JP2010138111A - Phospholipid derivative having thiol group and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new phospholipid derivative capable of easily modifying a metal surface and forming a monomolecular lipid film having high stability and to provide a method for synthesizing the derivative in high efficiency. <P>SOLUTION: The phospholipid derivative expressed by general formula (I) is produced by reacting a phospholipid derivative having an oxyamino group and expressed by formula (III) with one or more kinds of aldehydes expressed by formula (IV) and deprotecting the phosphate group. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a phospholipid derivative having a thiol group, a method for producing the same, and a biosensor using the phospholipid derivative.

  Phospholipids are main components of cell membranes and are important biological components responsible for various biological functions. For the expression of the action of a compound having physiological activity such as a drug, it is considered that the compound first needs to act on the cell membrane. Therefore, measuring the affinity between the phospholipid membrane and various compounds can be an important clue in discussing the physiological activity of various compounds. That is, a phospholipid derivative obtained by adding a functional functional group to a phospholipid can be used as a probe molecule useful for elucidating various biological functions.

  One experimental system for measuring the affinity between phospholipid membranes and various compounds is the quartz crystal microbalance method. The crystal oscillator microbalance method is a micro-mass spectrometry method that uses the piezoelectric effect of the crystal oscillator. From the amount of change in the frequency of the resonance vibration according to the mass change that accompanies the adsorption, dissociation, and dissolution of substances that occur on the crystal oscillator surface. The amount of mass change can be converted. Then, by modifying the gold electrode surface on the surface of the crystal oscillator, various biological reactions can be measured and quantified, and changes in substances due to interaction reactions can be analyzed kinetically. In order to measure the affinity between the phospholipid membrane and various compounds, it is necessary to fix the phospholipid on the gold electrode surface of the crystal oscillator surface. Nakayama et al. (Non-Patent Document 1) uses a DMPC (1,2-dimyristoyl-sn-glycerol-3-phosphocholine) lipid multilayer that can be easily immobilized on the gold electrode surface. There were many points to be resolved, such as sex.

  A phospholipid having a thiol group can be easily modified on a metal surface and forms a monomolecular film, and thus is useful as a sensor chip for a surface plasmon resonance method or a quartz crystal microbalance method. For example, a method is known in which a phospholipid bilayer is modified on the surface of a metal fine particle or metal substrate via a thiol group and used for biosensing (Patent Documents 1 to 3). In addition, a method of modifying a phospholipid bilayer membrane having a thiol group on a metal fine particle or a metal substrate surface and using it for biosensing is also known (Patent Document 4).

  However, since phospholipids having a thiol group are not commercially available, a simple production method thereof is required. In this regard, the conventional phospholipid synthesis methods, the amidite method and the multi-step synthesis method using a condensing agent, are complicated and unsuitable for practical use.

JP-A-9-236571 JP2007-40974 JP 2008-12444 A JP 2008-7815 Kamihira M. et al., "Interaction of tea catechins with lipid bilayers developing by a quartz-crystal microbalance analysis." Biosci. Biotechnol. Biochem. 2008, 72, 1372-1375.

An object of the present invention is to provide a novel phospholipid derivative capable of easily modifying a metal surface and forming a highly stable lipid monomolecular film, and an efficient synthesis method thereof.

  The inventors have succeeded in developing a synthesis method using an phospholipid having an oxyamino group in the head group as shown below as an intermediate (Org. Lett. 2008, 10, 4847.). This is a method in which various head groups can be easily introduced by oxime bond formation by utilizing the high nucleophilicity of the oxyamino group.

In synthesizing a novel phospholipid derivative having a thiol group, the inventors applied the above-described intermediate having an oxyamino group. That is, phospholipids having an oxyamino group in the tail group were synthesized and various tail groups were introduced by oxime bond formation (the following formula).

  Furthermore, in order to more firmly fix the lipid monolayer to the gold electrode surface, a phospholipid derivative in which a thiol group containing a sulfur atom having a high affinity for gold is introduced at the tail group end using the above synthesis method. The synthesis of was studied. Thus, the inventors have completed a method for easily synthesizing a phospholipid derivative having a thiol group by a unique thiol group introduction method. Then, a sensor chip using this phospholipid derivative was successfully produced, and the present invention was completed.

That is, the present invention provides a phospholipid derivative represented by the general formula (I).
(However, n1 and n2 are integers from 0 to 20, m1 and m2 are integers from 0 to 20,
R ₁ and R ₂ are independently selected from aryl, C 1-10 alkyl, alkenyl, and alkynyl;
X ^{_{is, CH 2 CH 2 -N + (}} CH 3) 3, CH 2 CHNH 3 + COO -, is selected from CH ₂ CHNH ₂ OH and C ₆ H ₁₁ O _5. )

The phospholipid derivative (I) is, in one embodiment, R ₁ and R ₂ are both C ₆ H ₄ .
In some embodiments, X is CH ₂ —N ⁺ (CH ₃ ) ₃ .
Further, in some embodiments, m1 and m2 are the same (eg, both m1 and m2 are 1).

As a suitable example of the phospholipid derivative of the present invention, for example, a phospholipid derivative represented by the following general formula (II) can be mentioned.
(However, n1 and n2 are integers from 0 to 20.)

  In one embodiment, in the phospholipid derivative (II), n1 and n2 are the same (for example, n1 and n2 are both 9).

The present invention also provides a method for producing a phospholipid derivative represented by the above general formula (I). The method comprises reacting a phospholipid derivative having an oxyamino group represented by formula (III) with one or two aldehydes represented by formula (IV), and then deprotecting the phosphate group. Features.
(Where n1 and n2 are integers from 0 to 20, m1 and m2 are integers from 0 to 20, and R1 and R2 are independently aryl, C1-10 alkyl, alkenyl, and alkynyl. X is selected from CH2CH2-N + (CH3) 3, CH2CHNH3 + COO-, CH2CHNH2OH and C6H11O5.)

  Here, the phospholipid derivative having an oxyamino group represented by the above formula (III) is obtained by, for example, amidating a diester represented by the formula (V) and then an alcohol represented by the formula (VI) by the phosphoramidite method. It can manufacture by deprotecting a phthalimide after coupling.

Here, the protecting group for the phosphoric acid moiety is not particularly limited, and examples thereof include a cyanoethyl group and a benzyl group.

In certain embodiments, R ₁ and R ₂ are both C ₆ H ₄ .
In some embodiments, X is CH ₂ —N ⁺ (CH ₃ ) ₃ .
Further, in some embodiments, m1 and m2 are the same (eg, both m1 and m2 are 1).

The present invention also provides a method for producing a phospholipid derivative represented by the above general formula (II). The production method is characterized in that the phospholipid derivative having the oxyamino group represented by the formula (III ′) is reacted with the aldehyde represented by the formula (IV ′), and then the phosphate group is deprotected. .
(However, n1 and n2 are integers from 0 to 20.)

Here, the phospholipid derivative having an oxyamino group represented by the above formula (III) is, for example, a diester represented by the formula (V) is amidated and then represented by the formula (VI ′) by the phosphoramidite method. After coupling with alcohol, it can be produced by deprotecting phthalimide.

  In one embodiment, n1 and n2 are the same (for example, n1 and n2 are both 9).

  The present invention further provides a sensor comprising the phospholipid derivative according to the present invention immobilized on a carrier having a metal surface.

  Here, the form of the carrier is not particularly limited, and examples thereof include fine particles and a substrate. Moreover, gold can be mentioned as a preferable example of a metal.

  According to the present invention, a phospholipid derivative having a thiol group can be efficiently synthesized. The phospholipid derivative of the present invention can easily modify a metal surface via a thiol group, and forms a highly stable lipid monomolecular film. The carrier on which the phospholipid derivative of the present invention is immobilized can be used as a biosensor useful for elucidating various biological functions.

1. Phospholipid derivative of the present invention The phospholipid derivative of the present invention is represented by the following general formula (I).
Here, n1 and n2 are each an integer of 0 to 20, preferably 5 to 20, more preferably 8 to 16, and may be the same or different. n1 and n2 are preferably the same in terms of ease of production.
M1 and m2 are each an integer of 0 to 20, preferably 0 to 10, more preferably 0 to 5, and may be the same or different. m1 and m2 are preferably the same in terms of ease of production, but when different, the difference between n1 + m1 and n2 + m2 is 3 or less, preferably 2 or less.

R ₁ and R _{2 in the} tail portion are independently selected from aryl such as phenyl (C ₆ H ₄ ), C 1-10, preferably C 1-5 alkyl, alkenyl, and alkynyl. R ₁ and R ₂ are preferably the same in terms of ease of production.

Head portion, choline, serine, ethanolamine, inositol (X ^{_{is, CH 2 CH 2 -N + (}} CH 3) 3, CH 2 CHNH 3 + COO -, CH 2 CHNH 2 OH, C 6 H 11 O 5 However, it is not limited to these as long as the purpose of the present invention is met.

A preferred example of the phospholipid derivative of the present invention is a phospholipid derivative represented by the following general formula (II).
(However, n1 and n2 are integers from 0 to 20.)

  In the examples described later, the production of the above phospholipid derivatives based on phosphatidylcholine was described (n1 = n2 = 9).

  Since the phospholipid derivative of the present invention has a thiol group at the end of the tail portion, it can be easily immobilized on metals such as gold, silver and copper.

2. Production method of phospholipid derivative of the present invention The phospholipid derivative of the present invention can be produced according to the following steps.

  That is, a phospholipid derivative in which the amino group of the tail group is protected with phthalimide is prepared, and this is deprotected to obtain an intermediate having an oxyamino group. Next, the intermediate is reacted with one or two aldehydes to synthesize a phospholipid derivative having a thiol group by an oxime bond forming reaction. Finally, the protecting group of the phosphate group is removed to obtain the desired phospholipid derivative.

2.1 Synthesis of intermediate having oxyamino group The intermediate having oxyamino group includes (1) synthesis of diester, (2) amidation, (3) synthesis of phospholipid derivative by phosphoramidite method, (4 ) It can be synthesized by a deprotection step.

(1) Synthesis of diester
As described above, N-hydroxyphthalimide is reacted with a carboxylic acid (fatty acid) having a carboxyl group protected with a tert-butyl group. The reason for protecting the carboxyl group with a tert-butyl group is to prevent a reaction between carboxylic acids from occurring as a side reaction. For protecting the carboxyl group, a methyl group, an ethyl group, a benzyl group or the like may be used in addition to the tert-butyl group. After the reaction, the tert-butyl group is deprotected in the presence of formic acid or the like to obtain a carboxylic acid having an amino group protected with phthalimide. Reaction conditions can be appropriately set by those skilled in the art with reference to the examples described later.

Next, two molecules of this carboxylic acid (which may be the same or different) are condensed with a diol protected with a p-methoxybenzyl (PMB) group, and then the PMB group is deprotected. To obtain the diester.

(2) Amidite formation Next, the obtained diester is induced to amidite. The amidite can be derived by coupling a diester with an organic phosphorus reagent in which an oxygen atom is protected with a protecting group such as a cyanoethyl group, according to a conventional method.

(3) Synthesis of phospholipid derivatives by phosphoramidite method The obtained amidite is coupled with amino acids such as choline, ethanolamine, serine and the like using 1H-tetrazole as an activator by phosphoramidite method. A phospholipid derivative having an amino group is synthesized.

(4) Deprotection Finally, the phthalimide group is deprotected (Org. Lett. 2008, 10, 4847.) to obtain an intermediate having the target oxyamino group.

2.2 Synthesis of phospholipid derivatives having a thiol group by an oxime bond formation reaction A phospholipid derivative having a thiol group is obtained by (1) coupling an intermediate having an oxyamino group and an aldehyde using an oxime bond formation reaction. (2) It can be obtained by deprotection.

(1) Oxime bond formation reaction Oxyamino groups are highly reactive with aldehydes, and oxime bond formation proceeds at room temperature without the need for special activators. Therefore, a phospholipid derivative having a target thiol group in the tail portion can be obtained efficiently and simply.

(2) Deprotection Finally, the protecting group of the phosphate group is removed. The deprotection conditions are appropriately determined according to the protecting group used in accordance with a conventional method.

3. Sensor Since the phospholipid derivative of the present invention has a thiol group in the Tail Group, metal surfaces such as gold can be easily modified. Phospholipids are the main components of cell membranes and are responsible for various biological functions. For the expression of the action of a compound having physiological activity such as a drug, it is first necessary that the compound acts on the cell membrane. If the phospholipid derivative of the present invention is immobilized on the surface of a sensor chip such as a quartz crystal microbalance or surface plasmon resonance, it can be used as a biosensor for probing biological functions through the affinity between the phospholipid membrane and various compounds. Available. When the sensor chip surface is an inorganic material or the like, a gold film may be formed on the surface to immobilize the phospholipid derivative.

The shape of the carrier having a metal surface for modifying the phospholipid derivative of the present invention is not particularly limited, and may be a fine particle or a substrate. In addition, fine particles on which a phospholipid derivative is fixed may be further aligned on a substrate of metal or the like (see JP 2008-7815 A). The metal surface used is not particularly limited as long as it has a high affinity for thiol groups, and examples thereof include gold.
The following are technologies that can be used as the sensor of the present invention.

3.1 Crystal Oscillator Microbalance Method Crystal Oscillator Microbalance Method is a micro mass analysis method that uses the piezoelectric effect of crystal oscillators, and changes in mass due to adsorption, dissociation, and dissolution of substances that occur on the surface of the crystal oscillator. The amount of mass change can be converted from the amount of change in frequency of resonance vibration according to the above. By modifying the gold electrode surface of the crystal oscillator surface, various interactions such as DNA, proteins, receptors, and drugs can be measured and quantified with high sensitivity and kinetics. Can be analyzed. The phospholipid derivative of the present invention is easily immobilized on the gold electrode surface on the surface of the crystal oscillator, whereby the affinity between the phospholipid membrane and various compounds can be measured.

3.2 Surface Plasmon Resonance Method A surface plasmon resonance sensor is a biosensor capable of analyzing intermolecular interactions in real time on a sensor chip using surface plasmon resonance. Already commercially available surface plasmon resonance sensors (for example, Sensor Film Au for SPR measurement by BIAcore) are readily available. On the sensor chip, the phospholipid derivative of the present invention is stably adsorbed with high binding properties. Thus, a microchannel is provided on a sensor chip to which a phospholipid derivative is bound, and a sample containing an analyte is fed at a constant flow rate. If the phospholipid derivative interacts with the analyte in the sample, binding and dissociation are detected optically by the surface plasmon phenomenon and can be monitored in real time as a sensorgram. The surface plasmon resonance sensor has an advantage that it can detect a minute amount of sample with high sensitivity and can analyze the kinetics of interaction in real time. Therefore, the surface plasmon resonance sensor to which the phospholipid derivative of the present invention is bound is useful as a biosensor for analyzing the interaction between various substances and the phospholipid membrane.

  The phospholipid derivative of the present invention can also be applied to two-dimensional surface plasmon resonance (SPR). Formation of a gold film on a finely processed inorganic material (semiconductor material) can be easily performed by a conventional method. A phospholipid derivative is immobilized on the gold film thus formed and used as a biosensor.

3.3 Field-Effect Transistor (FET)
A field-effect transistor (FET) is a technology widely used in biosensors using electrical detection. When the current flowing from the source electrode to the drain electrode is controlled by the gate electrode, which is the third electrode, and the protein-protein interaction is caused on the surface of the gate electrode, the charge of the gate electrode changes, so the current response changes. . If this gate electrode is covered with a gold film, the phospholipid derivative of the present invention can be easily immobilized and used as a biosensor using FET technology.

  In order to detect the interaction between the phospholipid membrane and the analyte by the FET, the reaction needs to occur as close to the surface of the gate electrode as possible. In the sensor of the present invention, since the affinity between the phospholipid derivative immobilized on the electrode surface and the analyte is observed, detection with extremely high sensitivity can be expected.

  Without being limited to the above examples, the phospholipid derivative of the present invention can also be applied to biosensors using transmitted light, reflected light, and near-field light of electromagnetic waves such as infrared rays. In addition, well-known techniques used for other immunodetection: development of biosensors using solid-phase immunization methods such as immunoprecipitation, western blotting, dot blotting, slot blotting, ELISA, and RIA as appropriate Is also possible.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to these Examples.

Example 1 Synthesis of Phospholipid Derivative <Materials and Equipment>
For NMR, [ ¹ H NMR (270 MHz), ¹³ NMR (68 MHz)] spectrum is JEOL EX-270, [ ¹ H NMR (500 MHz), ¹³ C NMR (125 MHz)] spectrum is JEOL ECA ³¹ P NMR (202 MHz) spectra were measured using JEOL ECA-500 using -500 and JEOL (-500. ¹ H NMR chemical shift was 1 million from the internal standard tetramethylsilane (δ). the indicated each, coupling constants are given in Hz each abbreviation is as follows:.. s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad of ¹³ C NMR Chemical shifts are given in ppm relative to the centerline of the ( ² H) chloroform triplet at 77.0 ppm ³¹ P NMR chemical shifts are given in ppm with 60% H ₃ PO ₄ as internal standard.

  High performance mass spectrometry (HRMS) was measured using a JEOL Mstation 700. Fast atom bombardment (FAB) mass spectra were measured using 3-nitrobenzylalcohol as a matrix.

Thin layer chromatography (TLC) was performed using Merck pre-coated plates (thickness 0.25 mm, silica gel 60 F ₂₅₄ ). Preliminary TLC separation was performed using a 7 × 20 cm plate (thickness 0.50 mm, Merck silica gel 60 F ₂₅₄ ) using 10% methanol in chloroform as a solvent.

  Flash chromatography was performed using KANTO CHEMICAL Silica Gel 60 (spherical) 40-50 (m, Silica Gel 60 (spherical) 63-210 (m or Silica Gel 60 N (spherical, neutral) 63-210 (m .

  Commercially available products and solvents were used as they were except for the following. Dichloromethane, diethyl ether, n-hexane, tetrahydrofuran, toluene: Used after drying with molecular sieves 4A. Methanol, ethanol, acetonitrile: Used after drying with molecular sieves 3A. All reactions sensitive to oxygen or water were performed under an argon atmosphere.

<Abbreviation>
Ac: acetyl
aq .: aqueous
DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
DIBAL: diisobuthylalminium hydride
DMAP: 4-dimethylaminopyridine
DMF: N, N-dimethylformamide
DMSO: dimethylsulfoxide
Et: ethyl
Et ₃ N: triethylamine
h: hour
i-: iso-
Me: methyl
min: minute
n-: normal-
p-: para
PMB: p-methoxybenzyl
Pr: propyl
pTLC: preparative TLC
quant .: quantative
t-: tertiary-
TBHP: tert-butyl hydroperoxide
TLC: thin-layer chromatography
WSC: water soluble carbodiimide 1-ethyl-3- (3-dimethylaminoproryl) carbodiimide hydrochloride

Method for producing phospholipid derivative
DMSO (120 mL), 2 (8.00 g, 24.9 mmol) and K ₂ CO ₃ (8.60 g, 62.3 mmol) were added to N-hydroxyphthalimide 1 (3.39 g, 20.8 mmol) at 0 ° C, and 30 at 80 ° C. Stir for minutes. Water was added, and the mixture was extracted 3 times with CH ₂ Cl _{2. The} organic layer was washed with saturated brine, dried over Na ₂ SO ₄ and concentrated under reduced pressure. Thereafter, the product was purified by silica gel column chromatography (n-hexane: EtOAc = 10: 1) to obtain 3 (7.98 g, 95%) of a white solid.
¹ H NMR (500 MHz, CDCl ₃ ): d 1.24-1.38 (m, 10H), 1.43 (s, 9H), 1.52-1.65 (m, 4H), 1.79 (quint, J = 6.7 Hz, 2H), 2.20 (t, J = 6.7 Hz, 2H), 4.20 (t, J = 6.7Hz, 2H), 7.70-7.77 (m, 2H), 7.80-7.87 (m, 2H).

HCOOH (30 mL) was added to 3 (7.98 g, 20.6 mmol) at room temperature, and the mixture was stirred at room temperature for 2 hours. Then, it concentrated under pressure reduction and obtained 4 (6.86 g, quant) of the white solid.
¹ H NMR (500 MHz, CDCl ₃ ): d 1.24-1.38 (m, 10H), 1.48 (quint, J = 7.3 Hz, 2H), 1.64 (quint, J = 7.3 Hz, 2H), 1.79 (quint, J = 7.3 Hz, 2H), 2.35 (t, J = 7.3 Hz, 2H), 4.20 (t, J = 7.3 Hz, 2H), 7.70-7.78 (m, 2H), 7.80-7.88 (m, 2H).

Under Ar atmosphere, add 4 (3.00 g, 8.65 mmol) to toluene (40 mL), 5 (873 mg, 4.12 mmol), WSC (3.16 g, 16.5 mmol), DMAP (101 mg, 0.828 mmol) at room temperature, The mixture was stirred at 60 ° C for 13 hours. A saturated aqueous ammonium chloride solution was added, and the mixture was extracted 3 times with AcOEt. The organic layer was washed with saturated brine, dried over Na ₂ SO ₄ and concentrated under reduced pressure. Thereafter, the residue was purified by silica gel column chromatography (n-hexane: EtOAc = 3: 2) to obtain 6 (2.89 g, 81%) of a yellow oil.
¹ H NMR (500 MHz, CDCl ₃ ): d 1.24-1.38 (m, 20H), 1.48 (quint, J = 7.3 Hz, 4H), 1.60 (quint, J = 7.3 Hz, 4H), 1.78 (quint, J = 7.3 Hz, 4H), 2.28 (t, J = 7.3 Hz, 2H), 2.32 (t, J = 7.3 Hz, 2H), 3.50-3.60 (m, 2H), 3.80 (s, 3H), 4.10-4.25 (m, 5H), 4.33 (dd, J = 4.0, 11.6 Hz, 1H), 4.45 (d, J = 11.6 Hz, 1H), 4.49 (d, J = 11.6 Hz, 1H), 5.15-5.25 (m, 1H), 6.87 (d, J = 8.6 Hz, 2H), 7.23 (d, J = 8.6 Hz, 2H), 7.70-7.78 (m, 4H), 7.80-7.88 (m, 4H).

CH ₂ Cl ₂ / H ₂ O = 10/1 (33 mL) and DDQ (0.90 g, 3.98 mmol) were added to 6 (2.89 g, 3.32 mmol) at room temperature, and the mixture was stirred at room temperature for 4 hours. After addition of 30% aqueous sodium thiosulfate solution, extracted three times with CH ₂ Cl _2, a saturated aqueous sodium hydrogen carbonate solution the organic layer was added, extracted three times with CH ₂ Cl _2, the organic layer was washed with brine , Dried over MgSO ₄ and concentrated under reduced pressure. Then, it was purified by silica gel column chromatography (n-hexane: EtOAc = 1: 1) to obtain 7 (2.32 g, 93%) as a yellow solid.
¹ H NMR (500 MHz, CDCl ₃ ): d 1.24-1.38 (m, 20H), 1.42-1.55 (m, 4H), 1.58-1.69 (m, 4H), 1.79 (quint, J = 7.9 Hz, 4H) , 2.16 (br s, 1H), 2.33 (t, J = 7.3 Hz, 2H), 2.35 (t, J = 7.3 Hz, 2H), 3.67-3.75 (m, 2H), 4.20 (t, J = 6.7 Hz , 4H), 4.24 (dd, J = 5.5, 12.5 Hz, 1H), 4.32 (dd, J = 4.5, 12.5 Hz, 1H), 5.03-5.11 (m, 1H), 7.23 (d, J = 8.6 Hz, 2H), 7.70-7.80 (m, 4H), 7.80-7.90 (m, 4H).

Under Ar atmosphere at room temperature 7 (1.00 g, 1.33 mmol) CH ₂ Cl ₂ (10 mL), 2-cyanoethyl-N, N, N ', N'-tetraisopropylphosphorodiamidite (8) (602 mg, 2.00 mmol), 1H-tetrazole (75.0 mg, 1.07 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. A saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted 3 times with CH ₂ Cl ₂ , dried over MgSO ₄ , and concentrated under reduced pressure. Concentrated under reduced pressure. Then, it was purified by silica gel column chromatography (n-hexane: EtOAc: Et ₃ N = 22: 11: 1) to obtain 9 (1.16 g) as a yellow solid.

Crude product containing 9 at room temperature under Ar atmosphere CH ₂ Cl ₂ / CH ₃ CN = 1/1 (10 mL), choline tosylate (336 mg, 1.22 mmol), 1H-tetrazole (172 mg, 2.46 mmol) And stirred at room temperature for 1.5 hours. After confirming the disappearance of 9 by TLC, the mixture was concentrated under reduced pressure. Then, under _{Ar, CH 2 Cl 2 (10 mL} ), TBHP (185 mL, 1.85 mmol) and the mixture was stirred for 1 hour at room temperature. After addition of 30% aqueous sodium thiosulfate solution, and extracted ten times with CH ₂ Cl _2, a saturated aqueous sodium hydrogen carbonate solution the organic layer was added, and the mixture was extracted ten times with CH ₂ Cl _2, the organic layer was washed with brine , Dried over MgSO ₄ and concentrated under reduced pressure. Then, silica gel column chromatography _{(CHCl 3: MeOH: H 2} O = 65: 25: 4) to give the 10 (412 mg) as a yellow solid.
¹ H NMR (500 MHz, CDCl ₃ ): d 1.21-1.38 (m, 20H), 1.38-1.53 (m, 4H), 1.53-1.70 (m, 4H), 1.70-1.85 (m, 4H), 2.20- 4.50 (m, 27H), 4.60-4.72 (m, 2H), 5.20-5.30 (m, 1H), 7.70-7.80 (m, 4H), 7.80-7.90 (m, 4H).

Under an Ar atmosphere, CH ₂ Cl ₂ (3 mL) and methyl hydrazine (38.0 mL, 0.726 mmol) were added to 10 (320 mg, 0.330 mmol) at 0 ° C., and the mixture was stirred at 0 ° C. for 1.5 hours. The reaction mixture was filtered through celite and concentrated under reduced pressure to give 11 as a colorless liquid.

At room temperature, add CHCl ₃ / CH ₃ OH = 1/1 (4 mL), 4- (mercaptomethyl) benzaldehyde (12) (120 mg, 0.805 mmol) to 11 (272 mg, 0.414 mmol), and then at room temperature for 14 hours Stir. Then concentrated under reduced pressure, silica gel column chromatography _{(CHCl 3: MeOH = 10:} 1 to CHCl 3: MeOH: H 2 O = 65: 25: 4) to give to give a yellow oil 13 (52.6 mg) It was.
¹ H NMR (500 MHz, CDCl ₃ ): d 1.20-3.00 (m, 38H), 3.32-4.80 (m, 27H), 5.14-5.35 (m, 1H), 7.10-8.10 (m, 10H).

At room temperature, CH ₂ Cl ₂ (1 mL) and tert-butylamine (0.1 mL) were added to 13 (39.7 mg, 40.6 mmol), and the mixture was stirred at room temperature for 12 hours. Then, it concentrated under pressure reduction and 14 of the yellow solid was obtained.
¹ H NMR (500 MHz, CDCl ₃ ): d 1.20-1.80 (m, 54H), 1.80-4.45 (m, 16H), 5.10-5.25 (m, 1H), 7.20-8.30 (m, 10H).

The above reaction is summarized as follows.

  Here, synthesis of a phospholipid derivative based on phosphatidylcholine was examined, but phospholipid derivatives based on phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, etc. can be easily synthesized by the same method.

[Example 2] Analysis of interaction between green tea catechins and phospholipid membrane using a sensor chip to which a phospholipid monolayer is bound <Method and result>
(1) 3 μL of the phospholipid derivative (0.1 mg / mL) synthesized in Example 1 was added to a gold electrode and left for 30 minutes.
(2) After washing with a solvent, it was dried.
(3) The mass increase (decrease in the number of Hz) was confirmed by using a quartz crystal microbalance (AFFINIX Q4 manufactured by Initiative), and it was confirmed that a sensor chip was created (Fig. 1).
(4) After mounting the sensor chip on the quartz crystal microbalance, PBS was added to familiarize the sensor chip.
(5) As a result of adding 500 μL of epicatechin gallate (10 mM), which is one of green tea catechins, an increase in weight (decrease in Hz number) was observed. That is, the interaction between the phospholipid monolayer and the catechins by using the sensor chip could be detected (FIG. 2).

  According to the present invention, a phospholipid derivative having a thiol group can be efficiently synthesized. This phospholipid derivative can easily modify the metal surface via a thiol group and forms a highly stable lipid monolayer. Since the metal carrier on which the phospholipid derivative of the present invention is immobilized can be easily modified to a sensor chip such as a quartz crystal microbalance method or a surface plasmon resonance method, it is useful as a biosensor useful in elucidating various biological functions. Available.

FIG. 1 shows an increase in mass of a gold electrode on which phospholipid derivative 1 is immobilized by quartz crystal microbalance. FIG. 2 shows the result of analyzing the interaction between a phospholipid monomolecular film and catechins using a sensor chip on which phospholipid derivative 1 is immobilized.

Claims (21)

Phospholipid derivatives represented by general formula (I):
However, n1 and n2 are integers from 0 to 20, m1 and m2 are integers from 0 to 20,
R ₁ and R ₂ are independently selected from aryl, C 1-10 alkyl, alkenyl, and alkynyl;
X ^{_{is, CH 2 CH 2 -N + (}} CH 3) 3, CH 2 CHNH 3 + COO -, is selected from CH ₂ CHNH ₂ OH and C ₆ H ₁₁ O _5. The phospholipid derivative according to claim 1, wherein R ₁ and R ₂ are both C ₆ H ₄ . The phospholipid derivative according to claim 1 or 2, wherein X is CH ₂ -N ⁺ (CH ₃ ) ₃ .   The phospholipid derivative according to any one of claims 1 to 3, wherein m1 and m2 are the same.   The phospholipid derivative according to any one of claims 1 to 3, wherein both m1 and m2 are 1. A phospholipid derivative represented by the general formula (II):
However, n1 and n2 are integers from 0 to 20.   The phospholipid derivative according to any one of claims 1 to 6, wherein n1 and n2 are the same.   The phospholipid derivative according to any one of claims 1 to 6, wherein n1 and n2 are both 9. A process for producing a phospholipid derivative represented by the general formula (I):
A method comprising reacting a phospholipid derivative having an oxyamino group represented by the formula (III) with one or two aldehydes represented by the formula (IV) and then deprotecting the phosphate group .
However, n1 and n2 are integers from 0 to 20, m1 and m2 are integers from 0 to 20,
R ₁ and R ₂ are independently selected from aryl, C 1-10 alkyl, alkenyl, and alkynyl;
X ^{_{is, CH 2 CH 2 -N + (}} CH 3) 3, CH 2 CHNH 3 + COO -, is selected from CH ₂ CHNH ₂ OH and C ₆ H ₁₁ O _5. After the phospholipid derivative having an oxyamino group represented by the formula (III) amidates the diester represented by the formula (V) and then coupled with the alcohol represented by the formula (VI) by the phosphoramidite method, The method according to claim 9, which is produced by deprotecting phthalimide.
The method according to claim 9 or 10, wherein R ₁ and R ₂ are both C ₆ H ₄ . X is ^{_{CH 2 -N + (CH 3)}} 3, The method according to any one of claims 9-11.   13. A method according to any one of claims 9 to 12, wherein m1 and m2 are the same.   The method according to any one of claims 9 to 12, wherein m1 and m2 are both 1. A process for producing a phospholipid derivative represented by the general formula (II):
A method comprising reacting a phospholipid derivative having an oxyamino group represented by the formula (III ′) with an aldehyde represented by the formula (IV ′) and then deprotecting the phosphate group.
However, n1 and n2 are integers from 0 to 20. A phospholipid derivative having an oxyamino group represented by the formula (III ′) amidated the diester represented by the formula (V), and then coupled with the alcohol represented by the formula (VI ′) by the phosphoramidite method. The process according to claim 15, which is subsequently produced by deprotecting the phthalimide.
  The method according to any one of claims 9 to 16, wherein n1 and n2 are the same.   The method according to any one of claims 9 to 16, wherein n1 and n2 are both 9.   A sensor comprising the phospholipid derivative according to claim 1 immobilized on a carrier having a metal surface.   The sensor according to claim 19, wherein the carrier is a fine particle or a substrate.   21. A sensor according to claim 20, wherein the metal is gold.