of tissue calculated as an average of the INPP4B (− / −) PTEN (+/−)) measurement, together with the standard deviation. For each diacylglycerol type, the results for wild type, INPP4B (− / −), PTEN (+/−), and INPP4B (− / −) PTEN (+/−) mice are shown from the left. FIGS. 16A to 16C show the change in phosphoinositide composition for each diacylglycerol and the total amount of each phosphoinositide in the thyroid gland (organ) of the mouse when the mouse is genetically modified from the measurement results of the chiral column-mass spectrometer. It is shown as a quantitative value. In the graphs of FIGS. 16A and B, the horizontal axis represents the type of diacylglycerol in phosphoinositide, and the vertical axis represents 3 times (wild type), 4 times (INPP4B (− / −), PTEN (+/−), And the amount (pmol) of each phosphoinositide contained in 1 mg of tissue calculated as an average of the INPP4B (− / −) PTEN (+/−)) measurement, together with the standard deviation. For each diacylglycerol type, the results for wild type, INPP4B (− / −), PTEN (+/−), and INPP4B (− / −) PTEN (+/−) mice are shown from the left. FIGS. 16A to 16C show changes in the phosphoinositide composition for each diacylglycerol and the total amount of each phosphoinositide in the thyroid gland (organ) of the mouse when the mouse is genetically modified from the measurement results of the chiral column-mass spectrometer. It is shown as a quantitative value. In the graph of FIG. 16C, the vertical axis represents the average of three times (wild type), four times (INPP4B (− / −), PTEN (+/−), and INPP4B (− / −) PTEN (+/−)) measurements. The total amount (pmol) of phosphoinositides having the same phosphate substitution contained in 1 mg of tissue calculated as follows is expressed with standard deviation, and the horizontal axis from the left is wild type, INPP4B (− / −), PTEN (+/− ) And INPP4B (− / −) PTEN (+/−) mice. 3 (wild type), 4 times (INPP4B (− / −), PTEN (+/−), and INPP4B (− / −) PTEN (+/−)) repeated measurement results, the average of repeated measurements , With standard deviation (error bar). ANOVA, when statistical processing using the multiple comparison test of ^Tukey, that ^*** is p ^<0.001, that ^** is p ^{<0.01, *,} p < Each represents 0.05. FIG. 17 is a diagram showing that it is possible to separate phosphoric acid position isomers of lysophospholipid by column chromatography. In each graph, the horizontal axis represents the retention time, and the vertical axis represents the relative signal intensity when the peak top signal intensity (cps) is 100%. The left column shows the results of lyso PI (3) P, lyso PI (4) P, and lyso PI (5) P at 17: 0 from the top. The right column shows the results of lyso PI (3, 5) P2, lyso PI (3,4) P2, and lyso PI (4, 5) P2 at 17: 0 from the top.

Hereinafter, the present invention will be described while showing the best mode. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

Hereinafter, definitions of terms particularly used in this specification and / or basic technical contents will be described as appropriate.

(Phospholipid)
The present invention provides a novel phospholipid and a technology for measuring, detecting or identifying phospholipids phosphoinositide and lysophosphoinositide separated by subclass.

In the present specification, “phosphatidylinositol phosphate” or “phosphatidylinositol phosphate” are used interchangeably and refer to the sn-1 position and sn− of glycerol (also referred to as propane-1,2,3-triol, glycerol). An inositol bonded to the sn-3 position is phosphorylated with X inositol bonded to the 2-position. One, two or three phosphate groups can be attached, in this case referred to as “phosphatidylinositol X phosphate” (where X is one, two or three). In this specification, various phosphatidylinositol X phosphates are collectively referred to as “phosphoinositide”, “phosphatidylinositol phosphates”, or “PIPs”. The phosphate group can be indicated at a position on inositol, such as phosphatidylinositol-3-monophosphate, phosphatidylinositol-4-monophosphate, phosphatidylinositol-5-monophosphate, phosphatidylinositol-3,4 -Described as diphosphate, phosphatidylinositol-3,5-diphosphate, phosphatidylinositol-4,5-diphosphate, phosphatidylinositol-3,4,5-triphosphate, etc.

For the first time in the present invention, a method for identifying the position of the phosphate group of the PIPs while retaining the acyl group was provided. In the present invention, it was shown that the detailed information of the phosphate group of PIPs in the living body can be understood by using the identification method provided by the present invention. In particular, since the information on the phosphate group of PIPs can be analyzed while retaining the acyl group, the present invention is a detailed information that cannot be analyzed by the prior art in the sense that information in the living body can be analyzed in a true sense. Have the potential to provide In the present invention, unless otherwise specified, inositol is myo-inositol and the fatty acid bond type is an ester type. PIPs with arbitrary acyl and phosphate group positions (on inositol) can be prepared, see, for example, Stuart J. et al. Conway et al. , Org. Biomol. Chem. , 2010, 8, 66-76, any kind of PIP can be manufactured. So far, phosphoinositides have been analyzed by separation of RI-label or non-label deacylated glycerophosphoinositides, or by using molecular probes and antibodies with specific binding proteins. In that sense, there are still no analytical methods that can measure intact phosphoinositide. Conventionally, an analysis method using RPLC-MS / MS has been reported for PIP3, which is a type of phosphoinositide, but PIP1 and PIP2, which have three isomers, cannot be separated in this analysis method. Is measured as the sum of

In the present specification, “lysophosphatidylinositol phosphate” or “lysophosphatidylinositol phosphate” is used interchangeably, and one acyl group is removed from phosphatidylinositol and inositol bonded to the sn-3 position is X Individual phosphorylated. One, two or three phosphate groups can be attached, in this case referred to as “lysophosphatidylinositol X phosphate” (where X is one, two or three). In the present specification, various lysophosphatidylinositol X phosphates are collectively referred to as “lysophosphoinositide”, “lysophosphatidylinositol phosphates”, or “LPIPs”. The phosphate group can be represented by a position on inositol, and is expressed as, for example, lysophosphatidylinositol-3,4,5-triphosphate. For the first time in the present invention, a method for identifying these LPIPs was provided, and a method for synthesis was also provided. In the present invention, it was shown that LPIPs are also present in the living body by using the identification method provided by the present invention. Similarly to PIPs, the position of the phosphate group could be identified for LPIPs. In the present invention, unless otherwise specified, inositol is myo-inositol and the fatty acid bond type is an ester type.

In this specification, the term “sn-” is an abbreviation for “Stereospecifically Numbered”, and is used when the carbon atoms of a glycerin derivative are represented by stereospecific numbering. If the glycerin derivative is racemic, add rac-; if the stereochemistry is unknown, add X-. There are two types of LPIPs, sn-1-LPIPs or sn-2-LPIPs, depending on the position of the acyl group binding. For the actual display, the position of the acyl group is used as it is, such as 1-acyl-phosphatidylinositol X phosphate, 2-acyl-phosphatidylinositol X phosphate. When there is no information regarding sn in this specification, it is the case where it explains without distinguishing in particular or uses it as a generic name (superordinate concept).

If only the desired fatty acid is available, the corresponding PIPs can be produced using this and applied to the synthesis method of the present invention to produce LPIPs having the desired fatty acid group.

Specific embodiments of the present invention include the following.
Monophosphate, sn-1-position 1-butanyl-lysophosphatidylinositol 4-monophosphate;
1-hexanyl-lysophosphatidylinositol 4-monophosphate;
1-octanyl-lysophosphatidylinositol 4-monophosphate;
1-hexadecanyl-lysophosphatidylinositol 4-monophosphate <palmityl>;
1-9Z-hexadecenyl-lysophosphatidylinositol 4-monophosphate <palmitreniinyl>;
1-heptadecanyl-lysophosphatidylinositol 4-monophosphate;
1-octadecanyl-lysophosphatidylinositol 4-monophosphate <stearyl>;
1-9Z-octadecenyl-lysophosphatidylinositol 4-monophosphate <Oleinyl>;
1-9Z, 12Z-octadecandienyl-lysophosphatidylinositol 4-monophosphate <linoleic acid>;
1-6Z, 9Z, 12Z-octadecandienyl-lysophosphatidylinositol 4-monophosphate <γ-linolenic acid>;
1-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 4-monophosphate;
1-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 4-monophosphate <arachidonic acid>;
1-7Z, 10Z, 13Z, 16Z-docosatetraenyl-lysophosphatidylinositol 4-monophosphate;
1-7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenyl-lysophosphatidylinositol 4-monophosphate;
1-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol 4-monophosphate;
Monophosphate, sn-2-position 2-butanyl-lysophosphatidylinositol 4-monophosphate;
2-hexanyl-lysophosphatidylinositol 4-monophosphate;
2-octanyl-lysophosphatidylinositol 4-monophosphate;
2-hexadecanyl-lysophosphatidylinositol 4-monophosphate <palmityl>;
2-9Z-hexadecenyl-lysophosphatidylinositol 4-monophosphate <palmitreniinyl>;
2-heptadecanyl-lysophosphatidylinositol 4-monophosphate;
2-octadecanyl-lysophosphatidylinositol 4-monophosphate <stearyl>;
2-9Z-octadecenyl-lysophosphatidylinositol 4-monophosphate <Oleinyl>;
2-9Z, 12Z-octadecandienyl-lysophosphatidylinositol 4-monophosphate <linoleic acid>;
2-6Z, 9Z, 12Z-octadecanedienyl-lysophosphatidylinositol 4-monophosphate <γ-linolenic acid>;
2-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 4-monophosphate;
2-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 4-monophosphate <arachidonic acid>;
2-7Z, 10Z, 13Z, 16Z-docosatetraenyl-lysophosphatidylinositol 4-monophosphate;
2-7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenyl-lysophosphatidylinositol 4-monophosphate;
2-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol 4-monophosphate;
Diphosphate, sn-1-position 1-butanyl-lysophosphatidylinositol 4,5-diphosphate;
1-hexanyl-lysophosphatidylinositol 4,5-diphosphate;
1-octanyl-lysophosphatidylinositol 4,5-diphosphate;
1-hexadecanyl-lysophosphatidylinositol 4,5-diphosphate <palmityl>;
1-9Z-hexadecenyl-lysophosphatidylinositol 4,5-diphosphate <palmitreniinyl>;
1-heptadecanyl-lysophosphatidylinositol 4,5-diphosphate;
1-octadecanyl-lysophosphatidylinositol 4,5-diphosphate <stearyl>;
1-9Z-octadecenyl-lysophosphatidylinositol 4,5-diphosphate <Oleinyl>;
1-9Z, 12Z-octadecandienyl-lysophosphatidylinositol 4,5-diphosphate <linoleic acid>;
1-6Z, 9Z, 12Z-octadecanedienyl-lysophosphatidylinositol 4,5-diphosphate <γ-linolenic acid>;
1-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 4,5-diphosphate;
1-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 4,5-diphosphate <arachidonic acid>;
1-7Z, 10Z, 13Z, 16Z-docosatetraenyl-lysophosphatidylinositol 4,5-diphosphate;
1-7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenyl-lysophosphatidylinositol 4,5-diphosphate;
1-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol 4,5-diphosphate;
Diphosphate, sn-2 position 2-butanyl-lysophosphatidylinositol 4,5-diphosphate;
2-hexanyl-lysophosphatidylinositol 4,5-diphosphate;
2-octanyl-lysophosphatidylinositol 4,5-diphosphate;
2-hexadecanyl-lysophosphatidylinositol 4,5-diphosphate <palmityl>;
2-9Z-hexadecenyl-lysophosphatidylinositol 4,5-diphosphate <palmitreniinyl>;
2-heptadecanyl-lysophosphatidylinositol 4,5-diphosphate;
2-octadecanyl-lysophosphatidylinositol 4,5-diphosphate <stearyl>;
2-9Z-octadecenyl-lysophosphatidylinositol 4,5-diphosphate <Oleinyl>;
2-9Z, 12Z-octadecandienyl-lysophosphatidylinositol 4,5-diphosphate <linoleic acid>;
2-6Z, 9Z, 12Z-octadecanedienyl-lysophosphatidylinositol 4,5-diphosphate <γ-linolenic acid>;
2-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 4,5-diphosphate;
2-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 4,5-diphosphate <arachidonic acid>;
2-7Z, 10Z, 13Z, 16Z-docosatetraenyl-lysophosphatidylinositol 4,5-diphosphate;
2-7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenyl-lysophosphatidylinositol 4,5-diphosphate;
2-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol 4,5-diphosphate;
Triphosphate, sn-1-position 1-butanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-hexanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-octanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-hexadecanyl-lysophosphatidylinositol 3,4,5-triphosphate <palmityl>;
1-9Z-hexadecenyl-lysophosphatidylinositol 3,4,5-triphosphate <palmitreniinyl>;
1-heptadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-octadecanyl-lysophosphatidylinositol 3,4,5-triphosphate <stearyl>;
1-9Z-octadecenyl-lysophosphatidylinositol 3,4,5-triphosphate <Oleinyl>;
1-9Z, 12Z-octadecandienyl-lysophosphatidylinositol 3,4,5-triphosphate <linoleic acid>;
1-6Z, 9Z, 12Z-octadecanedienyl-lysophosphatidylinositol 3,4,5-triphosphate <γ-linolenic acid>;
1-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate <arachidonic acid>;
1-7Z, 10Z, 13Z, 16Z-docosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol 3,4,5-triphosphate;
Triphosphate, sn-2 position 2-butanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-hexanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-octanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-hexadecanyl-lysophosphatidylinositol 3,4,5-triphosphate <palmityl>;
2-9Z-hexadecenyl-lysophosphatidylinositol 3,4,5-triphosphate <palmitreniinyl>;
2-heptadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-octadecanyl-lysophosphatidylinositol 3,4,5-triphosphate <stearyl>;
2-9Z-octadecenyl-lysophosphatidylinositol 3,4,5-triphosphate <Oleinyl>;
2-9Z, 12Z-octadecandienyl-lysophosphatidylinositol 3,4,5-triphosphate <linoleic acid>;
2-6Z, 9Z, 12Z-octadecanedienyl-lysophosphatidylinositol 3,4,5-triphosphate <γ-linolenic acid>;
2-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate <arachidonic acid>;
2-7Z, 10Z, 13Z, 16Z-docosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol 3,4,5-triphosphate These fatty acids or acyl groups based on fatty acids are based on It is understood that the lysophosphatidylinositol phosphates of the invention are available in a bound form at either the sn-1 or sn-2 position.

As used herein, “acyl (group)” is used in the ordinary sense in the art and refers to a group formed by removing a hydroxyl group from an organic acid (carboxylic acid; fatty acid). In a broad sense, formyl group HCO—, acetyl group CH ₃ CO—, malonyl group —COCH ₂ CO—, benzoyl group C ₆ H ₅ CO—, cinnamoyl group C ₆ H ₅ CH═CHCO—, etc. are included, and ketone derivatives And so on. Acyl groups contained in phosphatidylinositol phosphates and lysophosphatidylinositol phosphates are also referred to as fatty acid groups because they form fatty acids in a preferred embodiment. As used herein, a fatty acid can be represented by its carbon number and the number of double bonds, for example, arachidonic acid can be represented as (20: 4). When further specifying the position of the double bond, it is possible to specify all positions for the display of cis and trans, or the display of E or Z, or display in a system such as ω3 system, ω6 system, or the like.

In the present specification, the following fatty acids and acyl groups based thereon are generally used, but the present invention is not limited thereto, and it is understood that fatty acids having any chain length and any double bond can be used. For example, as for the number of carbon atoms, one or more, typically 1 to 30, usually 4 to 30 may be mentioned, 1, 2, 3, 4, 5, 6, 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23 24, 25, 26, 27, 28, 29, 30 and the like, but is not limited thereto. The number of double bonds may be any number that can be allowed according to the number of carbon atoms, such as 0, 1, 2, 3, 4, 5, 6, 7, and the like. The position of the double bond is typically the ω-3 system, ω-6 system, ω-9 system, etc. In addition, the ω-5 system, the ω-7 system, etc. have been confirmed. Any available one can be used. The fatty acid may contain a triple bond, and the number thereof is 0, 1, 2, 3, 4, 5, 6, 7, etc. Numbers can be employed.

(Separation techniques: chromatography, mass spectrometry, ion mobility separation)
As used herein, “chromatography” is used in the ordinary sense used in the art, and when the medium passes (permeates) uniformly through a column or capillary passage of a substance, one or more in the medium. Means a process in which the components of are selectively delayed. The delay is caused by the distribution of the mixture components between one or more stationary phases and this medium as the bulk medium (ie, mobile phase) moves relative to the stationary phase (s). Examples of “chromatography” include liquid chromatography (LC) and gas chromatography (GC). In one embodiment, chromatography can be used to identify the binding position of a phosphate group.

As used herein, “gas chromatography” or “GC” is used in the ordinary sense used in the art, and when a gas uniformly passes (permeates) through a column or capillary passage of a substance, Means a process in which one or more components are selectively delayed. The delay is caused by the distribution of the mixture components between one or more stationary phases and this gas as the bulk gas (ie, mobile phase) moves relative to the stationary phase (s).

As used herein, “liquid chromatography” or “LC” is used in the ordinary sense used in the art, and when a fluid passes uniformly (permeates) through a column or capillary passage of matter, Means a process in which one or more components are selectively delayed. The delay is caused by the distribution of the mixture components between one or more stationary phases and this fluid as the bulk fluid (ie, mobile phase) moves relative to the stationary phase (s). Examples of “liquid chromatography” include reverse phase liquid chromatography (RPLC), ion chromatography (IC), high performance liquid chromatography (HPLC), and turbulent liquid chromatography (TFLC) (high turbulent liquid chromatography). (Also referred to as (HTLC) or high-throughput liquid chromatography). As used herein, the term “high performance liquid chromatography” or “HPLC” (also referred to as “high pressure liquid chromatography”) is used under pressure through a stationary phase, typically a dense packed column. It refers to liquid chromatography whose resolution is improved by extruding the mobile phase. In a preferred embodiment, HPLC is utilized. Separation modes of the filler include normal phase, reverse phase, partition, chiral, ion exchange, molecular sieve, affinity column, etc., and any column chromatography that separates hydrophilic groups and hydrophobic groups can be used. Of these, chiral, reverse phase, normal phase, and ion exchange columns are preferred. In one preferred embodiment, it has been shown in the present invention that when a chiral column is used, all the binding positions of phosphate groups can be identified without using another column.

Examples of chiral columns include polysaccharide derivative chiral columns, protein-bonded chiral columns, chemically bonded optically active crown ether chiral columns, crown ether chiral columns, zwitterionic molecular column columns, anion exchange chiral columns, ligand exchange chiral columns, poly Examples thereof include, but are not limited to, methacrylate-type chiral columns and polysaccharide derivative chiral columns (for example, those sold by Daicel Corporation (Osaka, Japan)).

In analysis by liquid chromatography, the sample or sample components may be applied to the LC column at the inlet, eluted with the solvent or solvent mixture, and discharged at the outlet. Various solvent modes can be selected to elute the analyte (s) of interest. For example, liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polymorphic (ie, mixed) mode. During chromatography, the separation of materials is affected by variables such as the choice of eluent (also referred to as “mobile phase”), elution mode, gradient conditions, temperature, and the like.

In this specification, “ion mobility separation (IMS)” is a technique for separating ionized compounds by their bulkiness (collision cross section), and is used alone or in combination with mass spectrometry or chromatography. . This technology was developed in the 1950s to separate ions based on mobility and is widely used for detecting explosives. In IMS, typically, when ions move in a gas cell at a relatively high pressure (from atmospheric pressure to several mbar) by an electric field, depending on the mobility determined by collision of ions and neutral gas molecules. Perform separation. As an example, a DMS type (Differential ion Mobility Spectrometry) may also be used. In an exemplary embodiment, when a gaseous ion passes through an IMS cell filled with N ₂ gas, a bulkier compound has more frequent collisions with N ₂ molecules and lower mobility. The compounds can be separated by the difference in the transit time (Drift Time) caused thereby. By combining with mass spectrometry (MS), separation analysis of components having the same molecular weight and analysis of ion steric structure can be performed.

In this specification, “mass spectrometry” or “MS” is used in the ordinary sense used in the field, and refers to an analytical method for identifying a compound by its mass, and is a particle such as an atom, molecule, or cluster. Is a technique for separating and detecting ions according to the mass-to-charge ratio by making them into gaseous ions by some method (ionization), moving them in a vacuum and using electromagnetic force, etc., or by a time-of-flight difference. MS refers to a method of filtering, detecting, and measuring ions based on this mass-to-charge ratio, or “m / z”. With the dramatic improvement in detection sensitivity and mass resolution in recent years, the range of application is increasingly widened and used in many fields. Typically, the method exemplified in Clark J et al., Nat Methods. 2011 March; 8 (3): 267-272.doi: 10.1038 / nmeth.1564. Can be used. MS techniques generally include (1) ionizing a compound to form a charged compound: and (2) detecting the molecular weight of the charged compound and calculating a mass to charge ratio. The compound can be ionized and detected by appropriate means. A “mass spectrometer” generally includes an ionizer, a mass analyzer, and an ion detector. In general, the molecule or molecules of interest are ionized and the ions are then introduced into a mass spectrometer where the ions are subject to mass (“m”) and charge (for combination of magnetic and electric fields). Follow a path in space that depends on "z"). For a review of mass spectrometers, see, for example, Jurgen H, “Mass Spectrometry”, Maruzen Publishing (2014). Examples of the mass spectrometer include a magnetic field type, a quadrupole type, and a time-of-flight type, and it is preferable to use a quadrupole type that has good quantitativeness, a wide dynamic range, and good linearity. For the detection of ions in the quantification, one of the ion species purified by the selected ion monitoring or the first mass analysis unit that selectively detects only the target ions is selected as the precursor ion, and the second ion is detected. And selective reaction monitoring (SRM) that detects product ions generated by cleavage of the precursor ions in the mass spectrometer. In SRM, the signal / noise ratio is improved by increasing selectivity and reducing noise.

As used herein, the term “resolution” or “resolution (FWHM)” (also known in the art as “m / Δm50%”) is the width of the mass peak at 50% of the maximum height. Refers to the observed mass to charge ratio divided by (full width half maximum, “FWHM”). Also in the present invention, it is preferable to use an analyzer having a high resolution. The higher the resolution, the better the qualitative and quantitative properties.

In an actual arrangement, careful selection of valves and connector piping allows two or more chromatographic columns to be passed as needed so that material passes from one to the next without the need for any manual steps. It may be connected. In the preferred embodiment, the selection of valves and piping is controlled by a computer preprogrammed to perform the necessary steps. Most preferably, the chromatography system is also connected to a detection system, such as an MS system, in such an online manner. Thus, the operator can place a tray of samples into the autosampler and the remaining operations are performed under computer control, resulting in the purification and analysis of all selected samples.

In this specification, “chromatography-mass spectrometry (C-MS)” means an analysis method performed using an apparatus combining a chromatograph (eg, gas chromatograph, liquid chromatograph, etc.) and a mass spectrometer. To do. For example, tandem mass spectrometry (C-MS / MS) in which a plurality of mass spectrometry units are combined may be used. When performing ionization in C-MS, for example, atmospheric pressure chemical ionization, ESI, atmospheric pressure photoionization, or the like can be used.

In this specification, “liquid column chromatography-mass spectrometry (LC-MS)” means an analysis method performed using an apparatus in which a liquid chromatograph and a mass spectrometer are combined. For example, tandem mass spectrometry (LC-MS / MS) in which a plurality of mass spectrometry units are combined can also be used. When performing ionization in LC-MS, for example, atmospheric pressure chemical ionization, ESI, atmospheric pressure photoionization, or the like can be used.

In this specification, “ion mobility separation-mass spectrometry (IMS-MS)” means an analysis method performed using an apparatus combining ion mobility separation (IMS) and a mass spectrometer. For example, tandem mass spectrometry (IMS-MS / MS) in which a plurality of mass spectrometry units are combined may be used. When performing ionization in IMS-MS, for example, atmospheric pressure chemical ionization, ESI, atmospheric pressure photoionization, or the like can be used. IMS-MS enables separation of interfering components having the same m / z, which is impossible with a mass spectrometer alone, and analysis of the three-dimensional structure of ions, and can provide more specific and detailed information to scientists.

In this specification, “measurement” is used in the normal sense used in the field, and refers to determining how much a certain object is measured. In this specification, “detection” is used in the usual meaning used in the field, refers to finding out a substance, component, etc., and “identification” refers to an existing object related to itself. This refers to the act of finding the attribution of the classification from the classification of the substance, and when used in the chemical field, it refers to determining the identity of the target substance as a chemical substance (for example, determining the chemical structure), “Quantitative” refers to determining the amount of a target substance.

In this specification, the term “run” is used in the ordinary meaning used in the art, and the sample is loaded in a separation means such as mass spectrometry, chromatography, and ion mobility separation to separate components in the sample. , Refers to a series of steps until washing as necessary. Usually, different samples are measured in different runs to avoid confusion. When there are multiple measurement objects, it is advantageous to be able to measure all measurement objects in the same run, but the run is considered in consideration of adaptability to the measurement system for each measurement object in the sample, dynamic range, and separation degree. You may divide into several.

As used herein, the “amount” of an analyte in a body fluid sample generally refers to an absolute value that reflects the mass of the analyte that can be detected in the volume of the sample. However, an amount also contemplates a relative amount compared to another analyte amount. For example, the amount of analyte in the sample may be an amount greater than the control or normal level of analyte normally present in the sample.

The term “about” refers to the indicated value plus or minus 10% as used herein in connection with a quantitative measurement that does not include a measurement of the mass of an ion. Mass spectrometers can be slightly different in determining the mass of a given analyte. The term “about” in the context of ion mass or ion mass / charge ratio refers to +/− 0.5 atomic mass units.

As used herein, “alkylamine” refers to an alkyl group to which an amine (NH ₂ —) group is bonded. Typical examples include, but are not limited to, methylamine, ethylamine, propanamine and the like. Also referred to as aliphatic amine or aminoalkane.

In the present specification, the term “anion exchange resin” is used in the ordinary sense in the art, and typically has a property in which a basic group is bonded to the surface of the resin and binds to an anion. Can be concentrated.

As used herein, “acidic phospholipid” refers to a phospholipid in which the negative charge of the phosphate group is not canceled by a base, and includes phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol ( PI).

In the present specification, as the “protecting group” for the phosphate group, any substance used in the art can be used. Protecting groups are exemplified in, for example, Clark J et al, Nat Methods. 2011 March; 8 (3): 267-272.doi: 10.1038 / nmeth.1564. Benzyl group, p-methoxybenzyl group, Examples thereof include a tert-butyl group.

In the present specification, “the conditions under which phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate are not decomposed” means a condition in which components such as acyl groups are not decomposed and eliminated from phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate. Say. “Conditions under which phosphatidylinositol phosphate is not decomposed” refers to conditions in which components such as acyl groups are decomposed from phosphatidylinositol phosphate and do not leave, and “Conditions under which lysophosphatidylinositol phosphate is not decomposed” refers to lysophosphatidyl. The conditions under which components such as acyl groups are not decomposed and eliminated from inositol phosphate, and the conditions under which phosphatidylinositol phosphate and lysophosphatidylinositol phosphate are not decomposed are the conditions under which phosphatidylinositol phosphate and lysophosphatidylinositol phosphate This is a condition in which components such as are decomposed and do not desorb. Such conditions can be applied to chromatography, ion mobility separation, or MS by applying other treatments under conditions that do not include deacylation, in addition to applying the sample directly to chromatography, ion mobility separation, or MS. In addition to the preparation of the sample, for example, the treatment of denaturing the sample is not performed, and the treatment of introducing a protecting group into the phosphate group is included.

In this specification, “no deacylation treatment” means deacylation, for example, treatment with phospholipases A (phospholipase A ₁ , A ₂ etc.), treatment in the presence of alkylamine such as methylamine ( Serunian, et al., Methods in Enzymol. Vol., 198, 1991, pp. 78-87, especially pp. 82-83, and Fujii et al. Folia Pharmacol Jpn. 2013, pp. 236-240, especially pp. 238-239).

In this specification, “intact (sample)” means that when a sample to be analyzed is assumed, the sample is not denatured. Examples of sample denaturation include denaturation by heating, oxidation in the air, moisture, heat, light, metal ions, microorganisms or enzymes, and hydrolysis in an aqueous solvent. In order to be in an intact state, for example, the sample is frozen immediately after preparation and stored at −80 ° C. until the subsequent operation is performed.

In this specification, “not labeled” and “without labeling” means that a sample to be analyzed is not labeled. In this specification, the “label” refers to a presence (for example, a substance, energy, electromagnetic wave, etc.) for distinguishing a target molecule or substance from others. Examples of such a labeling method include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like. When a plurality of markers of the present invention or a factor or means for capturing them are labeled by the fluorescence method, the labeling is performed with fluorescent substances having different fluorescence emission maximum wavelengths. In the present invention, by using such a label, the target object can be modified so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can appropriately carry out such methods depending on the label and the target object.

As used herein, “diagnosis” identifies various parameters related to a disease, disorder, condition (eg, a disease, disorder, condition, etc. caused by a lipid mediator) in a subject, and such a disease, disorder , Refers to determining the current state or future of the state. By using the methods, devices, and systems of the present invention, conditions within the body can be examined, and such information can be used to formulate a disease, disorder, condition, treatment to be administered or prevention in a subject. Alternatively, various parameters such as methods can be selected. In the present specification, “diagnosis” in a narrow sense means diagnosis of the current state, but in a broad sense includes “early diagnosis”, “predictive diagnosis”, “preliminary diagnosis”, and the like. The diagnostic method of the present invention is industrially useful because, in principle, the diagnostic method of the present invention can be used from the body and can be performed away from the hands of medical personnel such as doctors. In this specification, in order to clarify that it can be performed away from the hands of medical personnel such as doctors, in particular, “predictive diagnosis, prior diagnosis or diagnosis” may be referred to as “support”. The technique of the present invention can be applied to such a diagnostic technique. By using the technique of the present invention, the presence of phosphoinositide and / or lysophosphoinositide or the position of the phosphate group thereof can be specified and applied to such various diagnoses.

As used herein, “treatment” refers to a certain disease or disorder (for example, a disorder such as cancer, a disease caused by a lipid mediator, a disorder, etc.), when such a condition or Prevents the deterioration of the disorder, preferably maintains the current state, more preferably reduces, and even more preferably eliminates the disorder, or exhibits the symptom improving effect or preventing effect of one or more symptoms associated with the disease. It includes what can be done. Diagnosing in advance and performing appropriate treatment is referred to as “companion treatment”, and the diagnostic agent therefor is sometimes referred to as “companion diagnostic agent”. The ability to identify the presence of phosphoinositide and / or lysophosphoinositide or the binding position of its phosphate group using the techniques of the present invention can be associated with a particular disease state and thus such companion therapy or companion diagnosis May be useful.

As used herein, “prevention” refers to preventing a certain disease or disorder (for example, cancer, a disease or disorder related to lipid mediators, etc.) from entering such a state before such a state occurs. That means. Possibility that diagnosis can be performed using the PIPs and / or LPIPs of the present invention, and if necessary, for example, prevention of diseases or the like can be taken using the drug of the present invention. There is. In the present specification, the term “prophylactic agent (agent)” refers to any agent that can prevent a target condition (for example, cancer, diseases and disorders related to lipid mediators, etc.).

As used herein, the term “prognosis” means predicting the possibility of death or progression due to a disorder such as cancer, a disease caused by a lipid mediator, or a disorder. Prognostic factors are variables related to the natural course of the disease, and these affect the recurrence rate of patients who have once developed the disease. Clinical indicators associated with worse prognosis include, for example, any cellular indicator used in the present invention. Prognostic factors are often used to classify patients into subgroups with different pathologies. If the presence of phosphatidylinositol and / or lysophosphatidylinositol or the binding position of its phosphate group can be identified using the technology of the present invention, it is useful as a technology for providing a prognostic factor because it can be associated with a specific disease state. obtain.

In this specification, “detection device (device)” means, in a broad sense, any device that can detect or inspect a target object. In the present invention, a chromatography, an ion mobility separation device, a mass analyzer, and the like are also included in the detection device.

In this specification, the term “diagnostic device (device)” means, in a broad sense, any drug capable of diagnosing a target condition (for example, cancer, diseases and disorders related to lipid mediators, etc.).

In this specification, “drug”, “agent” or “factor” (both corresponding to “agent” in English) are used interchangeably in a broad sense, and so long as they can achieve their intended purpose. It may also be a substance or other element (eg energy such as light, radioactivity, heat, electricity). Examples of such substances include proteins (including antibodies, etc.), polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (for example, DNA such as cDNA and genomic DNA, mRNA and the like) RNA), polysaccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals, etc.) However, the present invention is not limited thereto.

In this specification, “detection agent (agent)” or “test agent (agent)” refers to any agent that can detect or inspect a target object in a broad sense.

In this specification, “diagnostic agent (agent)” broadly refers to any agent capable of diagnosing a target condition (for example, cancer, diseases and disorders related to lipid mediators, etc.).

In this specification, the term “therapeutic agent (agent)” refers to any agent that can treat a target condition (for example, cancer, diseases or disorders related to lipid mediators, etc.).

As used herein, “cancer” refers to any cancer detectable with the marker of the present invention, such as hepatocellular carcinoma, squamous cell carcinoma of the esophagus, breast cancer, pancreatic cancer, and squamous cells of the head and neck. Cancer or adenocarcinoma, colorectal cancer, kidney cancer, brain cancer (tumor), prostate cancer, small and non-small cell lung cancer, bladder cancer, bone or joint cancer, uterine cancer, child Cervical cancer, multiple myeloma, hematopoietic malignancy, lymphoma, Hodgkin disease, non-Hodgkin lymphoma, skin cancer, melanoma, squamous cell cancer, leukemia, lung cancer, ovarian cancer, gastric cancer, Kaposi sarcoma, laryngeal cancer Endocrine cancer, thyroid cancer, parathyroid cancer, pituitary cancer, adrenal cancer, cholangiocarcinoma, endometriosis, esophageal cancer, liver cancer, NSCLC, osteosarcoma, pancreatic cancer, Includes but is not limited to SCLC, soft tissue tumors, AML, and CML. .

As used herein, “inflammation” refers to any inflammation detectable with the marker of the present invention, and activation of blood cells related to inflammation such as macrophages (inflammatory cell chemotaxis, active oxygen production, phagocytosis, enzyme A secretory reaction, etc.). Exemplary inflammations include, for example, arthritis, tendinitis, bursitis, psoriasis, cystic fibrosis, Sjogren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), spondylitis, multiple Dermatomyositis, dermatomyositis, pemphigus, pemphigoid, Hashimoto's thyroiditis, cholangitis, inflammatory bowel disease (IBD, eg Crohn's disease, ulcerative colitis), colitis, inflammatory skin disease, pneumonia, asbestosis, Silicosis, bronchiectasis, talc lung, pneumoconiosis, sarcoidosis, delayed hypersensitivity reaction (eg poison ivy dermatitis), respiratory tract inflammation, adult respiratory distress syndrome (ARDS), encephalitis, immediate hypersensitivity reaction, asthma, hay fever, allergy , Acute anaphylaxis, reperfusion injury, rheumatoid arthritis, glomerulonephritis, pyelonephritis, cellulitis, cystitis, cholecystitis, allograft rejection, host versus graft rejection, appendicitis, arteritis, blepharitis, bronchiolitis ,bronchitis Cervicitis, cholangitis, chorioamnionitis, conjunctivitis, lacrimal inflammation, dermatomyositis, endocarditis, endometritis, enteritis, total enteritis, epicondylitis, epididymis, fasciitis, connective tissue inflammation Gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, umbilitis, ovitis, testicularitis, osteomyelitis, otitis media, pancreatitis, parotitis, pericarditis Pharyngitis, pleurisy, phlebitis, pneumonia, proctitis, prostatitis, rhinitis, fallopianitis, sinusitis, stomatitis, synovitis, testicularitis, tonsillitis, urethritis, cystitis, uveitis, vagina Includes, but is not limited to, diseases or disorders with acute or chronic inflammation, such as inflammation, vasculitis, vulvitis, vulvovaginitis, vasculitis, osteomyelitis, optic neuritis, myelitis, and fasciitis .

In the present specification, the “cancer marker” is also referred to as a tumor marker, and refers to a biological substance that is effective for cancer diagnosis, follow-up after treatment, discovery of recurrence or metastasis, or a substance found in the living body. Typically, cancer diagnosis and the like can be performed by measuring substances in the blood. In this case, the substance in the blood corresponds to the cancer marker.

As used herein, the term “marker” is in a certain state (for example, functionality, transformation state, disease state, disorder state, proliferative ability, level of differentiation state, presence / absence, etc.) or is there a risk thereof? A substance that serves as an indicator for tracking whether or not. As used herein, “disease marker” refers to a substance that serves as an indicator for tracking whether a disease state is present or at risk. In the present invention, detection, diagnosis, preliminary detection, prediction, or prior diagnosis for a certain state (for example, a disease state, a health state, a disease such as a cell or tissue differentiation disorder) is the detection method provided by the present invention. Further, it can be realized by using a marker-specific drug, agent, factor or means, or a composition, kit or system containing the same.

As used herein, a “purified” substance or biological agent (eg, a nucleic acid or protein such as a DNA vaccine, nucleic acid construct, plasmid DNA, etc.) refers to an agent that naturally accompanies the substance or biological agent. This means that at least a part has been removed. Thus, typically, the purity of a biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present. The term “purified” as used herein is preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight, Means the presence of the same type of biological agent. The substance or biological agent used in the present invention is preferably a “purified” substance. As used herein, an “isolated” substance or biological agent (such as a nucleic acid or protein) is substantially free of the factors that naturally accompany the substance or biological agent. Say things. The term “isolated” as used herein does not necessarily have to be expressed in purity, as it will vary depending on its purpose, but is preferably at least 75% by weight, more preferably if necessary. Means that there is at least 85%, more preferably at least 95%, and most preferably at least 98% by weight of the same type of biological agent. The materials used in the present invention are preferably “isolated” materials or biological agents.

In general, the compositions, medicaments, agents (therapeutic agents, prophylactic agents, etc.) of the present invention comprise a therapeutically effective amount of a medicament or active ingredient, and a pharmaceutically acceptable carrier or excipient. As used herein, “pharmaceutically acceptable” refers to a licensed or otherwise recognized pharmacopoeia of a government for use in animals, and more particularly in humans, by a government supervisory authority. It means that it is enumerated.

In one embodiment of the present invention, a “patient” is mainly assumed to be a human, but may be a mammal other than a human as long as it is applicable.

As used herein, “subject (person)” refers to a subject to be used for the prevention or treatment of the present invention, and is a human or a mammal other than a human (eg, mouse, guinea pig, hamster, rat, rat, Including one or more of a rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, or chimpanzee).

In this specification, the “kit” is a unit provided with a portion to be provided (eg, a test agent, a diagnostic agent, a therapeutic agent, a reagent, a label, an instruction, etc.) usually divided into two or more compartments. Say. This kit form is preferred when it is intended to provide a composition that should not be provided in admixture for stability or the like, but preferably used in admixture immediately before use. Such kits preferably include instructions describing how to use or how to handle the provided moiety (eg, test, diagnostic, therapeutic, reagent, label, etc.). In the present specification, when the kit is used as a reagent kit, the kit describes how to use a test agent, a diagnostic agent, a therapeutic agent, a reagent, a label, and the like. This includes instructions, etc. When combining test equipment, diagnostic equipment, etc., a “kit” can be provided as a “system”.

In the present specification, the “instruction sheet” describes the method for using the present invention for a doctor or other user. This instruction manual includes a word indicating that the detection method of the present invention, how to use a diagnostic agent, or administration of a medicine or the like is given. In addition, the instruction sheet may include a word indicating a method for detecting and determining the marker. This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the United States) where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has been received. The instruction sheet is a so-called package insert and is usually provided in a paper medium, but is not limited thereto, and is in a form such as an electronic medium (for example, a homepage or an e-mail provided on the Internet). But it can be provided.

(Preferred embodiment)
Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification. It will also be appreciated that the following embodiments of the invention may be used alone or in combination.

<Lysophosphatidylinositol phosphates (LPIPs)>
In one aspect, the present invention relates to lyso-phosphatidylinositol monophosphate (LPIP1), lysophosphatidylinositol diphosphate (LPIP2) or lysophosphatidylinositol triphosphate (lyso-phosphatidylinositol monophosphate (LPIP2)). A compound that is phosphatidylinositol trisphosphate (LPIP3)) is provided. Studies on phosphatidylinositol phosphates have been made relatively early, and in recent years a considerable amount of content has been found (Sasaki et al., History of Medicine Vol. 248, No. 13, (2014) pp.1039-1049). ). Phosphoinositide / inosyl phospholipids (PIPs) are said to constitute plasma membranes and organelle membranes, and their head group is significantly larger than other glycerophospholipids due to the six-membered carbon ring and polyvalent phosphate groups. In addition, the PIPs metabolic system rich in electric charge is considered to be involved in a wide range of life phenomena such as proliferation, differentiation, movement, aging, and death. Therefore, the lipids (LPIPs) of the present invention identified for the first time in the present invention, their metabolic enzymes, receptors, and the like can be useful as targets for various therapeutic drugs and as disease markers. Since lipid metabolism is one of the important research subjects, these lipids are used as experimental reagents as reagents.

For lyso form of PIPs (LPIPs), since no conventional measurement was possible, there was no report identified. In the present invention, the inventors have concentrated acidic phospholipids with an anion exchange resin such as DEAE cellulose, then protected phosphate groups with TMS diazomethane, and then combined the SRM method with a triple quadrupole mass spectrometer. Analytical method was developed. By using this method, the inventors succeeded in finding LPIP1, LPIP2, and LPIP3 of the present invention, which are structures different from known inositol phospholipids. Also, LPIPs could not be produced because the synthesis method was not known. Therefore, although it can be described by a general naming rule for chemical substances, LPIPs have not been confirmed as substances and can be said to have not been known. In the present invention, by developing a technique capable of detecting and identifying LPIPs, it is found that LPIPs exist in a living body (here, those existing in a living body are also referred to as “natural LPIPs”). Also, synthetic methods can be developed, and in addition to providing LPIPs existing in vivo, LPIPs including “non-natural type” LPIPs can be provided by synthesis.

LPIPs are also shown in the present invention to be present in vivo. For example, as shown in the Examples, it is found in various mammalian cell lines and mouse prostate cancer tissues, and seems to be preferentially expressed in cancer cells in specific molecular species such as LPIP3. It became clear. It was also found that some of the found LPIPs are specifically or preferentially expressed in cancer. PIPs having a structure similar to LPIPs have been analyzed in the past. For example, it is known that mutations and decreased expression of the tumor suppressor gene PTEN are abnormalities that are frequently observed in about half of human cancers. ing. At present, it is thought that fluctuations in PIP3 (which is a substrate for PTEN), which degrades PTEN, are related to carcinogenesis and progression, but there are few reports that actually showed fluctuations in PIP3 (Thanh-Trang). T. Vo and David A. Fruman, Cancer Discov; 5 (7); 697-700, 2015; Kofuji et al., Cancer Discov; 5 (7); 730-9. 2015), LPIPs of the present invention are The possibility of working as a lipid mediator can also be considered. Moreover, it was found by high sensitivity measurement that LPIPs exist not only in cells but also outside cells. Measuring these lipids with a sample that is relatively easy to collect, such as blood and urine, is also useful in laboratory medicine.

Many changes in PIPs (for example, metabolism, degradation, etc.) remain unexplained because measurement techniques are immature. Since LPIPs have been found as novel substances in the present invention, and it has been found that some LPIPs are preferentially expressed in cancer, blood, urine in diseases associated with cancer or other lipid mediators It can be applied to medical examinations and diagnosis using specimens such as. In general, it is known that phospholipases A (phospholipases A ₁ , A _2, etc.) are involved in the metabolism of phospholipids to the corresponding lyso form, but phospholipases A ₁ and A ₂ have extremely high substrate specificity, It does not respond to different types of phospholipids. For example, it is said that a subtype having phosphatidylcholine as a substrate does not react with phosphatidylserine. Moreover, what uses PIPs as a substrate is not known. In accordance with the present invention, lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate, and lysophosphatidylinositol triphosphate have been found. As for “lysophosphatidylinositol monophosphate” found in the living body, a phosphate group is located at the 4-position, and a species in which the following fatty acids are bonded to the sn-1 position or the sn-2 position was found. : 16: 0, 16: 1 (9Z), 18: 0, 18: 1 (9Z), 18: 2 (9Z, 12Z), 20: 3 (8Z, 11Z, 14Z), 20: 4 (5Z, 8Z) , 11Z, 14Z), 22: 4 (7Z, 10Z, 13Z, 16Z), 22: 5 (7Z, 10Z, 13Z, 16Z, 19Z), 22: 6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z) . As for “lysophosphatidylinositol diphosphate” found in vivo, the phosphate group is located at the 4th and 5th positions, and the following fatty acids are found bound to the sn-1 or sn-2 position. 16: 0, 16: 1 (9Z), 18: 0, 18: 1 (9Z), 18: 2 (9Z, 12Z), 18: 3 (6Z, 9Z, 12Z), 20: 3 (8Z) , 11Z, 14Z), 20: 4 (5Z, 8Z, 11Z, 14Z). For “lysophosphatidylinositol triphosphate” found in vivo, the following fatty acids were found to bind to the sn-1 or sn-2 position: 16: 0, 16: 1 ( 9Z), 18: 0, 18: 1 (9Z), 20: 4 (5Z, 8Z, 11Z, 14Z). These molecular species are similar to the fatty acid species found in PIPs, suggesting the presence of a previously unknown phospholipase A ₁ or phospholipase A ₂ that has specificity for phosphatidylinositol phosphates. The The fatty acid molecular species are likely to be found in both sn-1 and sn-2 in vivo, although there are many abundances.

In one embodiment, the invention provides 1-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol monophosphate, 1-acyl-lysophosphatidylinositol diphosphate, 2-acyl-lysophosphatidylinositol. Compounds are provided that are diphosphate, 1-acyl-lysophosphatidylinositol triphosphate, or 2-acyl-lysophosphatidylinositol triphosphate. The acyl group may be any kind of acyl group as long as the corresponding fatty acid is available. It has been found in the present invention that the lysophosphatidylinositol phosphates of the present invention can be produced starting from phosphatidylinositol phosphates. Therefore, by introducing the obtained fatty acid into phosphatidylinositol phosphates based on the Conway method cited in this specification, it can be used as a starting material for the production method of lysophosphatidylinositol phosphates of the present invention.

In one preferred embodiment, the present invention provides 2-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl-lysophosphatidylinositol triphosphate or 2-acyl-lysophosphatidyl. A compound that is inositol triphosphate is provided.

As the compound of the present invention, lysophosphatidylinositol phosphates can be used in which the phosphate group is bonded to any position in inositol, and preferably one of 2, 3, and 5 positions. Or what was couple | bonded with three positions can be used. Therefore, 3-monophosphate type, 4-monophosphate type, 5-monophosphate type, 3,4-diphosphate type, 3,5-diphosphate type, 4,5-diphosphate type, The 3,4,5-triphosphate type is exemplified as a preferred example. In one embodiment, the present invention provides 1-acyl-lysophosphatidylinositol-3-monophosphate, 1-acyl-lysophosphatidylinositol-4-monophosphate, 1-acyl-lysophosphatidylinositol-5-monophosphate Acid, 2-acyl-lysophosphatidylinositol-3-monophosphate, 2-acyl-lysophosphatidylinositol-4-monophosphate, 2-acyl-lysophosphatidylinositol-5-monophosphate, 1-acyl-lysophosphatidyl Inositol-3.4-diphosphate, 1-acyl-lysophosphatidylinositol-3.5-diphosphate, 1-acyl-lysophosphatidylinositol-4.5-diphosphate, 2-acyl-lysophosphatidylinositol- 3,4-diphosphate, 2-acyl-lysophosphatidylinositol -3,5-diphosphate, 2-acyl-lysophosphatidylinositol-4,5-diphosphate, 1-acyl-lysophosphatidylinositol-3,4,5-triphosphate, or 2-acyl-lysophosphatidyl Provided is a compound that is inositol-3,4,5-triphosphate.

In a specific embodiment, the present invention illustratively provides the following compound, but the specific compound of the present invention is not limited to the following.

(Chemical 1)

<Uses of LPIPs>
In another aspect, the present invention provides a composition for use as a disease marker comprising a compound of the present invention (such as lysophosphatidylinositol phosphates). It is understood that as such a compound, any embodiment described in <lysophosphatidylinositol phosphates (LPIPs)> in this specification may be employed in combination of one or more. Examples of such may include, for example, lysophosphatidylinositol monophosphate (LPIP1), lysophosphatidylinositol diphosphate (LPIP2) or lysophosphatidylinositol triphosphate (LPIP3), in particular, LPIP3 has been shown to have a significantly increased expression level in cancer tissues, and the compound of the present invention should be used as an inflammation marker or a cancer marker, particularly in the case of substances present in vivo. It is understood that a synthetic product is also useful as a control or standard for detection of an inflammatory marker or a cancer marker.

When the compound of the present invention is used as a cancer marker, the target cancer is not limited. For example, hepatocellular carcinoma, esophageal squamous cell carcinoma, breast cancer, pancreatic cancer, and squamous cells in the head and neck Or adenocarcinoma, colorectal cancer, kidney cancer, brain cancer (tumor), prostate cancer, small and non-small cell lung cancer, bladder cancer, bone or joint cancer, uterine cancer, cervical cancer Cancer, multiple myeloma, hematopoietic malignancy, lymphoma, Hodgkin disease, non-Hodgkin lymphoma, skin cancer, melanoma, squamous cell cancer, leukemia, lung cancer, ovarian cancer, gastric cancer, Kaposi sarcoma, laryngeal cancer, Endocrine cancer, thyroid cancer, parathyroid cancer, pituitary cancer, adrenal cancer, cholangiocarcinoma, endometriosis, esophageal cancer, liver cancer, NSCLC, osteosarcoma, pancreatic cancer, SCLC , Soft tissue tumors, AML, and CML. Not, and preferably, it is understood that prostate cancer, and the like.

When the compound of the present invention is used as an inflammation marker, the target inflammation is not limited, for example, arthritis, tendinitis, bursitis, psoriasis, cystic fibrosis, Sjogren's syndrome, giant cell arteritis , Progressive systemic sclerosis (scleroderma), spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, Hashimoto's thyroiditis, cholangitis, inflammatory bowel disease (IBD such as Crohn's disease, ulcerative) Colitis), colitis, inflammatory skin disease, pneumonia, asbestosis, silicosis, bronchiectasis, talc lung, pneumoconiosis, sarcoidosis, delayed type hypersensitivity reaction (eg poison ivy dermatitis), airway inflammation, adult respiratory distress syndrome (ARDS), encephalitis, immediate hypersensitivity reaction, asthma, hay fever, allergy, acute anaphylaxis, reperfusion injury, rheumatoid arthritis, glomerulonephritis, pyelonephritis, cellulitis, cystitis, cholecystitis, allograft rejection, Main graft rejection, appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis, lacrimal inflammation, dermatomyositis, endocarditis, endometritis, Enteritis, panenteritis, epicondylitis, epididymis, fasciitis, connective tissue inflammation, gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, umbilitis, Ovariitis, testitis, osteomyelitis, otitis media, pancreatitis, parotitis, pericarditis, pharyngitis, pleurisy, phlebitis, pneumonia, proctitis, prostatitis, rhinitis, fallopianitis, sinusitis, stomatitis, Synovitis, testicular inflammation, tonsillitis, urethritis, cystitis, uveitis, vaginitis, vasculitis, vulvitis, vulvovaginitis, vasculitis, osteomyelitis, optic neuritis, myelitis, fasciitis, etc. Including, but not limited to, diseases or disorders associated with acute or chronic inflammation.

When used as a disease marker such as an inflammation marker or a cancer marker, the LPIPs of the present invention can be used for the detection method and other methods disclosed in the present specification (for example, immunological techniques, cytological techniques using radioactive substances, etc.) ) Can be used. When radioactive materials are used, intracellular LPIPs labeled with radioactive isotopes (RI) following the PIPs method (see Sasaki et al., Ayumi Sci. Vol. 248, No. 13, (2014) pp. 1039-1049) Are separated by high performance liquid chromatography (HPLC) to obtain radioactivity.

The detection, identification, quality control, and the like of the LPIPs of the present invention can be realized by using a substance or an interacting molecule that binds to LPIPs serving as a marker, in addition to using the detection method described in the present invention. In the context of the present invention, a “substance that binds” or “interacting molecule” to a marker substance binds and preferably binds to a molecule, such as a substance that is at least temporarily marker (eg, LPIPs). A molecule or substance that can indicate (eg, is labeled or is ready to be labeled). Substances that bind to molecules such as LPIPs may be receptors for molecules such as LPIPs or transferases (if known), and other examples include antibodies, antisense oligonucleotides, siRNAs, low molecular weight molecules ( LMW), binding peptides, aptamers, ribozymes, peptidomimetics, and the like, including, for example, binding proteins specific for molecules such as LPIPs. As used herein, “binding protein” for a molecule such as LPIPs refers to a type of protein that binds (preferably specifically) to a molecule such as LPIPs, and is induced to a molecule such as LPIPs. Including but not limited to antibodies such as polyclonal or monoclonal antibodies, antibody fragments and protein backbones. A typical example of such a protein is an antibody, and such an antibody can be carried out using a technique known in the art.

<Manufacturing method of LPIPs>
In another aspect, the present invention relates to a compound of the present invention (lysophosphatidylinositol) comprising the step of deacylating phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate by contacting with alkylamine. (Phosphate) production method.

In a preferred embodiment, the alkylamine is methylamine.

In a more preferred embodiment, the deacylation is carried out at a temperature (eg, room temperature to about 80 ° C.) at which the deacylation promotes in the presence of an appropriate concentration (eg, 10.7% or less) of an alkylamine such as methylamine. Incubate for an appropriate period of time (typically, about 53 ° C. can be used.) As a reference, Serunian, et al., Methods in Enzymol disclosing the dediacylation conditions Vol., 198, 1991, pp. 78-87, especially pp. 82-83, Fujii et al. Folia Pharmacol Jpn. Vol. 142, 2013, pp. 236-240, especially pp. 238-239. These documents only disclose conditions under which both sn-1 and sn-2 acyl groups are cleaved, and may be referred to herein as "dediacylation"). For example, under these conditions, the reaction is carried out at a methylamine concentration of about 10.7% for 50 to 120 minutes. In the present invention, only the acyl group of sn-1 or sn-2 is unexpectedly cleaved by changing the Serunian or Fujii conditions (dediacylation conditions) to be relaxed (this specification). (Referred to as “demonoacyl”). In the present invention, if the methylamine concentration is 10.7%, which is the same as that of Serunian and Fujii, the monoacyl is unexpectedly cleaved by using it for a short period of time such as about 30 minutes or less, and sn-1 We found that LPIPs or sn-2 LPIPs are generated. Alternatively, it was found that when the concentration of methylamine was 2.13%, only monoacyl was cleaved unexpectedly under the reaction conditions of 120 minutes to produce LPIP.

In the present specification, “deacylation” means that the fatty acid bond at the 1-position and / or 2-position in phosphatidylinositol phosphate (which may be monophosphate, diphosphate, or triphosphate, also referred to as PIPs). It means that the acyl group is cleaved and cleaved (eliminated). Unless otherwise specified herein, “deacylation” includes “demonoacylation” in which one acyl group is cleaved (eliminated) and “dediacylation” in which two acyl groups are cleaved (eliminated). Both reactions can be included. As used herein, it includes “demonoacylation”. “Demonoacylation” can be either of the sn-1 position or of the sn-2.

In one specific embodiment, methylamine / H ₂ O / methanol / n-butanol = 1.92 / 38.08 / 40/10 (deacylation reaction solution) was added to the dried PIPs, and 53 Incubate at 5 ° C for 5 minutes. An ice-cooled deacylation reaction solution is added to stop the reaction (an exemplary result obtained by allowing to stand on ice for 1 minute and then drying to measure the product is shown in FIG. 1). One or more (or all) of various conditions such as solvent composition, reaction temperature, reaction time, catalyst (typically methylamine) concentration, etc. may be appropriately changed as long as demonoacylation occurs. can do. In a preferred embodiment, it is understood that the object of the present invention can be achieved if the amount of methylamine is 1-3%.

The starting material used in the production method of the present invention, phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate, can be produced by a method known in the art or commercially available Can be used. For example, any kind of PIP can be manufactured by Stuart J. Conway et al., Org. Biomol. Chem., 2010, 8, 66-76.

Also provided is a method for easily separating a lysozyme having a desired acyl group at a desired position by making raw materials separately. In another aspect, the present invention provides (A) providing phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate having different mass acyl groups at the 1-position and 2-position, An acyl group having a desired mass is present at a desired position (position 1 or position 2); (B) the phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate with an alkylamine Deacylation by contact with (usually performed under the demonoacylation conditions described herein), and (C) lysophosphatidylinositol monophosphate having a desired mass of acyl groups, lyso Phosphatidylinositol diphosphate or lysophosphatidylinos A method for producing lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate having a desired acyl group at a desired position, which comprises a step of taking out the triphosphate. )I will provide a.

In the method of making the present invention, phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate having different masses of acyl groups at the 1-position and the 2-position are obtained from Stuart J. Conway et al., Org. Biomol. Chem., 2010, 8, 66-76 can be used as a reference and will be described in detail below. For the deacylation, the above-mentioned conditions can be appropriately applied. The desired lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate can be removed by any means that can be separated by differences in mass, hydrophobicity, and the like. For example, chromatography can be used. Of course, the purification step is not essential if it can be used without purification.

The following is a general production scheme for starting materials (PIPs and the like) having desired fatty acid groups and phosphate groups. Starting materials (PIPs, etc.) are comprehensively described in Stuart J. Conway et al., Org. Biomol. Chem., 2010, 8, 66-76, and phosphoramidite precursors (diacylglycerol phosphorus). Acid derivatives) and the like.

(Example of general production of phosphoramidite precursor)

(Where R ¹ and R ² are each independently any fatty acid residue, and R ^B and R ^C are each independently hydrogen or an alkyl group (for example, 1 to 12 carbon atoms such as methyl, ethyl, etc. an alkyl group) and the ^like, PG a, ^{PG P1} and ^{PG P2} as are each independently a protecting group. protecting groups such as benzyl group, p- methoxybenzyl group, be mentioned tert- butyl group (However, it is not limited to these. When a benzyl group is used, it is preferably not used when a fatty acid residue containing a double bond is introduced because hydrogenation is carried out upon elimination.)
Compound P-1 (eg (+)-1-O- (4-methoxybenzyl) -2-O-methoxymethyl-glycerol) and carboxylic acid halide under appropriate conditions (eg P-1 Compound P-2 is obtained by adding dimethylaminopyridine (DMAP) and acetyl chloride to a dichloromethane solution at 0 ° C. and stirring at room temperature. By subjecting compound P-2 to appropriate conditions (for example, by using trifluoroacetic acid), the protecting group PG ^P1 (for example, methoxymethyl group) of the hydroxy group is deprotected to obtain compound P-3. Compound P-3 and a carboxylic acid halide are subjected to appropriate conditions (for example, dimethylaminopyridine (DMAP) and octadecanoyl chloride are added to a dichloromethane solution of compound P-3 at 0 ° C. and stirred at room temperature. Compound P-4 is obtained. By subjecting compound P-4 to appropriate conditions (for example, by using 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) or ammonium hexanitrerium (IV) acid (CAN)) The protecting group PG ^P2 (for example, 4-methoxybenzyl group) of the hydroxy group is deprotected to give compound P-5. Compound 1-5 and aminophosphine (eg (4-methoxybenzyloxy) bis (N, N-diisopropylamino) phosphine) are subjected to appropriate conditions (eg 1H-tetrazole is added at room temperature in dichloromethane solution). To give phosphoramidite P-6.

(General production example of inositol precursor)

(PG ¹ , PG ^A , and PG ² are each independently a protecting group. Examples of the protecting group include, but are not limited to, methoxybenzyl and the like.)
Compound I-1 with subjecting to suitable conditions (e.g. the compound I-1 of DMF was added 4-methoxybenzyl chloride and sodium hydride was added at 0 ° C., by stirring at room temperature) protecting group PG ^A Compound I-2 into which (for example, 4-methoxybenzyl group) is introduced is obtained. By subjecting compound I-2 to appropriate conditions (for example, by adding chloromethyl methyl ether and sodium hydride to a DMF solution of compound I-2 at 0 ° C. and stirring at room temperature), protecting group PG ¹ ( For example, compound I-3 into which a methoxymethyl group is introduced is obtained. By subjecting compound I-3 to appropriate conditions (for example, by adding diisobutylaluminum hydride (DIBAL) to a dichloromethane / hexane solution of compound I-3 at 0 ° C. and stirring at room temperature), compound I-4 obtain. By subjecting compound I-4 to appropriate conditions (for example, by adding 4-methoxybenzyl chloride and sodium hydride to a DMF solution of compound I-4 at 0 ° C. and stirring at room temperature), the protecting group PG ² Compound (I-5) into which (for example, 4-methoxybenzyl group) is introduced is obtained. By subjecting compound I-5 to appropriate conditions (for example, adding hydrochloric acid to a methanol solution of compound I-5 and refluxing), compound I-6 is obtained. By subjecting compound I-6 to appropriate conditions (for example, a solution of compound I-6 in dichloromethane with toluenesulfonic acid monohydrate)

And then refluxing, followed by optical resolution, followed by toluenesulfonic acid monohydrate and dichloromethane solution of the compound obtained after optical resolution.

To react with compound I-7 (for example,

) By subjecting compound I-7 to appropriate conditions, compound I-8 (l, l) having ¹ hydroxy group, m hydroxy groups protected with protecting group PG ¹ and n hydroxy groups protected with PG ² is obtained. m and n are integers of 0 or more and 6 or less, and the sum of l, m, and n is 6.

(Example of general production of phosphatidylinositol)

(In the formula, l, m and n are integers of 0 or more and 6 or less, and the total of l, m and n is 6.)
Suitable for the hydroxy group of inositol precursor PI-1 (eg 1D-2,3,4,5,6-penta-O- (4′-methoxybenzyl) -1-O- (methoxymethyl) -myo-inositol) A suitable phosphorus reagent (eg bis (4-methoxybenzyloxy) (N, N-diisopropylamino) phosphine) under appropriate conditions (eg adding 1H-tetrazole to a solution of PI-1 in dichloromethane, PI-2 modified with a phosphate group (for example, 1D-2,3,4,5,6-penta-O- (4′-methoxybenzyl) -1-) by stirring at room temperature for 10 to 14 hours O- (methoxymethyl) -myo-inositol-4- (bis (4-methoxybenzyloxy) phosphate)) is obtained. By subjecting PI-2 to appropriate conditions (for example, by using trifluoroacetic acid), the protective group PG ¹ (for example, methoxymethyl group) is deprotected PI-3 (for example, 1D-2,3,4,5, 6-Penta-O- (4′-methoxybenzyl) -myo-inositol-4- (bis (4-methoxybenzyloxy) phosphate)) is obtained. By subjecting PI-3 and a phosphoramidite precursor (eg (1-acetyloxy-2′-octadecanoyloxypropyl) (4-methoxybenzyl) diisopropyl phosphoramidite) to the appropriate conditions (eg PI— 1-tetrazole and (1-acetyloxy-2-octadecanoyloxypropyl) (4-methoxybenzyl) diisopropyl phosphoramidite) were added to a dichloromethane solution of No. 3 and stirred at room temperature for 10-14 hours, then metachloroperbenzoic acid PI-4 is obtained by adding acid (mCPBA) at −78 ° C. and stirring for 20 minutes at room temperature. The protective group PG ² (eg 4-benzyl) is added to PI-4 under appropriate conditions (for example, by adding ammonium hexanitracelium (IV) to a solution of PI-4 in acetonitrile and stirring for 45 minutes at room temperature). PI-5 (eg, 1D-myo-inositol-1- (1′-O-acetyl-2′-O-octadecanoyl-sn-glycera-3-yl phosphate)) is obtained by deprotecting the group.

Phosphatidylinositol phosphate produced by the above production method includes, for example, the following formula

However, it is not limited to these.

(General production example of Lyso-phosphatidylinositol phosphate)

A solution of phosphatidylinositol 1 (eg 1D-myo-inositol-1- (1′-O-acetyl-2′-O-octadecanoyl-sn-glycera-3-yl phosphate) (eg chloroform / methanol (9 / 1) A solution may be mentioned, but the ratio of the solvent for preparing the solution can be appropriately changed, and another solvent can be used as long as the reaction is promoted.) Is dried with an inert gas such as N 2. After solidification, a suitable reagent (for example, but not limited to, a methylamine solution (example solvents include methylamine / water / methanol / 1-butanol (4.8 / 35.2 / 40/10). The ratio of the solvent for the preparation and methylamine can be appropriately changed, and another solvent can be used as long as the reaction is accelerated. It can be added at a temperature (eg, 53 ° C.) and exemplified for a suitable time (eg, 5 minutes), but can be stretched as long as it is longer or shorter as long as demonoacylation is achieved. It has been observed that demonoacylation occurs in 30 seconds.) Suitable temperatures (eg, 53 ° C. are exemplified, but can be raised or lowered as long as demonoacylation is achieved at higher or lower temperatures. The reaction is quenched (for example, with ice cooling) and the work-up is appropriately performed to obtain the desired Lyso-phosphatidylinositol, where the acyl group elimination reaction proceeds regioselectively. even not, if the acyl group is introduced into phosphatidylinositol 1 COR ¹ and COR ² is different (e.g. COR ¹ is an acetyl group, COR ² If a octadecanoyl group), 2 and 3 acyl group is a compound desorbed only one, since the molecular weight are different, it is possible to separate and purify each compound.

<Detection method>
In another aspect, the present invention provides a method for detecting, identifying or quantifying lysophosphatidylinositol phosphate, wherein (A) concentrating a phospholipid by contacting a sample with an anionic resin; B) protecting the phosphate group in the phospholipid with a protecting group; and (C) detecting, identifying or quantifying lysophosphatidylinositol phosphate in the phospholipid by mass spectrometry. Methods for detecting, identifying or quantifying phospholipids are provided. Phosphoinositides, including lysophosphatidylinositol phosphate, have many phosphate groups and are traced to ions by other lipids in the sample due to their trace amounts (abundance in biological lipid extracts) Therefore, it has become the most difficult membrane phospholipid that has been impossible to analyze in the past, but by using the method of the present invention, lysophosphatidylinositol phosphate can be detected, identified and quantified. It became so. The method of the present invention was able to set several hundred amol to several fmol as the lower limit of quantification, and was able to measure many types of LPIPs that were unknown whether they existed in vivo. Thus, the present invention provides a mass spectrometry based method capable of absolute quantification with various biological trace samples.

In the conventional measurement technique, PIPs can be measured, but it has been found in the present invention that lysophosphatidylinositol phosphates can be identified and detected. Therefore, the present invention provides for the first time a technique for identifying and detecting lysophosphatidylinositol phosphates.

Preferably, any protecting group for the phosphate group of the present invention can be used as long as it can be protected, but preferably it does not become an obstacle in mass spectrometry or is desorbed before mass spectrometry. For example, an alkyl group (for example, a methyl group) can be exemplified. Preferably, the protecting group contains an alkyl group, more preferably a methyl group.

Although any technique can be used for the mass spectrometry used in the present invention, it preferably includes a selective reaction monitoring method (SRM) using a triple quadrupole mass spectrometer.

The SRM used in the present invention further includes a step of detecting, identifying or quantifying the fatty acid side chain and / or phosphate group of the lysophosphatidylinositol phosphate using reverse phase column chromatography.

Preferably, in the reverse phase column chromatography, diacylglycerol can be selected as a product ion (fragment ion) to detect, identify or quantify the fatty acid side chain and / or phosphate group.

Specific examples of the phosphoinositide measurement technique are as follows.

As a result of examining this method, it was found that LPIPs can also be measured.

In one aspect, PIP1 and PIP2 are each quantified as the sum of three types of isomers (structural isomers with different positions of phosphorylated hydroxyl groups). The procedure is outlined below.

1. The total lipid fraction obtained by the high recovery Bligh-Dyer method of phosphoinositide such as lysophosphatidylinositol phosphates in vivo is further fractionated using an anion exchange column (DEAE cellulose column). Gradually separate and elute according to the difference in ion exchange capacity of each phospholipid polar site, and fractions rich in phosphoinositide can be recovered at a high rate, and lysophosphatidylinositol phosphates should be recovered at a high rate as well. Can do.

2 Stabilization of molecules Since phosphoinositides with many phosphate groups in the inositol ring are unstable and easily adsorb to many materials including metals, lysophosphatidylinositol phosphates are also the same. It is thought that it has the property of. Therefore, phosphoinositide is rapidly damaged in the analytical vial during the waiting time from collection to measurement, and tailing is seen in the detected elution peak. The present inventors perform a robust analysis by producing a stable derivative that suppresses decomposition / adsorption by methylating a phosphate group after high recovery of phosphoinositide. Such a technique can also be applied to lysophosphatidylinositol phosphates.

3 Sensitive Analytical Method For the measurement of phosphoinositide and lysophosphatidylinositol phosphates, a selective reaction monitoring method (SRM) using a triple quadrupole mass spectrometer, which is a highly sensitive and highly quantitative analytical method, is used. . Phosphoinositide is easy to detect as a negative ion due to its structure, but in the analysis method of the present invention, it is easy to detect as a positive ion because the phosphate group is protected such as methylation. Lysophosphatidylinositol phosphates also show similar properties. Each molecular species selects diacylglycerol (DG) as a characteristic fragment ion. Analysis is carried out by reverse phase column (C8) using the difference in hydrophobicity of fatty acid side chains to separate and elute each molecular species. Each phosphoinositide of the same molecular species elutes in the order of PIP3, PIP2, and PIP1 due to the difference in the number of phosphate groups, and therefore, analysis is performed using this difference in elution time. For quantitative analysis, the peak area of the chromatogram of each molecular species that has been smoothed by the Gaussian method is digitized, and this is applied to a calibration curve prepared using various 17: 0/20: 4 synthetic products that are hardly present in the living body. To calculate. This analysis method of the present invention is highly sensitive and excellent in quantification, and can be analyzed with human clinical specimens and animal tissue-derived samples in addition to cultured cells. The same analysis can be performed for lysophosphatidylinositol phosphates. In addition to the differences in molecular weight, LPIP3, LPIP2, and LPIP1 are eluted in this order. The peak area of the chromatogram of each molecular species subjected to smoothing can be digitized, compared with a standard product, and applied to the prepared calibration curve for calculation.

Since the present invention provides a technique for distinguishing and detecting lysophosphatidylinositol phosphates, which can also be disease markers, from other substances, it can also diagnose cancer.

Metabolic abnormalities of diacyl-type inositol phospholipids (PIPs), which are considered to be LPIPs precursors, are known to be associated with various pathological conditions, and their usefulness as therapeutic targets and diagnostic markers have been found. Therefore, the LPIPs of the present invention, which are structurally closely related to diacyl-type inositol phospholipids (PIPs), are also known to be related to various pathological conditions, and have usefulness as therapeutic targets and diagnostic markers. Expected to have. Specifically, the following uses are assumed. Also, Biochemistry Vol. 83, No. 6, pp. As described in 525-535, 201, it is known to have various activities including lysophosphatidylinositol as a lipid mediator and a G protein receptor such as its receptor GPR55. Therefore, the compound of the present invention in which lysophosphatidylinositol is phosphorylated in various forms is expected to be directly or indirectly related to various physiological uses in relation to lysophosphatidylinositol and its receptor. The Also, as described in Fujii et al. Folia Pharmacol Jpn. Vol. 142, 2013, pp. 236-240, the inositol polyphosphate group produced from PIPs by the action of phospholipase C controls intracellular calcium kinetics and the like. In addition, abnormal calcium dynamics are associated with a wide variety of conditions including mental illness, muscle disease, and heart disease. Therefore, the LPIPs of the present invention that are structurally closely related to the inositol polyphosphate group are also expected to have utility as therapeutic targets and diagnostic markers.

(Measurement, detection and identification of phosphatidylinositol phosphate and lysophosphatidylinositol phosphate)
In one aspect, the present invention provides a method for measuring, detecting or identifying phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample. The method comprises A) applying the sample to mass spectrometry (MS); and B) identifying the position of the phosphate group of the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate by the elution position of the peak in the MS Is included. In one embodiment, the sample is further applied to chromatography. In one embodiment, the sample is further applied to ion mobility separation (IMS).

In one embodiment, the position of the phosphate group of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate can be specified by comparison with a standard product. Alternatively, the elution position and elution time of a standard product can be calculated and set in advance, and the identification can be performed by comparing with the calculation. In addition, as a method for identifying PIPs and LPIPs, for example, whether stable isotopes of phosphoric acid and inositol ( ^13C and deuterium) can be added to a sample in advance can be replaced with natural ones. There is a method to investigate.

In another aspect, the present invention provides a method for measuring, detecting or identifying phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample. The method comprises A) applying the sample to ion mobility separation-mass spectrometry (IMS-MS); and B) depending on the elution position of the peak in the IMS-MS, the phosphorous of the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate. Including the step of identifying the position of the acid group.

In one embodiment, the presence or absence of an acyl group contained in the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is specified in the specifying step in the present invention. Some phospholipids in the sample do not have an acyl group, but they are not deacylated by the measurement of the present invention, and therefore can be measured in an intact state. Therefore, the presence or absence of an acyl group is also specified. can do.

In one embodiment, the total molecular weight of acyl groups contained in the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is also specified in the specifying step in the present invention. In the measurement of the present invention, the molecular weight is specified by mass spectrometry. Therefore, once it is specified that the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is specified, the total molecular weight of the acyl group bonded is specified from the molecular weight.

In a preferred embodiment, the type of acyl group contained in the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is also specified in the specifying step in the present invention. In the measurement of the present invention, the molecular weight is specified by mass spectrometry. Therefore, once it is identified that it is phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate, the total molecular weight of the bound acyl group is determined from its molecular weight, and in the case of phosphatidylinositol phosphate, In order to be specified, the molecular weight of one of the acyl groups is specified. When one molecular weight is specified, the other molecular weight is also specified, and the type of acyl group is specified from the molecular weight. A method for identifying the type of acyl group from the molecular weight based on the results of measurement by MS and MS / MS is described, for example, in Clark J et al. , Nat Methods. 2011 Mar; 8 (3): 267-72. Doi: 10.1038 / nmeth can be referred to.

In one embodiment, the measurement, detection or identification in the present invention is characterized in that phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is distinguished from other inositol-containing phospholipids. This is because the measurement, detection or identification method of the present invention can be carried out while retaining the acyl group. More specifically, phosphatidyl phosphate and lysophosphatidylinositol phosphate can be used. And inositol-containing phosphoric acid such as glycerophosphoinositol can be distinguished from each other in that the molecular weight of the acyl group is different by measuring the retention of the acyl group.

In one embodiment, the sample to be measured according to the present invention is prepared under conditions where phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate are not decomposed.

Therefore, in the measurement, detection or identification of the present invention, the conditions in which phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate are not degraded can include any conditions in which degradation is not performed, and in particular, deacylation treatment is performed. In addition to this, there are other conditions such as not performing a treatment for denaturing the sample and performing a treatment for introducing a protecting group into a phosphate group.

As the liquid column chromatography used in the present invention, a chiral column, a reverse phase column, a normal phase column, an ion exchange column or the like can be used, and any column chromatography which separates a hydrophilic group and a hydrophobic group can be used. .

As used in the present invention, chromatography (gas chromatography or liquid column chromatography) can be combined with ion mobility separation. Without wishing to be bound by theory, the present invention shows that ion mobility separation can more clearly separate the position of the phosphate group of phosphoinositide and / or lysophosphoinositide. Any chromatography (eg, liquid column chromatography) combined with ion mobility separation and further mass spectrometry can be used to analyze the phosphate group of phosphoinositide and / or lysophosphoinositide while retaining the acyl group. The position can be separated and measured, detected or identified. Alternatively, even when ion mobility separation is not used, the position of the phosphate group of phosphoinositide can be separated and measured, detected or identified while retaining the acyl group by using mass spectrometry (MS), and Furthermore, the position of the phosphate group can be separated and measured, detected or identified in combination with chromatography such as liquid chromatography using a chiral column. Or when using ion mobility separation, you may combine with mass spectrometry as it is, without using chromatography.

In a preferred embodiment of the invention, the method of the invention further comprises the step of quantifying phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate.

In one embodiment, the sample used in the present invention is an intact sample. Here, that the sample is intact includes conditions under which acyl groups of phosphoinositide and lysophosphoinositide are retained, such as denaturation by heating, oxygen in the air, moisture, heat, light, metal ions, Examples thereof include, but are not limited to, oxidation by the action of microorganisms or enzymes, hydrolysis in an aqueous solvent, deacylation in the presence of an alkylamine such as methylamine, and the like.

In one embodiment, in the present invention, the position of the phosphate group of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is determined without labeling the sample. Since it is not necessary to label with a fluorescent label or radioisotope, it is possible to perform analysis under the condition of the living body as it is or similar conditions to the sample in the living body, reflecting more living body information Analysis can be performed.

(Measurement, detection or identification device)
In one aspect, the present invention provides an apparatus for measuring, detecting or identifying the positions of acyl and phosphate groups of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample. Here, the apparatus comprises a mass spectrometer, preferably further comprising a chromatography apparatus or an ion mobility separation apparatus, for example the chromatography apparatus is at least one for gas chromatography and liquid chromatography. One device may be included. Without wishing to be bound by theory, it is possible to measure phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in this specification using liquid column chromatography and a mass spectrometer. It is understood that the separation can be similarly performed and can be measured. In order to more accurately separate phosphoric acid position isomers, it is preferable to combine with a chromatographic apparatus or an ion mobility separation apparatus. As a specific embodiment, the present invention includes a mass spectrometer (MS) apparatus and an application unit that applies the sample to the MS under a condition that the phosphatidylinositol phosphate is not decomposed. As a more specific embodiment, the present invention includes a liquid column chromatography-mass spectrometry (LC-MS) apparatus and an application unit that applies the sample to the LC-MS under conditions in which phosphatidylinositol phosphate is not decomposed. Including.

In another aspect, the present invention provides an apparatus for measuring, detecting or identifying the positions of acyl groups and phosphate groups of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample. Here, the apparatus includes an ion mobility separation-mass spectrometry (IMS-MS) apparatus and an application unit that applies the sample to the IMS-MS under a condition in which phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is not decomposed. Including.

In the present invention, when LC-MS is used, means for performing ion mobility separation may be included as necessary.

The liquid column chromatography used in the present specification is selected from the group consisting of chiral columns, reverse phase columns, normal phase columns, ion exchange columns, columns that separate hydrophilic groups and hydrophobic groups.

In one embodiment, it further comprises means for detecting or quantifying the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate. As such a viewpoint or means for quantifying, for example, a method of calculating and quantifying the obtained detection peak in comparison with an internal standard can be mentioned.

(Separation and purification of phosphoinositide and / or lysophosphoinositide and production of pure products)
In one aspect, the present invention provides a method for separating or purifying phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample depending on the position of a phosphate group while retaining an acyl group. The method comprises A) applying the sample to mass spectrometry (MS); and B) from the eluate of the MS, a phosphatidylinositol phosphate having a phosphate group at the target position identified by the elution position of the peak and / or Or collecting lysophosphatidylinositol phosphate. In one embodiment, the sample is further applied to chromatography or ion mobility separation (IMS).

In one aspect, the present invention provides a mixture of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate contained in the mixture from a mixture of two or more of the phosphate groups of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate. Provided is a method for producing a sample comprising phosphatidylinositol phosphates and / or lysophosphatidylinositol phosphates having phosphate groups at fewer (eg, one) positions than the number of acid group positions. The method includes: A) applying the sample to chromatography, ion mobility separation (IMS) or mass spectrometry (MS); and B) identifying the elution from the chromatography, IMS or MS by the elution position of the peak. Collecting a fraction containing a phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate having a phosphate group at a desired target position. In one embodiment, the sample is applied to any combination of chromatography, ion mobility separation and mass spectrometry.

In the present invention, any technique known in the art may be used as a technique for collecting phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate having a phosphate group at a target position specified by the elution position of the peak. For example, an appropriate device such as an autosampler can be used, or the data can be collected manually.

Here, phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate mixture containing two or more phosphate groups include biological samples, foods, metabolites and any other phosphoinositide and / or lysophosphatidylinositol phosphate. Any sample containing or presumed to contain can be mentioned. In particular, in the present invention, the mixture is composed of three isomers of monophosphate phosphatidylinositol (phosphatidylinositol-3-monophosphate, phosphatidylinositol-4-monophosphate, phosphatidylinositol-5-monophosphate). 2 or 3 isomers, as well as 2 or 3 isomers of the 3 isomers of the monophosphate lysophosphatidylinositol, and the diphosphate phosphatidylinositol (phosphatidylinositol-3 , 4-diphosphate, phosphatidylinositol-3,5-diphosphate, phosphatidylinositol-4,5-diphosphate), as well as diphosphate lysates Of the three isomers of phosphatidylinositol One or three isomers, which are separated and less than the number of types of phosphatidylinositol phosphates and / or lysophosphatidylinositol phosphates phosphate groups contained in the mixture (eg, one) Samples containing phosphatidylinositol phosphates and / or lysophosphatidylinositol phosphates with phosphate groups in position can be generated. In this specification, the phosphatidylinositol phosphate having a phosphate group at one position and the lysophosphatidylinositol phosphate having a phosphate group at one position each have a binding pattern of seven kinds of phosphate groups. These are phosphatidylinositol-3-monophosphate, phosphatidylinositol-4-monophosphate, phosphatidylinositol-5-monophosphate, phosphatidylinositol-3,4-diphosphate, phosphatidylinositol- 3,5-diphosphate, phosphatidylinositol-4,5-diphosphate, phosphatidylinositol-3,4,5-triphosphate, and their lysates. Techniques that can be separated in part from the mixture are also the subject of the present invention.

In the present specification, “the number of phosphatidylinositol phosphates and / or lysophosphatidylinositol phosphates contained in the mixture is less than the number of types of phosphate groups of the lysophosphatidylinositol phosphates” means phosphatidylinositol phosphates and / or lysophosphatidylinositol phosphates contained in the mixture When the number of types of the position of the phosphoric acid group is n, it means n-1 or less. Preferably, the number of phosphatidylinositol phosphates and / or lysophosphatidylinositol phosphates contained in the mixture is less than the number of types of the phosphate groups of the phosphatidylinositol phosphate, but is not limited thereto, but is not limited to one, two, three There may be four types. In particular, three isomers of monophosphate phosphatidylinositol (phosphatidylinositol-3-monophosphate, phosphatidylinositol-4-monophosphate, phosphatidylinositol-5-monophosphate), monophosphate lysophosphatidylinositol Three isomers of the diphosphate phosphatidylinositol (phosphatidylinositol-3,4-diphosphate, phosphatidylinositol-3,5-diphosphate, phosphatidylinositol-4,5-diphosphate) Alternatively, it may be advantageous to be able to separate the three isomers of the diphosphate lysophosphatidylinositol.

(General technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, for example, Sambrook J. et al. (1989). Molecular Cloning. : A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987) .Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience; Innis, MA (1990) .PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub.Associates; Ausubel, FM (1995) .Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Innis, MA et al. (1995) .PCR Strategies, Academic Press; Ausubel, FM (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999). PCR Applications: Protocols for It is described in Functional Genomics, Academic Press, a separate volume of experimental medicine "Gene Transfer & Expression Analysis Experimental Method" Yodosha, 1997, etc., and related portions (which may be all) are incorporated herein by reference. The

For DNA synthesis technology and nucleic acid chemistry to produce artificially synthesized genes, see, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Chapman & Chapman & Chapman & Chapman & Chapman & Shap et al. (1994) .Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996) .Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, Tech (jud. Which are incorporated herein by reference in the relevant part.

In this specification, “or” is used when “at least one or more” of the items listed in the sentence can be adopted. The same applies to “or”. In this specification, when “within range” of “two values” is specified, the range includes two values themselves.

References such as scientific literature, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety to the same extent as if they were specifically described.

As described above, the present invention has been described by showing preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of the claims.

Examples are described below. There has been little research focusing on the diversity (molecular species) of fatty acid chains that bind to the sn-1,2-hydroxyl group of glycerol, which is one structural characteristic of phosphoinositide. As a method for measuring intracellular phosphoinositide levels, an HPLC method for analyzing deacylated phosphoinositide labeled with a radioisotope is the mainstream. However, this semi-quantitative analysis method is limited in the target sample (difficult in vivo), and above all, measurement at the molecular species level is impossible. Therefore, the inventors established a new analysis method with excellent quantitativeness using LC-MS / MS. This analysis method enables phosphoinositide analysis at the molecular species level for a wide range of biological samples.

When necessary, handling of animals used in the following examples was carried out in accordance with the guidelines for animal experiments at Akita University for all animal experiments (biological sample preparation, etc.). In addition, in the case of targeting humans, it was conducted based on the declaration of Helsinki. The reagents described in the examples were used specifically for the reagents, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemicals, Nakarai, R & D Systems, USCN Life Science INC, etc.) can be substituted.

(Abbreviation)
In addition to the abbreviations described in this specification, the following abbreviations are also used.

PI: phosphatidylinositol PI3P or PI (3) P: phosphatidylinositol-3-monophosphate PI4P or PI (4) P: phosphatidylinositol-4-monophosphate PI5P or PI (5) P: phosphatidylinositol-5-mono Phosphoric acid (PI3P or PI4P or PI5P is expressed as PIP1)
PI (3,4) P ₂ : Phosphatidylinositol-3,4-diphosphate PI (3,5) P ₂ : Phosphatidylinositol-3,5-diphosphate PI (4,5) P ₂ : Phosphatidylinositol- 4,5 diphosphate (PI (3,4) _{P 2} or PI (3,5) _{P 2} or PI (4,5) _{P 2} and referred to as PIP2)
PI (3,4,5) P ₃ : Phosphatidylinositol-3,4,5-triphosphate (PI (3,4,5) P ₃ is expressed as PIP3)
PIP1, PIP2, and PIP3 are collectively referred to as PIPs.

Structures bonded to fatty acids or hydrocarbons through only one of the sn-1 hydroxyl group and sn-2 hydroxyl group of glycerol of PIPs are collectively referred to as LPIPs.

(Example 1: Production and identification of lysophosphatidylinositol phosphates)
In this example, lysophosphatidylinositol phosphates were produced, and a novel identification method for the new substance was also developed.

(Chemical substance)
The following samples were used as standard samples and starting materials.

(Materials and methods)
(Materials used)
Phosphatidylinositol phosphates used as starting materials are 17: 0/20: 4-PI3P, 17: 0/20: 4-PI4P, 17: 0/20: 4-PI5P, 17: 0/20: 4-PI ( 3,4) P ₂ , 17: 0/20: 4-PI (3,5) P ₂ , 17: 0/20: 4-PI (4,5) P ₂ , 17: 0/20: 4-PI (3,4,5) _{P 3} and other glycerophospholipids standard synthetic were purchased from Avanti Polar lipids (Alabaster, AL, USA). Trimethylsilyldiazomethane (TMS-diazomethane) was purchased from Tokyo Kasei. Ultrapure water was obtained from Kanto Chemicals (Tokyo, Japan). All other solvents were HPLC or LC-MS grade and other chemical reagents were analytical grade. These were obtained from Wako Pure Chemicals.

(Synthesis method)
Using phosphatidylinositol phosphate (monophosphate, diphosphate and triphosphate) as a starting material, deacylation was carried out by O → N transacylation reaction with methylamine. Deacylation was set up with the following conditions so that only one acyl can be deacylated:

(Experimental conditions)
100 pmol 1-heptadecanoyl-2-arachidonoyl-sn-glycero-3-phospho-1'-myo-inositol-4 'monophosphate dissolved in chloroform / methanol (1/1) (1-heptadecanoyl-2-arachidonoyl-) sn-glycero-3-phospho-1'-myo-inositol-4'-monophosphate) (17: 0/20: 4-PI4P) to dryness with N ₂ gas, 0.3 mL of 2.13% methylamine Solution (methylamine / water / methanol / 1-butanol (1.92 / 38.08 / 40/10)) or 10.7% methylamine solution (methylamine / water / methanol / 1-butanol (9.6 / 30.4 / 40/10)) at 53 ° C. or 37 ° C., and after stirring, let stand at 53 ° C. or 37 ° C. for 0.5, 10, 10, 30, 60, 120 minutes, and add ice-cold methanol solution Queuing the reaction by I started. The reaction product was dried with N ₂ gas, the target lysophosphatidylinositol monophosphate (LPIP1: yield 0-40%) was protected by methylation of the phosphate group, and triple quadrupole mass spectrometry The measurement was carried out by the following method.

(Measurement and detection)
The “measurement and detection method by SRM with a triple quadrupole mass spectrometer” is described below.

LPIPs phosphate group methylation reaction: LPIPs phosphate group methylation protection reaction was performed for highly sensitive measurement with a mass spectrometer. A specimen containing LPIPs was dissolved in 800 μL of chloroform / methanol (1/1), 150 μL of trimethylsilyldiazomethane was added, and the mixture was reacted at room temperature for 5 minutes. The reaction was quenched by adding 10 μL of glacial acetic acid. After adding 800 μL of methanol / water / chloroform (48: 47: 3) and 400 μL of chloroform and stirring, the organic layer was dried with N ₂ gas. Methanol / 70% ethylamine (1: 0.13%), v / v) 27 μL and water 9 μL were added and stirred.

LC-ESIMS / MS system: The Ultimate-Mate 3000 LC system (Tm) coupled LC-ESIMS / MS analysis with a HTC PAL autosampler (CTC Analytics, Zwingen, Switzerland) with a non-metal injector portion and a sample loop of 25 μL PEEK tube. -Using TSQ-Vantage (Thermo-Fisher Scientific) together with Fisher Scientific, Waltham, MA. The PIPs fraction was added to mobile phase A (acetonitrile / H ₂ O = 4/1, 0.13% ethylamine (70% solution), v / v)) / mobile phase B (2-propanol / acetonitrile = 4 / 1,0). .13% ethylamine (70% solution), v / v)) ratio 70% / 30% (0-1 min), 10% / 90% gradient over 1-3 min, followed by 10% / 90% (3-7.5 minutes), separated by gradient using 70% / 30% (7.5-13 minutes). The flow rate was 220 μL / min and chromatography was performed at 60 ° C. using a GL sciences Inertsil Bio C18 150 × 1.0 (particle size 1.9 μm) column. Typically, 10 μL of sample was injected.

Conditions for mass spectrometry The ion spray voltage was set to 3.0 kV. The heated capillary temperature was set at 270 ° C. Other parameters were set according to manufacturer's recommendations. The MS system was calibrated using Xcalibur software. LPIPs were measured by the selective reaction monitoring (SRM) method in positive ion mode. Table 2 summarizes the measurement conditions of individual LPIPs in the SRM mode. LPIP1, LPIP2, and LPIP3 were identified by the combination of the m / z value (Q1) of the parent ion and the m / z value (Q2) of monoacylglycerol measured as the daughter ion. A schematic diagram is shown in FIG. 1-1.

(result)
The abscissa represents the reaction time, and the ordinate represents the production amount of 17: 0 LPIP1 or 20: 4 LPIP1 (the yield obtained from the peak area ratio of the raw material to the reaction product on the chromatogram). In the conventional method (incubation for 120 minutes with a methylamine concentration of 10.7%), LPIP1 was not detected, even when the reaction time was shortened to 60 minutes. LPIP1 was detected only when the reaction time was further reduced to 5, 10, and 30 minutes. Both 17: 0 LPIP1 and 20: 4 LPIP1 were generated. A similar reaction time-dependent tendency was observed in the reaction with a methylamine concentration of 2.14%, which is lower than that of the conventional method. In this case, a small amount of LPIP1 was detected even at reaction times of 60 minutes and 120 minutes, and more LPIP1 was detected at 5, 10, and 30 minutes.

As shown in FIG. 1-2, the reaction depends on the temperature. Incubation at 10.7% methylamine at 37 ° C. for 10 minutes yielded LPIP1 in a higher yield than the reaction at 53 ° C.

(Discussion)
This method using methylamine does not affect the hydrophilic groups of phospholipids, including inositol phospholipids, but binds to inositol as is widely used for the complete deacylation of two acyl groups. It is known that the position of phosphoric acid does not change (Serunian LA, et al. Methods Enzymol. 1991; 198: 78-87, Fujii et al. Folia Pharmacol Jpn. 2013; 142: 236-240) If the conditions are more relaxed, the position and number of phosphate groups are not changed. By adjusting the degree of deacylation by shortening the reaction time, reducing the methylamine concentration, reducing the reaction temperature, and a combination thereof, only one acyl can be deacylated, corresponding to diacyl-type PIPs used as starting materials LPIPs can be synthesized. Next, Table 3 shows examples of LPIPs that can be synthesized using a commercially available PIPs synthetic product as a raw material in this example, and Table 4 shows an example in which the position of the phosphate group can be determined. The numbers in the table indicate the number of carbon atoms and the number of double bonds. For those with double bonds, 16: 1 = 9-hexadecenoic acid (palmitoleic acid); 18: 1 = cis-9-octadecenoic acid (oleic acid); 20: 4 = 5,8,11,14-icosatetraene Acid (arachidonic acid); 22: 6 = 4,7,10,13,16,19-docosahexaenoic acid.

In FIG. 2 (FIGS. 2A to 2G), the results of LPIPs produced from seven kinds of PIPs raw materials (acyl groups are 17: 0, 20: 4) having different phosphorylation patterns are illustrated. 17: 0/20: 4-PI3P, 17: 0/20: 4-PI4P, 17: 0/20: 4-PI5P, 17: 0/20: 4-PI (3,4) P ₂ as starting materials, 17: 0/20: 4-PI (3,5) P ₂ , 17: 0/20: 4-PI (4,5) P ₂ , 17: 0/20: 4-PI (3,4,5) with P _3, 10.7% methylamine presence 53 ° C., after incubation for 10 min the reaction was quenched by addition of ice-cold methanol. The reaction product was dried with N2 gas, the methylation protection of the phosphate group of the target LPIPs was performed, and measurement was performed by the above method using a triple quadrupole mass spectrometer. Using any PIPs as a raw material, the production of LPIPs was observed by the methylamine reaction.

(Identification of compounds)
In order to identify the compounds of the present invention, accurate mass measurements of specific fragments supporting the structure of LPIPs were performed. The “measurement and detection method using a hybrid quadrupole-orbitrap mass spectrometer” is described below. The instrument used is Orbitrap Q-Exclusive Plus, which has higher mass accuracy than the above-described triple quadrupole mass spectrometer, and the use conditions are as follows.

LC-ESI MS / MS system: LC-ESI MS / MS analysis is performed using Ultimate 3000 LC system (Thermo-Fisher Scientific, Waltham, MA) and Orbitrap Q-Exactive Plus (Thermo-SciMA). I went there. The PIPs fraction was added to mobile phase A (acetonitrile / H ₂ O = 4/1, 0.13% ethylamine (70% solution), v / v)) / mobile phase B (2-propanol / acetonitrile = 4 / 1,0). .13% ethylamine (70% solution), v / v)) ratio 70% / 30% (0-1 min), 10% / 90% gradient over 1-3 min, followed by 10% / 90% (3-7.5 minutes), separated by gradient using 70% / 30% (7.5-13 minutes). The flow rate was 220 μL / min and chromatography was performed at 60 ° C. using a GL sciences Inertsil Bio C18 150 × 1.0 (particle size 1.9 μm) column.

In addition, about the analysis of FIG. 5C and FIG. 5D, it carried out on the following conditions instead. The PIPs fraction was added to mobile phase A (methanol / H ₂ O / 70% ethylamine (20: 80: 0.13, v / v)) / mobile phase B (methanol / H ₂ O / 2-propanol / 70% ethylamine ( 5: 5: 90: 0.13, v / v)) ratio of 90% / 10% (0-0.1 min), with a gradient of 70% / 30% over 0.1-3 min, followed by 10 Separation by a gradient with% / 90% (3-15 min). The flow rate was 30 μL / min and chromatography was performed at 30 ° C. using a Waters X-Bridge C8 (3.5 mm, 1.0 × 150 mm) column.
Typically, 10 μL of sample was injected. As conditions for mass spectrometry, the ion spray voltage was set to 3.0 kV. The heated capillary temperature was set to 250 ° C. Other parameters were set according to manufacturer's recommendations. The MS system was calibrated using Xcalibur software. LPIPs were measured by selected ion monitoring (SIM) and parallel reaction monitoring (PRM) in positive ion mode.

17: 0/20: 4-PI4P, 17: 0/20: 4-PI (4,5) P ₂ , 17: 0/20: 4-PI (3,4,5) P ₃ Thus, it was confirmed that the compounds of the present invention were synthesized using LPIPs prepared by incubation at 53 ° C. for 10 minutes in the presence of 10.7% methylamine as a sample. The theory that the structure of the present invention can be identified by identifying specific fragments of LPIP1, LPIP2, and LPIP3 is as follows.

The identification of LPIP1 will be described with reference to the chromatogram in FIG. 3A and the structural formulas of the starting material, product, and fragment in FIG. 3B. Two types of LPI (4) P produced by mild deacylation of 37: 4 PI (4) P (sn-1 17: 0, sn-2 20: 4 PI (4) P) (see FIG. 1) The results of accurate mass spectrometry of LPI (4) P and fragments derived from LPI (4) P are shown as representative examples of LPIP1 synthesis reaction. In FIG. 3A, the accurate mass measurement results of 17: 0 LPIP ₁ (first stage) and its fragment ion (second stage) and 20: 4 LPIP ₁ (third stage) and its fragment ion (fourth stage) are determined. Together with the molecular formula.

In the reaction product, a substance having m / z = 754.902 (first stage) and a substance having m / z = 788.3749 (third stage) were detected. The error between these values and the theoretical m / z value obtained from the molecular formulas of 17: 0 LPIP1 (3-methylated product, ethylamine adduct) and 20: 4 LPIP1 (3-methylated product, ethylamine added) is 0.03 ppm. (5 ppm or less) and 0.47 ppm (5 ppm or less), it is clear that substances having the same composition formula as 17: 0 LPIP1 and 20: 4 LPIP1 were produced by the deacylation reaction. It became.

Next, the results of detecting substances (fragment ions) generated by decomposing substances that can be detected in the range of m / z = 754.902 ± 1.0 including the substances detected in the first stage are shown in the second stage. The fragment ion of the substance that can be detected in the range of m / z = 788.3749 ± 1.0 including the substance detected in the third stage is shown in the fourth stage.

The substance m / z = 754.902 shown in the first stage has m / z 327.2892 and m / z 383.0507 detected in the second stage as its partial structure. m / z 327.2892 has an error of 0.48 ppm (5 ppm or less) from the theoretical value of m / z determined from the molecular formula of 17: 0 monoacylglycerol (17: 0 MG) (monovalent positive ion with one hydroxyl group missing). M / z 383.0507 had an error of 1.16 ppm (5 ppm or less) from the theoretical value of m / z determined from the molecular formula of inositol diphosphate (trimethylated, detected as a proton adduct). . Therefore, the structural formula of the first-stage substance was determined, and m / z = 754.902 was confirmed to be 17: 0 LPIP1 (detected as a 3-methylated product and an ethylamine adduct).

On the other hand, the substance m / z = 788.3749 shown in the third stage has m / z 361.2736 and m / z 383.0511 detected in the fourth stage as its partial structure. m / z 361.22736 has an error of 0.45 ppm (5 ppm or less) from the theoretical value of m / z determined from the molecular formula of 20: 4 monoacylglycerol (20: 4 MG) (monovalent positive ion from which one hydroxyl group is missing). M / z 383.0511 had an error of 2.18 ppm (5 ppm or less) from the theoretical value of m / z determined from the molecular formula of inositol diphosphate (detected as trimethylated and protonated adducts). . Therefore, the structural formula of the third-stage substance was confirmed, and m / z = 788.3749 was confirmed to be 20: 4 LPIP1 (detected as a 3-methylated product and an ethylamine adduct).

The identification of LPIP2 will be described with reference to the chromatogram in FIG. 3C and the structural formulas of the reaction starting material, product, and fragment in FIG. 3D. FIG. 3C shows two types of LPIs produced by a mild deacylation reaction of 37: 4 PI (4,5) P2 (sn-1 17: 0, sn-2 20: 4 PI (4,5) P2) ( As a representative example of LPIP2 synthesis reaction, the results of accurate mass spectrometry of fragments derived from LPI (4,5) P2 and LPI (4,5) P2 are shown for 4,5) P2. 17: 0 LPIP ₂ (first step) and its fragment ion (second step), 20: 4 LPIP ₂ (third step) and its fragment ion (fourth step) accurate mass measurement results together with the determined molecular formula Show.

In the reaction product, a substance with m / z = 862.3916 (first stage) and a substance with m / z = 8966.3695 (third stage) were detected. The error between these values and the theoretical m / z values obtained from the molecular formulas of 17: 0 LPIP2 (5-methylated product, ethylamine adduct) and 20: 4 LPIP2 (detected as 5-methylated product, ethylamine adduct) is Since it was 4.35 ppm (5 ppm or less) and 3.00 ppm (5 ppm or less), it was clear that substances having the same composition formula as 17: 0 LPIP2 and 20: 4 LPIP2 were produced by the above reaction. It became.

Next, the results of detecting substances (fragment ions) generated by decomposing substances that can be detected in the range of m / z = 862.3916 ± 1.0 including the substances detected in the first stage are shown in the second stage. The fragment ion of the substance that can be detected in the range of m / z = 896.3695 ± 1.0 including the substance detected in the third stage is shown in the fourth stage.

The substance m / z = 862.3916 shown in the first stage has m / z = 327.2894 and m / z = 491.0487 detected in the second stage as its partial structure. m / z = 327.2894 has an error of 0.01 ppm from the theoretical value of m / z determined from the molecular formula of 17: 0 monoacylglycerol (17: 0 MG) (monovalent positive ion from which one hydroxyl group is missing) ( M / z = 491.0487 has an error of 1.69 ppm (5 ppm or less) from the theoretical value of m / z determined from the molecular formula of inositol triphosphate (5-methylated, detected as proton adduct). )Met. Therefore, the structural formula of the first-stage substance was confirmed, and m / z = 862.3916 was confirmed to be 17: 0 LPIP2 (detected as a 5-methylated product and an ethylamine adduct).

On the other hand, the substance m / z = 8966.3695 shown in the third stage has m / z = 361.737 and m / z = 491.0488 detected in the fourth stage as its partial structure. m / z = 361.737 has an error of 0.11 ppm from the theoretical value of m / z determined from the molecular formula of 20: 4 monoacylglycerol (20: 4 MG) (monovalent positive ion from which one hydroxyl group is missing). M / z = 491.0488 is 1.88 ppm (5 ppm or less) from the theoretical value of m / z determined from the molecular formula of inositol triphosphate (5-methylated, detected as proton adduct). )Met. Therefore, the structural formula of the third-stage substance was confirmed, and m / z = 896.3695 was confirmed to be 20: 4 LPIP2 (detected as a 3-methylated product and an ethylamine adduct).

The identification of LPIP3 will be described with reference to the chromatogram in FIG. 3E and the structural formulas of the reaction starting material, product, and fragment in FIG. 3F. FIG. 3E shows the results of a mild deacylation reaction of 37: 4 PI (3,4,5) P3 (sn-1 17: 0, sn-2 20: 4 PI (3,4,5) P3). The result of carrying out the accurate mass spectrometry of the fragment derived from LPI (3,4,5) P3 and LPI (3,4,5) P3 is shown for the species LPI (3,4,5) P3. 17: 0 LPIP ₃ (first step) and its fragment ion (second step), 20: 4 LPIP ₃ (third step) and its fragment ion (fourth step) accurate mass measurement results together with the determined molecular formula Show. A substance with m / z = 970.3843 (first stage) and a substance with m / z = 1004.3726 (third stage) were detected in the reaction product. The error between these values and the theoretical m / z value obtained from the molecular formulas of 17: 0 LPIP3 (7 methylated product, ethylamine adduct) and 20: 4 LPIP3 (7 methylated product, ethylamine adduct) is 1. Since it was 18 ppm (5 ppm or less) and 2.72 ppm (5 ppm or less), it became clear that substances having the same composition formula as 17: 0 LPIP3 and 20: 4 LPIP3 were produced by the above reaction. It was.

Next, the result of detecting the substance (fragment ion) generated by decomposing the substance that can be detected in the range of m / z = 970.3843 ± 1.0 including the substance detected in the first stage is shown in the second stage. The fragment ions of the substance that can be detected in the range of m / z = 1004.3726 ± 1.0 including the substance detected in the third stage are shown in the fourth stage.

The substance m / z = 970.3843 shown in the first stage has m / z = 327.2891 and m / z = 599.0455 detected in the second stage as its partial structure. m / z = 327.2894 has an error of 0.85 ppm from the theoretical value of m / z obtained from the molecular formula of 17: 0 monoacylglycerol (17: 0 MG) (a monovalent positive ion from which one hydroxyl group is missing) ( M / z = 599.455 is 0.01 ppm (5 ppm or less) from the theoretical value of m / z obtained from the molecular formula of inositol tetraphosphate (7-methylated, detected as proton adduct). )Met. Therefore, the structural formula of the first-stage substance was confirmed, and m / z = 970.3843 was confirmed to be 17: 0LPIP3 (detected as a 7-methylated product and an ethylamine adduct).

On the other hand, the substance m / z = 1004.3726 shown in the third stage has m / z = 361.735 and m / z = 599.444 detected in the fourth stage as its partial structure. m / z = 361.735 has an error of 0. 0 from the theoretical value of m / z determined from the molecular formula of 20: 4 monoacylglycerol (20: 4 MG) (detected as a monovalent positive ion with one hydroxyl group missing). 62 ppm (5 ppm or less), and m / z = 599.444 has an error of 1.94 ppm from the theoretical value of m / z determined from the molecular formula of inositol tetraphosphate (7-methylated, detected as proton adduct). 5 ppm or less). Therefore, the structural formula of the third-stage substance was confirmed, and m / z = 1004.3726 was confirmed to be 20: 4LPIP3 (detected as a 7-methylated product and an ethylamine adduct).

3A to 3F are proof of identification of LPIPs by accurate mass measurement.

(Discussion)
By determining the exact mass of the structure of the product and product-derived fragment (fragment), the structure of the product was uniquely determined as noted in the above results. The results obtained by “Measurement and detection method using hybrid quadrupole-orbitrap mass spectrometer” are the same as those of “Measurement and detection method by SRM with triple quadrupole mass spectrometer”. It has been shown that LPIPs are generated by a proper methylamine treatment, and both measurement methods were considered to be suitable for detection and measurement of LPIPs.

(NMR analysis of LPIPs)
The structure of the synthesized LPIPs was determined by NMR. Regarding the structure determination of lipids by NMR, those skilled in the art can appropriately determine the structure, for example, ¹ H chemical shift is described in Ong et al. Mol. Biosys. (2009) 5, 288-298 can be referred to.

In the glycerol skeleton, the first position gives a chemical shift of about 3.7 ppm, the second position about 5.2 ppm, and the third position about 4.1 ppm, so that it is possible to distinguish between sn-1 and sn-2.

As for the a-position and b-position of the fatty acid, aCH ₂ gives a chemical shift of about 2.3 ppm and bCH ₃ gives a chemical shift of about 1.6 ppm, so that it is possible to distinguish between sn-1 and sn-2.

Since the polyunsaturated portion of the polyunsaturated fatty acid gives a chemical shift of about 2.8 ppm and about 5.3 ppm, it can be used to estimate the type of fatty acid in the lysophospholipid.

The phosphorus atom gives a chemical shift of 0 to 2 ppm ( ³¹ P, about 0.5 ppm low magnetic field shift with lysophospholipidation), and can be used for investigation of reaction progress.

For example, the lipid structure can be determined from NMR as shown in FIG. 4A.

NMR measurement was performed on the following samples. Brain PI4P (Avanti Polar Lipids, Alabama, USA) 1 mg NMR measurement was also performed.
Sample 1) 16: 0 LPI4P 5-7 nmol
Sample 2) 17: 0 LPI (4,5) P2 4-6 nmol
Sample 3) 18: 0 LPI (4,5) P2 4-6 nmol
Sample 4) 32: 0 (16: 0/16: 0) PI4P 10 nmol
Sample 5) 37: 4 (17: 0/20: 4) PI (4,5) P2 10 nmol
Sample 6) Brain PI (4,5) P2 (mostly 38: 4 (18: 0/20: 4)) 10 nmol
Samples 1-3 were prepared according to the above examples. Samples 4-6 were purchased from Avanti Polar Lipids (Alabama, USA).

About 500 mL of CDCl ₃ : CD ₃ OD = 1: 1 was added to each of samples 1 to 6 and dissolved in Vortex. ¹ H-1D, TOCSY spectra were measured (37 ° C., about half a day). 100 mL of an EDTA-2Cs solution in D ₂ O at pH = 6.0 was added (J. Lipids Res. (1988) 29, 679-689). ³¹ P-NMR spectrum was measured (37 ° C., about 2 days). Here, for sample 6 only, ¹ H-1D and TOCSY spectra were measured at 25 ° C. The NMR measurement time of 1 mg of Brain PI4P was about 1/10 of that of the other samples, and about 1/100 of ³¹ P-1D.

In order to assign the inositol carbon position, NMR analysis was performed on Brain PI4P, and the inositol carbon position could be assigned.

In sample 4 (32: 0 PI4P), signals of C1, C2, and C3 of fatty acid and glycerol skeleton were observed, but signals of polyunsaturated fatty acid were not observed.

In sample 6 (Brain PI (4, 5) P2), signals of fatty acids and C1, C2, and C3 of the glycerol skeleton were observed, and signals of polyunsaturated fatty acids were hardly observed.

As a result of the measurement, it was confirmed that most of the signals were derived from the target compound. The structures confirmed by NMR for Samples 1 to 3 and 5 are shown in FIGS. 4B to 4F.
(Conclusion)
In NMR, it was possible to observe signals of at least part of the fatty acid and glycerol skeleton of lysophospholipid with a sample amount of about 5 to 10 nmol, and the structure of lysophospholipid could be determined. Moreover, if there is a sample amount of about 50 nmol, it is possible to observe signals of all hydrogen atoms and phosphorus atoms.

(Example 2: Analysis in biological sample and separation of LPIPs (cultured cells))
In this example, experiments were conducted on the separation and analysis of LPIPs in biological samples. A human cultured cell line was used as the biological sample.

(Materials and methods)
(Cultivated cell line) HEK293T (human embryo-derived kidney epithelial cell line, Open biosystems catalog number HCL4517), Jurkat (human acute T-cell leukemia cell-derived cell line, ATCC catalog number TIB-152) under the conditions recommended by the provider Cultured and maintained.

(Phospholipid extraction) Lipid extraction was performed based on the Bligh & Dyer method (EG Bligh et al, Canadian Journal of Biochemistry and Physiology, 1959, 37 (8): 911-917, 10.1139 / o59-099). .

For analysis of PIPs, cultured cells and mouse tissue were scraped or homogenized with 1.5 mL of ice-cold methanol with a cell scraper and 1 nmol / μL 8: 0/8: 0-PI (4,5) as an adsorption inhibitor. P ₂ (2 μL) was added. Then, 200 fmol / μL 17: 0/20: 4-PI4P, 17: 0/20: 4-PI (4,5) P _2, 17: 0/20: 4-PI (3,4,5) P ₃ 17: 0/20: 4-PI, 17: 1-LPI and 17: 0/20: 4-PS  50 μL was added as an internal standard, 2N HCl (0.75 mL), water (0.75 mL), 1M NaCl (0.2 mL) and chloroform (3 mL) were added, then stirred and centrifuged. After centrifugation, the lower layer was collected.

(Separation by DEAE cellulose)
Before adding the phospholipid extracted by the above procedure to the DEAE-cellulose column, 1.5 mL of methanol was added to each sample. The lipids adsorbed on the column were chloroform / methanol (1: 1, v / v) (3 mL) and chloroform / methanol / 28% aqueous ammonia / acetic acid (200: 100: 3: 0.9, v / v) (3 mL). ) To wash sequentially. Highly acidic lipids containing LPIPs were then eluted with chloroform / methanol / HCl / water (12: 12: 1: 1, v / v) (1.5 mL). After the addition of water (0.75 mL) and 1 M NaCl (0.1 mL), the solution was stirred and centrifuged to collect the lower layer.

(Methylation of phosphate group)
After the addition of 0.6 M TMS-diazomethane in hexane (150 μL), the sample was incubated at room temperature for 10 minutes (Nature Methods 8, 267-272 (2011)). Glacial acetic acid (15 μL) and washings (chloroform / methanol / water (3:48:47, v / v) 0.7 mL) were then added to each solution. The solution was stirred and centrifuged to collect the lower layer. Each sample was dried under nitrogen gas, redissolved in 24 μL of methanol / 70% ethylamine (100: 0.065, v / v), and 8 μL of water was added. The resulting fractions rich in acidic phospholipids containing LPIPs were analyzed within one day of preparation.

(Analysis method)
The measurement was carried out according to the “measurement and detection method by SRM with a triple quadrupole mass spectrometer” having excellent quantitative properties. Based on molecular species analysis of diacyl-type PIPs, selective reaction monitoring (SRM) was performed on LPIPs molecular species expected to be contained in biological samples. Using TSQ Vantage, Q1: m / z of parent ion and Q3: m / z of monoacylglycerol as product ion. Mass spectrometric data was calculated based on LPI quantified peak areas and the abundance of each molecular species added as an internal standard. The adduct ion is [M + CH ₃ CH ₂ NH ₃ ] ⁺ . Table 5 shows a list of m / z values designated by Q1 and Q3 used for detecting each LPIP in this embodiment.

(result of analysis)
FIG. 5A shows the presence of LPIP1, LPIP2, and LPIP3 in HEK293T cells and FIG. 5B in Jurkat cells. The horizontal axis represents molecular species having different fatty acid structures, and the vertical axis represents the abundance in cell-derived phospholipids containing PS 1 nmol. LPIPs having various acyl groups having 16 to 22 carbon atoms and 0 to 6 double bonds were found in cells. LPIP1 and LPIP2 were trace amounts of phospholipids present at a level of 1 / hundred to several tenths of PS, and LPIP3 was present at a level of 1 / hundred to several thousandths.

5K and 5D show the results of detection of 18: 0 LPIP3 and its specific fragments in the analysis of HEK293T cell lipid extract by “Measurement and detection method using hybrid quadrupole-orbitrap mass spectrometer”. Shown in

As shown in the upper part of FIG. 5C, a peak at m / z = 9844.424 was detected, which was the m / z theoretical value (9844.401) of 18: 0 LPIP3 (7 methylated product, methylamine addition). The error was 1.2673 ppm (5 ppm or less), and thus it became clear that a substance having the same composition formula as 18: 0 LPIP3 was contained in the lipid extract of HEK293 cells. As shown in the lower part of FIG. 5D, peaks of m / z 341.3047 and m / z 599.0449 were detected as ion fragments detected in the range of m / z = 9844.424 ± 0.2. The former is 18: 0 monoacylglycerol (monovalent positive ion with one hydroxyl group missing) and the latter is consistent with the m / z of inositol tetraphosphate (7-methylated, protonated). Error of 5 ppm or less (1.0460 and 1.0539) (see (FIG. 3E), the latter peak is the same ion as the fragment ion generated from the LPIP3 synthesized product). If m / z 341.3047 and m / z 599.449 in the lower part of FIG. 5C are ion fragments derived from m / z = 9844.424 in the upper part of FIG. 5D, the times when these peaks are detected coincide. FIG. 5D shows that m / z 341.3047 and m / z 599.449 are detected, consistent with an elution time of m / z = 9844.424.

The CH ₃ CH ₂ NH ₃ ⁺ adduct is shown below.

Before the addition, it is as follows.

The dehydroxylated product is shown below.

The pre-dehydration oxidation is shown below.

H ⁺ adducts are shown below.

Before the addition, it is as follows.

Example 3: Analysis and separation of LPIPs in biological samples (mouse tissue and human blood samples)
Next, in this example, an experiment for separation and analysis of LPIPs in a biological sample was performed using another sample. As biological samples, LPIPs in mouse or human tissues or serum or plasma were used.

Mouse: C57BL / 6J <purchased from Clea Japan Co., Ltd.>, 12-16 weeks old organ / tissue: Brain, heart, liver, large intestine were prepared as follows. Mice were dislocated from the cervical vertebrae, fixed on their back and then laparotomized. Organs isolated after perfusion and blood removal with PBS were immediately frozen in liquid nitrogen. Thawed immediately before lipid extraction and homogenized with a homogenizer. Extraction of phospholipid, separation with DEAE cellulose and methylation reaction were performed according to Example 2. The analysis was also carried out in the same manner as in Example 2, “Measurement and detection method by SRM with triple quadrupole mass spectrometer”. Serum: An anesthetic (ketamine) was opened 3 minutes after intraperitoneal administration, and blood was collected from the heart. The collected blood was allowed to stand at room temperature for 15 minutes and then allowed to stand at 4 ° C. for 12 hours. The clot was removed by centrifugation (600 g, 30 minutes), and serum was prepared. Extraction of phospholipid, separation with DEAE cellulose and methylation reaction were performed according to Example 2. The analysis was also carried out in the same manner as in Example 2, “Measurement and detection method by SRM with triple quadrupole mass spectrometer”.

Plasma: The abdominal cavity was opened 3 minutes after administration of anesthetic (ketamine) and 0.5 ml of blood was collected from the lower abdominal aorta. The collected blood was collected in a 1.5 mL tube containing 2 mg of EDTA-2Na cooled with ice, and a plasma sample was prepared from the supernatant after stirring and centrifugation (600 g, 30 minutes). Extraction of phospholipid, separation with DEAE cellulose and methylation reaction were performed according to Example 2. The analysis was also carried out in the same manner as in Example 2, “Measurement and detection method by SRM with triple quadrupole mass spectrometer”.

Human serum: Blood was collected from a peripheral vein into a test tube, and the collected blood was allowed to stand at room temperature for 15 minutes, and then allowed to stand at 4 ° C. for 12 hours. The clot was removed by centrifugation (600 g, 30 minutes), and serum was prepared. Extraction of phospholipid, separation with DEAE cellulose and methylation reaction were performed in the same manner as in Example 3. The analysis was carried out in the same manner as in Example 2, “Measurement and detection method by SRM with triple quadrupole mass spectrometer”.

(result)
As shown in FIG. 6A, it was revealed that LPIPs were present in the mouse tissue. Furthermore, since LPIPs are present as liquid components in the living body such as the serum shown in FIG. 6B and the plasma shown in FIG. It was suggested that the production of LPIP2 by phosphorylation of the protein also occurs outside the cell. LPIPs were also present in human serum as shown in FIG. 6D.

(Discussion)
In addition to samples derived from mice and humans, other experiments have found that LPIPs are also present in yeast and Drosophila, and it has been found that LPIPs exist intracellularly and extracellularly in many biological species. .

(Example 4: Use as a cancer marker)
In this example, the usefulness of novel LPIPs was examined.

(Materials and methods)
PTEN ^{flox / flox} mice (subject mice) and PB-Cre4: PTEN ^{flox / flox} mice (prostate-specific Pten deficient mice) ♂, 16 wk
Organ: Prostate Samples were prepared as follows. Mice were dislocated from the cervical spine, fixed on the back and then laparotomized, and the prostate isolated under a stereomicroscope was immediately frozen in liquid nitrogen. Thawed immediately before lipid extraction and homogenized with a homogenizer. Extraction of phospholipid, separation with DEAE cellulose and methylation reaction were performed according to Example 2. The analysis was also carried out in the same manner as in Example 2, “Measurement and detection method by SRM with triple quadrupole mass spectrometer”.

(result)
FIG. 7A shows the tissue weight in the upper row and the tissue image by HE staining in the lower row of the prostate of a normal mouse (Ctrl) or a mouse (PTEN KO) that specifically lacks the tumor suppressor gene Pten. It can be seen that Pten deficiency causes prostate cancer. As shown in FIG. 7B, it was revealed that 16: 0 LPIPs were increased in prostate cancer tissue as compared to the normal prostate as a control. In particular, in LPIP3, a significant increase was found with tumor formation when the normal tissue was below the detection limit. Since the fluctuation of LPIPs correlates with carcinogenesis, the usefulness of LPIPs as biomarkers reflecting cancer treatment targets and cancer is expected.

(Example 5: Use as an inflammation marker)
(Materials and methods)
C57BL / 6J <purchased from Clea Japan Co., Ltd.>, 12-16 week old mice were dissected after dislocation of the cervical vertebrae to obtain the foot femur. Myelospheres and neutrophils were collected from the obtained femur and stimulated with complement component C5a, which is an inflammatory substance. Extraction of phospholipid, separation with DEAE cellulose and methylation reaction were carried out in the same manner as in Example 2. The analysis was also carried out in the same manner as in Example 2, “Measurement and detection method by SRM with triple quadrupole mass spectrometer”.

(result)
As shown in FIG. 8, 18: 0 LPIP3 is increased with stimulation of complement component C5a involved in the activation of blood cells related to inflammation such as neutrophils and macrophages. The possibility that LPIP3 is involved in chemotaxis of inflammatory cells, active oxygen production, phagocytosis, enzyme secretion reaction, etc. is presented, and it can be expected to be useful as a therapeutic target for inflammation.

(Example 6) Separation of phosphoinositide by column In this example, it was demonstrated that separation by the position of the phosphate group of phosphoinositide (PIPs) in LC-MS was possible.

(Materials and methods)
(Materials used)
Phosphatidylinositol phosphate (phosphoinositide) used as a sample for analysis below was purchased from Avanti Polar Lipids (Alabama, USA).
17: 0-20: 4 PI (3) P (LM-1900)
17: 0-20: 4 PI (4) P (LM-1901)
17: 0-20: 4 PI (5) P (LM-1902)
17: 0-20: 4 PI (3,4) P2 (LM-1903)
17: 0-20: 4 PI (4,5) P2 (LM-1904)
17: 0-20: 4 PI (3, 5) P2 (LM-1905)
17: 0-20: 4 PI (3,4,5) P3 (LM-1906)
17: 0-20: 4 PI (LM-1502)
Other Glycerophospholipid Standard Synthetic Products All solvents utilized were HPLC or LC-MS grade and other chemical reagents were analytical grade. Trimethylsilyldiazomethane (TMS-diazomethane) was purchased from Tokyo Kasei. Ultrapure water was obtained from Kanto Chemicals (Tokyo, Japan).

Each phosphoinositide was dissolved in chloroform: methanol = 1: 1 so as to have a concentration of 100 μM in a glass vial (4 mL size), and stored in a freezer at −30 ° C.

The following samples were prepared from this stock solution.

Each phosphoinositide alone (100 μM 1 μL)
PI (3) P + PI (4) P + PI (5) P (each 100 μM 1 μL)
PI (3,4) P2 + PI (4,5) P2 + PI (3,5) P2 (each 100 μM 1 μL)
Each sample was treated with TMS diazomethane to protect the phosphate groups. Specifically, each sample was treated with 150 μL of 0.6 M TMS diazomethane (22-25 ° C.), and after 5 minutes, 15 μL of glacial acetic acid was added to stop the methylation reaction. Thereafter, 700 μL of a mixed solution of chloroform: methanol: water (3:48:47) was added to each sample, and after centrifugation, the organic layer (lower layer) was dispensed into a Spitz test tube and evaporated with a nitrogen gas concentrator. Each sample was dried and redissolved in 100 μL of acetonitrile to prepare a sample for LC-MS measurement.

(Measurement condition)
The measurement by LC-MS was performed under the following conditions.

(Used equipment)
Mass spectrometer: QTRAP6500 with SelexION System (ABSciex, Tokyo, Japan)
Pump: Nexera X2 system (Shimadzu Corporation, Kyoto, Japan)
Autosampler: PAL HTC-XT (AMR, Tokyo, Japan)
Column: CHIRALPAK IC-3, 2.1 mm x 250 mm, particle size 3 μm (DAICEL corporation, Osaka, Japan)
Flow rate: 0.1 mL / min Injection sample volume: 10 μL
(Chromatographic conditions)
Mobile phase A: acetonitrile + 5 mM ammonium acetate Mobile phase B: methanol + 5 mM ammonium acetate * All these reagents were LC-MS grades purchased from Wako Pure Chemical (Tokyo, Japan).

Gradient condition (linear gradient)
0 minutes, mobile phase B = 40%
1 minute, mobile phase B = 40%
3 minutes, mobile phase B = 85%
14 minutes, mobile phase B = 85%
15 minutes, mobile phase B = 40%
20 minutes, mobile phase B = 40%
・ Mass spectrometer conditions Ionization method: Electrospray (positive ion mode)
Measurement method: Multiple reaction monitoring (MRM)
Control software: Analyst 1.6.2 (Sciex, Tokyo, Japan)
The following measurement methods were used.

Software: Software Version: Analyst 1.6.2 Figure 1 shows the chromatogram of C17: 0 / C20: 4-PI (3) P, -PI (4) P, -PI (5) P as internal surrogate standards Data of the chromatogram obtained by measuring Gram and C17: 0 / C20: 4-PI (3,5) P2, -PI (3,4) P2, and -PI (4,5) P2 are shown. The three isomers of PIP1 were eluted and separated in the order of PI (3) P, PI (4) P, and PI (5) P. The three isomers of PIP2 were eluted and separated in the order of PI (3,5) P2, PI (3,4) P2, and PI (4,5) P2.

The combination of the precursor ion of Q1 and the product ion of Q3 used below is shown.

<Measurement conditions for mass spectrometry>
Parameter Table (Period1 Experiment1)
CUR: 20.00
CAD: 9.00
IS: 5500.00
TEM: 100.00
GS1: 50.00
GS2: 50.00
DP 100.00
EP 10.00
CXP 12.00
Resolution tables
Quad1 Positive Unit Scan Speed = 10 Da / s
(Calibration conditions)
When polypropylene glycol (PPG) was used as a calibration reagent, the following results were obtained.
IE1 1.800
Mass (Da) Offset Value
59.050 0.010
175.133 -0.035
500.380 -0.194
616.464 -0.268
906.673 -0.421
1254.925 -0.617
1545.134 -0.782
1952.427 -1.013
Quad3 Positive Unit Scan Speed = 10 Da / s
IE3 1.000
Mass (Da) Offset Value
59.050 0.005
175.133 -0.012
500.380 -0.085
616.464 -0.105
906.673 -0.183
1254.925 -0.270
1545.134 -0.329
1952.427 -0.429
Calibration tables
Quad1 Positive Unit Resolution Scan Speed = 10 Da / s
Mass (Da) Dac Value
175.133 18682
500.380 53678
616.464 66162
906.673 97393
1254.925 134868
1545.134 166099
1952.427 209933
Quad3 Positive Unit Resolution Scan Speed = 10 Da / s
Mass (Da) Dac Value
59.050 6189
175.133 18637
500.380 53564
616.464 66028
906.673 97195
1254.925 134603
1545.134 165759
1952.427 209490
(Device parameter)
Detector Parameters (Positive):
CEM 1800.0
(software version)
software version: Analyst 1.6.2
As described above, the acquired data was analyzed using Analyst (Sciex, Tokyo, Japan) or Multi Quant (Sciex, Tokyo, Japan).

FIG. 1 shows the results of isomer separation using a 17: 0/20: 4 synthetic product. Phosphoinositides with different numbers of phosphate groups were eluted with different retention times. Even when three types of isomers differing only in the position of the phosphate group were mixed (mixture of PIP or PIP2), they could be separated well by a 20-minute measurement method using a column. As described above, the separation of the phosphoinositide by the phosphate group positional isomer enables regioisomer-specific analysis.

It was also confirmed that it is possible to specify the combination of acyl group types of phosphoinositide by using the measurement results of MS and MS / MS (for the specification of the acyl group type, Clark J et al., Nat Methods. 2011 Mar; 8 (3): 267-72. Doi: 10.1038 / nmeth can also be referred to). Therefore, the position of the phosphoric acid group and the kind of acyl group of phosphoinositide can be specified using the method of the present invention.

(Example 7) Quantification of phosphoinositide In order to examine the accuracy of quantification of phosphoinositide by LC-MS / MS, the effects of various sample amounts and repeated measurements were examined.

Sample Preparation A synthetic 17: 0/20: 4 phosphoinositide sample was prepared as in Example 6.

This sample was subjected to the following methylation treatment. The sample was treated with 150 μL of 0.6 M TMS diazomethane (22-25 ° C.), and after 5 minutes, 15 μL of glacial acetic acid was added to stop the methylation reaction. Thereafter, 700 μL of a mixed solution of chloroform: methanol: water (3:48:47) was added to each sample, and after centrifugation, the organic layer (lower layer) was dispensed into a Spitz test tube and evaporated with a nitrogen gas concentrator. Each sample was dried and redissolved in 100 μL of acetonitrile to prepare a sample for LC-MS measurement.

Eight types of 17: 0/20: 4 phosphoinositide (PI (3) P, PI (4) P, PI (5) P, PI (3,4) P2, PI (4,5) P2, PI ( 3,5) P2, PI (3,4,5) P3 and PI), respectively, 0.02, 0.05, 0.1, 0.2, 5, 10 and 50 pmol (4 times at each injection volume) The signal intensity (peak area) when LC-MS measurement was performed by injecting repeated measurement) was compared. The result of the calibration curve is shown in FIG.

In addition, the calculation formula of each calibration curve is shown in the following table.

As a result of the calibration curve of C17: 0 / C20: 4-PIPs, linearity was obtained in the range of 0.002 pmol to 50 pmol. The limit of detection (LOD) was 0.005 to 0.033 pmol, and the limit of quantification (LOQ) was 0.014 to 0.099 pmol.

From these results, it was confirmed that good quantitative results could be obtained even for trace amounts of phosphoinositide at or below the pmol level.

Similarly, FIG. 3 shows the results of 50 repeated measurements (injection sample amount 10 pmol) for each sample in order to investigate the variation between the measurement runs for each phosphoinositide. The dispersion coefficient was 8.4 to 12.0%, indicating that the variation between measurement runs was small.

ホ ス ホ Phosphoinositide is stable in the prepared sample, and it is expected that the influence on the quantitative result due to the difference in measurement timing between samples is small. In other words, these results indicate that the developed analytical method is excellent in quantification and reproducibility.

Example 8 Separation of Phosphoinositide by Ion Mobility Separation Example 8 demonstrates that separation by the position of the phosphate group of phosphoinositide by ion mobility separation is possible.

The PIP2 isomer was separated by a method other than a chiral column (mass spectrometer only). Attempts were made to separate intact PIP2 (C17: 0 / C20: 4-PI (3,5) P2, -PI (3,4) P2 and -PI (4,5) P2) by using ion mobility. Details are shown below.

The following phosphoinositide purchased from Avanti Polar Lipids (Alabama, USA);
17: 0-20: 4 PI (3,4) P2 (LM-1903)
17: 0-20: 4 PI (4,5) P2 (LM-1904)
17: 0-20: 4 PI (3, 5) P2 (LM-1905)
Thus, a sample for MS analysis was prepared in the same manner as in Example 6.

The measurement was performed under the following conditions.
MS:
Equipment: Q TRAP 6500, SelexION system (ABSciex, Tokyo, Japan)
Control software: Analyst 1.6.2, PeakView (Sciex, Tokyo, Japan)
Ionization / ion source: ESI / IonDrive (Sciex, Tokyo, Japan)
Syringe pump: 7 μL / min Ion source parameters:
Curtain gas: 20
Collision gas: Medium
Ion spray voltage: 5500V (positive ion mode)
Temperature: 500 ° C
Ion source gas 1: 70 psi
Ion source gas 2: 50 psi
The results are shown in FIG. As shown in FIG. 12, PI (4,5) P2 could be separated from PI (3,5) P2 and PI (3,4) P2 due to the difference in compensation voltage (COV). Thus, it was shown that regioisomers of phosphoinositide can be separated without using a column. C17: 0 / C20: 4-PIP2 used for the measurement was not methylated.

Furthermore, further experiments combining ion mobility with LC revealed that phosphoinositide could be detected with higher selectivity.

(Example 9: Analysis of evaluation of change in phosphoinositide composition)
This example shows that changes in phosphoinositide composition in drug-treated cells can be evaluated using the method of the present invention. As in Example 6, the elution order of the positional isomers of phosphate groups was confirmed in advance for each diacylglycerol species.

HEK293T cells (human embryonic kidney epithelial cell line, Open biosystems (Colorado, USA) catalog number HCL4517) were cultured under the conditions recommended by the supplier until day 1 × 10 ⁶ cells / 10 cm dish (day 0). On the second day, H ₂ O ₂ was added to the culture medium to a final concentration of 10 mM, and the cells were scraped and collected with a cell scraper after 0 minutes, 2 minutes, 5 minutes, and 15 minutes, respectively. After centrifuging for 3 minutes, the pellet was recovered, and further washed with PBS (−) to recover the cells.

Thereafter, the following lipid extraction and methylation treatment were performed, and measurement was performed by chiral LC-MS / MS.
Phospholipid extraction For PIPs analysis, 50 μL of 0.2 pmol / μL 17: 0/20: 4-PI3P, 17: 0/20: 4-PI4P, 17: 0/20: 4-PI5P 17: 0/20: 4-PI (3,5) P2, 17: 0/20: 4-PI (3,4) P2, 17: 0/20: 4-PI (4,5) P2, 17 : 0/20: 4-PI (3,4,5) P3, 2 pmol / μL 17: 0/20: 4-PI, and 17: 0/20: 4-PS, which were added to the internal surrogate standard did. Then 2 μL of 1 nmol / μL 8: 0/8: 0-PI (4,5) P2 (as an adsorption inhibitor), 0.75 mL of 2N HCl, 0.75 mL of water, 0.2 mL of 1M NaCl and 3 mL of chloroform was added and shaken. After centrifugation, the lower layer was collected and 1.5 mL of methanol was added to each sample before being applied to a DEAE cellulose column (Santa Cruz Biotechnology, Texas, USA). Lipids adsorbed on the column were mixed with chloroform: methanol (1: 1, v / v) (3 mL) and chloroform: methanol: 28% aqueous ammonia: acetic acid (200: 100: 3: 0.9, v / v) ( 3 mL). Highly acidic lipids including PIPs, PI and PS were then eluted with chloroform: methanol: HCl: water (12: 12: 1: 1, v / v) (1.5 mL). After adding 0.75 mL of water and 0.1 mL of 1M NaCl, the solution was shaken, centrifuged and the lower layer was collected. After the addition of 150 μL of 0.6M TMS-diazomethane (in hexane), the sample was incubated for 10 minutes at room temperature (Nature Methods). Next, 20 μL glacial acetic acid and 0.7 mL wash solution (chloroform: methanol: water (3:48:47, v / v)) were added to each solution. The solution was shaken and centrifuged, and the lower layer was collected. Each sample was dried under nitrogen gas and redissolved in acetonitrile. The resulting PIPs-enriched fraction was analyzed within 1 day of preparation.

The methylation treatment was performed by treating each sample with TMS diazomethane in the same manner as in Example 6.
LC-ESI MS / MS system The sample was analyzed under the following conditions in the same manner as in Example 6.
Mass spectrometer: QTRAP 6500 (ABSciex, Tokyo, Japan)
Pump: Nexera X2 system (Shimadzu Corporation, Kyoto, Japan)
Autosampler: PAL HTC (CTC Analytics, Zwingen, Switzerland)
Column: CHIRALPAK IC-3, 2.1 mm x 250 mm, particle size 3 μm (DAICEL corporation, Osaka, Japan)
Injector: Non-metal injector unit Sample loop: 25 μL PEEK tube flow rate: 0.1 mL / min Injection sample volume: 10 μL
Temperature: Room temperature (chromatographic conditions)
Mobile phase A: acetonitrile + 5 mM ammonium acetate Mobile phase B: methanol + 5 mM ammonium acetate * All these reagents were LC-MS grades purchased from Wako Pure Chemical (Tokyo, Japan).

Gradient condition (linear gradient)
0 minutes, mobile phase B = 40%
1 minute, mobile phase B = 40%
3 minutes, mobile phase B = 85%
14 minutes, mobile phase B = 85%
15 minutes, mobile phase B = 40%
20 minutes, mobile phase B = 40%
・ Mass spectrometer conditions Ionization method: Electrospray (positive ion mode)
Measurement method: Multiple reaction monitoring (MRM)
The measurement results are shown in FIGS.

The results in FIG. 13 show an extracted ion chromatogram of 38: 4 phosphoinositide. It is shown that PI (4,5) P2 decreases and PI (3,4) P2 increases as the H ₂ O ₂ treatment time increases.

The results in FIGS. 14A-D show how PIPs with different diacylglycerols change with H ₂ O ₂ treatment. Statistical processing used one-way analysis of variance and Tukey's multiple comparison test. PIPs with different diacylglycerols were also shown to decrease PI (4,5) P2 and increase PI (3,4) P2 with increasing H ₂ O ₂ treatment time.

Thus, by using the method of the present invention, changes in the phosphoinositide composition (the type of diacylglycerol, the number and position of phosphate groups in PIPs) in the drug-treated cells can be observed in detail.

(Example 10: Evaluation of changes in phosphoinositide composition in genetically engineered mice)
This example shows that changes in phosphoinositide composition in genetically engineered mice can be evaluated using the method of the present invention. As in Example 6, the elution order of the positional isomers of phosphate groups was confirmed in advance for each diacylglycerol species.

Changes in phosphoinositide composition were observed when two genes (INPP4B and PTEN) involved in phosphoinositide metabolism were manipulated.

INPP4B has the following dephosphorylation reaction PI (3,4,5) P3 → PI (3,4) P2 → PI (3) P
PTEN promotes the following dephosphorylation reaction PI (3,4,5) P3 → PI (4,5) P2
(Kofuji S et. Al., Cancer Discov. 2015 Jul; 5 (7): 730-9, Li Chew C et.al., Cancer Discov. 2015 Jul; 5 (7) : 740-51 and VoTT et.al., Cancer Discov. 2015 Jul; 5 (7): 697-700).

Mice generated according to previous papers (Kofuji S et. Al., Cancer Discov. 2015 Jul; 5 (7): 730-9) were used in the experiments. Specifically, mice were prepared as follows. A conditional targeting vector was constructed, and the gene fragment containing the 21st coding exon of the mouse Inpp4b gene was deleted by homologous recombination. One loxP site was introduced into intron 20 and two loxP sites were introduced into intron 21. The LacZ-PGK-Neo ^r cassette was inserted between the two lox sites in intron 21 in the antisense orientation relative to the transcription of Inpp4b. The linear construct was introduced into 1 × 10 ⁷ E14K mouse embryonic stem (ES) cells by electroporation (Sasaki T et. Al., Science 2000; 287: 1040-6). ES cell colonies resistant to G418 (0.3 mg / mL; Life Technologies) were screened by standard PCR to select colonies that had undergone homologous recombination. Recombinant clones were confirmed by standard Southern blots of HindIII digested genomic DNA fragments using a 590 bp probe. Targeted ES cells were injected into C57BL / 6J blastocysts (CLEA Japan, Tokyo, Japan). Chimeric male mice were mated with C57BL / 6JJ female mice to achieve germline inheritance. Inpp4b ^{+ / flox} mice in which the selection cassette was deleted were produced by mating with MeuCre40 transgenic mice (Leneuve P et. Al., Nucleic Acids Res 2003; 31: e21). Similarly, Inpp4b ^{+ / Δ} mice were produced by crossing Inpp4b ^{+ / flox} mice with MeuCre40 mice. PCR confirmed that Inpp4b ^{Δ / Δ} mice were different from Inpp4b ^{+ / Δ} mice and Inpp4b ^{+ / +} mice. An oligo primer common to the Inpp4A ⁺ allele and the Inpp4A ^Δ allele (5′-CCTGCCATGGGGTAGTTTTCT-3 ′), a primer specific for the Inpp4A ⁺ allele (5′-GTTTACATTTGACAGGGTGGTTGG-3 ′), and the Inpp4A ^Δ allele Primers specific for '-TGCTGTCGCCGAAGAAGTTA-3') were combined in the same PCR reaction. Inpp4b ^{+ / Δ} Pten ^+/− mice were generated by crossing Inpp4b ^{+ / Δ} mice with Pten ^+/− mice (Suzuki A et al., Curr Biol 1998; 8: 1169-78). To create Inpp4b ^{Δ / Δ} Pten ^+/− mice, Inpp4b ^{+ / Δ} Pten ^+/− mice were mated with Inpp4b ^{+ / Δ} mice. Triple mutants were made by crossing Inpp4b ^{+ / Δ} Pten ^+/− mice with Akt1 ^{− / −} mice or Akt2 ^{− / −} mice (purchased from Jackson Laboratory). All animal experiments were conducted with the review and approval of the Akita University Animal Experiment Committee.

Of the mice prepared in this way, the thyroid gland obtained from the following mice was used as a sample.
C57 / B6J mice (male or female, 5-6 months old) (N = 3)
INPP4B (− / −) mice (male or female, 5-8 months old) (N = 4)
PTEN (+/-) mice (male or female, 5-7 months old) (N = 4)
INPP4B (− / −) PTEN (+/−) mice (male or female, 5-7 months old) (N = 4)
Each sample was subjected to the following lipid extraction and methylation treatment, and measured by chiral LC-MS / MS.
Phospholipid extraction For PIPs analysis, each mouse tissue was homogenized with 1.5 mL ice-cold methanol and 50 μL 0.2 pmol / μL 17: 0/20: 4-PI3P, 17: 0/20: 4. PI4P, 17: 0/20: 4-PI5P, 17: 0/20: 4-PI (3,5) P2, 17: 0/20: 4-PI (3,4) P2, 17: 0/20 : 4-PI (4,5) P2, 17: 0/20: 4-PI (3,4,5) P3, 2 pmol / μL 17: 0/20: 4-PI, and 17: 0/20: 4 -PS was added and these served as internal surrogate standards. Then 2 μL of 1 nmol / μL 8: 0/8: 0-PI (4,5) P2 (as an adsorption inhibitor), 0.75 mL of 2N HCl, 0.75 mL of water, 0.2 mL of 1M NaCl and 3 mL of chloroform was added and shaken. After centrifugation, the lower layer was collected and 1.5 mL of methanol was added to each sample before being applied to a DEAE cellulose column (Santa Cruz Biotechnology, Texas, USA). Lipids adsorbed on the column were mixed with chloroform: methanol (1: 1, v / v) (3 mL) and chloroform: methanol: 28% aqueous ammonia: acetic acid (200: 100: 3: 0.9, v / v) ( 3 mL). Highly acidic lipids including PIPs, PI and PS were then eluted with chloroform: methanol: HCl: water (12: 12: 1: 1, v / v) (1.5 mL). After adding 0.75 mL of water and 0.1 mL of 1M NaCl, the solution was shaken, centrifuged and the lower layer was collected. After the addition of 150 μL of 0.6M TMS-diazomethane (in hexane), the sample was incubated for 10 minutes at room temperature (Nature Methods). Next, 20 μL glacial acetic acid and 0.7 mL wash solution (chloroform: methanol: water (3:48:47, v / v)) were added to each solution. The solution was shaken and centrifuged, and the lower layer was collected. Each sample was dried under nitrogen gas and redissolved in acetonitrile. The resulting PIPs-enriched fraction was analyzed within 1 day of preparation.

The methylation treatment was performed by treating each sample with TMS diazomethane in the same manner as in Example 6.
LC-ESI MS / MS system The sample was analyzed under the following conditions in the same manner as in Example 6.
Mass spectrometer: QTRAP 6500 (ABSciex, Tokyo, Japan)
Pump: Nexera X2 system (Shimadzu Corporation, Kyoto, Japan)
Autosampler: PAL HTC (CTC Analytics, Zwingen, Switzerland)
Column: CHIRALPAK IC-3, 2.1 mm x 250 mm, particle size 3 μm (DAICEL corporation, Osaka, Japan)
Injector: Non-metal injector unit Sample loop: 25 mL PEEK tube flow rate: 0.1 mL / min Injection sample volume: 10 μL
Temperature: Room temperature (chromatographic conditions)
Mobile phase A: acetonitrile + 5 mM ammonium acetate Mobile phase B: methanol + 5 mM ammonium acetate * All these reagents were LC-MS grades purchased from Wako Pure Chemical (Tokyo, Japan).

Gradient condition (linear gradient)
0 minutes, mobile phase B = 40%
1 minute, mobile phase B = 40%
3 minutes, mobile phase B = 85%
14 minutes, mobile phase B = 85%
15 minutes, mobile phase B = 40%
20 minutes, mobile phase B = 40%
・ Mass spectrometer conditions Ionization method: Electrospray (positive ion mode)
Measurement method: Multiple reaction monitoring (MRM)
The measurement results are shown in FIGS.

15 shows the extracted ion chromatogram of 38: 4 phosphoinositide. PI (3,4) P2 was not observed in wild type and INPP4B (− / −) mice, but production was observed in PTEN (+/−) mice and INPP4B (− / −) PTEN (+/−) ) Further enhancement of production was observed in mice. This result is consistent with the reported activity of PTEN.

The results in FIGS. 16A-D show how PIPs with different diacylglycerols are altered by genetic manipulation. Statistical processing used one-way analysis of variance and Tukey's multiple comparison test. In mice knocked out of a gene that promotes PIP3 degradation, the amount of PIP3 was greatly increased as expected, but a decrease in PIP2 and PIP was not observed. PI (3,4) P2 was not observed in wild type and INPP4B (− / −) mice, but production was observed in PTEN (+/−) mice and INPP4B (− / −) PTEN (+/−) ) Further enhancement of production was observed in mice.

Thus, by using the method of the present invention, changes in the phosphoinositide composition (type of diacylglycerol, the number and position of phosphate groups in PIPs) in genetically engineered mice can be observed in detail.

(Example 11: Separation and quantification of phosphate regioisomers of LPIPs)
This example shows that the phosphate regioisomers of LPIPs can be separated and quantified.

17: 0/20: 4 Using lysed PIPs as a starting material, according to the method of Example 1, 17: 0 lysoPI (3) P, lysoPI (4) P, lysoPI (5) P, Lyso PI (3, 5) P2, lyso PI (3,4) P2, and lyso PI (4, 5) P2 were prepared, respectively.

The measurement conditions were the same as in Example 6 except that the combination of the precursor ion of Q1 and the product ion of Q3 was set as follows.
17: 0 Reso PIP1
Q1: 726.5 (Da) Q3: 327.3 (Da)
17: 0 lyso PIP2
Q1: 834.5 (Da) Q3: 327.3 (Da)
The result of analysis in the same manner as in Example 6 is shown in FIG. Three isomers differing only in the position of the phosphate group were observed with different retention times. From this, quantitative regioisomer-specific analysis becomes possible by separation of the lysophosphoinositide with a phosphate group positional isomer. Therefore, it is possible to analyze the position of the phosphate group and the type of acyl group for lysophosphoinositide using the method of the present invention.

(Note)
As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood. This application claims priority to Japanese Patent Application No. 2016-144177 filed on July 22, 2016 and Japanese Patent Application No. 2017-51354 filed on March 16, 2017. And the entirety of these applications is incorporated herein by reference.

The present invention can be used for pharmaceuticals and their development.

Claims (48)

A compound which is lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate. 1-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol monophosphate, 1-acyl-lysophosphatidylinositol diphosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl-lysophosphatidyl The compound according to claim 1, which is inositol triphosphate or 2-acyl-lysophosphatidylinositol triphosphate. 1-acyl-lysophosphatidylinositol-3-monophosphate, 1-acyl-lysophosphatidylinositol-4-monophosphate, 1-acyl-lysophosphatidylinositol-5-monophosphate, 2-acyl-lysophosphatidylinositol- 3-monophosphate, 2-acyl-lysophosphatidylinositol-4-monophosphate, 2-acyl-lysophosphatidylinositol-5-monophosphate, 1-acyl-lysophosphatidylinositol-3.4-diphosphate, 1-acyl-lysophosphatidylinositol-3.5-diphosphate, 1-acyl-lysophosphatidylinositol-4.5-diphosphate, 2-acyl-lysophosphatidylinositol-3,4-diphosphate, 2- Acyl-lysophosphatidylinositol-3,5-diphosphate, 2-acyl -With lysophosphatidylinositol-4,5-diphosphate, 1-acyl-lysophosphatidylinositol-3,4,5-triphosphate, or 2-acyl-lysophosphatidylinositol-3,4,5-triphosphate A compound according to claim 1. The 2-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl-lysophosphatidylinositol triphosphate or 2-acyl-lysophosphatidylinositol triphosphate Compound. 1-butanyl-lysophosphatidylinositol 4-monophosphate;
1-hexanyl-lysophosphatidylinositol 4-monophosphate;
1-octanyl-lysophosphatidylinositol 4-monophosphate;
1-hexadecanyl-lysophosphatidylinositol 4-monophosphate;
1-9Z-hexadecenyl-lysophosphatidylinositol 4-monophosphate;
1-heptadecanyl-lysophosphatidylinositol 4-monophosphate;
1-octadecanyl-lysophosphatidylinositol 4-monophosphate;
1-9Z-octadecenyl-lysophosphatidylinositol 4-monophosphate;
1-9Z, 12Z-octadecandienyl-lysophosphatidylinositol 4-monophosphate;
1-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 4-monophosphate;
1-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 4-monophosphate;
1-7Z, 10Z, 13Z, 16Z-docosatetraenyl-lysophosphatidylinositol 4-monophosphate;
1-7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenyl-lysophosphatidylinositol 4-monophosphate;
1-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol 4-monophosphate;
2-butanyl-lysophosphatidylinositol 4-monophosphate;
2-hexanyl-lysophosphatidylinositol 4-monophosphate;
2-octanyl-lysophosphatidylinositol 4-monophosphate;
2-hexadecanyl-lysophosphatidylinositol 4-monophosphate;
2-9Z-hexadecenyl-lysophosphatidylinositol 4-monophosphate;
2-heptadecanyl-lysophosphatidylinositol 4-monophosphate;
2-octadecanyl-lysophosphatidylinositol 4-monophosphate;
2-9Z-octadecenyl-lysophosphatidylinositol 4-monophosphate;
2-9Z, 12Z-octadecanedienyl-lysophosphatidylinositol 4-monophosphate;
2-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 4-monophosphate;
2-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 4-monophosphate;
2-7Z, 10Z, 13Z, 16Z-docosatetraenyl-lysophosphatidylinositol 4-monophosphate;
2-7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenyl-lysophosphatidylinositol 4-monophosphate;
2-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol 4-monophosphate;
1-butanyl-lysophosphatidylinositol 4,5-diphosphate;
1-hexanyl-lysophosphatidylinositol 4,5-diphosphate;
1-octanyl-lysophosphatidylinositol 4,5-diphosphate;
1-hexadecanyl-lysophosphatidylinositol 4,5-diphosphate;
1-9Z-hexadecenyl-lysophosphatidylinositol 4,5-diphosphate;
1-heptadecanyl-lysophosphatidylinositol 4,5-diphosphate;
1-octadecanyl-lysophosphatidylinositol 4,5-diphosphate;
1-9Z-octadecenyl-lysophosphatidylinositol 4,5-diphosphate;
1-9Z, 12Z-octadecandienyl-lysophosphatidylinositol 4,5-diphosphate;
1-6Z, 9Z, 12Z-octadecanedienyl-lysophosphatidylinositol 4,5-diphosphate;
1-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 4,5-diphosphate;
1-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 4,5-diphosphate;
2-butanyl-lysophosphatidylinositol 4,5-diphosphate;
2-hexanyl-lysophosphatidylinositol 4,5-diphosphate;
2-octanyl-lysophosphatidylinositol 4,5-diphosphate;
2-hexadecanyl-lysophosphatidylinositol 4,5-diphosphate;
2-9Z-hexadecenyl-lysophosphatidylinositol 4,5-diphosphate;
2-heptadecanyl-lysophosphatidylinositol 4,5-diphosphate;
2-octadecanyl-lysophosphatidylinositol 4,5-diphosphate;
2-9Z-octadecenyl-lysophosphatidylinositol 4,5-diphosphate;
2-9Z, 12Z-octadecandienyl-lysophosphatidylinositol 4,5-diphosphate;
2-6Z, 9Z, 12Z-octadecanedienyl-lysophosphatidylinositol 4,5-diphosphate;
2-8Z, 11Z, 14Z-icosatrienyl-lysophosphatidylinositol 4,5-diphosphate;
2-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 4,5-diphosphate;
1-butanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-octanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-hexadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-9Z-hexadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-heptadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-octadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-9Z-octadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-butanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-octanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-hexadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-9Z-hexadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-heptadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-octadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-9Z-octadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-5Z, 8Z, 11Z, 14Z-icosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate; and 2-4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenyl-lysophosphatidylinositol Selected from the group consisting of 3,4,5-triphosphate,
The compound of claim 1. A composition comprising the compound according to any one of claims 1 to 5 for use as a disease marker. The composition according to claim 6, wherein the disease marker is an inflammation marker or a cancer marker. The composition of claim 7, wherein the cancer comprises prostate cancer. The method for producing a compound according to claim 1, comprising a step of deacylating phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate by contacting with alkylamine. The production method according to claim 9, wherein the alkylamine is methylamine. The production method according to claim 9, wherein the deacylation is a milder condition than a condition for dediacylation. The method according to claim 11, wherein the mild condition is achieved by shortening a reaction time, reducing a concentration of the alkylamine, a reaction temperature, or a combination thereof among the conditions for dediacylation. The mild condition is that methylamine is used as the alkylamine, the reaction time is 10 minutes or less, or the concentration of the methylamine is about 11% or less, the reaction temperature is 53 ° C. or less, or the reaction time, the concentration, and the reaction temperature. The method of claim 11, which is a combination. (A) a step of providing phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate having different mass acyl groups at the 1-position and the 2-position, wherein the acyl group having a desired mass is Existing in a desired position selected from the 1st or 2nd position;
(B) deacylating the phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate by contacting with an alkylamine, and (C) an acyl having a desired mass, if necessary. Removing lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate having a group, lysophosphatidylinositol monophosphate having a desired acyl group at a desired position, lysophosphatidylinositol A process for producing diphosphate or lysophosphatidylinositol triphosphate. Concentrating the phospholipid by contacting the sample with an anion exchange resin;
Protecting a phosphate group in the acidic phospholipid with a protecting group; and detecting, identifying or quantifying lysophosphatidylinositol phosphate in the phospholipid by mass spectrometry;
For detecting, identifying or quantifying lysophosphatidylinositol phosphate. 16. The method of claim 15, further comprising detecting, identifying or quantifying phosphatidylinositol phosphate in the phospholipid. The method of claim 15 or 16, wherein the protecting group comprises an alkyl group. The method according to claim 15 or 16, wherein the protecting group comprises a methyl group. 17. The method according to claim 15 or 16, wherein the mass spectrometry includes a selective reaction monitoring method (SRM) with a triple quadrupole mass spectrometer. The method according to claim 19, further comprising the step of detecting, identifying or quantifying fatty acid side chains and / or phosphate groups of the lysophosphatidylinositol phosphate using liquid column chromatography or ion mobility separation. . 21. The method according to claim 20, further comprising locating a phosphate group. In the mass spectrometry, acylglycerol is selected as a product ion (fragment ion) to detect, identify or quantify the fatty acid side chain and / or phosphate group. the method of. A method for diagnosing cancer by detection, identification or quantification according to any one of claims 15 to 22. A method for measuring, detecting or identifying phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample, the method comprising: A) applying the sample to mass spectrometry (MS); and B) a peak in the MS And locating the phosphate group of the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate according to the elution position of the phosphatidylinositol phosphate. The method according to claim 24, further comprising applying the sample to chromatography in the step A). The mass spectrometry is liquid column chromatography-mass spectrometry (LC-MS);
The method according to claim 24, wherein the specifying step B) is a step of specifying the position of the phosphate group of the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate by the elution position of the peak in the LC-MS. The method according to any one of claims 24 to 26, wherein the total molecular weight of acyl groups contained in the phosphatidylinositol phosphate is also specified in the specifying step. The method according to any one of claims 24 to 26, wherein the type of acyl group contained in the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is also specified in the specifying step. The method according to any one of claims 24 to 26, wherein the position of the phosphate group of the phosphatidylinositol phosphate and the lysophosphatidylinositol phosphate is specified in the same run. The method according to any one of claims 24 to 29, wherein the measurement, detection or identification is carried out by distinguishing phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate from other inositol-containing phospholipids. The method according to any one of claims 24 to 30, wherein the sample is prepared under conditions that do not degrade phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate. 32. The method of claim 31, wherein the condition comprises not performing a deacylation process. The method according to any one of claims 26 to 32, wherein the liquid column chromatography is selected from the group consisting of a chiral column, a reverse phase column, and column chromatography for separating a hydrophilic group and a hydrophobic group. The method according to any one of claims 24 to 33, wherein the liquid column chromatography is combined with ion mobility separation. The method according to any one of claims 24 to 34, further comprising quantifying the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate. The method according to any one of claims 24 to 35, wherein the sample is an intact sample. The method according to any one of claims 24 to 36, wherein the identification is performed without labeling the sample. The mass spectrometry is ion mobility separation-mass spectrometry (IMS-MS);
The specifying step B) is a step of specifying the position of the phosphate group of the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate by the elution position of the peak in the IMS-MS.
25. A method according to claim 24. The method of claim 38, comprising any one or more of the features of claims 27-33, 35-37. An apparatus for measuring, detecting or identifying the position of acyl group and phosphate group of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample under conditions where mass spectrometry (MS) and phosphatidylinositol phosphate are not decomposed And an application unit that applies the sample to the MS. 41. The device of claim 40 further comprising a chromatography device. The mass spectrometry is liquid column chromatography-mass spectrometry (LC-MS), and the application section is an application section that applies the sample to the LC-MS under a condition in which phosphatidylinositol phosphate is not decomposed. 40. The apparatus according to 40. 43. The apparatus according to claim 42, wherein the liquid column chromatography is selected from the group consisting of all column chromatography that separates hydrophilic groups and hydrophobic groups, such as chiral columns and reverse phase columns. The apparatus according to any one of claims 40 to 43, further comprising means for detecting or quantifying the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate. 41. The mass spectrometry is ion mobility separation-mass spectrometry (IMS-MS), and the application section is an application section that applies the sample to the IMS-MS under conditions in which phosphatidylinositol phosphate is not decomposed. The device described in 1. A method for separating or purifying phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample according to the position of a phosphate group while retaining an acyl group, the method comprising: A) Steps applied to graphy-mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS); and B) the elution position of the peak from the eluate of LC or IMS in the LC-MS or IMS-MS Collecting phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate having a phosphate group at the position of interest specified by. From a mixture of two or more of the phosphate group positions of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate, from the number of types of phosphate groups of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate contained in the mixture A method for producing a sample comprising a phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate having a phosphate group at a small number of positions, comprising:
A) applying the sample to liquid column chromatography-mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS); and B) LC or IMS in the LC-MS or IMS-MS Collecting a fraction containing phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate having a phosphate group at a target position specified by the elution position of the peak from the eluate. 48. The method according to claim 47, wherein the number of the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate contained in the mixture is less than the number of types of the phosphate group.

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